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Inhibition of Lipid Synthesis of Bacteria, Yeast and Animal Cells by Anacardic Acids, Glycerol-3-phosphate Dehydrogenase Inhibitors from Ginkgo
Lebensm.-Wiss. u.-Technol., 30, 458–463 (1997)
Inhibition of Lipid Synthesis of Bacteria, Yeast and Animal Cells by Anacardic Acids, Glycerol-3-phosphate Dehydrogenase Inhibitors from Ginkgo Masatsune Murata*, Junko Irie† and Seiichi Homma Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112 (Japan) (Received August 1, 1996; accepted September 16, 1996)
The effects of anacardic acids (Ana), glycerol-3-phosphate dehydrogenase (GPDH) inhibitors from ginkgo, on growth and lipid synthesis of Bacillus subtilis, Lipomyces starkeyi and 3T3-L1 cells were examined. Ana inhibited growth of B. subtilis, and the inhibition was reversed by phosphatidic acid or monoacylglycerol. Ana-a (100 µg/mL) inhibited lipid accumulation in L. starkeyi, but not growth, when it was added after 40 h of incubation. Ana did not inhibit the adipose conversion of 3T3-L1 cells, as their inhibitory activity against GPDH of the cells was weak.
©1997 Academic Press Limited Keywords: ginkgo; anacardic acid; glycerol-3-phosphate dehydrogenase inhibitor; Lipomyces starkeyi; lipogenesis
Introduction Nuts of ginkgo (Ginkgo biloba) are popular and traditional foods in Japan, often steamed in eggs with vegetable. Recently, we isolated anacardic acids (Ana) from ginkgo as inhibitors of glycerol-3-phosphate dehydrogenase (EC 1.1.1.8, GPDH) (1). GPDH converts glycerol-3-phosphate to glyceraldehyde-3-phosphate in the presence of NADH and is a key enzyme in the synthesis of triacylglycerol (TG). Therefore, GPDH inhibitors are interesting from the point of antiobesity. The 50% inhibitory concentrations (IC50s) of Ana-a (6-tridecylsalicylic acid), Ana-b (6-[(8Z)-pentadecenyl]salicylic acid), Ana-c (6-[(9Z, 12Z)-heptadeca-dienyl]salicylic acid), and Ana-d (6-[(8Z)-heptadecenyl]salicylic acid) against GPDH from rabbit muscle were 9.4, 4.0, 4.6 and 2.4 µmol/L, respectively (Fig. 1). Ana-d was the main component among Ana in ginkgo; the sarcotesta contained about 2.2 mg of Ana-d per 100 g. Ana-c (about 0.02 mg/100 g) was detected in the edible part of ginkgo, ‘Ginnan’ (1). We expected that Ana would have some effects on lipid metabolism in cells as they definitely inhibited GPDH in vitro. Here we examined the effect of Ana on lipid *To whom correspondence should be addressed. †Present address: Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., 1188 Shimotogari, Nagaizumi, Sunto-gun, Shizuoka 411, Japan.
synthesis in three kinds of cells, i.e. bacteria (Bacillus subtilis), yeast accumulating TG (Lipomyces starkeyi), and animal cells converting to adipose cells (3T3-L1 cells). Cells of Bacillus subtilis grow on a synthetic medium without lipid constitutes. Growth of the bacterium is inhibited by Ana, because Ana inhibit lipid synthesis. Various lipid-related compounds were added to the medium in the presence of Ana. Elimination of growth inhibition by Ana was examined. After multiplication of Lipomyces starkeyi cells has COOH HO
R
Ana-a, R =
Ana-b, R =
8 9
Ana-c, R =
Ana-d, R =
8
Fig. 1 Structures of anacardic acids
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stopped, the yeast cells accumulate TG on exhaustion of nitrogen or phosphorus and in an excess of carbon (2, 3). The effect of Ana on this lipid accumulation was examined by observing the formation of oil globules in the yeast and measuring the lipid content by gas chromatography (GC). Swiss/3T3 fibroblasts were transformed to adipose cells under certain culture conditions and the GPDH activity increased several hundred times during this conversion (4, 5). The effect of Ana on this conversion was also examined.
Growth inhibition of B. subtilis by Ana and its reversal Ana-a, -b, -c or -d was dissolved in methanol and the solution added to the medium at a rate of 1.0 mL/100 mL. At this concentration, methanol had no effect on growth of B. subtilis. Glucose, glycerol or lipids (g/L; 20 glucose, 0.1 glycerol, 0.1 hexanoic acid, 0.1 stearic acid, 0.1 linoleic acid, 0.1 linolenic acid, 0.1 monoacylglycerol (glycerol α-monooleate), 0.1 L- α-phosphatidic acid, or 0.1 lecithin (from soybean)) was added at the same time. Growth of B. subtilis was monitored by measuring absorbance at 550 nm as described above.
Materials and Methods
Animal cell culture and adipose conversion Swiss 3T3 L-1 cells were provided by Dr Y. Kitagawa (Nagoya University, Nagoya, Japan). The cells were cultured by the method of Morita et al. (7) with some modifications. The cells were maintained in Dulbecco’s modified Eagle’s medium (Nissui Seiyaku Co., Tokyo) containing 100 mL/L foetal calf serum (Gibco), penicillin G (50,000 units/L) and streptomycin (25 mg/L). This medium is referred to as the shared medium. For adipose conversion, cells reaching confluence were fed a fresh standard medium supplemented with 10 mg/L insulin (Sigma), 0.25 mol/L dexamethasone, 0.5 mol/L methyl-isobutyl-xanthine and 0.2 mol/L L-ascorbic acid 2-phosphate magnesium salt (induction medium) (8). Three days later the medium was changed to the standard medium. The medium was changed every 2 d. Cells were incubated for about 10 d. Each Ana was dissolved in ethanol, and the solution added to the induction medium and fresh media after induction at a rate of 1.0 mL/L. At this concentration, ethanol had no effect on growth and conversion of the cells.
Lipomyces starkeyi. IFO10381 was kept on MY medium (g/L; 5.0 peptone, 3.0 yeast extract, 3.0 malt extract, and 10.0 glucose, pH 6.0). The yeast was transferred to a test tube containing 3 mL of MY medium and incubated for 2 d to give a seed culture. This seed culture was transferred at the rate of 1.0 mL to a test tube containing medium with the following constituents (g/L); 30 glucose, 7.0 KH2PO4, 5.0 Na2HPO4 . 12H2O, 1.5 MgSO4 . 12H2O, 0.5 NH4Cl, 0.1 CaCl2 . H2O, 0.08 FeCl3 . 6H2O, 0.01 ZnSO4 . 7H2O, 0.00007 MnSO4 . H2O, 0.0001 CuSO4 . 5H2O, and 0.000063 Co(NO3)2 (3). Ana was added to the medium at the start of incubation or after 48 h. The yeast was shaken at 27 °C and 110 oscillation per min in a reciprocating shaker. Growth was monitored by measuring absorbance at 550 nm. The number of viable cells was determined by measuring the number of yeast not stained with 1 g/100 mL methylene blue in 0.1 mol/L potassium phosphate buffer (pH 4.6). The cellular dry weight was determined after washing the cells (5 mL) with distilled water and drying in a freeze-dryer (Tozai Tsusho VFD-520, Tokyo). The cellular fatty acid composition and content were determined by directly treating dried cells with BF3/methanol (6) (Nacalai Tesque, Tokyo) and by GC (Shimazu GC-7A, Kyoto; column, Silar 10% Uniport 100/120, GL Science, Tokyo). Each measurement was taken twice for two cultures.
0.8
0.6 Absorbance at 550 nm
Strains and media Bacillus subtilis. PCI219 was kept on nutrient agar (Eiken Chemical Co., Tokyo, Japan). The bacterium was transferred to a test tube containing 3 mL of Spizizen medium (g/L; 2.0 (NH4)2SO4, 14 K2HPO4, 16 KH2PO4, 0.1 sodium citrate . 2H2O, 0.2 MgSO4 . 2H2O, and 5.0 glucose) and incubated for 11 h at 37 °C to give a seed culture. This seed culture was transferred at the rate of 1.0 mL/100 mL to a test tube containing a Spizizen medium supplemented with Ana or/and lipid related-compounds. The test tubes were shaken at 37 °C and 120 oscillation per min in a reciprocating shaker (Iwashiya Bio Science, Tokyo). Growth was monitored by measuring the absorbance at 550 nm with a Shimadzu Spectronic 201 (Kyoto, Japan) spectrometer.
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4
8 Incubation time (h)
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Fig. 2 Reversal by monoacylglycerol and phosphatidic acid of growth inhibition of Bacillus subtilis on Spizizen medium by anacardic acid. Ana-d (0.4 µg/mL), monooleine (1 g/L) and phosphatidic acid (1 g/L) were added to the medium at the start of incubation. (s) = no addition; (d) = Ana-d; (m) = Ana-d plus monoolein; (h) = Ana-d plus phosphatidic acid
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The cells were collected and washed by phosphate buffered saline (mmol/L); 137 NaCl, 2.7 KCl, 8.1 Na2HPO4 and 1.5 KH2PO4 (pH 7.2). GPDH was extracted with 5 mol/L tris-HCl buffer (pH 7.5) containing 1 mol/L EDTA and 1 mol/L 2-mercaptoethanol. After centrifugation enzyme activity and protein content (9) were determined. Content of triacylglycerol (TG) in cells was determined by a TG measuring kit (Wako, Osaka, Japan). Each measurement was taken twice for two cultures.
Oil red O staining (10) Lipomyces starkeyi or 3T3-L1 cells were stained with oil red O solution (50 g/L oil red O in isopropanol/ water (6:4)). Oil globules stained red or orange and were observed under microscopy (Olympus Vanox, Tokyo).
Glycerol-3-phosphate dehydrogenase (GPDH) assay The enzyme assay was performed as already described (1). The reaction mixture contained the following constituents (mol/L); 100 tris-HCl buffer (pH 7.5), 2.5 EDTA, 0.12 NADH, 0.2 dihydroxyacetone phosphate, 0.1 2-mercaptoethanol and the enzyme. The reaction was followed by optical density at 340 nm and 30 °C. Each measurement was taken twice.
Results and Discussion Effect of Ana on the growth of Bacillus subtilis At first, the inhibitory effect of Ana against the growth of Bacillus subtilis on Spizizen medium, which does not contain TG and phospholipid, was examined. All Ana inhibited growth. For example, growth was repressed by 0.05 µg/mL of Ana-d for 14 h. Various lipid-related compounds (glucose, glycerol, hexanoic acid, stearic acid, linoleic acid, linolenic acid, monoacylglycerol, phosphatidic acid and lecithin) were added to the medium with 0.4 µg/mL of Ana-d. After 14 h of incubation, growth was turbidimetrically estimated. Monoacylglycerol, phosphatidic acid and lecithin restored growth of the bacterium but the others did not. Figure 2 showed the growth inhibition by Ana-d and its reversal by monoacylglycerol and phosphatidic acid. Growth was considerably restored by phosphatidic acid and monoacylglycerol. Ana-a, -b and -c also inhibited growth and the inhibition was restored by phosphatidic acid or monoacylglycerol (data not shown). It was considered that GPDH of the bacterium was inhibited by Ana, and therefore TG and phospholipid were not synthesized and cells did not grow. Supplementation with glycerides reversed this inhibition.
Effect of Ana on lipid accumulation of Lipomyces starkeyi After cell multiplication had stopped, oleoginous yeast such as Lipomyces starkeyi accumulate TG. Lipomyces starkeyi can accumulate lipids up to about 80 g/100 g of
Fig. 3 Lipomyces starkeyi IFO10381 stained with oil red O (A) 50 h after incubation, and (b) and (c) 150 h after incubation; in (c) Ana-a (10 µg/mL) was added to the medium at the start of incubation
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10
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0
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(a) Log cell number (1/mL)
Absorbance at 550 nm
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30 60 90 Incubation time (h)
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Fig. 4 Effect of anacardic acid on growth and lipid accumulation in L. starkeyi IFO10381. (a) OD550; (b) cell number; (c) lipid accumulation. Ana-a was added to the medium at the start of incubation. (j) = no addition; (m) = 1 µg/mL; (d) = 5 µg/mL
biomass relatively quickly (3). As shown in Fig. 3, L. starkeyi IFO10381 accumulated TG after 130 h of incubation in a synthetic medium. Oil globules were stained with oil red O. The effect of Ana on the formation of oil globules was observed under microscopy. Ana-a and -b (5–10 µg/mL) showed repression of lipid accumulation, while Ana-c and -d showed no effect at 100 µg/mL. Although we do not know why Ana-c and -d showed no effect, the effects of Ana-a on growth and lipid accumulation were examined in detail. The viability of the yeast was estimated by methylene blue staining, and the lipid content was determined by
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(a)
(b)
8 Lipid (pg/cell)
Log cell number (1/mL)
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GC. The cell number was almost constant after 50 h of incubation but OD550 increased after that, due to the accumulation of TG in the control culture without Anaa. When 5 µg/mL of Ana-a was added to the medium, the growth was delayed and lipid formation was repressed (Fig. 4). However, after 200 h of incubation, the lipid content reached the level of the control. It was considered that this delay of growth was caused by the inhibition of lipid synthesis. Next, the effect of Ana-a was examined when it was added after 50 h of incubation. As shown in Fig. 5, the number of viable cells was almost unchanged and lipid formation was
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Fig. 5 Effect of anacardic acid on (a) viability and (b) lipid accumulation in L. starkeyi IFO10381. Ana-a was added to the medium at 50 h after incubation (↓). (j) = no addition; (d) = Ana-a (100 µg/mL)
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3 Log GPDH (U/mg protein)
TG (% of control)
150
100
50
0
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4 6 Ana-d (µg/ml)
8
10
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5 10 Days after induction (d)
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Fig. 6 Effect of anacardic acid on (a) lipid accumulation and (b) glycerol-3-phosphate dehydrogenase (GPDH) in 3T3-L1 cells ((h) = no addition; (d) = Ana-d (10 µg/mL)). Ana-d was added to the induction medium and fresh media after induction
depressed when 100 µg/mL of Ana-a was added to the medium at the induction period of TG. This result suggests that Ana-a repressed the lipid accumulation of the yeast by inhibiting GPDH.
Effect of Ana on adipose conversion of 3T3-L1 cells Swiss 3T3-L1 cells have a high probability of conversion to adipose cells accumulating TG (5). Exponentially-growing 3T3-L1 cells have the morphological and biochemical properties of fibroblasts, but they differentiate into adipocytes, rounded cells containing a large droplet of TG after reaching confluence. In the course of this conversion, the activities of many lipogenic enzymes increases. Among them GPDH must have an important role in the conversion process. (4) The enzyme occupies a central position in the pathway of TG synthesis (11) at the branch point from the glycolytic pathway. Conversion is stimulated by insulin or methylisobutylxanthine plus dexamethasone (12) and the expression of GPDH is also induced by insulin, dexamethasone or c-AMP (13). Here, the effect of Ana on this adipose conversion was examined. At first, the morphological change was observed in the presence or absence of Ana or LiCl (14) under microscopy, the oil globule being stained with oil red O. Lithium ion inhibited inducer-stimulated adipose conversion of 3T3-L1 cells (14). Although LiCl (10 mol/L) definitely repressed differentiation in this experiment, Ana-d did not. The TG content in 3T3-L1 cells during the conversion was also measured. However, Ana-d did not inhibit the accumulation of TG and GPDH in the cells (Fig. 6), while 20 µg/mL of Ana-d inhibited 50% of the growth of the cells. Ana-b, -c and -d also did not inhibit the conversion of adipose cells. Furthermore, the effect of Ana on the activity of GPDH from the cells was examined. Ana did not inhibit the enzyme strongly. For example, the IC50 of Ana-d against GPDH from the cells was about 10 µg/mL, while the IC50 against the enzyme from rabbit muscle, which was used for screening of GPDH inhibitors, was 0.9 µg/mL (1). It was considered that Ana did not inhibit the accumula-
tion of TG and the conversion because it did not inhibit GPDH from 3T3-L1 cells. Not much is known about definite inhibitors of GPDH. Adipostatins A and B were isolated as inhibitors of GPDH from fermented broth of actinomycete in 1992 (15). Ana was also isolated from ginkgo as GPDH inhibitors in 1996 (1). Ana inhibited the growth of B. subtilis on a synthetic medium without lipids and its inhibition was restored by acylglycerol. Lipomyces starkeyi accumulated TG during the later part of incubation. When Ana was added to the medium at the induction period of TG accumulation, TG accumulation was repressed. On the other hand, Ana did not inhibit TG accumulation and GPDH of 3T3-L1 cells. It seemed that GPDH inhibition of Ana was variable depending on the origin of GPDH.
Acknowledgements We thank Dr Yasuo Kitagawa, Professor of Nagoya University, for providing 3T3-L1 cells and instructing the cell culture.
References 1 IRIE, J., MURATA, M. AND HOMMA, S. Glycerol-3-phosphate dehydrogenase inhibitors, anacardic acids, from Ginkgo biloba. Bioscience, Biotechnology, and Biochemistry, 60, 240–243 (1996) 2 STARKEY, R. L. Lipid production by a soil yeast. Journal of Bacteriology, 51, 33–50 (1945) 3 SUUTARI, M., PRIHA, P. AND LAALSO, S. Temperature shifts in regulation of lipids accumulated by Lipomyces starkeyi. Journal of American Oil Chemists’ Society, 70, 891–894 (1993) 4 WISE, L. S. AND GREEN, H. Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. The Journal of Biological Chemistry, 254, 273–275 (1979) 5 GREEN, H. AND KEHINDE, O. Sublines of mouse 3T3 cells that accumulate lipid. Cell, 1, 113–116 (1974) 6 KNAPP, D. R. Fatty acid esters from glycerides. In: Handbook of analytical derivatization reaction. New York: John Wiley & Sons, p. 166 (1979)
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7 MORITA, A., ARATANI, Y., SUGIMOTO, E. AND KITAGAWA, Y. Catecholamine-stimulated protein phosphorylation in 3T3-L1 preadipocytes and adipocytes. Journal of Biochemistry, 95, 743–750 (1984) 8 ONO, M., ARATANI, Y., KITAGAWA, I. AND KITAGAWA, Y. Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells. Experimental Cell Research, 187, 309–314 (1990) 9 LOWRY, O. H., ROSENBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. Protein measurement with Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275 (1951) 10 PREECE, A. Oil red O. In: Mannual for Histologic technicians. Boston: Little, Brown & Co., pp. 260–261 (1972) 11 STEINBERG, D., VAUGHAM, M. AND MARGOLIS, S. The Journal of Biological Chemistry, 236, 1631–1637 (1961)
12 RUBIN, C. S., HIRSCH, A., FUNG, C. AND ROSEN, O. M. Development of hormone receptors and hormonal responsiveness in vitro. The Journal of Biological Chemistry, 253, 7570–7578 (1978) 13 BHANDARI, B., SAINI, K. S. AND MILLER, R. E. Glycerol 3-phosphate dehydrogenase gene expression in cultured 3T3-L1 adipocytes: regulation by insulin, dexamethasone and dibutyryl cAMP at the level of mRNA abundance, transcription and mRNA stability. Molecular and Cellular Endocrinology, 76, 71–77 (1991) 14 ARATANI, Y., SUGIMOTO, E. AND KITAGAWA, Y. Lithium ion reversibly inhibits inducer-stimulated adipose conversion of 3T3-L1 cells. FEBS Letters, 218, 47–51 (1987) 15 TSUGE, N., MIZOKAMI, M., IMAI, S., SHIMAZU, A. AND SETO, H. Adipostatins A and B, new inhibitors of glycerol3-phosphate dehydrogenase. The Journal of Antibiotics, 45, 886–891 (1992)
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Inhibition of Lipid Synthesis of Bacteria, Yeast and Animal Cells by Anacardic Acids, Glycerol-3-phosphate Dehydrogenase Inhibitors from Ginkgo Masatsune Murata*, Junko Irie† and Seiichi Homma Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112 (Japan) (Received August 1, 1996; accepted September 16, 1996)
The effects of anacardic acids (Ana), glycerol-3-phosphate dehydrogenase (GPDH) inhibitors from ginkgo, on growth and lipid synthesis of Bacillus subtilis, Lipomyces starkeyi and 3T3-L1 cells were examined. Ana inhibited growth of B. subtilis, and the inhibition was reversed by phosphatidic acid or monoacylglycerol. Ana-a (100 µg/mL) inhibited lipid accumulation in L. starkeyi, but not growth, when it was added after 40 h of incubation. Ana did not inhibit the adipose conversion of 3T3-L1 cells, as their inhibitory activity against GPDH of the cells was weak.
©1997 Academic Press Limited Keywords: ginkgo; anacardic acid; glycerol-3-phosphate dehydrogenase inhibitor; Lipomyces starkeyi; lipogenesis
Introduction Nuts of ginkgo (Ginkgo biloba) are popular and traditional foods in Japan, often steamed in eggs with vegetable. Recently, we isolated anacardic acids (Ana) from ginkgo as inhibitors of glycerol-3-phosphate dehydrogenase (EC 1.1.1.8, GPDH) (1). GPDH converts glycerol-3-phosphate to glyceraldehyde-3-phosphate in the presence of NADH and is a key enzyme in the synthesis of triacylglycerol (TG). Therefore, GPDH inhibitors are interesting from the point of antiobesity. The 50% inhibitory concentrations (IC50s) of Ana-a (6-tridecylsalicylic acid), Ana-b (6-[(8Z)-pentadecenyl]salicylic acid), Ana-c (6-[(9Z, 12Z)-heptadeca-dienyl]salicylic acid), and Ana-d (6-[(8Z)-heptadecenyl]salicylic acid) against GPDH from rabbit muscle were 9.4, 4.0, 4.6 and 2.4 µmol/L, respectively (Fig. 1). Ana-d was the main component among Ana in ginkgo; the sarcotesta contained about 2.2 mg of Ana-d per 100 g. Ana-c (about 0.02 mg/100 g) was detected in the edible part of ginkgo, ‘Ginnan’ (1). We expected that Ana would have some effects on lipid metabolism in cells as they definitely inhibited GPDH in vitro. Here we examined the effect of Ana on lipid *To whom correspondence should be addressed. †Present address: Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., 1188 Shimotogari, Nagaizumi, Sunto-gun, Shizuoka 411, Japan.
synthesis in three kinds of cells, i.e. bacteria (Bacillus subtilis), yeast accumulating TG (Lipomyces starkeyi), and animal cells converting to adipose cells (3T3-L1 cells). Cells of Bacillus subtilis grow on a synthetic medium without lipid constitutes. Growth of the bacterium is inhibited by Ana, because Ana inhibit lipid synthesis. Various lipid-related compounds were added to the medium in the presence of Ana. Elimination of growth inhibition by Ana was examined. After multiplication of Lipomyces starkeyi cells has COOH HO
R
Ana-a, R =
Ana-b, R =
8 9
Ana-c, R =
Ana-d, R =
8
Fig. 1 Structures of anacardic acids
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stopped, the yeast cells accumulate TG on exhaustion of nitrogen or phosphorus and in an excess of carbon (2, 3). The effect of Ana on this lipid accumulation was examined by observing the formation of oil globules in the yeast and measuring the lipid content by gas chromatography (GC). Swiss/3T3 fibroblasts were transformed to adipose cells under certain culture conditions and the GPDH activity increased several hundred times during this conversion (4, 5). The effect of Ana on this conversion was also examined.
Growth inhibition of B. subtilis by Ana and its reversal Ana-a, -b, -c or -d was dissolved in methanol and the solution added to the medium at a rate of 1.0 mL/100 mL. At this concentration, methanol had no effect on growth of B. subtilis. Glucose, glycerol or lipids (g/L; 20 glucose, 0.1 glycerol, 0.1 hexanoic acid, 0.1 stearic acid, 0.1 linoleic acid, 0.1 linolenic acid, 0.1 monoacylglycerol (glycerol α-monooleate), 0.1 L- α-phosphatidic acid, or 0.1 lecithin (from soybean)) was added at the same time. Growth of B. subtilis was monitored by measuring absorbance at 550 nm as described above.
Materials and Methods
Animal cell culture and adipose conversion Swiss 3T3 L-1 cells were provided by Dr Y. Kitagawa (Nagoya University, Nagoya, Japan). The cells were cultured by the method of Morita et al. (7) with some modifications. The cells were maintained in Dulbecco’s modified Eagle’s medium (Nissui Seiyaku Co., Tokyo) containing 100 mL/L foetal calf serum (Gibco), penicillin G (50,000 units/L) and streptomycin (25 mg/L). This medium is referred to as the shared medium. For adipose conversion, cells reaching confluence were fed a fresh standard medium supplemented with 10 mg/L insulin (Sigma), 0.25 mol/L dexamethasone, 0.5 mol/L methyl-isobutyl-xanthine and 0.2 mol/L L-ascorbic acid 2-phosphate magnesium salt (induction medium) (8). Three days later the medium was changed to the standard medium. The medium was changed every 2 d. Cells were incubated for about 10 d. Each Ana was dissolved in ethanol, and the solution added to the induction medium and fresh media after induction at a rate of 1.0 mL/L. At this concentration, ethanol had no effect on growth and conversion of the cells.
Lipomyces starkeyi. IFO10381 was kept on MY medium (g/L; 5.0 peptone, 3.0 yeast extract, 3.0 malt extract, and 10.0 glucose, pH 6.0). The yeast was transferred to a test tube containing 3 mL of MY medium and incubated for 2 d to give a seed culture. This seed culture was transferred at the rate of 1.0 mL to a test tube containing medium with the following constituents (g/L); 30 glucose, 7.0 KH2PO4, 5.0 Na2HPO4 . 12H2O, 1.5 MgSO4 . 12H2O, 0.5 NH4Cl, 0.1 CaCl2 . H2O, 0.08 FeCl3 . 6H2O, 0.01 ZnSO4 . 7H2O, 0.00007 MnSO4 . H2O, 0.0001 CuSO4 . 5H2O, and 0.000063 Co(NO3)2 (3). Ana was added to the medium at the start of incubation or after 48 h. The yeast was shaken at 27 °C and 110 oscillation per min in a reciprocating shaker. Growth was monitored by measuring absorbance at 550 nm. The number of viable cells was determined by measuring the number of yeast not stained with 1 g/100 mL methylene blue in 0.1 mol/L potassium phosphate buffer (pH 4.6). The cellular dry weight was determined after washing the cells (5 mL) with distilled water and drying in a freeze-dryer (Tozai Tsusho VFD-520, Tokyo). The cellular fatty acid composition and content were determined by directly treating dried cells with BF3/methanol (6) (Nacalai Tesque, Tokyo) and by GC (Shimazu GC-7A, Kyoto; column, Silar 10% Uniport 100/120, GL Science, Tokyo). Each measurement was taken twice for two cultures.
0.8
0.6 Absorbance at 550 nm
Strains and media Bacillus subtilis. PCI219 was kept on nutrient agar (Eiken Chemical Co., Tokyo, Japan). The bacterium was transferred to a test tube containing 3 mL of Spizizen medium (g/L; 2.0 (NH4)2SO4, 14 K2HPO4, 16 KH2PO4, 0.1 sodium citrate . 2H2O, 0.2 MgSO4 . 2H2O, and 5.0 glucose) and incubated for 11 h at 37 °C to give a seed culture. This seed culture was transferred at the rate of 1.0 mL/100 mL to a test tube containing a Spizizen medium supplemented with Ana or/and lipid related-compounds. The test tubes were shaken at 37 °C and 120 oscillation per min in a reciprocating shaker (Iwashiya Bio Science, Tokyo). Growth was monitored by measuring the absorbance at 550 nm with a Shimadzu Spectronic 201 (Kyoto, Japan) spectrometer.
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0
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8 Incubation time (h)
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Fig. 2 Reversal by monoacylglycerol and phosphatidic acid of growth inhibition of Bacillus subtilis on Spizizen medium by anacardic acid. Ana-d (0.4 µg/mL), monooleine (1 g/L) and phosphatidic acid (1 g/L) were added to the medium at the start of incubation. (s) = no addition; (d) = Ana-d; (m) = Ana-d plus monoolein; (h) = Ana-d plus phosphatidic acid
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The cells were collected and washed by phosphate buffered saline (mmol/L); 137 NaCl, 2.7 KCl, 8.1 Na2HPO4 and 1.5 KH2PO4 (pH 7.2). GPDH was extracted with 5 mol/L tris-HCl buffer (pH 7.5) containing 1 mol/L EDTA and 1 mol/L 2-mercaptoethanol. After centrifugation enzyme activity and protein content (9) were determined. Content of triacylglycerol (TG) in cells was determined by a TG measuring kit (Wako, Osaka, Japan). Each measurement was taken twice for two cultures.
Oil red O staining (10) Lipomyces starkeyi or 3T3-L1 cells were stained with oil red O solution (50 g/L oil red O in isopropanol/ water (6:4)). Oil globules stained red or orange and were observed under microscopy (Olympus Vanox, Tokyo).
Glycerol-3-phosphate dehydrogenase (GPDH) assay The enzyme assay was performed as already described (1). The reaction mixture contained the following constituents (mol/L); 100 tris-HCl buffer (pH 7.5), 2.5 EDTA, 0.12 NADH, 0.2 dihydroxyacetone phosphate, 0.1 2-mercaptoethanol and the enzyme. The reaction was followed by optical density at 340 nm and 30 °C. Each measurement was taken twice.
Results and Discussion Effect of Ana on the growth of Bacillus subtilis At first, the inhibitory effect of Ana against the growth of Bacillus subtilis on Spizizen medium, which does not contain TG and phospholipid, was examined. All Ana inhibited growth. For example, growth was repressed by 0.05 µg/mL of Ana-d for 14 h. Various lipid-related compounds (glucose, glycerol, hexanoic acid, stearic acid, linoleic acid, linolenic acid, monoacylglycerol, phosphatidic acid and lecithin) were added to the medium with 0.4 µg/mL of Ana-d. After 14 h of incubation, growth was turbidimetrically estimated. Monoacylglycerol, phosphatidic acid and lecithin restored growth of the bacterium but the others did not. Figure 2 showed the growth inhibition by Ana-d and its reversal by monoacylglycerol and phosphatidic acid. Growth was considerably restored by phosphatidic acid and monoacylglycerol. Ana-a, -b and -c also inhibited growth and the inhibition was restored by phosphatidic acid or monoacylglycerol (data not shown). It was considered that GPDH of the bacterium was inhibited by Ana, and therefore TG and phospholipid were not synthesized and cells did not grow. Supplementation with glycerides reversed this inhibition.
Effect of Ana on lipid accumulation of Lipomyces starkeyi After cell multiplication had stopped, oleoginous yeast such as Lipomyces starkeyi accumulate TG. Lipomyces starkeyi can accumulate lipids up to about 80 g/100 g of
Fig. 3 Lipomyces starkeyi IFO10381 stained with oil red O (A) 50 h after incubation, and (b) and (c) 150 h after incubation; in (c) Ana-a (10 µg/mL) was added to the medium at the start of incubation
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Fig. 4 Effect of anacardic acid on growth and lipid accumulation in L. starkeyi IFO10381. (a) OD550; (b) cell number; (c) lipid accumulation. Ana-a was added to the medium at the start of incubation. (j) = no addition; (m) = 1 µg/mL; (d) = 5 µg/mL
biomass relatively quickly (3). As shown in Fig. 3, L. starkeyi IFO10381 accumulated TG after 130 h of incubation in a synthetic medium. Oil globules were stained with oil red O. The effect of Ana on the formation of oil globules was observed under microscopy. Ana-a and -b (5–10 µg/mL) showed repression of lipid accumulation, while Ana-c and -d showed no effect at 100 µg/mL. Although we do not know why Ana-c and -d showed no effect, the effects of Ana-a on growth and lipid accumulation were examined in detail. The viability of the yeast was estimated by methylene blue staining, and the lipid content was determined by
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GC. The cell number was almost constant after 50 h of incubation but OD550 increased after that, due to the accumulation of TG in the control culture without Anaa. When 5 µg/mL of Ana-a was added to the medium, the growth was delayed and lipid formation was repressed (Fig. 4). However, after 200 h of incubation, the lipid content reached the level of the control. It was considered that this delay of growth was caused by the inhibition of lipid synthesis. Next, the effect of Ana-a was examined when it was added after 50 h of incubation. As shown in Fig. 5, the number of viable cells was almost unchanged and lipid formation was
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Fig. 5 Effect of anacardic acid on (a) viability and (b) lipid accumulation in L. starkeyi IFO10381. Ana-a was added to the medium at 50 h after incubation (↓). (j) = no addition; (d) = Ana-a (100 µg/mL)
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Fig. 6 Effect of anacardic acid on (a) lipid accumulation and (b) glycerol-3-phosphate dehydrogenase (GPDH) in 3T3-L1 cells ((h) = no addition; (d) = Ana-d (10 µg/mL)). Ana-d was added to the induction medium and fresh media after induction
depressed when 100 µg/mL of Ana-a was added to the medium at the induction period of TG. This result suggests that Ana-a repressed the lipid accumulation of the yeast by inhibiting GPDH.
Effect of Ana on adipose conversion of 3T3-L1 cells Swiss 3T3-L1 cells have a high probability of conversion to adipose cells accumulating TG (5). Exponentially-growing 3T3-L1 cells have the morphological and biochemical properties of fibroblasts, but they differentiate into adipocytes, rounded cells containing a large droplet of TG after reaching confluence. In the course of this conversion, the activities of many lipogenic enzymes increases. Among them GPDH must have an important role in the conversion process. (4) The enzyme occupies a central position in the pathway of TG synthesis (11) at the branch point from the glycolytic pathway. Conversion is stimulated by insulin or methylisobutylxanthine plus dexamethasone (12) and the expression of GPDH is also induced by insulin, dexamethasone or c-AMP (13). Here, the effect of Ana on this adipose conversion was examined. At first, the morphological change was observed in the presence or absence of Ana or LiCl (14) under microscopy, the oil globule being stained with oil red O. Lithium ion inhibited inducer-stimulated adipose conversion of 3T3-L1 cells (14). Although LiCl (10 mol/L) definitely repressed differentiation in this experiment, Ana-d did not. The TG content in 3T3-L1 cells during the conversion was also measured. However, Ana-d did not inhibit the accumulation of TG and GPDH in the cells (Fig. 6), while 20 µg/mL of Ana-d inhibited 50% of the growth of the cells. Ana-b, -c and -d also did not inhibit the conversion of adipose cells. Furthermore, the effect of Ana on the activity of GPDH from the cells was examined. Ana did not inhibit the enzyme strongly. For example, the IC50 of Ana-d against GPDH from the cells was about 10 µg/mL, while the IC50 against the enzyme from rabbit muscle, which was used for screening of GPDH inhibitors, was 0.9 µg/mL (1). It was considered that Ana did not inhibit the accumula-
tion of TG and the conversion because it did not inhibit GPDH from 3T3-L1 cells. Not much is known about definite inhibitors of GPDH. Adipostatins A and B were isolated as inhibitors of GPDH from fermented broth of actinomycete in 1992 (15). Ana was also isolated from ginkgo as GPDH inhibitors in 1996 (1). Ana inhibited the growth of B. subtilis on a synthetic medium without lipids and its inhibition was restored by acylglycerol. Lipomyces starkeyi accumulated TG during the later part of incubation. When Ana was added to the medium at the induction period of TG accumulation, TG accumulation was repressed. On the other hand, Ana did not inhibit TG accumulation and GPDH of 3T3-L1 cells. It seemed that GPDH inhibition of Ana was variable depending on the origin of GPDH.
Acknowledgements We thank Dr Yasuo Kitagawa, Professor of Nagoya University, for providing 3T3-L1 cells and instructing the cell culture.
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