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)     

Classifying amino acids as gluco(glyco)genic, ketogenic, or both

Eleven popular and recent biochemistry textbooks were examined and compared concerning the classification of amino acids as being gluco(glyco)genic, ketogenic, or both. The main differences concerned mostly two amino acids, threonine, which in some cases is considered only gluco(glyco)genic and in others both gluco(glyco)-ketogenic, and lysine, which is considered chiefly ketogenic, but in two cases is reported as being gluco(glyco)-ketogenic. Some more mistakes, discrepancies and curiosities among different textbooks are also reported. Biochemistry textbook authors and teachers should be aware of this situation to avoid any confusion, especially among students.     

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)     

Hormonal and lipogenic and gluconeogenic enzymatic responses in LA/N-corpulent rats

Genetically obese normotensive rats, LA/N-corpulent (cp), were fed ad libitum diets containing either 54% sucrose or cooked corn starch for 12 weeks. Twenty-four rats were used for the study; half were corpulent (cp/cp) and half were lean (cp/+ or +/+). Fasting levels of plasma insulin, glucose, corticosterone, glucagon and growth hormone, and activities of liver and epididymal fat pad glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), and liver and kidney glucose-6-phosphatase (G6Pase), fructose 1,6-diphosphatase (FDPase), and phosphoenolpyruvate carboxykinase (PEPCK) were measured. A significant phenotype effect was observed in insulin, corticosterone, growth hormone, and liver G6PD, ME, FDPase, and kidney PEPCK, G6Pase, FDPase, and epididymal fat pad G6PD and ME (corpulent greater than lean), and glucagon (lean greater than corpulent). Diet effect (sucrose greater than starch) was significant for plasma glucose, liver ME, and kidney G6Pase. Although not significant at the P less than 0.05 level, insulin, corticosterone, liver G6PD and FDPase and kidney FDPase tended to be higher in sucrose-fed rats. This study suggests that the corpulent rat is more lipogenic and gluconeogenic than the lean, and that the hormones responsible are effective in keeping both the lipogenic and gluconeogenic enzyme activity elevated.     

Development of intra- and intermuscular adipose tissue in growing large white and Meishan pigs

Lipogenic enzyme activities of porcine intra- and intermuscular adipose tissues were determined in growing lean (Large White) and fat (Meishan) pigs. The activities of acetyl-CoA-carboxylase (ACX), malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH) were compared in both breeds and at both adipose sites. All three enzyme activities were much lower in the intramuscular adipose tissue than in the intermuscular site. Although the lipogenic activity of the intramuscular adipose site was low, it appeared, however, to possess adequate levels of enzymes for in situ lipid synthesis. The highest differences in lipogenic enzyme activities between Meishan and Large White pigs were found in intramuscular adipose tissue, and essentially concerned the activity of malic enzyme which was much higher in Meishan pigs. A close relationship between ME activity and lipid content of intramuscular adipose tissue was observed in both breeds. It was concluded that ME appeared to be a major factor affecting the incidence of higher intramuscular fat in the pig.     

Scaling effects on metabolism of a teleost

Scaling effects on citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), RNA. RNA/DNA ratio and protein contents of brain, liver and skeletal muscle were studied in a teleost, Clarias batrachus. The activity of white skeletal muscle CS decreased significantly as a function of increasing body mass of the fish. It shows that the fulfilment of energy demand in white skeletal muscle is not dependent on aerobic metabolism. The activity of liver G6-PDH decreased with the increasing body mass showing reduction in NADPH generation for lipogenic activity. However, increase in G6-PDH activity showed enhancement in reductive synthesis in skeletal muscle of the larger-sized individuals. A positive scaling of RNA, RNA/DNA ratio and protein contents reflects changes in macromolecular turnover for ATP-supplying enzymes and proteins.     

HISTOCHEMICAL STUDY OF ENZYME ACTIVITY IN THE SHOOT APICAL MERISTEM OF BRASSICA CAMPESTRIS DURING TRANSITION TO FLOWERING. IV. GLUCOSE-6-PHOSPHATASE

Glucose-6-phosphatase (G6P) activity was determined in fresh-frozen, cryostat sections in the shoot apical meristem of Brassica campestris L. Enzymatic activity was differentially distributed in a zonate pattern in the vegetative meristem, but not in the transition and floral meristem. Vegetative apices showed a heterogenous localization with the highest activity in the central zone and the pith-rib meristem zone. At the early transition stage of development, G6P activity in the peripheral zone increased slightly. At the late transitional (prefloral) stage, G6P activity was not localized within the peripheral zone in island-like areas of activity. This is the first demonstration of G6P in shoot apical meristem at the vegetative, transition, and floral stage. The results indicate that G6P activity 1) is an accompanying event of evocation, but 2) does not mark incipient floral primordia. G6P may play an important role in the maintenance of glucose-6-phosphate homeostasis in an evoked shoot apical meristem.     

NADP-dependent dehydrogenases in rat liver parenchyma

Summary The activities of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME) and isocitrate dehydrogenase (ICDH) were investigated with optimized histochemical methods (Rieder et al. 1978), and the activity of 3-hydroxybutyrate dehydrogenase (3HBDH) and neutral fat content with conventional techniques in the liver of male rats under the following experimental dietary conditions: (A) Fasting for 0, 12 and 84 h; (B) 84-h fasting followed by refeeding with a low-fat, high-carbohydrate diet for 6 h and for 2, 3, 5, 7, 11 and 14 nights; (C) refeeding with standard diet for 5 nights; (D) low-fat, high-carbohydrate diet for 7 and 14 nights.The activities of G6PDH, 6PGDH and ME decreased slightly during fasting primarily in zone 1 and increased dramatically on refeeding with a low-fat, high-carbohydrate diet. This activity increase was confined mainly to zone 3 during the first 3 days and was accompanied by a deposition of neutral fats that began in zone 3 and progressed to zone 1. Neutral fat accumulation was maximal after 3 nights, with a uniform accumulation of large droplets in all the hepatocytes; this was followed by a release that started in zone 3 and proceeded in a periportal direction. On the other hand, G6PDH, 6PGDH and ME attained their maximum activities after 5 and 7 nights of the low-fat diet, the activities being nearly homogeneously distributed over the liver acinus in a few cases. Subsequently the activities fell mainly in zone 1, causing the activity patterns and levels to approach those of the animals in group (D). In contrast to this, the activity of ICDH increased during fasting principally in zone 1, so that the otherwise steep activity gradient in favor of zone 3 lessened. Refeeding led at first to a fall of activity below the initial value, but later the normal distribution pattern was restored. The activity of 3HBDH showed a behavior similar to that of ICDH. The findings are discussed with reference to the functional heterogeneity of the liver perenchyma, and the existence of a liponeogenic area in zone 3 is proposed.Essential parts of this study have been presented to the Medical Faculty of the University of Freiburg/Br. as an inaugural dissertationSupported by grants from the Deutsche Forschungsgemeinschaft (Sa 127/7) and SFB 46     

Graduated resistance to G418 leads to differential selection of cultured mammalian cells expressing the neo gene

The effects of Geneticin (G418) selection on the growth and survival of cultured mammalian cells expressing the neomycin-resistance gene (neo) were studied by the analysis of cell clones from two retroviral neo vector-infected populations. We found a correlation between the neo expression level and growth rates in medium containing varying G418 concentrations. This relationship permits the use of differential selection schemes for the isolation of rare cells with increased expression. Comparison, by clone sampling, of vector-positive populations before and after selection with a G418 concentration in the range usually used for selection, showed different expression level and vector copy number distributions for the population infected with the vector of lower LTR activity, but not for the other. Such biasing effects of G418 selection may be important when selected cells are used for quantitative studies of gene expression.     

Construction of a neo fusion gene for expression in both prokaryotic and eukaryotic cells

A high-copy-number plasmid, pLink, was constructed to allow the direct selection in Escherichia coli of a neo fusion gene capable of conferring Geneticin (G418) resistance on mouse L cells. pLink was derived from pdMmtneo by insertion of a KpnI linker within the 5'-coding region of the neo gene. This created a minus-one frameshift mutation resulting in a translational termination within the N-terminal region of the protein. The Neo activity was restored by insertion into the modified neo gene of a piece of coding sequence derived from human HPRT cDNA. The resulting plasmid, pAH, was microinjected into mouse A9 cells and shown to confer resistance to G418.     

High dietary sodium intake increases white adipose tissue mass and plasma leptin in rats

Objective: Salt restriction has been reported to increase white adipose tissue (WAT) mass in rodents. The objective of this study was to investigate the effect of different sodium content diets on the lipogenic and lipolytic activities of WAT. Research Methods and Procedures: Male Wistar rats were fed on normal‐sodium (NS; 0.5% Na⁺), high‐sodium (HS; 3.12% Na⁺), or low‐sodium (LS; 0.06% Na⁺) diets for 3, 6, and 9 weeks after weaning. Blood pressure (BP) was measured using a computerized tail‐cuff system. At the end of each period, rats were killed and blood samples were collected for leptin determinations. The WAT from abdominal and inguinal subcutaneous (SC), periepididymal (PE) and retroperitoneal (RP) depots was weighed and processed for adipocyte isolation, rate measurement of lipolysis and d ‐[U‐¹⁴C]‐glucose incorporation into lipids, glucose‐6‐phosphate dehydrogenase (G6PDH) and malic enzyme activity evaluation, and determination of G6PDH and leptin mRNA expression. Results: After 6 weeks, HS diet significantly increased BP; SC, PE, and RP WAT masses; PE adipocyte size; plasma leptin concentration; G6PDH activity in SC WAT; and PE depots and malic activity only in SC WAT. The leptin levels correlated positively with WAT masses and adipocyte size. An increase in the basal and isoproterenol‐stimulated lipolysis and in the ability to incorporate glucose into lipids was observed in isolated adipocytes from HS rats. Discussion: HS diet induced higher adiposity characterized by high plasma leptin concentration and adipocyte hypertrophy, probably due to an increased lipogenic capacity of WAT.     

Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.     

High fructose-containing drinking water-induced steatohepatitis in rats is prevented by the nicotinamide-mediated modulation of redox homeostasis and NADPH-producing enzymes

An imbalance in the redox state, increased levels of lipid precursors and overactivation of de novo lipogenesis determine the development of fibrosis during nonalcoholic steatohepatitis (NASH). We evaluated the modulation of NADPH-producing enzymes associated with the antifibrotic, antioxidant and antilipemic effects of nicotinamide (NAM) in a model of NASH induced by excess fructose consumption. Male rats were provided drinking water containing 40% fructose for 16 weeks. During the last 12 weeks of fructose administration, water containing NAM was provided to some of the rats for 5 h/day. The biochemical profiles and the ghrelin, leptin, lipoperoxidation and TNF-α levels in serum and the glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and NADP⁺-dependent isocitric dehydrogenase (IDP) levels, the reduced/oxidized glutathione (GSH/GSSG) and reduced/oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H/NAD(P)⁺) ratios, and the levels of various lipogenic and fibrotic markers in the liver were evaluated. The results showed that hepatic fibrosis induced by fructose consumption was associated with weight gain, hunger-satiety system dysregulation, hyperinsulinemia, dyslipidemia, lipoperoxidation and inflammation. Moreover, increased levels of hepatic G6PD and ME activity and expression, the NAD(P)H/NAD(P)⁺ ratios, and GSSG concentration and increased expression of lipogenic and fibrotic markers were detected, and these alterations were attenuated by NAM administration. Specifically, NAM diminished the activity and expression of G6PD and ME, and this effect was associated with a decrease in the NADPH/NADP⁺ ratios, increased GSH levels and decreased lipoperoxidation and inflammation, ameliorating fibrosis and NASH development. NAM reduces liver steatosis and fibrosis by regulating redox homeostasis through a G6PD- and ME-dependent mechanism.

     

Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis

Prior work has reported that cotransfecting a gene of interest with the selectable marker neo can seriously perturb a number of cellular processes. In this study the influence of the neo gene on the growth, death, and metabolism of a murine myeloma NS0 cell line, expressing a chimeric antibody, was investigated. A pool of neo transfectants, 6A1-NEO, was selected with 500 microg/mL G418. Quantitative PCR analysis revealed that 6A1-NEO contained, on average, three copies of the neo gene per cell. Batch cultivation of 6A1-NEO showed that there was a 36% increase in maximum viable cell concentration, a 20% increase in the maximum apparent growth rate, and a 134% increase in cumulative cell hours as compared with the parent, 6A1-(100)3. Batch cultivation of five randomly selected clones illustrated that 6A1-NEO's advantage over the parent was not due to clonal variation. Neither the use of G418 during the selection process nor the cultivation of cells in the presence of G418 were responsible. This implied that the neo gene product, APH(3')-II, was causing the changes in proliferative capacity. Analysis of the cell cycle revealed that there were no differences in the distribution of cells in the G(1), S, and G(2) phases. When cell growth was synchronized, there were no observed differences in cell-cycle duration. 6A1-NEO resisted the onset of apoptosis during the growth phase. Consequently, there was a larger viable population of 6A1-NEO cells available for proliferation as compared with the parent. However, 6A1-NEO died at the same rate as the parent when resuspended in spent media or after treatment with staurosporin. Expression of the anti-apoptotic protein Bcl-2 was upregulated in 6A1-NEO, indicating that APH(3')-II could be acting by modulating endogenous gene expression. Analysis of key metabolites showed that 6A1-NEO's specific glucose consumption rate was 133% higher, whilst its specific glutamate consumption rate was 45% lower than the parent. 6A1-NEO's efficient utilization of glutamate and shift towards glucose metabolism may have contributed to the rise in proliferative capacity. However, this was accompanied by a 70% drop in the specific antibody production rate. These results show that the increase in growth rate and proliferative capacity caused by the expression of recombinant APH(3')-II was associated with changes in metabolism, apoptosis, and endogenous gene expression.     

Comparative studies on lipogenic enzyme activities in brown adipose tissue and liver of the rat during starvation-refeeding transition and cold exposure

1. The effect of starvation-refeeding transition and cold exposure on the activity of lipogenic enzymes in brown adipose tissue (BAT) and liver from rats was compared. 2. Starvation caused a decrease of lipogenic enzyme activities in BAT and liver. 3. Refeeding of the animals with a high carbohydrate diet caused an increase of lipogenic enzymes in these tissues. 4. Cold exposure (4 degrees C for 30 days) led to the increase of BAT enzyme activities to the values observed in rats fed a high carbohydrate diet. 5. Under the same conditions the activity of hepatic lipogenic enzymes also increased but never reached the values observed in the liver of rats fed with a high carbohydrate diet. 6. Therefore BAT and liver lipogenic enzymes showed, in general, a similar pattern of variation under identical nutritional conditions, but substantial differences between these two organs occurred as far as the response to cold exposure was concerned. 7. The experiments also revealed that in the control animals BAT displayed a higher lipogenic potential than the liver.     

Mechanisms for recombination between stably integrated vector sequences in CHO cells

Possible mechanisms for homologous recombination in CHO cells have been investigated using a stably integrated vector, pIII-14gpt. The vector contains 2 inactive neo gene fragments in tandem arrangement. Functional neo gene activity can be restored by recombination between homologous regions in the 2 fragments. Cells in which this event has taken place become resistant to the antibiotic G418. Possible mechanisms for neo gene reactivation in this system are unequal exchange between chromatids, intra-chromatidal deletion and gene conversion.

DNA from a total of 74 G418-resistant cell clones have been isolated, and analyzed on Southern blots using neo-specific probes. Rearrangements of neo-specific restriction fragments were found to have occurred in all cell clones. In 50% of the revertants, these rearrangements can be explained by a deletion which brings the complementary regions in the 2 neo gene gragments together.

One single revertant (1.3%) shows a possible gene conversion event. The other isolated revertants (about 48%) contain more complex rearrangements. These results indicate that the predominating recombination mechanism for reactivation of the neo gene in this system is either a deletion within a chromatid or an unequal exchange between sister chromatids.     

Increased activity of glycerol 3-phosphate dehydrogenase and other lipogenic enzymes in human bladder cancer.

Common molecular changes in cancer cells are high carbon flux through the glycolytic pathway and overexpression of fatty acid synthase, a key lipogenic enzyme. Since glycerol 3-phosphate dehydrogenase creates a link between carbohydrates and the lipid metabolism, we have investigated the activity of glycerol 3-phosphate dehydrogenase and various lipogenic enzymes in human bladder cancer. The data presented in this paper indicate that glycerol 3-phosphate dehydrogenase activity in human bladder cancer is significantly higher compared to adjacent non-neoplastic tissue, serving as normal control bladder tissue. Increased glycerol 3-phosphate dehydrogenase activity is accompanied by increased enzyme activity, either directly (fatty acid synthase) or indirectly (through ATP-citrate lyase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and citrate synthase) involved in fatty acid synthesis. Coordinated upregulation of glycerol 3-phosphate dehydrogenase and lipogenic enzymes activities in human bladder cancer suggests that glycerol 3-phosphate dehydrogenase supplies glycerol 3-phosphate for lipid biosynthesis.