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.     

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.     

Metabolic zonation in liver of diabetic rats. Zonal distribution of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucose-6-phosphatase and succinate dehydrogenase

The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of diabetic rats. 48 h after alloxan treatment the following alterations were observed, intermediate values being reached after 24 h: Blood glucose, acetoacetate and beta-hydroxybutyrate were increased to more than 500%; liver glycogen was reduced to about 10%. Portal vein insulin was reduced to below 10%, portal glucagon was increased to almost 200%. The glucogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were enhanced to 320% and 150%, respectively. The glycolytic enzymes glucokinase and pyruvate kinase L (differentiated from the M2 isoenzyme with a specific anti-L-antibody) were lowered to 50% and 75%, respectively. The citrate cycle enzyme succinate dehydrogenase remained unchanged. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3:1, as measured in microdissected tissue samples, was enhanced to about 4:1 with activities elevated to 230% and 190%, respectively, in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7, as determined with the microdissection technique, was reduced to about 1:1.4 with levels lowered to 55% and 45%, respectively, in the two zones. The even zonal distribution of pyruvate kinase M2 remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Modulation by oxygen of zonal gene expression in liver studied in primary rat hepatocyte cultures

The different endowment with key enzymes and thus different metabolic capacities of periportal and perivenous cell types led to the model of "metabolic zonation." The periportal and perivenous hepatocytes receive different signals owing to the decrease of substrate concentrations including O2 and hormone levels during passage of blood through the liver sinusoids. These different signal patterns should be important for the short-term regulation of metabolism and also for the long-term induction and maintenance of the different enzyme pathways by control of gene expression. The periportal to perivenous drop in oxygen tension was considered to be a key regulator in the zonated expression of carbohydrate-metabolizing enzymes. In primary hepatocyte cultures, glucagon activated the phosphoenolpyruvate carboxykinase (PCK) gene to higher levels under arterial than under venous oxygen. The insulin-dependent activation of the glucokinase (GK) gene was reciprocally modulated by oxygen. Exogenously added hydrogen peroxide mimicked the effects of arterial oxygen on both the glucagon-dependent PCK gene and the insulin-dependent GK activation. Therefore, the oxygen sensor could be a hydrogen peroxide-producing oxidase which could contain a heme group for "measuring" the O2 tension. This notion was corroborated by the finding that CO mimicked the positive effect of O2 on PCK gene activation. Transfection of PCK promoter-CAT gene constructs into primary hepatocytes showed that the oxygen modulation of the PCK gene activation occurred in the region -281/+69. The modulation by O2 was not mediated by isolated cAMP-responsive elements. Nuclear protein extracts prepared from hepatocytes cultured under venous Po2 as compared to arterial Po2 showed an enhanced binding activity to the promoter fragment -149/-43. Oxidative conditions such as H2O2 reduced the DNA-binding activity, thus supporting the role of H2O2 as a mediator in the O2 response of the PCK and GK genes.     

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.     

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.     

Hepatocyte heterogeneity in the metabolism of fatty acids: discrepancies on zonation of acetyl-CoA carboxylase.

Lipid metabolism appears to be less zonated than carbohydrate and protein metabolism. Studies on the zonation of lipid metabolism have been centered in particular on fatty acid synthesis which, according to the concept of metabolic zonation, should be a predominantly perivenous process while fatty acid oxidation should be periportal. There are, however, conflicting data on the activity gradients of lipogenic enzymes as well as measurements of actual synthesis of fatty acid and very low density lipoprotein. Data obtained by microdissection show a 1.5- to 2-fold higher activity of acetyl-CoA carboxylase and citrate lyase in the perivenous zone in agreement with measurements of the actual rate of fatty acid synthesis in preparations of hepatocyte, enriched in periportal or perivenous cells. On the other hand, results obtained with the dual-digitonin-pulse perfusion technique demonstrate the opposite gradient in the form of a 2- to 3-fold higher specific activity of acetyl-CoA carboxylase in the periportal zone based on measurements of the acetyl-CoA carboxylase protein proper. This specific activity gradient, which applies to male and not female rats, disappears almost completely in the fasted-refed animal, were lipogenesis is strongly induced. In this review we attempt to rationalize these discrepancies in the results as methodological differences which in particular apply to the following parameters: (1) expression of results (reference substance); (2) selectivity of zonal sampling, and (3) differences in methodology of acetyl-CoA carboxylase measurements. It is concluded that these factors could account for the discrepancies, but further studies, in particular on the zonation acetyl-CoA carboxylase mRNA, are required in order to further understand the zonation of lipid metabolism and its possible role in the metabolic regulation of the liver.     

Gluconeogenesis in periportal and perivenous hepatocytes of rat liver, isolated by a new high-yield digitonin/collagenase perfusion technique.

A technique is described which allows preparations of hepatocytes, enriched in either periportal or perivenous hepatocytes ('PP-cells' and 'PV-cells' respectively), in a yield of about 30-50% compared with control cell preparations. The liver is first perfused for 40-60s with digitonin (4 mg/ml) to destroy selectively either the periportal or the perivenous part of the microcirculatory unit, and then the remaining hepatocytes are isolated by the ordinary collagenase perfusion technique. In periportal cells the activities of alanine aminotransferase and pyruvate kinase were 29.4 and 18.7 mumol/min per mg of DNA respectively. The rate of gluconeogenesis was 0.402 mumol/min per mg of DNA. In perivenous cells the corresponding values were 9.55, 22.1 and 0.244 mumol/min per mg of DNA respectively. These data support the concept of a zonation of glucose metabolism within the microcirculatory unit of the liver, with the afferent part (periportal zone) having a 2-fold, more active gluconeogenesis than the efferent part (perivenous zone).     

Regulation of glycogen synthesis from glucose and gluconeogenic precursors by insulin in periportal and perivenous rat hepatocytes.

Glycogen synthesis in hepatocyte cultures is dependent on: (1) the nutritional state of the donor rat, (2) the acinar origin of the hepatocytes, (3) the concentrations of glucose and gluconeogenic precursors, and (4) insulin. High concentrations of glucose (15-25 mM) and gluconeogenic precursors (10 mM-lactate and 1 mM-pyruvate) had a synergistic effect on glycogen deposition in both periportal and perivenous hepatocytes. When hepatocytes were challenged with glucose, lactate and pyruvate in the absence of insulin, glycogen was deposited at a linear rate for 2 h and then reached a plateau. However, in the presence of insulin, the initial rate of glycogen deposition was increased (20-40%) and glycogen deposition continued for more than 4 h. Consequently, insulin had a more marked effect on the glycogen accumulated in the cell after 4 h (100-200% increase) than on the initial rate of glycogen deposition. Glycogen accumulation in hepatocyte cultures prepared from rats that were fasted for 24 h and then re-fed for 3 h before liver perfusion was 2-fold higher than in hepatocytes from rats fed ad libitum and 4-fold higher than in hepatocytes from fasted rats. The incorporation of [14C]lactate into glycogen was 2-4-fold higher in periportal than in perivenous hepatocytes in both the absence and the presence of insulin, whereas the incorporation of [14C]glucose into glycogen was similar in periportal and perivenous hepatocytes in the absence of insulin, but higher in perivenous hepatocytes in the presence of insulin. Rates of glycogen deposition in the combined presence of glucose and gluconeogenic precursors were similar in periportal and perivenous hepatocytes, whereas in the presence of glucose alone, rates of glycogen deposition paralleled the incorporation of [14C]glucose into glycogen and were higher in perivenous hepatocytes in the presence of insulin. It is concluded that periportal and perivenous hepatocytes utilize different substrates for glycogen synthesis, but differences between the two cell populations in the relative utilization of glucose and gluconeogenic precursors are dependent on the presence of insulin and on the nutritional state of the rat.     

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.     

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.     

Gluconeogenic-glycolytic capacities and metabolic zonation in liver of rats with streptozotocin, non-ketotic as compared to alloxan, ketotic diabetes

Activities (mumol X min-1 X g liver) and zonal distributions of key enzymes of carbohydrate metabolism were studied in livers of streptozotocin-diabetic rats and compared to the values in alloxan-diabetes. Streptozotocin led to a non-ketotic diabetes with blood glucose being increased by more than fivefold but ketone bodies being in the normal range, while alloxan produced a ketotic diabetes with blood glucose, acetoacetate and beta-hydroxybutyrate being elevated by more than fivefold. Portal insulin was decreased to about 20% in streptozotocin- and more drastically to about 7% in alloxan-diabetes. Conversely, portal glucagon was increased in the two states to about 250% and 180%, respectively. The glucogenic key enzyme phosphoenolpyruvate carboxykinase (PEPCK) was enhanced in streptozotocin- and alloxan-diabetes to over 300%, while the glycolytic pyruvate kinase L (PKL) was lowered to 65% and 80%, respectively. The normal periportal to perivenous gradient of PEPCK of about 3:1, as measured in microdissected tissue samples, was maintained with elevated activities in the two zones. The normal periportal to perivenous gradient of PKL of 1:1.7 was diminished with lowered activities in the two zones. The glucogenic glucose-6-phosphatase (G6Pase) was increased in streptozotocin- and alloxan-diabetes to 130% and 140%, respectively, while the glucose utilizing glucokinase (GK) was decreased to 60% and 50%, respectively. The normal periportal to perivenous gradient of G6Pase, demonstrated histochemically, remained unaffected. Carnitine palmitoyltransferase (CPT) was increased to over 190% and acetyl-CoA carboxylase (ACC) was decreased to 60% in streptozotocin, non-ketotic diabetes, while the two enzymes were altered more drastically to 400% and 50%, respectively, in alloxan, ketotic diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deficiency of manganese superoxide dismutase in hepatocytes disrupts zonated gene expression in mouse liver

The liver acinus displays a physiological periportal to perivenous oxygen gradient. This gradient was implicated to use reactive oxygen species (ROS) as mediators for the zonal gene expression. Mitochondria use oxygen and produce ROS, therefore they may contribute to the zonation of gene expression. To further elucidate this, we used the Cre-loxP system to generate a hepatocyte-specific null mutation of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) in mice. We found that ROS levels were enhanced in livers of MnSOD(-/-) mice which were reduced in size and displayed signs of liver failure such as intracellular protein droplets, increased apoptotic bodies and Bax levels as well as multinuclear hepatocytes. Further, the zonation of glutamine synthetase, glucokinase and phosphoenolpyruvate carboxykinase was no longer preserved. We conclude that deficiency of mitochondrial MnSOD initiates a dysregulation of zonated gene expression in liver.     

Zonation of metabolism and gene expression in liver

Conclusion The zonation of gene expression resulting in the different cellular equipment of liver cells with enzymes, translocators and subcellular structures is regulated mainly at a pretranslational level. In the dynamic zonation of carbohydrate metabolizing enzymes, gradients in oxygen and in the glucagon/insulin ratio decreasing from the periportal to the perivenous area appear to be major effectors. In the stable zonation of ammonia-metabolizing enzymes local paracrine cell-cell interactions via diffusible mediators could play a major role (Fig. 6).This paper was presented at the symposium Metabolic Zonation of the Liver: New Answers to Old Questions, held in honour of Prof. Dr. D. Sasse's 60th birthday, 26 August 1994, in Basel     

Insulin resistance in the liver: Deficiency or excess of insulin?

In insulin-resistant states (obesity, pre-diabetes, and type 2 diabetes), hepatic production of glucose and lipid synthesis are heightened in concert, implying that insulin deficiency and insulin excess coexists in this setting. The fact that insulin may be inadequate or excessive at any one point in differing organs and tissues has many biologic ramifications. In this context the concept of metabolic compartmentalization in the liver is offered herein as one perspective of this paradox. In particular, we focus on the hypothesis that insulin resistance accentuates differences in periportal and perivenous hepatocytes, namely periportal glucose production and perivenous lipid synthesis. Subsequently, excessive production of glucose and accumulation of lipids could be expected in the livers of patients with obesity and insulin resistance. Overall, in this review, we provide our integrative perspective regarding how excessive production of glucose in periportal hepatocytes and accumulation of lipids in perivenous hepatocytes interact in insulin resistant states.     

Glucagon regulation of gluconeogenesis and ketogenesis in periportal and perivenous rat hepatocytes. Heterogeneity of hormone action and of the mitochondrial redox state.

Hepatocytes isolated from the periportal or perivenous zones of livers of fed rats were used to study the long-term (14 h) and short-term (2 h) effects of glucagon on gluconeogenesis and ketogenesis. Long-term culture with glucagon (100 nM) resulted in a greater increase (P less than 0.01) in gluconeogenesis in periportal than in perivenous cells (93 +/- 16 versus 30 +/- 14 nmol/h per mg of protein; 72% versus 30% increase), but short-term incubation (2 h) with glucagon resulted in similar stimulation in the two cell populations. Rates of ketogenesis (acetoacetate and D-3-hydroxybutyrate production) were not significantly higher in periportal cells cultured without glucagon, compared with perivenous cells. However, after long-term culture with glucagon, the periportal cells had a significantly higher rate of ketogenesis (from either palmitate or octanoate as substrate), but a lower 3-hydroxybutyrate/acetoacetate production ratio, suggesting a more oxidized mitochondrial NADH/NAD+ redox state despite the higher rate of beta-oxidation. Periportal hepatocytes had a higher activity of carnitine palmitoyltransferase but a lower activity of citrate synthase than did perivenous cells. These findings suggest that: (i) glucagon elicits greater long-term stimulation of gluconeogenesis in periportal than in perivenous hepatocytes maintained in culture; (ii) after culture with glucagon, the rates of ketogenesis and the mitochondrial redox state differ in periportal and perivenous hepatocytes.