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.     

Interactions of inhibitors of carnitine palmitoyltransferase I and fibrates in cultured hepatocytes.

Culture of rat hepatocytes with etomoxir, an inhibitor of carnitine palmitoyltransferase I (CPT I), for 48 h, resulted in increased carnitine acetyltransferase (CAT) activity (74%), a marked decrease in CPT activity (82%) measured in detergent extracts, and increased activities of glucose-6-phosphate dehydrogenase (227%) and fructose-1,6-bisphosphatase (65%). Changes in CAT and CPT activities were not observed after 4 h culture with etomoxir. When hepatocytes were cultured with etomoxir and benzafibrate (a hypolipidaemic analogue of clofibrate) for 48 h, etomoxir prevented the 5-fold increase in CAT activity caused by bezafibrate, whereas bezafibrate suppressed the increase in glucose-6-phosphate dehydrogenase and fructose-bisphosphatase caused by etomoxir. However, bezafibrate did not prevent the suppression of CPT activity by etomoxir. Etomoxir inhibited palmitate beta-oxidation and ketogenesis after short-term (0-4 h) and long-term (48 h) exposure, but it caused accumulation of triacylglycerol in hepatocytes only after short-term exposure (0-4 h). These effects of etomoxir on fatty acid metabolism and suppression of CPT (after 48 h) were similar in periportal and perivenous hepatocytes, but the increases in CAT and glucose-6-phosphate dehydrogenase activities were higher in periportal than in perivenous cells. The effects of CPT I inhibitors on CAT activity and long-term suppression of CPT activity are probably mediated by independent mechanisms.     

Localization of the fructose 1,6-bisphosphatase at the nuclear periphery

The localization of fructose 1,6-bisphosphatase (D-Fru-1,6-P₂-1-phosphohydrolase, EC 3.1.3.11) in rat kidney and liver was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from pig kidney. The immunohistochemical analysis revealed that the bisphosphatase was preferentially localized in hepatocytes of the periportal region of the liver and was absent from the perivenous region. Fructose-1,6-bisphosphatase was also preferentially localized in the cortex of the kidney proximal tubules and was absent in the glomeruli, loops of Henle, collecting and distal tubules, and in the renal medulla. As indicated by immunocytochemistry using light microscopy and confirmed with the use of reflection confocal microscopy, the enzyme was preferentially localized in a perinuclear position in the liver and the renal cells. Subcellular fractionation studies followed by enzyme activity assays revealed that a majority of the cellular fructose-1,6-bisphosphatase activity was associated to subcellular particulate structures. Overall, the data support the concept of metabolic zonation in liver as well as in kidney, and establish the concept that the Fructose-1,6-bisphosphatase is a particulate enzyme that can not be considered a soluble enzyme in the classical sense. © 1996 Wiley-Liss, Inc.     

Subcellular localization of aldolase B

The localization of the aldolase B isozyme was determined immunohistochemically in rat kidney and liver using a polyclonal antibody. Aldolase B was preferentially localized in a nuclear region of hepatocytes from the periportal region and was absent in those from the perivenous region. Aldolase B was also preferentially localized in the proximal tubules and was absent in other structures of the renal cortex as well as in the renal medulla. Using reflection confocal microscopy, the enzyme was preferentially localized in a nuclear position in liver and renal cells, which was similar to the cellular and intracellular location found for the gluconeogenic enzyme fructose-1,6-bisphosphatase (Sáez et al. [1996] J. Cell. Biochem. 63:453-462). Subcellular fractionation studies followed by enzyme activity assays revealed that aldolase activity was associated with subcellular particulate structures. Overall, the data suggest that different aldolase isoenzymes are needed in the glycolytic and gluconeogenic pathways.     

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.     

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。     

Effects of epinephrine, glucagon and insulin on the activity and degree of phosphorylation of fructose-1,6-bisphosphatase in cultured hepatocytes

The effects of epinephrine, glucagon and insulin on the activity and degree of phosphorylation of fructose-1,6-bisphosphatase in isolated hepatocytes maintained in cell culture for 24 h were investigated. Epinephrine caused a rapid decrease in the apparent Km monitored as the activity ratio between the activity at 12.5 and 83 microM fructose-1,6-bisphosphate, reaching a maximum after 5 min. Glucagon caused a slower and less pronounced activation, and insulin caused an equally slow increase in Km. The effect of epinephrine and glucagon was completely reciprocated by insulin and the action of insulin was totally erased by the other two. Glucagon stimulated the incorporation of [32P]phosphate into fructose-1,6-bisphosphatase from about 2.5 to 4.2 mol/mol enzyme and epinephrine to 3.5 mol/mol. The effect of the two hormones acting together was cumulative. Insulin brought about a decrease in the degree of phosphorylation to 2.0 mol/mol. The effect of epinephrine was shown to be caused by the beta-receptors, since it was completely blocked by propanolol (a beta-antagonist) and remained unaffected by the presence of phentolamine (an alpha-antagonist).     

Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism

Isolated rat hepatocytes convert 2,5-anhydromannitol to 2,5-anhydromannitol-1-P and 2,5-anhydromannitol-1,6-P2. Cellular concentrations of the monophosphate and bisphosphate are proportional to the concentration of 2,5-anhydromannitol and are decreased by gluconeogenic substrates but not by glucose. Rat liver phosphofructokinase-1 phosphorylates 2,5-anhydromannitol-1-P; the rate is less than that for fructose-6-P but is stimulated by fructose-2,6-P2. At 1 mM fructose-6-P, bisphosphate compounds activate rat liver phosphofructokinase-1 in the following order of effectiveness: fructose-2,6-P2 much greater than 2,5-anhydromannitol-1,6-P2 greater than fructose-1,6-P2 greater than 2,5-anhydroglucitol-1,6-P2. High concentrations of fructose-1,6-P2 or 2,5-anhydromannitol-1,6-P2 inhibit phosphofructokinase-1. Rat liver fructose 1,6-bisphosphatase is inhibited competitively by 2,5-anhydromannitol-1,6-P2 and noncompetitively by 2,5-anhydroglucitol-1,6-P2. The AMP inhibition of fructose 1,6-bisphosphatase is potentiated by 2,5-anhydroglucitol-1,6-P2 but not by 2,5-anhydromannitol-1,6-P2. Rat liver pyruvate kinase is stimulated by micromolar concentrations of 2,5-anhydromannitol-1,6-P2; the maximal activation is the same as for fructose-1,6-P2. 2,5-Anhydroglucitol-1,6-P2 is a weak activator. 2,5-Anhydromannitol-1-P stimulates pyruvate kinase more effectively than fructose-1-P. Effects of glucagon on pyruvate kinase are not altered by prior treatment of hepatocytes with 2,5-anhydromannitol. Pyruvate kinase from glucagon-treated hepatocytes has the same activity as the control pyruvate kinase at saturating concentrations of 2,5-anhydromannitol-1,6-P2 but has a decreased affinity for 2,5-anhydromannitol-1,6-P2 and is not stimulated by 2,5-anhydromannitol-1-P. The inhibition of gluconeogenesis and enhancement of glycolysis from gluconeogenic precursors in hepatocytes treated with 2,5-anhydromannitol can be explained by an inhibition of fructose 1,6-bisphosphatase, an activation of pyruvate kinase, and an abolition of the influence of phosphorylation on pyruvate kinase.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Distribution of hepatic glutaminase activity and mRNA in perivenous and periportal rat hepatocytes.

Perivenous and periportal hepatocytes were isolated by the digitonin/collagenase perfusion technique. The specific activity of phosphate-activated glutaminase was 2.33-fold higher in periportal cells than in perivenous cells. Similarly, the relative abundance of glutaminase mRNA was 2.6-fold higher in samples from periportal cells. The distribution of glutaminase activity and mRNA was compared with those for glutamine synthetase (predominantly perivenous) and phosphoenolpyruvate carboxykinase (predominantly periportal). The results suggest that phosphate-activated glutaminase is predominantly expressed in the periportal zone of the liver acinus.     

Ribose 1,5-bisphosphate inhibits fructose-1,6-bisphosphatase in rat kidney cortex

Fructose-1,6-bisphosphatase is one of the regulatory enzymes of gluconeogenesis in kidney cortex. The effect of ribose 1,5-bisphosphate on fructose-1,6-bisphosphatase purified from rat kidney cortex was studied. Rat kidney cortex, fructose-1,6-bisphosphatase exhibited hyperbolic kinetics with regard to its substrate, but the activity was inhibited by ribose 1,5-bisphosphate at nanomolar concentrations. The inhibitory effect of ribose 1,5-bisphosphate on the fructose-1,6-bisphosphatase was enhanced in the presence of AMP, one of the inhibitors of fructose-1,6-bisphosphatase. Fructose-2,6-bisphosphate, which is an inhibitor of fructose-1,6-bisphosphatase, inhibited rat kidney cortex fructose-1,6-bisphosphatase activities at a low concentration of fructose-1,6-bisphosphate but a high concentration of fructose-1,6-bisphosphate relieved fructose-1,6-bisphosphatase from fructose-2,6-bisphosphate-dependent inhibition. On the contrary, fructose-1,6-bisphosphate was not effective for the recovery of fructose-1,6-bisphosphatase from ribose 1,5-bisphosphate-dependent inhibition. These results suggest that ribose 1,5-bisphosphate is a potent inhibitor and is involved in the regulation of fructose-1,6-bisphosphatase in rat kidney cortex.     

Regulation of fructose-1,6-bisphosphatase activity in primary cultured astrocytes

In the gluconeogenic pathway, fructose-1,6-bisphosphatase (EC 3.1.3.11) is the last key-enzyme before the synthesis of glucose-6-phosphate. The extreme diversity of cells present in the whole brain does not facilitate in vivo study of this enzyme and makes it difficult to understand the regulatory mechanisms of the related carbohydrate metabolism. It is for instance difficult to grasp the actual effect of ions like potassium, magnesium and manganese on the metabolic process just as it is difficult to grasp the effect of different pH values and the influence of glycogenic compounds such as methionine sulfoximine. The present investigation attempts to study the expression and regulation of fructose-1,6-bisphosphatase in cultured astrocytes. Cerebral cortex of new-born rats was dissociated into single cells that were then plated. The cultured cells were flat and roughly polygonal and were positively immunostained by anti-glial fibrillary acidic protein antibodies. Cultured astrocytes are able to display the activity of fructose-1,6-bisphosphatase. This activity was much higher than that in brain tissue in vivo. Fructose-1,6-bisphosphatase in cultured astrocytes did not require magnesium ions for its activity. The initial velocity observed when the activity was measured in standard conditions was largely increased when the enzyme was incubated with Mn²⁺. This increase was however followed by a decrease in absorbance resulting in the induction, by the manganese ions, of a singular kinetics in the enzyme activity. Potassium ions also stimulated fructose-1,6-bisphosphatase activity. When the enzyme was exposed to different pH values ranging from 6 to 9 units, the highest activity was observed at pH 6. When the cultured astrocytes were incubated with methionine sulfoximine, the fructose-1,6-bisphosphatase activity increased. This increase was quick and depended on the dose of methionine sulfoximine. These results show that cultured astrocytes are able to maintain fructose-1,6-bisphosphatase activity. With the exception of the higher level activity associated acidic pH ranges, the properties of the enzyme resemble those of the in vivo enzyme. Methionine sulfoximine has a direct effect on astrocytes in its activation of fructose-1,6-bisphosphatase. It is concluded that the expression and the regulation of fructose-1,6-bisphosphatase activity in cultured astrocytes look like those in the brain. Astrocytes are probably the principal cells that express this activity in the brain in vivo.     

Studies on the regulation of chloroplast fructose-1,6-bisphosphatase. Activation by fructose 1,6-bisphosphate

Chloroplast fructose-1,6-bisphosphatase (D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) isolated from spinach leaves, was activated by preincubation with fructose 1,6-bisphosphate. The rate of activation was slower than the rate of catalysis, and dependent upon the temperature and the concentration of fructose 1,6-bisphosphate. The addition of other sugar diphosphates, sugar monophosphates or intermediates of the reductive pentose phosphate cycle neither replaced fructose 1,6-bisphosphate nor modified the activation process. Upon activation with the effector the enzyme was less sensitive to trypsin digestion and insensitive to mercurials. The activity of chloroplast fructose-1,6-bisphosphatase, preincubated with fructose 1,6-bisphosphate, returned to its basal activity after the concentration of the effector was lowered in the preincubation mixture. The results provide evidence that fructose-1,6-bisphosphatase resembles other regulatory enzymes involved in photosynthetic CO2 assimilation in its activation by chloroplast metabolites.     

Zonation of fatty acid metabolism in rat liver.

Fatty acid metabolism was studied in periportal and perivenous hepatocytes isolated by the method of Chen & Katz [Biochem. J. (1988) 255, 99-104]. The rate of fatty acid synthesis and the activity of acetyl-CoA carboxylase were markedly enhanced in perivenous hepatocytes as compared with periportal cells. However, the response of these two parameters to short-term modulation by cellular effectors such as the hormones insulin and glucagon, the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate and the xenobiotics ethanol and acetaldehyde was similar in the two zones of the liver. In addition, perivenous hepatocytes showed a higher capacity of esterification of exogenous fatty acids into both cellular and very-low-density-lipoprotein lipids. Nevertheless, no difference between the two cell sub-populations seemed to exist in relation to the secretion of very-low-density lipoproteins. On the other hand, the rate of fatty acid oxidation was increased in periportal cells. This could be accounted for by a higher activity of carnitine palmitoyltransferase I and a lower sensitivity of this enzyme to inhibition by malonyl-CoA in the periportal zone. No differences were observed between periportal and perivenous hepatocytes in relation to the short-term response of fatty acid oxidation and carnitine palmitoyltransferase I activity to the cellular modulators mentioned above. In conclusion, our results show that: (i) lipogenesis is achieved at higher rates in the perivenous zone of the liver, whereas the fatty-acid-oxidative process occurs with a certain preference in the periportal area of this organ; (ii) the short-term response of the different fatty-acid-metabolizing pathways to cellular effectors is quantitatively similar in the two zones of the liver.     

Evidence for non-vacuolar proteolytic catabolite inactivation of yeast fructose-1,6-bisphosphatase

Immunoblotting was used to study whether proteolytic degradation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in yeast cells during catabolite inactivation occurs intra- or extravacuolarly. The 40-kDa subunits of both the phosphorylated and the non-phosphorylated fructose-1,6-bisphosphatase are rapidly degraded by an extract from isolated vacuoles to a 32-kDa intermediate which accumulates and is then slowly further degraded. However, in intact cells, neither the 32-kDa nor any other intermediate reacting with the fructose-1,6-bisphosphatase antibodies is observed following glucose-induced degradation of the enzyme. These observations are discussed as evidence against intravacuolar degradation of fructose-1,6-bisphosphatase during proteolytic catabolite inactivation.     

Functioning of fructose-1,6-bisphosphatase--main enzyme of gluconeogenesis in microorganisms

The modern literature data about common characteristics, genetic and molecular-biological properties of main enzyme of gluconeogenesis (fructose-1,6-bisphosphatase) were analyzed. Regulation of fructose-1,6-bisphosphatase activity (stimulation and inhibition) by fructose-1,6-bisphosphate, fructose-2,6-bisphosphate, phosphoenolpyruvate, AMP and by metal ions are discussed. It was concluded that apart from the fact that fructose-1,6-bisphosphatase was intensively investigated, this enzyme from Mollicutes failed to be studied sufficiently.     

Quantitative histochemical study of glucose-6-phosphatase in periportal and perivenous human hepatocytes.

The glucose-6-phosphatase activity of periportal and perivenous human hepatocytes was studied with a quantitative method. The results obtained with histogram of light intensity distributions indicate that the enzyme reaction was 1.3 to 2.5 fold higher in periportal zone than in perivenous zone. The profiles of light intensity along portal----hepatic venous distances show a progressive decrease of enzyme activity with highest values in periportal hepatocytes.