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Effect of Eicosapentaenoic Acid on Glucose-Induced Diacylglycerol Synthesis in Cultured Bovine Aortic Endothelial Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
247, 473–477 (1998)
RC988814
Effect of Eicosapentaenoic Acid on Glucose-Induced Diacylglycerol Synthesis in Cultured Bovine Aortic Endothelial Cells Tatsuya Kuroki, Toyoshi Inoguchi, Fumio Umeda, and Hajime Nawata The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan
Received May 13, 1998
Hyperglycemia has been postulated to increase diacylglycerol (DAG) level through de novo synthesis pathway and subsequently activate protein kinase C (PKC) in vascular cells, possibly leading to vascular dysfunction associated with diabetes. In this study, we examined the effect of eicosapentaenoic acid (EPA) on high glucose-induced increase in DAG level in cultured aortic endothelial cells (ECs). In ECs, total DAG level was significantly increased in the cells cultured with high glucose levels (400 mg/dl) compared with the cells with normal glucose levels (100 mg/dl). The addition of EPA completely prevented high glucose-induced increase in total DAG level. In contrast, other common fatty acids such as palmitate and oleate significantly stimulated DAG syntheisis, although arachidonate did not affect it. High glucose level significantly stimulated the incorporation of 3H-palmitate into DAG, while it did not affect the incorporation of 3H-arachidonate into DAG. The addition of EPA completely prevented the high glucose-induced increase in 3H-palmitate incorporation into DAG, while it did not affect the 3Harachidonate incorporation. These findings suggest that EPA can prevent high glucose induced-increase in DAG level in ECs, probably by specifically inhibiting de novo synthesis at the step of acylation. EPA may be one of the candidates for clinical agents normalizing activation of DAG-PKC pathway in diabetic vascular tissues and preventing vascular complications associated with diabetes. q 1998 Academic Press Key Words: eicosapentaenoic acid; diacylglycerol; endothelial cells; high glucose level.
Hyperglycemia is a major pathogenic factor in the development of diabetic macro- and microvascular complications (1). We and other investigators have recently reported that diabetes or high glucose level increases diacylglycerol (DAG) level, subsequently activating protein kinase C (PKC) in vascular tissues or cultured vascular cells (2-5). The glucose-induced activation of
DAG-PKC pathway in the vascular wall could be probably linked to the dysfunction of vasculature in diabetes (6). This notion has been supported by the several studies that various abnormal functions observed in diabetic animals can be normalized by PKC inhibitors (7-10). All these findings have implied that normalization of hyperglycemia-induced activation of DAG-PKC pathway might be possible treatment for diabetic vascular complications. Eicosapentaenoic acid (EPA), which is one of n-3 polyunsaturated fatty acids, has been noted in its anti-atherosclerotic effects through the modulation of vascular cell functions related to atherosclerosis (11). One of anti-atherosclerotic mechanisms is the inhibitory effect of EPA on triglyceride synthesis through glycerol-phosphate shunt in glycolytic pathway in the liver (12). The increase in DAG level by high glucose is supposed to be mainly derived from the de novo synthesis pathway which is the same as triglyceride synthesis pathway (2-5). We therefore speculated that EPA might prevent the increase in DAG induced by high glucose level in vascular cells. In the present study, we examined the effect of EPA on the glucose-induced increase in DAG levels in vitro using cultured bovine aortic endothelial cells (ECs). In addition, the effect of other common fatty acids such as palmitate, oleate, or arachidonate on DAG synthesis was also examined. MATERIALS AND METHODS Cell culture. Vascular endothelial cells (ECs) were harvested from a bovine thoracic aorta by scraping the intimal surface and cultured with Dulbecco’s modified Eagle medium (DMEM, Gibco Lab., N.Y., USA) containing 10% fetal calf serum (FCS, Gibco Lab., N. Y., USA), 100 mU/ml penicillin and 100 mg/ml streptomycin (Gibco Lab., N. Y., USA). The ECs were cultured at 377C under an atmosphere of 95% O2/ 5% CO2 , as described previously (13). Every 5-10 days, the cells were subcultured by 0.05% (w/v) trypsin (Difco Lab., Detroit, Michigan, USA) harvesting. For all experiments, the third through seventh subcultured ECs were used. For the experiments, cells are allowed to reach confluency in 35-
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mm dishes, and then the medium was changed to DMEM containing either 100 mg/dl or 400 mg/dl glucose with or without EPA and 1% serum and incubated for 72 hrs, when the maximal increase in DAG level was observed (4). Medium was changed daily to maintain the desired glucose and EPA levels. EPA ethyl ester (95.2% of purity) was kindly provided from Mochida Pharmaceutical Co. Ltd. (Tokyo, Japan). EPA ethyl ester was dissolved in ethanol. The final concentration of ethanol in test medium was less than 0.1%, and the same amount of ethanol without EPA ethyl ester was also added to the control medium. Assay of fatty acid composition in phospholipids of cell membrane. After 3 days of incubation with or without EPA ester (5 mg/ml), the cell experiments were terminated by the addition of 2 ml of ice-cold methanol, and the samples were transferred to the tubes containing 2 ml of chloroform and 1 ml of water. Total lipids were extracted, and then immediately, 0.2% of butylated hydroxytoluen was added to the samples to protect from oxygenation. Assay of fatty acid composition in phospholipids was performed with gas chromatography. Extraction and assay of total DAG. After 3 days of incubation with or without EPA ester (5 mg/ml) in the presence of 100 mg/dl or 400 mg/dl glucose, total lipids were extracted as described above. Total DAG was determined by an enzymatic assay using DAG kinase, as previously reported (3, 4). Briefly, the resulting 32 P-phosphatidic acid which was converted from DAG by DAG kinase in vitro was separated on silica gel G thin layer plates, developed in chambers using a solvent of chloroform-acetone-methanolacetic acid-water (10:4:3:2:1). The spots of phosphatidic acid visualized by autoradiography were scraped from the plates into vials, and radioactivities were determined by liquid scintillation counting. In addition, to evaluate the effect of other common fatty acids on DAG synthesis, palmitate, oleate, or arachidonate at various concentrations was incubated with the cells for 24 h and total DAG level was measured as described above. Labeling of DAG with 3H-palmitate and 3H-arachidonate. After 3 days of incubation with or without EPA ester in the presence of either 100 mg/dl or 400 mg/dl glucose, the medium was changed to the test medium containing 3H-palmitate, or 3H-arachidonate at the concentration of 10 mCi/ml, respectively, and incubated for 12 h. The test medium did not contain EPA ester to avoid the competitive inhibition of fatty acid incorporation into the cells. After incubation with labeled-fatty acids, total lipids were extracted from the cells as described above. First, the radioactivity of the cells was measured to evaluate the effect of EPA on the incorporation of labeled-fatty acids into the cells. Next, labeled DAG was separated on silica gel G thin layer plates developed in hexane: ether: acetic acid (60:40:1) ,and the spots of DAG visualized by using iodine gas were scraped and radioactivities of samples were determined by liquid scintillation counting (3, 4). Assay of triglyceride levels. After 3 days of incubation with or without EPA ester (5 mg/ml) in the presence of 100 mg/dl or 400 mg/dl glucose, total lipids were extracted as described above. Total triglyceride level was determined by an enzymatic assay kit (Iatron, Tokyo, Japan using glycerokinase and L-a-glycerophosphate oxidase as previously reported (14). Statistical analysis. Statistical analyses were conducted using the Fisher’s PLSD test. P values less than 0.05 were considered as significantly differences. Data are shown as means { SE.
RESULTS Effect of EPA on fatty acid composition in phospholipids of vascular endothelial cells. After 3 days incubation of the cells with EPA (5 mg/ml), EPA contents in phospholipids of the cell membrane fractions were
FIG. 1. Effect of EPA on fatty acid composition in phospholipids of cultured ECs. Confluent ECs were cultured in DMEM containing 100 mg/dl glucose and 1% serum with or without EPA (5 mg/ml) and incubated for 72 h. Assay of fatty acid composition in phospholipids was performed with gas chromatography. EPA: eicosapentaenoic acid, GHLA: dihomo-g-linoleic acid, AA: arachidonic acid, DHA: docosahexaenoic acid. Results are shown as mean { SE of three different experiments.
significantly (P õ 0.001) increased by approximately 14-fold. On the other hand, both arachidonic acid and DHA (docosahexaenoic acid) contents in phospholipids were significantly reduced (P õ 0.01) (Fig.1). There were no differences in phospholipid composition between the cells cultured in 100 mg/dl and 400mg/dl glucose levels. Therefore, further studies were performed at the final concentration of 5 mg/ml of EPA. Effect of EPA on total DAG levels in vascular endothelial cells. After 3 days of increasing glucose level from 100 mg/dl to 400mg/dl, total DAG level was significantly increased by 13810 (P õ 0.05) in the ECs. The addition of EPA (5 mg/ml) completely prevented the increase in the total DAG level induced by high glucose level in the ECs (Fig. 2). In contrast, the addition of palmitate or oleate with bovine serum albumin (50 mM) significantly increased total DAG level in a time- and dose-dependency, respectively. The addition of palmitate at the concentration of 100 mg/ml for 24 h dramatically increased total DAG level by 8.21 { 0.29 fold (P õ 0.001) and the addition of oleate at the concentration of 100 mg/ml for 24 h significantly (P õ 0.001) increased total DAG level by 152 { 28% (Table 1). Arachidonate did not affect total DAG level. Effect of EPA on incorporation of 3H-palmitate and H-arachidonate into DAG. Increasing glucose level in the incubation medium from 100 mg/dl to 400mg/dl 3
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FIG. 2. Effect of EPA on high glucose-induced increase in DAG level in cultured ECs. Confluent ECs were incubated with a test medium with or without EPA (5 mg/dl) in the presence of either 100 mg/dl or 400 mg/dl glucose and 1 % serum for 72 h. Total DAG level was determined by an enzymatic assay using DAG kinase. Each experiment was done in triplicate with each using 3 confluent 60mm culture dishes. N; number of independent experiments. Results are shown as mean { SE.
for 3 days did not affect the accumulation of 3H-arachidonate-labeled DAG, whereas labeled DAG levels were significantly increased when the cells were incubated with 3H-palmitate (Fig. 3A). The addition of EPA did not affect the incorporation of either 3H-palmitate or 3 H-arachidonate into the cells. However, the addition of EPA completely prevented the increase in accumulation of 3H-palmitate-labeled DAG , while it did not affect accumulation of 3H-arachidonate-labeled DAG (Fig. 3B). Effect of EPA on triglyceride levels. After 3 days of increasing glucose level from 100 mg/dl to 400 mg/dl, total triglyceride level in ECs was not significantly affected by either high glucose level or EPA (Fig. 4).
FIG. 3. Effect of EPA on 3H-palmitate (A) and 3H-arachidonate (B) incorporation into DAG in cultured ECs. After 3 days of incubation with or without EPA ester (5 mg/dl) in the presence of either 100 mg/dl or 400 mg/dl glucose, the medium was changed to a test medium containing 3H-palmitate (10 mCi/ml) or 3H-arachidonate (10 mCi/ml) without EPA and incubated for 12 h. Each experiment was done in triplicate with each using 3 confluent 60-mm culture dishes. N; number of independent experiments. Results are shown as mean { SE.
TABLE 1
DISCUSSION Fatty acid
Palmitate
Oleate
Arachidonate
Total DAG level (% of control)
821 { 29* (N Å 4)
152 { 28* (N Å 4)
112 { 25 (N Å 4)
Note. Effect of palmitate, oleate, and arachidonate on total DAG level in cultured ECs. Confluent ECs were incubated with a test medium containing palmitate, oleate, or arachidonate (100 mg/dl) with bovine serum albumin (50 mM) for 24 h. Total DAG level was determined by an enzymatic assay using DAG kinase. Each experiment was done in triplicate with each using 3 confluent 60-mm culture dishes. N; number of independent experiments. Results are shown as mean { SE. *p õ 0.001.
Multiple studies, including ours, have demonstrated that high glucose level or diabetic state increases total DAG level, subsequently enhancing PKC activity in vascular tissues and cultured vascular cells such as endothelial cells and smooth muscle cells (2-5). This activation in DAG-PKC pathway are supposed to cause various cellular dysfunction associated with diabetes. Several studies have demonstrated that PKC inhibitors can normalize various abnormal vascular functions observed in diabetic an-
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FIG. 4. Effect of EPA on triglyceride level in cultured ECs. Confluent ECs were incubated with a test medium with or without EPA (5 mg/dl) in the presence of either 100 mg/dl or 400 mg/dl glucose and 1% serum for 72 h. Three 100-mm culture dishes were used for each preparation. Results are shown as mean { SE of three different experiments.
imals such as abnormalities in retinal hemodynamics, renal hemodynamics, capillary albumin permeability, and expression in fibronectin and TGF b (7-10). All these findings have implied that normalization of hyperglycemia-induced activation of DAGPKC pathway might be possible treatment for diabetic vascular complications. However, using PKC inhibitors in vivo has been hindered by the toxicity and nonspecificity (9). Recently, Kunisaki et al. reported that D-a-tocopherol, the most active form of vitamin E, can prevent the activation of DAG-PKC pathway induced by high glucose in aorta of diabetic rats and cultured rat smooth muscle cells (15). Specific PKC b inhibitor, which may be much less toxic than nonspecific PKC inhibitors, has been reported to normalize the hyperfiltration in renal hemodynamics (9) and increased expression of extracellular matrix components of kidney in diabetic rats (14). In the present study, we found that EPA could prevent the increase in DAG induced by high glucose levels in cultured aortic ECs. This effect is specific in EPA , since the present results showed that other common fatty acids such as palmitate or oleate significantly stimulated DAG synthesis in aortic ECs, although arachidonate did no affect DAG synthesis. We and other investigators have previously reported that glucose-induced increase in DAG may be derived from de novo synthesis pathway (4-6). Interestingly, fatty acid composition of DAG derived from de novo synthesis are supposed to be enriched in palmitate and oleate, while DAG derived from phosphatidyl inositol (PI) breakdown is enriched in arachidonate (4, 16) Taken to-
gether, it is likely that both palmitate and oleate may stimulate DAG synthesis through de novo synthesis pathway as well as high glucose level. This notion is consistent with previous reports that palmitate as well as glucose induce PKC activation in islets of pancreas (17-18). In addition, the present results also showed that EPA completely prevented high glucoseinduced increase in 3H-palmitate incorporation into DAG, whereas it did not affect 3H-arachidonate incorporation into DAG. These findings suggest that EPA may prevent high glucose level-induced increase in DAG level, probably by specifically inhibiting de novo synthesis at the step of incorporation of palmitate and oleate into DAG. It has been reported that EPA may reduce triglyceride synthesis from glycerol phosphate shunt in glycolytic pathway in the liver (12). However, the present study showed that triglyceride level was not affected by either high glucose level or EPA in ECs. Possible explanation for this finding is that it may be difficult to detect the difference since conversion from DAG to triglyceride is too small in ECs. It should be also excluded that EPA might inhibit DAG synthesis derived from phosphatidylcholine (PC) breakdown rather than de novo synthesis, since DAG derived from PC breakdown has also palmitate-rich fatty acid composition. Further studies should be done to reveal the mechanism for the effect of EPA on DAG synthesis in detail. In conclusion, the present results suggest that EPA may be one of the candidates for clinical agents normalizing the activation of DAG-PKC pathway and preventing vascular complications associated diabetes. REFERENCES 1. The DCCT Research Group (1993) N. Engl. J. Med. 323, 977– 986. 2. King, G. L., Johnson, S., and Wu, G. (1990) in Growth Factors in Health and Disease (Westermark, B., Betscholtz, C., and Hokfelt, B., Eds.), pp.303–317. Elsevier Science, Amsterdam. 3. Inoguchi, T., Battan. R., Handler, E, Sportsman, J. R., Heath, W., and King, G. L. (1992) Proc. Natl. Acad. Sci. USA 89, 11059– 11063. 4. Inoguchi, T., Xia, P., Kunisaki, M., Higashi, S., Feener, E. P., and King, G. L. (1994) Am. J .Physiol. 267, E369–379. 5. Xia, P., Inoguchi, T., Kem, T. S., Engerman, R. L., Oates, P. J., and King, G. L. (1994) Diabetes 43, 1122–1129. 6. Craven, P. A., DeRubertis, F. R. (1989) J. Clin. Invest. 83, 1667– 1675. 7. Shiba, T., Inoguchi, T., Sportsman, W. F., Heat, S., Bursell, S., and King, G. L. (1993) Am. J. Physiol. 265, E783-E793. 8. Wolf, B. A., Williamson, J. R., Easom, R. A., Chang, K., Sherman, W. R., Turk, J. (1991) J. Clin. Invest. 87, 31–38 9. Ishii, H., Jirousek, M. R., Koya, D., Takagi, C., Xia, P., Clermont, A., Bursell, S. E., Kern, T. S., Ballas, L. M., Heath, W. F., Stramm, L. E., Feener, E. P., and King, G. L. (1996) Science 272, 728–731.
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10. Koya, D., Jirousek, M. R., Lin, Y. W., Ishii, H., Kuboki, K., and King, G. L. (1997) J. Clin. Invest. 100, 115–26. 11. Bang, H. O., Dyeberg, J., and Nielsen, A. (1971) Lancet 1, 1143– 1145. 12. Mizuguchi, K., Yano, T., Ishibashi, M., Masada, A., Mizota, M., and Saito, Y. (1993) Eur. J. Pharmacol. 235, 221–227. 13. Inoguchi, T., Umeda, F., Ono, H., Kunisaki , M., Watanabe, J., and Nawata, H. (1989) Metabolism 38, 837–842. 14. Spayd, R. W., Bruchi, B., Burdic, B. A., Dappen, G. M., Eikenberry, J. N., Esders, T. W., Figureras, J., Goodhue, C. T., LaRossa,
15. 16. 17. 18.
D. D., Nelson, R. W., Rand, R. N., and Wu, T. W. (1978) Clin. Chem. 24, 1343–1350. Kunisaki , M., Umeda, F., Nawata , H., and King, G. L. (1996) Diabetes 45, 117–119. Wolf, B. A., Easom, R. A., Hughes, J. H., and McDaniel, M. L., Turk, J. (1989) Biochemistry 28, 4291–4301. Prentki, M., Vischer, S., Glennon, C. M., Regazzi, R, Deeney, J. T., and Corkey, B. E. (1992) J. Biol. Chem. 267, 5802–5810. Alcazar, O., Qiu-yue, Z, Gine, E., and Tamarit-Rodriguez, J. (1997) Diabetes 46, 1153–1158.
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RC988814
Effect of Eicosapentaenoic Acid on Glucose-Induced Diacylglycerol Synthesis in Cultured Bovine Aortic Endothelial Cells Tatsuya Kuroki, Toyoshi Inoguchi, Fumio Umeda, and Hajime Nawata The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan
Received May 13, 1998
Hyperglycemia has been postulated to increase diacylglycerol (DAG) level through de novo synthesis pathway and subsequently activate protein kinase C (PKC) in vascular cells, possibly leading to vascular dysfunction associated with diabetes. In this study, we examined the effect of eicosapentaenoic acid (EPA) on high glucose-induced increase in DAG level in cultured aortic endothelial cells (ECs). In ECs, total DAG level was significantly increased in the cells cultured with high glucose levels (400 mg/dl) compared with the cells with normal glucose levels (100 mg/dl). The addition of EPA completely prevented high glucose-induced increase in total DAG level. In contrast, other common fatty acids such as palmitate and oleate significantly stimulated DAG syntheisis, although arachidonate did not affect it. High glucose level significantly stimulated the incorporation of 3H-palmitate into DAG, while it did not affect the incorporation of 3H-arachidonate into DAG. The addition of EPA completely prevented the high glucose-induced increase in 3H-palmitate incorporation into DAG, while it did not affect the 3Harachidonate incorporation. These findings suggest that EPA can prevent high glucose induced-increase in DAG level in ECs, probably by specifically inhibiting de novo synthesis at the step of acylation. EPA may be one of the candidates for clinical agents normalizing activation of DAG-PKC pathway in diabetic vascular tissues and preventing vascular complications associated with diabetes. q 1998 Academic Press Key Words: eicosapentaenoic acid; diacylglycerol; endothelial cells; high glucose level.
Hyperglycemia is a major pathogenic factor in the development of diabetic macro- and microvascular complications (1). We and other investigators have recently reported that diabetes or high glucose level increases diacylglycerol (DAG) level, subsequently activating protein kinase C (PKC) in vascular tissues or cultured vascular cells (2-5). The glucose-induced activation of
DAG-PKC pathway in the vascular wall could be probably linked to the dysfunction of vasculature in diabetes (6). This notion has been supported by the several studies that various abnormal functions observed in diabetic animals can be normalized by PKC inhibitors (7-10). All these findings have implied that normalization of hyperglycemia-induced activation of DAG-PKC pathway might be possible treatment for diabetic vascular complications. Eicosapentaenoic acid (EPA), which is one of n-3 polyunsaturated fatty acids, has been noted in its anti-atherosclerotic effects through the modulation of vascular cell functions related to atherosclerosis (11). One of anti-atherosclerotic mechanisms is the inhibitory effect of EPA on triglyceride synthesis through glycerol-phosphate shunt in glycolytic pathway in the liver (12). The increase in DAG level by high glucose is supposed to be mainly derived from the de novo synthesis pathway which is the same as triglyceride synthesis pathway (2-5). We therefore speculated that EPA might prevent the increase in DAG induced by high glucose level in vascular cells. In the present study, we examined the effect of EPA on the glucose-induced increase in DAG levels in vitro using cultured bovine aortic endothelial cells (ECs). In addition, the effect of other common fatty acids such as palmitate, oleate, or arachidonate on DAG synthesis was also examined. MATERIALS AND METHODS Cell culture. Vascular endothelial cells (ECs) were harvested from a bovine thoracic aorta by scraping the intimal surface and cultured with Dulbecco’s modified Eagle medium (DMEM, Gibco Lab., N.Y., USA) containing 10% fetal calf serum (FCS, Gibco Lab., N. Y., USA), 100 mU/ml penicillin and 100 mg/ml streptomycin (Gibco Lab., N. Y., USA). The ECs were cultured at 377C under an atmosphere of 95% O2/ 5% CO2 , as described previously (13). Every 5-10 days, the cells were subcultured by 0.05% (w/v) trypsin (Difco Lab., Detroit, Michigan, USA) harvesting. For all experiments, the third through seventh subcultured ECs were used. For the experiments, cells are allowed to reach confluency in 35-
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mm dishes, and then the medium was changed to DMEM containing either 100 mg/dl or 400 mg/dl glucose with or without EPA and 1% serum and incubated for 72 hrs, when the maximal increase in DAG level was observed (4). Medium was changed daily to maintain the desired glucose and EPA levels. EPA ethyl ester (95.2% of purity) was kindly provided from Mochida Pharmaceutical Co. Ltd. (Tokyo, Japan). EPA ethyl ester was dissolved in ethanol. The final concentration of ethanol in test medium was less than 0.1%, and the same amount of ethanol without EPA ethyl ester was also added to the control medium. Assay of fatty acid composition in phospholipids of cell membrane. After 3 days of incubation with or without EPA ester (5 mg/ml), the cell experiments were terminated by the addition of 2 ml of ice-cold methanol, and the samples were transferred to the tubes containing 2 ml of chloroform and 1 ml of water. Total lipids were extracted, and then immediately, 0.2% of butylated hydroxytoluen was added to the samples to protect from oxygenation. Assay of fatty acid composition in phospholipids was performed with gas chromatography. Extraction and assay of total DAG. After 3 days of incubation with or without EPA ester (5 mg/ml) in the presence of 100 mg/dl or 400 mg/dl glucose, total lipids were extracted as described above. Total DAG was determined by an enzymatic assay using DAG kinase, as previously reported (3, 4). Briefly, the resulting 32 P-phosphatidic acid which was converted from DAG by DAG kinase in vitro was separated on silica gel G thin layer plates, developed in chambers using a solvent of chloroform-acetone-methanolacetic acid-water (10:4:3:2:1). The spots of phosphatidic acid visualized by autoradiography were scraped from the plates into vials, and radioactivities were determined by liquid scintillation counting. In addition, to evaluate the effect of other common fatty acids on DAG synthesis, palmitate, oleate, or arachidonate at various concentrations was incubated with the cells for 24 h and total DAG level was measured as described above. Labeling of DAG with 3H-palmitate and 3H-arachidonate. After 3 days of incubation with or without EPA ester in the presence of either 100 mg/dl or 400 mg/dl glucose, the medium was changed to the test medium containing 3H-palmitate, or 3H-arachidonate at the concentration of 10 mCi/ml, respectively, and incubated for 12 h. The test medium did not contain EPA ester to avoid the competitive inhibition of fatty acid incorporation into the cells. After incubation with labeled-fatty acids, total lipids were extracted from the cells as described above. First, the radioactivity of the cells was measured to evaluate the effect of EPA on the incorporation of labeled-fatty acids into the cells. Next, labeled DAG was separated on silica gel G thin layer plates developed in hexane: ether: acetic acid (60:40:1) ,and the spots of DAG visualized by using iodine gas were scraped and radioactivities of samples were determined by liquid scintillation counting (3, 4). Assay of triglyceride levels. After 3 days of incubation with or without EPA ester (5 mg/ml) in the presence of 100 mg/dl or 400 mg/dl glucose, total lipids were extracted as described above. Total triglyceride level was determined by an enzymatic assay kit (Iatron, Tokyo, Japan using glycerokinase and L-a-glycerophosphate oxidase as previously reported (14). Statistical analysis. Statistical analyses were conducted using the Fisher’s PLSD test. P values less than 0.05 were considered as significantly differences. Data are shown as means { SE.
RESULTS Effect of EPA on fatty acid composition in phospholipids of vascular endothelial cells. After 3 days incubation of the cells with EPA (5 mg/ml), EPA contents in phospholipids of the cell membrane fractions were
FIG. 1. Effect of EPA on fatty acid composition in phospholipids of cultured ECs. Confluent ECs were cultured in DMEM containing 100 mg/dl glucose and 1% serum with or without EPA (5 mg/ml) and incubated for 72 h. Assay of fatty acid composition in phospholipids was performed with gas chromatography. EPA: eicosapentaenoic acid, GHLA: dihomo-g-linoleic acid, AA: arachidonic acid, DHA: docosahexaenoic acid. Results are shown as mean { SE of three different experiments.
significantly (P õ 0.001) increased by approximately 14-fold. On the other hand, both arachidonic acid and DHA (docosahexaenoic acid) contents in phospholipids were significantly reduced (P õ 0.01) (Fig.1). There were no differences in phospholipid composition between the cells cultured in 100 mg/dl and 400mg/dl glucose levels. Therefore, further studies were performed at the final concentration of 5 mg/ml of EPA. Effect of EPA on total DAG levels in vascular endothelial cells. After 3 days of increasing glucose level from 100 mg/dl to 400mg/dl, total DAG level was significantly increased by 13810 (P õ 0.05) in the ECs. The addition of EPA (5 mg/ml) completely prevented the increase in the total DAG level induced by high glucose level in the ECs (Fig. 2). In contrast, the addition of palmitate or oleate with bovine serum albumin (50 mM) significantly increased total DAG level in a time- and dose-dependency, respectively. The addition of palmitate at the concentration of 100 mg/ml for 24 h dramatically increased total DAG level by 8.21 { 0.29 fold (P õ 0.001) and the addition of oleate at the concentration of 100 mg/ml for 24 h significantly (P õ 0.001) increased total DAG level by 152 { 28% (Table 1). Arachidonate did not affect total DAG level. Effect of EPA on incorporation of 3H-palmitate and H-arachidonate into DAG. Increasing glucose level in the incubation medium from 100 mg/dl to 400mg/dl 3
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FIG. 2. Effect of EPA on high glucose-induced increase in DAG level in cultured ECs. Confluent ECs were incubated with a test medium with or without EPA (5 mg/dl) in the presence of either 100 mg/dl or 400 mg/dl glucose and 1 % serum for 72 h. Total DAG level was determined by an enzymatic assay using DAG kinase. Each experiment was done in triplicate with each using 3 confluent 60mm culture dishes. N; number of independent experiments. Results are shown as mean { SE.
for 3 days did not affect the accumulation of 3H-arachidonate-labeled DAG, whereas labeled DAG levels were significantly increased when the cells were incubated with 3H-palmitate (Fig. 3A). The addition of EPA did not affect the incorporation of either 3H-palmitate or 3 H-arachidonate into the cells. However, the addition of EPA completely prevented the increase in accumulation of 3H-palmitate-labeled DAG , while it did not affect accumulation of 3H-arachidonate-labeled DAG (Fig. 3B). Effect of EPA on triglyceride levels. After 3 days of increasing glucose level from 100 mg/dl to 400 mg/dl, total triglyceride level in ECs was not significantly affected by either high glucose level or EPA (Fig. 4).
FIG. 3. Effect of EPA on 3H-palmitate (A) and 3H-arachidonate (B) incorporation into DAG in cultured ECs. After 3 days of incubation with or without EPA ester (5 mg/dl) in the presence of either 100 mg/dl or 400 mg/dl glucose, the medium was changed to a test medium containing 3H-palmitate (10 mCi/ml) or 3H-arachidonate (10 mCi/ml) without EPA and incubated for 12 h. Each experiment was done in triplicate with each using 3 confluent 60-mm culture dishes. N; number of independent experiments. Results are shown as mean { SE.
TABLE 1
DISCUSSION Fatty acid
Palmitate
Oleate
Arachidonate
Total DAG level (% of control)
821 { 29* (N Å 4)
152 { 28* (N Å 4)
112 { 25 (N Å 4)
Note. Effect of palmitate, oleate, and arachidonate on total DAG level in cultured ECs. Confluent ECs were incubated with a test medium containing palmitate, oleate, or arachidonate (100 mg/dl) with bovine serum albumin (50 mM) for 24 h. Total DAG level was determined by an enzymatic assay using DAG kinase. Each experiment was done in triplicate with each using 3 confluent 60-mm culture dishes. N; number of independent experiments. Results are shown as mean { SE. *p õ 0.001.
Multiple studies, including ours, have demonstrated that high glucose level or diabetic state increases total DAG level, subsequently enhancing PKC activity in vascular tissues and cultured vascular cells such as endothelial cells and smooth muscle cells (2-5). This activation in DAG-PKC pathway are supposed to cause various cellular dysfunction associated with diabetes. Several studies have demonstrated that PKC inhibitors can normalize various abnormal vascular functions observed in diabetic an-
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FIG. 4. Effect of EPA on triglyceride level in cultured ECs. Confluent ECs were incubated with a test medium with or without EPA (5 mg/dl) in the presence of either 100 mg/dl or 400 mg/dl glucose and 1% serum for 72 h. Three 100-mm culture dishes were used for each preparation. Results are shown as mean { SE of three different experiments.
imals such as abnormalities in retinal hemodynamics, renal hemodynamics, capillary albumin permeability, and expression in fibronectin and TGF b (7-10). All these findings have implied that normalization of hyperglycemia-induced activation of DAGPKC pathway might be possible treatment for diabetic vascular complications. However, using PKC inhibitors in vivo has been hindered by the toxicity and nonspecificity (9). Recently, Kunisaki et al. reported that D-a-tocopherol, the most active form of vitamin E, can prevent the activation of DAG-PKC pathway induced by high glucose in aorta of diabetic rats and cultured rat smooth muscle cells (15). Specific PKC b inhibitor, which may be much less toxic than nonspecific PKC inhibitors, has been reported to normalize the hyperfiltration in renal hemodynamics (9) and increased expression of extracellular matrix components of kidney in diabetic rats (14). In the present study, we found that EPA could prevent the increase in DAG induced by high glucose levels in cultured aortic ECs. This effect is specific in EPA , since the present results showed that other common fatty acids such as palmitate or oleate significantly stimulated DAG synthesis in aortic ECs, although arachidonate did no affect DAG synthesis. We and other investigators have previously reported that glucose-induced increase in DAG may be derived from de novo synthesis pathway (4-6). Interestingly, fatty acid composition of DAG derived from de novo synthesis are supposed to be enriched in palmitate and oleate, while DAG derived from phosphatidyl inositol (PI) breakdown is enriched in arachidonate (4, 16) Taken to-
gether, it is likely that both palmitate and oleate may stimulate DAG synthesis through de novo synthesis pathway as well as high glucose level. This notion is consistent with previous reports that palmitate as well as glucose induce PKC activation in islets of pancreas (17-18). In addition, the present results also showed that EPA completely prevented high glucoseinduced increase in 3H-palmitate incorporation into DAG, whereas it did not affect 3H-arachidonate incorporation into DAG. These findings suggest that EPA may prevent high glucose level-induced increase in DAG level, probably by specifically inhibiting de novo synthesis at the step of incorporation of palmitate and oleate into DAG. It has been reported that EPA may reduce triglyceride synthesis from glycerol phosphate shunt in glycolytic pathway in the liver (12). However, the present study showed that triglyceride level was not affected by either high glucose level or EPA in ECs. Possible explanation for this finding is that it may be difficult to detect the difference since conversion from DAG to triglyceride is too small in ECs. It should be also excluded that EPA might inhibit DAG synthesis derived from phosphatidylcholine (PC) breakdown rather than de novo synthesis, since DAG derived from PC breakdown has also palmitate-rich fatty acid composition. Further studies should be done to reveal the mechanism for the effect of EPA on DAG synthesis in detail. In conclusion, the present results suggest that EPA may be one of the candidates for clinical agents normalizing the activation of DAG-PKC pathway and preventing vascular complications associated diabetes. REFERENCES 1. The DCCT Research Group (1993) N. Engl. J. Med. 323, 977– 986. 2. King, G. L., Johnson, S., and Wu, G. (1990) in Growth Factors in Health and Disease (Westermark, B., Betscholtz, C., and Hokfelt, B., Eds.), pp.303–317. Elsevier Science, Amsterdam. 3. Inoguchi, T., Battan. R., Handler, E, Sportsman, J. R., Heath, W., and King, G. L. (1992) Proc. Natl. Acad. Sci. USA 89, 11059– 11063. 4. Inoguchi, T., Xia, P., Kunisaki, M., Higashi, S., Feener, E. P., and King, G. L. (1994) Am. J .Physiol. 267, E369–379. 5. Xia, P., Inoguchi, T., Kem, T. S., Engerman, R. L., Oates, P. J., and King, G. L. (1994) Diabetes 43, 1122–1129. 6. Craven, P. A., DeRubertis, F. R. (1989) J. Clin. Invest. 83, 1667– 1675. 7. Shiba, T., Inoguchi, T., Sportsman, W. F., Heat, S., Bursell, S., and King, G. L. (1993) Am. J. Physiol. 265, E783-E793. 8. Wolf, B. A., Williamson, J. R., Easom, R. A., Chang, K., Sherman, W. R., Turk, J. (1991) J. Clin. Invest. 87, 31–38 9. Ishii, H., Jirousek, M. R., Koya, D., Takagi, C., Xia, P., Clermont, A., Bursell, S. E., Kern, T. S., Ballas, L. M., Heath, W. F., Stramm, L. E., Feener, E. P., and King, G. L. (1996) Science 272, 728–731.
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D. D., Nelson, R. W., Rand, R. N., and Wu, T. W. (1978) Clin. Chem. 24, 1343–1350. Kunisaki , M., Umeda, F., Nawata , H., and King, G. L. (1996) Diabetes 45, 117–119. Wolf, B. A., Easom, R. A., Hughes, J. H., and McDaniel, M. L., Turk, J. (1989) Biochemistry 28, 4291–4301. Prentki, M., Vischer, S., Glennon, C. M., Regazzi, R, Deeney, J. T., and Corkey, B. E. (1992) J. Biol. Chem. 267, 5802–5810. Alcazar, O., Qiu-yue, Z, Gine, E., and Tamarit-Rodriguez, J. (1997) Diabetes 46, 1153–1158.
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