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Increased adenylate energy charges in rat liver or primary cultured rat hepatocytes by diisopropyl 1,3-dithiol-2-ylidenemalonate
Gen. Pharmac. Vol. 18, No. 1, pp. 13-15, 1987
0306-3623/87 $3.00 +0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
INCREASED ADENYLATE ENERGY CHARGES IN RAT LIVER OR PRIMARY CULTURED RAT HEPATOCYTES BY DIISOPROPYL 1,3-DITHIOL-2-YLIDENEMALONATE MITSUO ITAKURA,l MASAMI TSUCHIYA, 1KAMEJIROYAMASHITAI and TOSHITSUGU ODA2 tDivision of Endocrinology and Metabolism, Institute of Clinical Medicine, University of Tsukuba, Niihari-gun, Ibaraki, 305 and 2National Medical Center, Shinjuku, Tokyo, 160 Japan (Received 13 May 1986)
Abstract--1. Six hours after oral administration in rat liver, diisopropyl 1,3-dithiol-2-ylidenemalonate (malotilate) decreased AMP concentrations by 60% with unchanged total adenine nucleotide concentrations and significantly increased adenylate energy charges of 0.87 in comparison to those of 0.81 in vehicle administered control animals. 2. Twelve hours after incubation in primary cultured rat hepatocytes, malotilate decreased AMP concentrations by 18% and increased ATP and GTP concentrations by 13 and 18% respectively with significantly increased adenylate energy charges of 0.89 in comparison to 0.87 in vehicle control. 3. Increased adenylate energy charges by malotilate coincided with the initiation of increases in protein and de novo purine synthesis in vivo, and are associated with the increased rate of de novo purine synthesis in vitro.
INTRODUCTION Diisopropyl 1,3-dithiol-2-ylidenemalonate (malotilate) has been recently introduced in Japan for the treatment of chronic hepatitis and liver cirrhosis (Oda and Tygstrup, 1983). Malotilate increases rat liver R N A and protein synthesis (Imaizumi et al., 1982a, b) and cholesterol synthesis (Oda and Tygstrup, 1983). Malotilate accelerates liver regeneration in partially hepatectomized rats (Niwano et al., 1986), or in cirrhotic rat liver (Oda and Tygstrup, 1983). In addition, malotilate also protects liver from damage induced by ethionine or orotic acid (Oda and Tygstrup, 1983) which are known to deplete hepatic purines resulting in decreased A T P concentrations or adenylate energy charges (Shinozuka et al., 1968; Kelley et al., 1970). Based on the diversity of its effects and its protection against purine-depriving liver damages, it is hypothesized that one of the crucial functions of malotilate in liver is to increase A T P concentrations and/or adenylate energy charges which work as putative activators of multiple metabolic pathways (Atkinson, 1968). In this study this hypothesis was directly tested by studying purine ribonucleotide concentrations in rat liver in vivo after oral administration of malotilate and in primary cultured rat hepatocytes in vitro after incubation with malotilate. MATERIALS AND METHODS Materials
Malotilate was supplied by Nihon Nohyaku Co. Ltd, Tokyo, Japan. Eight week old male Wistar rats weighing 170-220 g were used. In the in vivo experiment, 250 mg/kg body wt of malotilate as 5% solution in 2% gum arabic or 2% gum arabic vehicle control were administered orally through a gastric tube at 10 a.m. In the in vitro experiment, malotilate was initially dissolved in dimethylsulfoxide because of its small solubility in water. The final concentration
of malotilate in the culture medium was 50 p g/ml with 0. 1% dimethylsulfoxide as vehicle control. Liver tissue sampling
Liver tissue sampling was performed under light open ether anesthesia by freeze-clamping the liver with metal tongs precooled with liquid nitrogen. The procedure was performed within 45 sec from skin incision to liver sampling. Liver tissue samples thus obtained were stored under liquid nitrogen. Isolation and culture o f hepatocytes
Hepatocytes were isolated from male Wistar rats by the two-step collagenase perfusion technique (Seglen, 1976), with a slight modification. After perfusion in Eagle's minimum essential medium (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan), the hepatocytes were obtained by centrifugation at 50 x g for 1 min. The viability of the hepatocytes determined by the trypan blue exclusion test was more than 90%. Hepatocytes were cultured in William's medium E (Life Technologies, Inc., New York) with 10% newborn calf serum (Life Technologies, Inc.), 10-TM insulin (Novo Industries, Copenhagen, Denmark), 10-6 M dexamethasone (Towa Pharmaceutical Co., Ltd, Osaka, Japan), 100 g/ml penicillin (Banyu Pharmaceutical Co., Ltd, Tokyo, Japan), and 100/~g/ml streptomycin (Meiji Pharmaceutical Co., Ltd, Tokyo, Japan). The cell density was initially 6 x 105 cells/35 mm plastic Corning dish and did not change after incubation for 12hr with malotilate. After incubating hepatocytes at 37°C for 4 hr in 95% 02 and 5% CO2, the medium was changed to hormone- and serum-free medium. The medium was again changed after 20 hr of the second culture. Malotilate at a final concentration of 50/zg/ ml was added to the culture medium after the second culture and incubated for 12 hr. Assay o f purine ribonucleotide concentrations
The purine ribonucleotide concentrations in liver including AMP, ADP, ATP, GMP, GDP and GTP were determined at 0, 6, 12 and 18 hr after administration of malotilate or vehicle. The methods for assay of purine ribonucleotides in liver were the same as those previously published (Itakura 13
14
MITSUO ITAKURA et al.
et al., 1981). In brief, the acid extract in 12% trichloroacetic acid was centrifuged at 12,000 × g for 2 min and the supernatant neutralized by an equal volume of 0.5 N tris-n-octylamine/l,l,2-trichlorotrifluoroethane. Twenty microliters of the neutralized sample were applied to a high pressure liquid chromatography system with an automatic sampler with a cooling unit (at 4°C) (obtained from Waters Associates, Milford, Mass.) containing an SAX 10 anion exchange column (obtained from Whatman, Clifton, N.J.). The sample was eluted by a linear gradient starting with 5 mM NH4PO 4 pH 2.8 and ending with 750 mM NH4PO 4 pH 3.9 at a rate of 2ml/min. Nucleotides were quantitated by absorbance unit at 254 nm and data expressed as /~mol/g wet liver weight of duplicate assay. Hepatocytes obtained from one rat were incubated for 12 hr in the presence or absence of 50/~g/ml of malotilate. After incubation, hepatocytes in 35 mm culture dish were rinsed with ice-cold phosphate buffered saline and purine ribonucleotides extracted from the cells by ice-cold 12% trichloroacetic acid for 30 rain and processed as described above. Purine ribonucleotide concentrations in hepatocytes were assayed in duplicate and data expressed as I0 mmol/ 105 cells. Calculation of adenylate energy charge
The adenylate energy charge was calculated as (1/2[ADP] + [ATP])/([AMP] + [ADP] + [ATP]) according to Atkinson (Atkinson and Fall, 1967). Statistical analysis
Statistical analysis was performed by Student's two-tailed t-test for unpaired samples. RESULTS AND DISCUSSION
Concentrations of adenine nucleotides and adenylate energy charges in rat liver at 0, 6 and 12 hr after administration of malotilate or vehicle control are summarized in Table 1. At 6 hr following administration, A M P concentration was significantly lower by 60% in malotilate-administered rat liver than that of control (P < 0.01). At this time point, the mean A D P concentration was 31% lower and the mean A T P concentration 14% higher than control without statistical significance (0.05 < P < 0.10). As a result the adenylate energy charges of 0.87 were significantly higher than control of 0.81 (P < 0.02). N o significant changes were observed in guanine nucleotide concentrations between the two groups. At 12 and 18 hr (the latter data not shown) following malotilate administration, there were no statistical differences between the purine ribonucleotide concentrations or adenylate energy charges of the two groups.
Table 2. Adenine nucleotide concentrations and adenylate energy charges in hepatocytes. Abbreviations and the signals for statistical analysis are the same as in Table I Vehicle Malotilate ( × 10 l0 mol/105 cells, Means _+ SEM) AMP ADP ATP TAN AEC (N)
0.33 _+0.02 3.22 _+0.07 11.62 _+0.24 15.21 _+0.30 0.873 _+0.00 (8)
0.27 _+0.02* 3.19 + 0.05 13.16 _+0.51 *~ 16.60 _+0.66 0.889 _+0.002** (8)
Concentrations of adenine nucleotides and adenylate energy charges in hepatocytes after incubation for 12 hr in the presence or absence of malotilate are summarized in Table 2. The A M P concentrations in malotilate-treated hepatocytes were significantly lower by 18% than control (P <0.05). The A T P concentrations in malotilate-treated hepatocytes were significantly higher by 13% than control (P < 0.02). Although not shown in this table, G T P concentration of 2.12 + 0.10 (n = 8) was also significantly higher by 13% than control of 1.79 _ 0.11 (n = 8) (P < 0.05), while no significant changes were observed in G M P or G D P concentrations between the two groups. The increased A T P and decreased A M P concentrations with the unchanged total adenine nucleotide concentration resulted in significantly increased adenylate energy charges of 0.889 in malotilate-treated hepatocytes in comparison to control values of 0.873 (P < 0.01). The changes in adenine nucleotide concentrations in liver in vivo at 6 hr after administration of malotilate and in malotilate-treated cells in vitro are both characterized by decreased A M P concentrations and unchanged total adenine nucleotide concentrations with increased adenylate energy charges. This suggests that these effects of malotilate are direct on hepatocytes. Increased adenylate energy charges have been suggested as the putative factor for accelerated metabolic pathways (Atkinson, 1968). As a typical example, 5-phosphoribosyl-l-pyrophosphate (PRPP) synthesis has been shown to be increased by increased adenylate energy charge (Atkinson and Fall, 1967), which will in turn work as an activator of the rate of purine synthesis de novo (Wyngaarden and Kelley, 1983). The increased adenylate energy charges in rat liver in vivo at 6 hr after malotilate administration coincided with the first apparent increase in protein synthesis at 5 or 8 hr, or in purine synthesis de novo at 4 hr after oral administration (Oda and Tygstrup, 1983;
Table 1. Adenine nucleotide concentrations and adenylate energy charges in liver. TAN, AEC and (N) represent total adenine nucleotides, adenylate energy charge and the number of animals, respectively. The results of statistical analysis by Student's unpaired t-test between 2 groups in rat liver 6 hr after administration are shown as follows; ~0.05 < P < 0.10, *P < 0.05, *++P < 0.02 and **P < 0.01
0
Time after administration (hr) (,umol/g wet liver weight, Means + SEM) 6 Vehicle
AMP ADP ATP TAN AEC (N)
0.13 +0.01 1.40+0.08 3.51_+0.10 5.05 ± 0.10 0.82_+0.02 (7)
0.20+0.03 1.56_+0.25 3.17_+0.13 4.82 _+ 0.26 0.81 -+0.02 (10)
Maiotilate 0.08_+0.01"* 1.08 + 0.07:[: 3.60 -+ 0.16:~ 4.75 + 0.18 0.87 -+ 0.01"~ (10)
12
Vehicle
Malotilate
0.19_+0.02 1.36_+0.11 3.35+0.13 4.88 + 0.17 0.82-+0.01 (15)
0.16_+0.02 1.22 -+ 0.09 3.33_+0.14 4.80 ± 0.18 0.83_+0.01 (17)
Malotilate and energy charges Itakura et al., 1986). The increase in protein synthesis reaches its peak at 12 or 15 hr and that for purine synthesis de novo at 12 hr after administration (Oda and Tygstrup, 1983; Itakura et al., 1986). In hepatocytes in vitro, the observed increases in adenylate energy charges and ATP concentrations are associated with the increased rate of purine synthesis de novo (Itakura et al., 1986). These results suggest that increased adenylate energy charges and/or ATP concentrations by malotilate may explain the accelerated rates in protein and purine synthesis de novo as have been shown for malotilate, and that they return to normal rates approximately 12 hr after administration, due to the consumption of ATP. The increases of adenylate energy charges and/or ATP concentrations are, although significant, limited in their magnitude both in vivo and in vitro. It is speculated that they could be even larger in diseased liver since they are tightly controlled in normal cells (Atkinson, 1968). We conclude that the increases in adenylate energy charges and/or ATP concentrations in vivo and in vitro by malotilate are one of the mechanisms for activation of multiple metabolic pathways by malotilate and is subject to further study.
REFERENCES
Atkinson D. E. (1968) The energy charge of the adenylate pool as a regulatory parameter: Interaction with feedback modifiers. Biochemistry 7, 4030-4034. Atkinson D. E. and Fall L. (1967) Adenosine triphosphate conservation in biosynthetic regulation. Escherichiaphosphoribosylpyrophosphate synthetase. J. biol. Chem. 242, 324i-3242.
15
Imaizumi Y., Katoh M. and Sugimoto T. (1982a) Effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate)on the synthesis and movement of RNA in rat liver. Folia Pharmac. Japan 79, 285-291. Imaizumi Y., Katoh M., Sugimoto T. and Kasai T. (1982b) Effect of malotilate (diisopropyl 1,3-dithiol-2-ylidene malonate) on the protein synthesis of the rat liver. Jap. J. Pharmac. 32, 369-375. Itakura M., Tsuchiya M., Yamashita K. and Oda T. (1986) Malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) increases liver de novo purine biosynthesis and amidophosphoribosyltransferase activity. J. Pharmac. exp. Ther. 273, 794-798. Itakura M., Sabina R. L., Heald P. W. and Holmes E. W. (1981) Basis for the control of purine biosynthesis by purine ribonucleotides. J. clin. Invest. 67, 994-1002. Kelley W. N., Greene M. L., Fox I. H., Rosenbloom F. M., Levy R. I. and SeegmillerJ. E. (1970) Effect of orotic acid on purine and lipoprotein metabolism in man. Metabolism 19, 1025-1035. Niwano Y., Katoh M., Uchida M. and Sugimoto T. (1986) Acceleration of liver regeneration by malotilate in partially hepatectomized rats. Jap. J. Pharmac. 40, 411-415. Oda T. and Tygstrup N. (1983) Malotilate, Hepatotropic Agent. Proc. Syrup. on Malotilate Held at the 7th Worm Cong. of Gastroenterology. Stockholm, June 1982, pp. 1-82, Excerpta Medica, Current Clinical Practice Series No. 10, Amsterdam-Princeton-Geneva-Tokyo. Seglen P. O. (1976) Preparation of isolated rat liver cells. Meth. Cell Biol. 13(4), 29-83. Shinozuka H., Goldblatt P. J. and Farber E. (1968) The disorganization of hepatic cell nucleoli induced by ethionine and its reversal by adenine. J. Cell. Biol. 36, 313-328. Wyngaarden J. B. and Kelley W. N. (1983) In The Metabolic Basis of Inherited Disease (Edited by Stanbury J. B., Wyngaarden J. B., Fredrickson D. S., Goldstein J. L. and Brown M. S.), 5th edn, Chap. 50, pp. 1043-1114. McGraw-Hill, New York.
0306-3623/87 $3.00 +0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
INCREASED ADENYLATE ENERGY CHARGES IN RAT LIVER OR PRIMARY CULTURED RAT HEPATOCYTES BY DIISOPROPYL 1,3-DITHIOL-2-YLIDENEMALONATE MITSUO ITAKURA,l MASAMI TSUCHIYA, 1KAMEJIROYAMASHITAI and TOSHITSUGU ODA2 tDivision of Endocrinology and Metabolism, Institute of Clinical Medicine, University of Tsukuba, Niihari-gun, Ibaraki, 305 and 2National Medical Center, Shinjuku, Tokyo, 160 Japan (Received 13 May 1986)
Abstract--1. Six hours after oral administration in rat liver, diisopropyl 1,3-dithiol-2-ylidenemalonate (malotilate) decreased AMP concentrations by 60% with unchanged total adenine nucleotide concentrations and significantly increased adenylate energy charges of 0.87 in comparison to those of 0.81 in vehicle administered control animals. 2. Twelve hours after incubation in primary cultured rat hepatocytes, malotilate decreased AMP concentrations by 18% and increased ATP and GTP concentrations by 13 and 18% respectively with significantly increased adenylate energy charges of 0.89 in comparison to 0.87 in vehicle control. 3. Increased adenylate energy charges by malotilate coincided with the initiation of increases in protein and de novo purine synthesis in vivo, and are associated with the increased rate of de novo purine synthesis in vitro.
INTRODUCTION Diisopropyl 1,3-dithiol-2-ylidenemalonate (malotilate) has been recently introduced in Japan for the treatment of chronic hepatitis and liver cirrhosis (Oda and Tygstrup, 1983). Malotilate increases rat liver R N A and protein synthesis (Imaizumi et al., 1982a, b) and cholesterol synthesis (Oda and Tygstrup, 1983). Malotilate accelerates liver regeneration in partially hepatectomized rats (Niwano et al., 1986), or in cirrhotic rat liver (Oda and Tygstrup, 1983). In addition, malotilate also protects liver from damage induced by ethionine or orotic acid (Oda and Tygstrup, 1983) which are known to deplete hepatic purines resulting in decreased A T P concentrations or adenylate energy charges (Shinozuka et al., 1968; Kelley et al., 1970). Based on the diversity of its effects and its protection against purine-depriving liver damages, it is hypothesized that one of the crucial functions of malotilate in liver is to increase A T P concentrations and/or adenylate energy charges which work as putative activators of multiple metabolic pathways (Atkinson, 1968). In this study this hypothesis was directly tested by studying purine ribonucleotide concentrations in rat liver in vivo after oral administration of malotilate and in primary cultured rat hepatocytes in vitro after incubation with malotilate. MATERIALS AND METHODS Materials
Malotilate was supplied by Nihon Nohyaku Co. Ltd, Tokyo, Japan. Eight week old male Wistar rats weighing 170-220 g were used. In the in vivo experiment, 250 mg/kg body wt of malotilate as 5% solution in 2% gum arabic or 2% gum arabic vehicle control were administered orally through a gastric tube at 10 a.m. In the in vitro experiment, malotilate was initially dissolved in dimethylsulfoxide because of its small solubility in water. The final concentration
of malotilate in the culture medium was 50 p g/ml with 0. 1% dimethylsulfoxide as vehicle control. Liver tissue sampling
Liver tissue sampling was performed under light open ether anesthesia by freeze-clamping the liver with metal tongs precooled with liquid nitrogen. The procedure was performed within 45 sec from skin incision to liver sampling. Liver tissue samples thus obtained were stored under liquid nitrogen. Isolation and culture o f hepatocytes
Hepatocytes were isolated from male Wistar rats by the two-step collagenase perfusion technique (Seglen, 1976), with a slight modification. After perfusion in Eagle's minimum essential medium (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan), the hepatocytes were obtained by centrifugation at 50 x g for 1 min. The viability of the hepatocytes determined by the trypan blue exclusion test was more than 90%. Hepatocytes were cultured in William's medium E (Life Technologies, Inc., New York) with 10% newborn calf serum (Life Technologies, Inc.), 10-TM insulin (Novo Industries, Copenhagen, Denmark), 10-6 M dexamethasone (Towa Pharmaceutical Co., Ltd, Osaka, Japan), 100 g/ml penicillin (Banyu Pharmaceutical Co., Ltd, Tokyo, Japan), and 100/~g/ml streptomycin (Meiji Pharmaceutical Co., Ltd, Tokyo, Japan). The cell density was initially 6 x 105 cells/35 mm plastic Corning dish and did not change after incubation for 12hr with malotilate. After incubating hepatocytes at 37°C for 4 hr in 95% 02 and 5% CO2, the medium was changed to hormone- and serum-free medium. The medium was again changed after 20 hr of the second culture. Malotilate at a final concentration of 50/zg/ ml was added to the culture medium after the second culture and incubated for 12 hr. Assay o f purine ribonucleotide concentrations
The purine ribonucleotide concentrations in liver including AMP, ADP, ATP, GMP, GDP and GTP were determined at 0, 6, 12 and 18 hr after administration of malotilate or vehicle. The methods for assay of purine ribonucleotides in liver were the same as those previously published (Itakura 13
14
MITSUO ITAKURA et al.
et al., 1981). In brief, the acid extract in 12% trichloroacetic acid was centrifuged at 12,000 × g for 2 min and the supernatant neutralized by an equal volume of 0.5 N tris-n-octylamine/l,l,2-trichlorotrifluoroethane. Twenty microliters of the neutralized sample were applied to a high pressure liquid chromatography system with an automatic sampler with a cooling unit (at 4°C) (obtained from Waters Associates, Milford, Mass.) containing an SAX 10 anion exchange column (obtained from Whatman, Clifton, N.J.). The sample was eluted by a linear gradient starting with 5 mM NH4PO 4 pH 2.8 and ending with 750 mM NH4PO 4 pH 3.9 at a rate of 2ml/min. Nucleotides were quantitated by absorbance unit at 254 nm and data expressed as /~mol/g wet liver weight of duplicate assay. Hepatocytes obtained from one rat were incubated for 12 hr in the presence or absence of 50/~g/ml of malotilate. After incubation, hepatocytes in 35 mm culture dish were rinsed with ice-cold phosphate buffered saline and purine ribonucleotides extracted from the cells by ice-cold 12% trichloroacetic acid for 30 rain and processed as described above. Purine ribonucleotide concentrations in hepatocytes were assayed in duplicate and data expressed as I0 mmol/ 105 cells. Calculation of adenylate energy charge
The adenylate energy charge was calculated as (1/2[ADP] + [ATP])/([AMP] + [ADP] + [ATP]) according to Atkinson (Atkinson and Fall, 1967). Statistical analysis
Statistical analysis was performed by Student's two-tailed t-test for unpaired samples. RESULTS AND DISCUSSION
Concentrations of adenine nucleotides and adenylate energy charges in rat liver at 0, 6 and 12 hr after administration of malotilate or vehicle control are summarized in Table 1. At 6 hr following administration, A M P concentration was significantly lower by 60% in malotilate-administered rat liver than that of control (P < 0.01). At this time point, the mean A D P concentration was 31% lower and the mean A T P concentration 14% higher than control without statistical significance (0.05 < P < 0.10). As a result the adenylate energy charges of 0.87 were significantly higher than control of 0.81 (P < 0.02). N o significant changes were observed in guanine nucleotide concentrations between the two groups. At 12 and 18 hr (the latter data not shown) following malotilate administration, there were no statistical differences between the purine ribonucleotide concentrations or adenylate energy charges of the two groups.
Table 2. Adenine nucleotide concentrations and adenylate energy charges in hepatocytes. Abbreviations and the signals for statistical analysis are the same as in Table I Vehicle Malotilate ( × 10 l0 mol/105 cells, Means _+ SEM) AMP ADP ATP TAN AEC (N)
0.33 _+0.02 3.22 _+0.07 11.62 _+0.24 15.21 _+0.30 0.873 _+0.00 (8)
0.27 _+0.02* 3.19 + 0.05 13.16 _+0.51 *~ 16.60 _+0.66 0.889 _+0.002** (8)
Concentrations of adenine nucleotides and adenylate energy charges in hepatocytes after incubation for 12 hr in the presence or absence of malotilate are summarized in Table 2. The A M P concentrations in malotilate-treated hepatocytes were significantly lower by 18% than control (P <0.05). The A T P concentrations in malotilate-treated hepatocytes were significantly higher by 13% than control (P < 0.02). Although not shown in this table, G T P concentration of 2.12 + 0.10 (n = 8) was also significantly higher by 13% than control of 1.79 _ 0.11 (n = 8) (P < 0.05), while no significant changes were observed in G M P or G D P concentrations between the two groups. The increased A T P and decreased A M P concentrations with the unchanged total adenine nucleotide concentration resulted in significantly increased adenylate energy charges of 0.889 in malotilate-treated hepatocytes in comparison to control values of 0.873 (P < 0.01). The changes in adenine nucleotide concentrations in liver in vivo at 6 hr after administration of malotilate and in malotilate-treated cells in vitro are both characterized by decreased A M P concentrations and unchanged total adenine nucleotide concentrations with increased adenylate energy charges. This suggests that these effects of malotilate are direct on hepatocytes. Increased adenylate energy charges have been suggested as the putative factor for accelerated metabolic pathways (Atkinson, 1968). As a typical example, 5-phosphoribosyl-l-pyrophosphate (PRPP) synthesis has been shown to be increased by increased adenylate energy charge (Atkinson and Fall, 1967), which will in turn work as an activator of the rate of purine synthesis de novo (Wyngaarden and Kelley, 1983). The increased adenylate energy charges in rat liver in vivo at 6 hr after malotilate administration coincided with the first apparent increase in protein synthesis at 5 or 8 hr, or in purine synthesis de novo at 4 hr after oral administration (Oda and Tygstrup, 1983;
Table 1. Adenine nucleotide concentrations and adenylate energy charges in liver. TAN, AEC and (N) represent total adenine nucleotides, adenylate energy charge and the number of animals, respectively. The results of statistical analysis by Student's unpaired t-test between 2 groups in rat liver 6 hr after administration are shown as follows; ~0.05 < P < 0.10, *P < 0.05, *++P < 0.02 and **P < 0.01
0
Time after administration (hr) (,umol/g wet liver weight, Means + SEM) 6 Vehicle
AMP ADP ATP TAN AEC (N)
0.13 +0.01 1.40+0.08 3.51_+0.10 5.05 ± 0.10 0.82_+0.02 (7)
0.20+0.03 1.56_+0.25 3.17_+0.13 4.82 _+ 0.26 0.81 -+0.02 (10)
Maiotilate 0.08_+0.01"* 1.08 + 0.07:[: 3.60 -+ 0.16:~ 4.75 + 0.18 0.87 -+ 0.01"~ (10)
12
Vehicle
Malotilate
0.19_+0.02 1.36_+0.11 3.35+0.13 4.88 + 0.17 0.82-+0.01 (15)
0.16_+0.02 1.22 -+ 0.09 3.33_+0.14 4.80 ± 0.18 0.83_+0.01 (17)
Malotilate and energy charges Itakura et al., 1986). The increase in protein synthesis reaches its peak at 12 or 15 hr and that for purine synthesis de novo at 12 hr after administration (Oda and Tygstrup, 1983; Itakura et al., 1986). In hepatocytes in vitro, the observed increases in adenylate energy charges and ATP concentrations are associated with the increased rate of purine synthesis de novo (Itakura et al., 1986). These results suggest that increased adenylate energy charges and/or ATP concentrations by malotilate may explain the accelerated rates in protein and purine synthesis de novo as have been shown for malotilate, and that they return to normal rates approximately 12 hr after administration, due to the consumption of ATP. The increases of adenylate energy charges and/or ATP concentrations are, although significant, limited in their magnitude both in vivo and in vitro. It is speculated that they could be even larger in diseased liver since they are tightly controlled in normal cells (Atkinson, 1968). We conclude that the increases in adenylate energy charges and/or ATP concentrations in vivo and in vitro by malotilate are one of the mechanisms for activation of multiple metabolic pathways by malotilate and is subject to further study.
REFERENCES
Atkinson D. E. (1968) The energy charge of the adenylate pool as a regulatory parameter: Interaction with feedback modifiers. Biochemistry 7, 4030-4034. Atkinson D. E. and Fall L. (1967) Adenosine triphosphate conservation in biosynthetic regulation. Escherichiaphosphoribosylpyrophosphate synthetase. J. biol. Chem. 242, 324i-3242.
15
Imaizumi Y., Katoh M. and Sugimoto T. (1982a) Effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate)on the synthesis and movement of RNA in rat liver. Folia Pharmac. Japan 79, 285-291. Imaizumi Y., Katoh M., Sugimoto T. and Kasai T. (1982b) Effect of malotilate (diisopropyl 1,3-dithiol-2-ylidene malonate) on the protein synthesis of the rat liver. Jap. J. Pharmac. 32, 369-375. Itakura M., Tsuchiya M., Yamashita K. and Oda T. (1986) Malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) increases liver de novo purine biosynthesis and amidophosphoribosyltransferase activity. J. Pharmac. exp. Ther. 273, 794-798. Itakura M., Sabina R. L., Heald P. W. and Holmes E. W. (1981) Basis for the control of purine biosynthesis by purine ribonucleotides. J. clin. Invest. 67, 994-1002. Kelley W. N., Greene M. L., Fox I. H., Rosenbloom F. M., Levy R. I. and SeegmillerJ. E. (1970) Effect of orotic acid on purine and lipoprotein metabolism in man. Metabolism 19, 1025-1035. Niwano Y., Katoh M., Uchida M. and Sugimoto T. (1986) Acceleration of liver regeneration by malotilate in partially hepatectomized rats. Jap. J. Pharmac. 40, 411-415. Oda T. and Tygstrup N. (1983) Malotilate, Hepatotropic Agent. Proc. Syrup. on Malotilate Held at the 7th Worm Cong. of Gastroenterology. Stockholm, June 1982, pp. 1-82, Excerpta Medica, Current Clinical Practice Series No. 10, Amsterdam-Princeton-Geneva-Tokyo. Seglen P. O. (1976) Preparation of isolated rat liver cells. Meth. Cell Biol. 13(4), 29-83. Shinozuka H., Goldblatt P. J. and Farber E. (1968) The disorganization of hepatic cell nucleoli induced by ethionine and its reversal by adenine. J. Cell. Biol. 36, 313-328. Wyngaarden J. B. and Kelley W. N. (1983) In The Metabolic Basis of Inherited Disease (Edited by Stanbury J. B., Wyngaarden J. B., Fredrickson D. S., Goldstein J. L. and Brown M. S.), 5th edn, Chap. 50, pp. 1043-1114. McGraw-Hill, New York.