Changes in activity and intra-acinar distribution of glucose-6-phosphate dehydrogenase and malic enzyme during pregnancy in rat liver

Using techniques of microdissection and microassay as well as qualitative histochemistry the activity and intra-acinar distribution of G6PDH and ME were studied on selected days of pregnancy in the rat. Both enzymes show distinct fluctuations during the course of pregnancy in keeping with changes in hepatic lipogenesis. Marked increases in activity are seen as early as the 4th day, while highest values are attained on day 20, with a predominant perivenous induction. On day 22, just before parturition a sharp decrease of both enzyme activities with a flattening of the periportal/perivenous gradient was detected. G6PDH shows proportionally considerably larger increases and more distinct changes in zonation. The perivenous fluctuations in G6PDH activity of late gestation are supposed to be caused primarily by insulin. Although estrogen is known to induce both enzymes, the temporal changes in enzyme activity in pregnancy cannot be related to the action of estrogen alone. The changes in enzyme activity, however, correspond well to those of progesterone, and although no direct action of progesterone on these enzymes has yet been proposed, further work on its effects on enzyme activity and distribution is indicated.     

Antagonistic reaction of the periportal and perivenous zone in the rat liver after castration and estrogen treatment

Summary G6PDH and ME activity was determined biochemically in homogenates and demonstrated histochemically in cryostat sections of rat liver. Control animals were sham-operated, the male and female rats of the experimental groups were castrated. After castration groups of rats were treated with daily doses of 3 or 6 g/estradiolbenzoate for 21 days. The results show that in the controls there is a sex-dependent distribution pattern of the two enzymes; in males the rather low activity is mainly located in the periportal area, in females the higher activity is demonstrable in the perivenous area. After castration G6PDH activity (and to a lesser extent ME activity) increases, mainly in the periportal zone. Estrogen treatment results in the high activity of both enzymes, which are exclusively located in the perivenous zone. In the periportal zone no G6PDH or ME activity is demonstrable histochemically. This zone-typical effect of estrogen is interpreted in terms of the concept of Metabolic Zonation, according to which it is supposed that the NADPH generating enzymes in the perivenous area have a lipogenic function whereas the periportal activity contributes to bile acid production.Parts of this study were presented as an Inaugural Dissertation to the Medical Faculty of the University of FreiburgSupported by grants from the Deutsche Forschungsgemeinschaft (Sa 127/7) and the SFB 46 (Molgrudent)     

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.     

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)     

Carbohydrate deprivation reduces NADPH-production in fish liver but not in adipose tissue

Little is known about the way in which carnivorous fish such as salmonids mobilise and metabolise dietary carbohydrates, which are essential to lipid metabolism. Thus we have studied changes caused by the absence of dietary carbohydrates to the kinetics and molecular behaviour of the four cellular NADPH-production systems [glucose 6-phosphate dehydrogenase (G6PDH); 6-phosphogluconate dehydrogenase (6PGDH); malic enzyme (ME); and isocitrate dehydrogenase NADP-dependent (NADP-IDH)] in the liver and adipose tissue of rainbow trout (Oncorhynchus mykiss). We used spectrophotometry to study enzyme kinetics and nucleic acid concentrations, and immunoblot analysis to determine specific protein concentrations. The absence of carbohydrate reduced specific enzyme activity, maximum rate and catalytic efficiency by almost 65% in G6PDH and 6PGDH, by more than 50% in ME, and by almost 25% in NADP-IDH but caused no significant changes in the K(m) values or activity ratios in any of these hepatic enzymes. Molecular analysis clearly showed that this kinetic behaviour reflected concomitant changes in intracellular enzyme concentrations, produced by protein-induction/repression processes rather than changes in the activity of pre-existing enzymes. We conclude that the absence of carbohydrates significantly reduces intracellular concentrations of G6PDH, ME and NADP-IDH in trout liver in percentages similar to those recorded for enzyme activity. We found no such variations in the concentrations of any of these enzymes in adipose tissue and no change in the levels of their activity, suggesting that the liver and adipose tissues are subject to different regulation systems with regard to carbohydrates and play distinct roles in lipid metabolism.     

Postnatal adaptation of gluconeogenic and liponeogenic areas in the liver parenchyma

The activity of G6Pase points to the gluco(neo)genic function of hepatocytes, whereas the activity of G6PDH and malic enzyme, both yielding NADPH, are involved in liponeogenesis. Histochemical studies of the distribution patterns and quantitative measurements concerning the postnatal weeks of rats show that after the 6th week sex differences occur. In contrast to males in female rat liver a lipogenic area emerges in the perivenous zone. This lipogenic zone is situated opposite to the gluco(neo)genic area which corresponds to the periportal zone.     

Enzyme activity patterns of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucose-6-phosphate-dehydrogenase and malic enzyme in human liver

The distribution patterns of the enzyme activities of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucose-6-phosphate-dehydrogenase and malic enzyme were determined in the liver acini of men and women by microquantitative means. The activity of PEPCK was higher in men compared to the activity in women. In both sexes no heterotopic distribution pattern was observed. PK activity of men was higher, but in both sexes no heterotopic distribution was detectable. G6PDH and ME showed relatively low activity. The distribution of G6PDH and ME activity was to some extent different in men and women. Yet their heterotopic patterns were not particularly distinct.     

Enzyme activity patterns of phosphoenolpyruvate carboxykinase,pyruvate kinase,glucose-6-phosphate-dehydrogenase and malic enzyme in human liver

Summary The distribution patterns of the enzyme activities of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucose-6-phosphate-dehydrogenase and malic enzyme were determined in the liver acini of men and women by microquantitative means. The activity of PEPCK was higher in men compared to the activity in women. In both sexes no heterotopic distribution pattern was observed. PK activity of men was higher, but in both sexes no heterotopic distribution was detectable. G6PDH and ME showed relatively low activity. The distribution of G6PDH and ME activity was to some extent different in men and women. Yet their heterotopic patterns were not particularly distinct.     

Microfluorometric study of glucose-6-phosphate and malate dehydrogenases in histologically defined areas of the uterus in cyclic and pregnant ewes

Activities of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49; G6PDH) and malate dehydrogenase (E.C. 1.1.1.37; MDH) were determined fluorometrically in freeze-dried sections of the sheep uterus during the estrous cycle and pregnancy. Samples (0.2–0.3 μg) from the luminal epithelium, uterine glands, maternal caruncles, fetal cotyledons and intercotyledonary trophoblast were assayed in a small aliquot (5 μl) of the reaction medium under oil.Activity of G6PDH in the luminal epithelium, uterine glands and maternal caruncles did not change during the estrous cycle. Activity of MDH in the uterine glands did not change during the cycle, but in the luminal epithelium and maternal caruncles highest activities were found on day 9 and day 2 post-estrus, respectively.The enzyme activities in the fetal tissues were lower than in the maternal tissues. In all maternal tissues, MDH and G6PDH activities decreased during early pregnancy, but after implantation, the activities increased significantly. In fetal tissues G6PDH activity increased, whereas MDH activity decreased during the second half of gestation. These results suggest an increased rate of pentose shunt activity in both maternal and fetal tissues, and an increased rate of Krebs' cycle activity in the maternal but not in the fetal tissues.     

Variations in the kinetic behaviour of the NADPH-production systems in different tissues of the trout when fed on an amino-acid-based diet at different frequencies

We have studied the effects of feeding an amino-acid-based diet (ABD) at different frequencies upon growth and several NADPH-production systems in the rainbow trout (Oncorhynchus mykiss). The kinetic behavior of glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME) and NADP-linked isocitrate dehydrogenase (NADP-IDH) was followed in the liver, kidney and adipose tissue.The kinetic parameters of NADP-IDH alone remained unaltered by either ABD or changes in feeding frequency. Maximum-velocity and catalytic-efficiency values of hepatic G6PDH and ME increased significantly when fed four times a day compared to twice a day with both the control diet and ABD, although these parameters for ME were significantly lower with ABD than with the control diet at both frequencies. In the kidney the activity and catalytic efficiency of G6PDH and 6PGDH increased significantly with high-frequency feeding on ABD. The activities of these enzymes in adipose tissue were much lower than in hepatic tissue. In the liver, maximum velocity and the catalytic efficiency of G6PDH, 6PGDH and ME increased significantly with the control diet at high-frequency feeding whereas they decreased significantly with ABD, especially with high-frequency feeding. Neither the Michaelis constant nor the activity ratios varied.Both feeding frequency and free amino acid altered the activity of the most important cytosolic NADPH-production systems. The varying response to nutritional stimuli of NADP-linked enzymes in fish tissues shows that they have independent physiological and metabolic roles and that their regulatory mechanisms respond to changes in nutritional and metabolic factors.     

The effect of hydrophobization of glucose-6-phosphate dehydrogenase with progesterone on its thermal inactivation

Thermal inactivation of glucose-6-phosphate dehydrogenase (G6PDH) and its conjugates with progesterone containing 3, 7 and 35 molecules of the modifier was studied in bidistilled water over a temperature range 35-47 degrees. At different temperatures and initial concentrations of the enzyme and its modified forms, thermal inactivation is described by the equation of the first order up to a significant degree of enzyme deactivation. The effective Kin values are decreased with the increase of the native G6PDH concentration and changed in a complicated manner with the increase of the conjugate concentration depending on the enzyme modification degree, which reflects a great role of the enzyme hydrophobicity in its inactivation. The role of hydrophobicity of the modified G6PDH in changes of its specific activity is discussed.     

Presence of uterine peroxidase activity in the rat early pregnancy

Peroxidase has been associated with estrogen action in the uterus. This enzyme plays an important role in the control of hydrogen peroxide levels and in catechol estrogen production. Since the uterus, during early pregnancy, is subjected to estrogen and progesterone regulation, we analyzed the changes of peroxidase activity in relation to receptivity and uterine early response to the embryo. Soluble and microsomal peroxidase activity were determined in the rat uterus during the estrus phase and early pregnancy (days 3 through 6). Soluble peroxidase activity increased significantly (p < 0.01) from day 3 (1.50 +/- 0.24) to day 4 (3.5 +/- 0.3) and 5 (5 +/- 0.5 U/mg protein, mean +/- S.D., n = 6) of pregnancy. During day 6, a significant decrease was noted in both the implantation site and the nonimplantation uterine tissue. Microsomal calcium-extractable peroxidase showed a similar pattern, with lower specific activity than, the soluble peroxidase. During estrus, the uterine tissue showed the highest activity of calcium-extracted peroxidase (8.7 +/- 1.35 U/mg protein), statistically greater when compared with days 3, 4, 5 and 6 of pregnancy. In conclusion, high peroxidase activity was associated with uterine receptivity. The decrease of activity on day 6 might be due to a progesterone-estrogen interaction, and consequently, hydrogen peroxide can be utilized for hydroxile production by means of the Fenton reaction. Lipoperoxidation may be necessary for changes in membrane fluidity for embryo attachment to endometrial epithelium.     

Difference in activation capacity between oocytes of Pleurodeles waltl matured in vivo and in vitro.

Oocytes of Pleurodeles waltl were activated after in vivo maturation by needle pricking or electric shock. After in vitro maturation, the oocytes were not activated by these stimuli. Coelomic oocytes and the oocytes which began their maturation in vivo could be activated by electric shock. During in vivo oocyte maturation, the activity of glucose-6-phosphate dehydrogenase (G6PDH), the key enzyme of the pentose phosphate cycle, increased while that of phosphofructokinase, the key enzyme of glycolysis, remained unchanged. During progesterone-induced in vitro oocyte maturation, the activity of both enzymes remained unchanged. Oocytes of Misgurnus fossilis matured in vivo and in vitro were activated spontaneously. No changes in the activity of G6PDH were observed during their maturation. These results suggest a relationship between G6PDH activity in the oocyte and oocyte capacity for activation by needle pricking or electric shock.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Cyclic nucleotides and protein phosphorylation in mouse mammary glands: effects of estrogen and progesterone administered in vivo

Ovariectomized mice were injected daily for 20 days with saline, 17 beta-estradiol (1 microgram/day), progesterone (1 mg/day), or estrogen + progesterone. Mammary glands were removed, homogenized, and analyzed for DNA, cAMP, cGMP, cAMP-dependent protein kinase (kinase A), cGMP-dependent protein kinase (kinase G), tyrosyl kinase (kinase T), and epidermal growth factor-stimulated tyrosyl kinase (EGF-T). Estrogen and progesterone, administered singly, increased DNA, cAMP, kinase A, kinase T, and EGF-T. In addition, progesterone, administered alone or with estrogen, decreased kinase G activity. cGMP concentrations were not altered by estrogen or progesterone. No evidence of a synergism between estrogen and progesterone on the levels of the cyclic nucleotides and the activities of kinase enzyme was observed, although an additive effect of these steroids was seen. These data indicate that ovarian steroid-induced growth of mouse mammary glands is accompanied by significant changes in protein phosphorylation, i.e., increased cAMP-dependent protein phosphorylation and tyrosyl phosphorylation and decreased cGMP-dependent protein phosphorylation.     

Daily and annual variations of the hepatic glucose 6-phosphate dehydrogenase activity and seasonal changes in the body fats of the gilthead seabream Sparus aurata

In mammalians, Glucose 6-phosphate dehydrogenase (G6PDH) is clearly related with the daily cycle lipogenesis/lipolysis and therefore with the photoperiod and activity. In fish the temporally changes on the activity of this enzyme have not been described. In this work, annual and diurnal activity of G6PDH was investigated in the liver of Sparus aurata in different seasonal conditions of temperature and photoperiod. During experiment the fish were kept in cages under natural temperature and photoperiod while consuming a commercial diet. The months studied were chosen to establish if there is any influence on the G6PDH activity, because of the change in temperature and photoperiod (equal photoperiod and different temperature, March and October; different photoperiod and equal temperature, May and November; and different photoperiod and temperature, June and January). To study the enzyme activity along the day, the livers of six fish were removed every 3 hr during 24 hr. Annual activity was determined as the average activity during 1 day. Also the annual changes on fat content in muscle and digestive system were studied. The Vmax (maximum velocity) showed seasonal changes in period and phase of the rhythms and for the Km (Michaelis-Menten constant) a period of 24 hr was found in all sampled months, except March, the phase time moves depending on the month. In addition, the annual variations in muscular fat and G6PDH-specific activity seem to be more related to the reproductive stage than any other variable considered.     

Molecular characterization of a novel glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.)

We describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox-regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C-terminal Strep tag, the enzyme retained G6PDH activity upon affinity purification. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein-mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The Km values determined for both substrates were significantly lower compared to those of P1. The high Vmax and Ki [NADPH] values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid-localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features.