Jiro Hoshino

Nicotinamide methylation and its relation to NAD synthesis in rat liver tissue culture. Biochemical basis for the physiological activities of 1-methylnicotinamide.

The mode of [14C]nicotinamide conversion to NAD and 1-methylnicotinamide and the effects of exogenous 1-methylnicotinamide on this metabolic conversion were studied using rat liver slices incubated in a chemically defined culture medium. It was shown that at the physiological nicotinamide concentrations tested (11-500 microM), 1-methylnicotinamide is preferentially produced, rather than NAD. Upon increasing nicotinamide concentration to the levels that cause cytotoxicity (1-10 mM and higher), the rate of NAD synthesis dramatically increased and reached a level 6-fold higher than that of 1-methylnicotinamide. A dose-dependent inhibition (up to 60%) of NAD synthesis was seen by the exogenous addition of 1-methylnicotinamide; the degree of inhibition is affected also by the concentration of nicotinamide present as a precursor. A large depletion of intracellular ATP, associated with a marked accumulation of NAD, occurred in slices in response to the addition of high amounts of nicotinamide. However, the loss of ATP was overcome, when nicotinamide was given together with 1-methylnicotinamide. Finally, 1-methylnicotinamide per se was proven active in regulating cell growth by comparing the cytosolic activity of 1-methylnicotinamide oxidation of cultured RLC cells with that of rat liver. Thus, the previously observed growth stimulation of hepatic cells by 1-methylnicotinamide can reasonably been explained by its ATP-sparing effect due to the inhibition of NAD synthesis, a reaction which requires ATP.

Methylation of nicotinamide in rat liver cytosol and its correlation with hepatocellular proliferation

The changes in the activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase) were studied in rat liver which was subjected to different rates of cellular proliferation. The cytosolic enzyme activity increased 3-4-fold in the first 24-48 h after partial hepatectomy and decreased again to the basal levels until 4 days post-operatively, whereas it remained unchanged in the livers of sham-operated animals. A single administration of thioacetamide at a dose of 50-250 mg/kg body weight, a treatment which induces hepatocellular proliferation as well, also enhanced the enzyme activity 2-3-fold 24 h after drug administration. This activity increase was associated with a marked lowering of intracellular NAD content of as much as 50% of the control levels. D-Galactosamine, a known hepatotoxic agent causing acute hepatitis in experimental animals and preventing DNA synthesis in regenerating liver, blocked the activity increase in regenerating rat liver. The rate of 1-methylnicotinamide synthesis, as measured by incubating liver slices in the culture medium supplemented with [14C]nicotinamide as a precursor, was found to be 2-4 times higher in the slices from regenerating liver and thioacetamide-treated rat liver than those from non-proliferating control liver. These results, together with our previous finding on the enhancement by 1-methylnicotinamide of the growth of cultured rat liver cells (Hoshino, J., Kühne, U. and Kröger, H. (1982) Biochem. Biophys. Res. Commun. 105, 1446-1452), support the view that nicotinamide methylase and its product, 1-methyl-nicotinamide, are involved in the control of hepatocellular DNA synthesis and proliferation.

Nicotinamide methylation. Tissue distribution, developmental and neoplastic changes.

The distribution of cytosolic activity of nicotinamide:S-adenosylmethionine methyltransferase (nicotinamide methylase, EC 2.1.1.1) in normal tissues from adult rat and mouse and in tumors and the change in the enzyme activity during the development of rat tissues were studied. (1) Rat liver exhibited the highest nicotinamide methylase activity among all adult tissues tested; other rat tissues, like adrenal, pancreas, kidney, brain and mouse tissues, had only less than 15% of the adult rat liver activity. (2) 3 days before birth, fetal liver showed a very low nicotinamide methylase activity (2% of adult rat liver), which, however, increased already 1 day before birth and reached the adult level on the day 28 after birth. (3) In a variety of hepatomas and ascites tumors, an inverse correlation, with some exceptions, between tumor growth rate and nicotinamide methylase activity could be seen. In all hepatomas, with the exception of Morris hepatoma 5123tc, nicotinamide methylase activity was significantly decreased in comparison to normal adult rat liver. The highly malignant Zajdela hepatoma, Yoshida sarcoma, sarcoma 180 and Ehrlich ascites tumor methylated nicotinamide only at a negligibly low rate. (4) Cultured RLC cells (an established rat liver cell line) from the stationary growth phase or G1-arrested RLC cells had about half of the adult rat liver activity, yet the activity was 70% higher than that of the logarithmically growing RLC cells.

Enhancement of DNA synthesis and cell proliferation by 1-methylnicotinamide in rat liver cells in culture: Implication for its in vivo role

Abstract 1-Methylnicotinamide, a direct methylation product of nicotinamide, stimulates the DNA synthesis and proliferation of rat liver cells (RLC) in culture at concentrations higher than 20 μM. The effect of nicotinamide, which is a potent inhibitor of DNA synthesis and proliferation, is counteracted by 1-methylnicotinamide. The intracellular NAD concentration decreases within 2 h under 1-methylnicotinamide, whereas it increases in the presence of nicotinamide. The poly(ADP-ribose) synthesizing activity in the isolated nuclei remained unchanged. These results suggest a physiological role of 1-methylnicotinamide in the cell growth through a lowering of intracellular NAD level.

3-aminobenzamide protects the mouse thymocytes in vitro from dexamethasone-mediated apoptotic cell death and cytolysis without changing DNA strand breakage

Exposure of mouse thymocytes to 1 microM dexamethasone (Dex) resulted in a dramatic increase in the degree of DNA strand breakage up to 80% between 4 to 6 h postincubation. During incubation a marked decrease in the number of total and viable cells as well as an increase in the release of lactate dehydrogenase into medium were detectable, indicating a strong cytotoxicity of Dex on the mouse thymocytes. Agarose gel electrophoresis of DNA from cells exposed to Dex for 6 h clearly demonstrated an increased laddering of DNA fragments multiple of approx. 200 base pairs as a characteristic feature of an apoptosis or programmed cell death. The cytotoxicity of Dex, as judged by the decrease in the viability and increase in the cell lysis, was effectively prevented by 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) synthesis. Furthermore, the lowering of intracellular NAD levels, which was observable in the present study most probably as a result of activation of poly(ADP-ribose) synthesis due to Dex-mediated DNA strand breakage, was also specifically prevented by the inhibitor, although the DNA strand breakage itself was not affected under these conditions. Our present results indicate that the Dex-mediated thymocyte death and cytolysis and probably intrathymic apoptotic thymocytolysis could be attributable primarily to the loss of intracellular NAD.

l-serine dehydrataste from rat liver. Purification and some properties

1. 1. l-Serine dehydratase l-serine hydrolyase (deaminating), EC 4.2.1.13] from rat liver was purified to electrophoretic homogeneity by a new method. 2. 2. After the last purification step two isoenzymes can be distinguished by DEAE-cellulose chormatography as well as by analytical disc electrophoresis. The same isoenzymes can be demonstrated after electrophoresis of a crude extract from rat liver. The smalles subunit of both isoenzymes has a mol. wt of about 35 000. This is in agremment with previous reports (Inoue, H., Kasper, C. B. and Pitot, H. C. (1971) J. Biol. Chem. 246, 2626–2632). 3. 3. From gel filtration experiments in the presence of the substrate l-serine it is concluded that the active form of l-serine dehydratase consists of two subunits. At low enzyme concentrations, the enzyme dissociates into its subunits; K+ and NH4+ counteract this process.

Induction in vivo of rat liver L-serine:pyruvate aminotransferase by N6, O2′-dibutyryl cyclic AMP and its inhibition by cortisone

Abstract 1. 1. a single injection of N 6 , O 2 ′-dibutyryl cyclic AMP (dibutyryl cyclic AMP) to intact and adrenalectomized rats causes a nearly 4–5-fold increase in the activity of hepatic serine-pyrovate aminotransferase within 6 h. The level of phosphoserine aminotransferase does not change. 2. Administration of actinomycin D along with dibutyryl cyclic AMP completely prevents the dibutyryl cyclic AMP-stimulated increase in serine-pyruvate aminotransferase. However, the antibiotic is no longer effective, if it is given one hour after the drug administration. When actinomycin D was given 3 h after the cyclic AMP, a marked stimulation in the enzyme level over thecontrol one was observed. Cycloheximide inhibits the dibutyryl cyclic AMP stimulation throughout the course of dibutyryl cyclic AMP stimulation. 3. Cortisone acetate, given shortly prior to, or simultaneously with dibutyryl cyclic AMP, partially prevents the rise in serine-pyruvate aminotransferase activity. But cortisone, administered 1 h after dibutyryl cyclic AMP, does not influence any more.

Potentiating effect of a physiological dose of cortisone acetate on the dibutyryl cyclic amp‐mediated induction of tyrosine aminotransferase in rat liver

The stimulatory effect of glucocorticoids and cyclic AMP (or glucagon) on the induction of hepatic tyrosine aminotransferase (EC 2.6.1.5) (TAT) has well been documented and its de novo synthesis was evidenced in rat liver [ 1,2] and in cultured cells [3]. Given together with glucocorticoids to animals or to cultured cells, cyclic AMP exerts a more than additive effect in the induction of TAT [2-41. Recent work of Krone et al. [5] suggests a role of the pharmacological dose of hydrocortisone in maintaining the induced level of TAT and phosphenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) brought about by I@, 02’-dibutyryl cyclic AMP (DBcAMP) in rat liver. Early works from our laboratory revealed that administration of a physiological dose of glucocorticoids is essential to the substrateor L-tryptophan-mediated stimulation of TAT in the liver of adrenalectomized . rats [6-81. In contrast to TAT the DBcAMP-mediated induction of L-serine:pyruvate aminotransferase (EC 2.6.1.5 1) is inhibited by cortisone acetate at a dose which stimulates TAT in rat liver [9,10]. The present study demonstrates that a physiological dose of cortisone acetate restores the reduced response of the enzyme to the DBcAMP-mediated induction in adrenalectomized rats nearly to the level of intact animals.

Identification of One of the L-Serine Dehydratase Isoenzymes from Rat Liver as L-Homoserine Dehydratase,

Abstract The higher molecular weight L-serine dehydratase was purified from rat liver and fractionated into two active components by Sephadex G-200 gel chromatography. On the basis of molecular weight, ultracentrifugal pattern and substrates specificity of the major component, it was concluded that this component is identical to L-homoserine dehydratase. Some misinter-pretations which have been derived from the work on L-serine dehydratase in animal tissues are pointed out.

Indirect involvement of glucocorticoids in the dibutyryl cyclic AMP-induction of tyrosine aminotransferase in cultured rat liver cells: Analysis using gonadal anti-glucocorticoids

Experiments were designed to study whether dibutyryl cyclic AMP (DBcAMP) requires glucocorticoids for the induction of tyrosine aminotransferase (TAT; EC 2.6.1.5) in a cultured rat liver cell line (RLC). For this purpose anti-glucocorticoids, 17α-methyltestosterone and progesterone, that are known to interfere with the action of glucocorticoids on the hepatic TAT, were given to the culture. As previously reported [3H]-dexamethasone binding by the cells was almost completely prevented by 17α-methyltestosterone in large excess. A large part of the previously bound dexamethasone was also depleted from the cells. Under these glucocorticoids-depleted conditions TAT was efficiently induced by DBcAMP. The induction was cordycepin-sensitive. However, the inducibility of TAT by DBcAMP rapidly increased following exposure of cells to dexamethasone. This “sensitization” of cells by the glucocorticoid was prevented by 17α-methyltestosterone and progesterone as well as by cycloheximide and cordycepin. The DBcAMP-induction of TAT even in the maximally sensitized cells was also not prevented by a large excess of 17α-methyltestosterone. These results clearly demonstrate that glucocorticoids are only indirectly involved in the DBcAMP-induction of TAT in RLC cells.