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Effects of FR145237, an Acyl-CoA:cholesterol acyltransferase inhibitor, on diet-induced hypercholesterolemia in diabetic rats
Life Sciences, Vol. 60, No. 6, pp. 351~X56,19!J7 Gxpytight 0 1997 Ekevier s&M Inc. Printed in the USA. All fights rtscmd 0024-32Qs/w $17.00 t .oo ELSEVIER
PI1 SOO24-3205(96)00458-3
EFFECTS OF FR145237, AN Acyl-CoA:CHOLESTEROL ACYLTRANSFERASE INHIBITOR, ON DIET-INDUCED HYPERCHOLESTEROLEMIA IN DIABETIC RATS Yuri Sakuma, Hiroyuki Hagihara, Akira Nagayoshi, Kazuhiko Ohne, Seitaro Mutoh*, Yoshikuni Ito, Kunio Nakahara, Yoshitada Notsu and Masakuni Okuhara Exploratory Research Laboratories, Fujisawa Pharmaceutical 5-2-3 Tokodai, Tsukuba, Ibaraki 300-26, Japan
Co., Ltd.
(Received in find form November l&19%)
Summary Recent studies have shown that acyl-CoA:cholesterol acyltransferase (ACAT) plays an important role in the initiation of diabetes-associated hypercholesterolemia. To confirm this hypothesis, effects of a potent ACAT inhibitor, FR145237, on diet-induced hypercholesterolemia were examined in streptozotocin (STZ)-induced diabetic rats. One-week feeding of 1 % cholesterol and 0.5 % cholic acid to normal rats and STZ-induced diabetic rats increased plasma cholesterol levels in both groups, and the response was more remarkable in the STZ rats than in the normal ones (1266f476 mg/dl and 146*7 mg/dl, respectively). FR145237 dose-dependently reduced the rise in plasma cholesterol levels in the STZ rats and the levels were almost normalized by treatment with 1 mg/kg/day of the compound. These results suggest that hyperresponse to dietary cholesterol was induced in the STZ rats and that ACAT is involved in the hyperresponse. The effects of FR145237 on other plasma lipids such as high density lipoprotein (HDL) cholesterol and triglyceride (TG) levels were also examined. Key Words: acyI-CoAxholesterol acykransferase,ACAT inhibitor,diabetes, atherosderosis
Hypercholesterolemia is one of complications frequently observed in patients with diabetes mellitus (l-5) and certainly contributes to the development of atherosclerosis in these patients. To clarify the mechanism by which the diabetic state induces hypercholesterolemia, many studies have been performed using diabetic animal models such as streptozotocin (STZ)-induced diabetic rats (6,7), and recent studies have shown an increased cholesterol absorption in STZ rats (8-l 1). Recently, Acyl-CoA:cholesterol acytransferase (ACAT, EC 2.3.1.26), the rate-limiting enzyme for cholesterol absorption, was reported to be increased in STZ rats (12). In addition, Maechler et al. clearly demonstrated that ACAT plays a major role in the initiation of hypercholesterolemia in STZ rats (13,14). In their studies, CL-277082, an ACAT inhibitor, was used to see the involvement of intestinal ACAT in the hypercholesterolemia, and the inhibitor completely improved it in the dose of 5Omg/kg/day. To confirm that ACAT is involved in the development of diet-induced hypercholesterolemia the diabetic state, we examined the effects of a potent ACAT inhibitor, FR145237, on
in
352
ACAT in Diabetic Hypercholesterolemia
Vol. 60, No. 6, 1997
hypercholesterolemia induced by cholesterol feeding to STZ rats. Additionally, we examined the effects of the compound on the changes of plasma triglyceride and HDL cholesterol levels in the cholesterol-fed STZ rats. FR145237 has potent inhibitory activities on ACAT activities in the intestine, aorta and liver (15). The ICso values are around 1x10-*M. In addition, the compound attenuated aortic cholesterol depositions in cholesterol-fed rabbits and Watanabe heritable hyperlipidemic (WHHL) rabbits (16).
Methods Chemicals FR145237 (N-Benzyl-N-[3-(4-chorophenyl)-5-methyl-2-benzofuranyl] trifluorophenyl) urea) was synthesized at Exploratory Research Pharmaceutical Co., Ltd. (Ibaraki, Japan).
methyl-N’-(2,4,6Laboratories, Fujisawa
Animals Male Sprague-Dawley rats (180-220 g body weight, 6 weeks, Clea, Tokyo, Japan) were used. Rats were acclimated for 1 week at a controlled temperature of 22-25 “C under illumination from 0800 to 2000 h with free access to standard chow (Clea, Tokyo, Japan) and tap water. After 16 hours fasting, diabetes was induced by injection to the tail vein of streptozotocin (Sigma, St.Louis, MO, USA) dissolved in 5 mM citrate buffer, 0.1 M NaCl, pH 4.5 (60 mg/kg). Nondiabetic control rats were injected with the buffer only. One week after the injection, about 100 pl of blood was taken from the tail vein and glucose was measured to ensure the induction of diabetes. The rats were then fed for 1 week a high-fat diet prepared with the standard chow to which 1 % cholesterol and 0.5 % cholic acid (w/w) were added. The total amount of food intake of each cage (5 rats) was measured everyday for 1 week starting from cholesterol feeding, and the average of daily food intake was calculated on the basis of the 1 week observation. The rats were killed by collecting blood from the abdominal aorta under ether anesthesia. All animal procedures were carried out as approved by the Animal Care and Use Committee at Fujisawa Pharmaceutical Co., Ltd. Drum Treatment FR145237 was dissolved in ethanol and mixed with the high-fat diet at final concentrations of 0.00001 %, 0.0001 % and 0.001 % (w/w) in order to obtain daily doses of approximately 0.01 mg/kg, 0.1 mg/kg and 1 mg/kg, respectively. Biochemical Analvsis of Serum Plasma glucose, cholesterol, triglyceride (TG) and free fatty acid were determined enzymatically using kits purchased, respectively, from Wako Pure Chemicals (Osaka, Japan). High density lipoprotein (HDL) cholesterol was measured after removal of very low density lipoprotein (VLDL) and LDL cholesterol by heparin-manganese precipitation. Statistical Analvsis Data were expressed as mean&SEM. Statistical differences between two groups were evaluated by Student’s t-test. For evaluation of effects of FR145237, analysis of variance (ANOVA) followed by Dunnett’s t-test was used. P values less than 0.05 were considered significant.
Results Hyperresuonse to high cholesterol diet in diabetic rats As shown in Table 1, plasma glucose levels were higher than 600 mg/dl in the STZ rats, indicating a diabetic state. Compared to the normal rats, the food intake was increased in the STZ
Vol. 60, No. 6, 1997
ACAT in Diabetic Hypercholesterolemia
353
rats, but the body weight was significantly reduced. One-week feeding of cholesterol had no effect on plasma glucose levels, food intake or body weight in either the normal or the STZ rats. One-week feeding of a high cholesterol diet to the normal rats significantly increased plasma cholesterol levels compared to those in the normal rats fed the standard chow (Fig. 1A) The same treatment markedly increased plasma cholesterol levels in the STZ rats and the level was about 8.7 fold higher than that of the cholesterol-fed normal rats (1266&476 mg/dl and 146f7 mg/dl, respectively). Plasma TG levels were not affected significantly by cholesterol feeding to the STZ rats, but a significant decrease in plasma HDL cholesterol levels was induced (Fig.lB,lC). TABLE
I
Effect of a High Cholesterol Diet on Plasma Glucose, Food Intake and Body Weight of Normal and Diabetic rats Rat
Normal Normal STZ STZ STZ
Diet
Plasma Glucose (mg/dU
Food Intake (g/day/rat)
Final Body Weight
141f3 131s 625+32** 614&39**
25 26 41 39
327&l 1 328f6 271+8** 271f17**
607f25 572k43 514*41
38 36 30
274f7 27M3 263tlO
Standard Cholesterol Standard Cholesterol Cholesterol + FR145237 O.Olmg/kg 0.1 1
Data are expressed as mea&SEM
Cholesterol
-
+
Normal rak
-
+
STZ rats
(8)
(n=5). **PcO.Ol versus respective normal controls.
Cholesterol
-
+
Normal rats
-
+
STZ rak
Cholesterol
-
+
Normal rak
-
+
STZ rak
Fig.1 Cholesterol, TG and HDL cholesterol levels in the plasma of cholesterol-fed normal and diabetic rats. Data represent mea&SEM (n=5). The rats were fed a high cholesterol diet or a standard chow for 1 week. *Pc0.05,***Pc0.001 versus the standard chow-fed normal rats. ##PcO.Ol versus the standard chow-fed STZ rats.
Vol. 60, No. 6, l!N7
ACAT in Diabetic Hypercholesterolemia
354
Hvpocholesterolemic acti ‘ty of FR145237 in cholesterol-fed diabetic rats As shown in Table 1, ‘Al45237 dose-dependently decreased plasma glucose concentrations, the food intake and the final body weight in the STZ rats fed a high cholesterol diet, but those values were not significant. As mentioned above, a marked increase in plasma cholesterol levels was observed in diabetic rats fed a high cholesterol diet for 1 week (Fig. 1A). FR145237 dose-dependently reduced this increase and at a dose of 1 mg/kg/day the levels were almost the same as those of the standard chow-fed control (Fig.2A). Treatment of the diabetic rats with FR145237 did not affect significantly the changes of plasma TG (Fig.2B) and HDL cholesterol (Fig.2C) levels although the tendency for those values to return towards normal was observed.
Cholesterol FR145237
0
+
+
+
0
0.01
0.1
+
Cholesterol-
1
FR145237 0
(wWday)
CholesterolFR145237 0
+
+
+
+
0
0.01
0.1
1
bg/Wday)
+ 0
+
+
0.01
0.1
+ 1
(mg/k%day)
Fig.2 Effects of FR145237 on plasma cholesterol, TG and HDL cholesterol levels in cholesterol-fed diabetic rats. Data represent meamtSEM (n=5). The rats were fed a high cholesterol diet for 1 week and FR145237 was administered as a dietary admixture. **P
Vol. 60, No. 6, 1997
ACAT in Diabetic Hypereholesterolemia
355
hyperresponse to dietary cholesterol in the STZ rats is due to the increase in food intake because the hyperresponse was too great. This speculation is supported by previous reports that hyperresponse to dietary cholesterol in STZ rats is induced independently of hyperphagia and that ACAT plays an important role in the initiation of the condition (13,14). Since intestinal ACAT is involved in cholesterol absorption, the remarkable increase in plasma cholesterol levels in the STZ rats suggests that the diabetic state may enhance the response of intestinal ACAT to cholesterol feeding. The involvement of ACAT in the hyperresponse was further supported by our finding that FR145237 effectively normalized hypercholesterolemia in the STZ rats. These results coincided well with those reported by Maechler et al. who used CL-277082 as an ACAT inhibitor (13,14). Since FR145237 is a specific inhibitor of ACAT, there is no doubt that the reduction of plasma cholesterol levels was due to inhibition of ACAT. The mechanism of the enhancement of ACAT activity in STZ rats is unknown. It is reported that intestinal hypertrophy is induced in STZ rats and it is assumable that the hypertrophy contributes to the enhancement of ACAT activity (17). However, it was reported that cholesterol hyperabsorption due to ACAT activation occurs in the absence of intestinal hypertrophy in STZ rats (13,14). Further studies are needed to clarify this point. FR145237 did not show clear effects on plasma TG and HDL cholesterol levels in comparison with those on plasma cholesterol levels in the cholesterol-fed STZ, but additional studies are suggested by the tendency for values of TG and HDL cholesterol to return towards normal, even if the changes were not statistically significant in this limited study. TG is incorporated into chylomicrons in the intestine together with cholesterol esters, phospholipids and apolipoproteins, and esterification of cholesterol is critical for the assembly of chylomicrons (18-2 1). Therefore, inhibition of cholesterol ester formation by ACAT inhibitors might interfere with the assembly of chylomicrons, and possibly result in the reduction of plasma TG levels. It is known that circulating concentrations of TG and HDL cholesterol are inversely related (22). This inverse relationship was also observed in the diabetic rats treated with lmg/kg of FR145237 although the precise mechanism is unknown. FR145237 dose-dependently reduced the food intake and body weight in the STZ rats. This reduction is associated with the slight decrease in plasma glucose levels. Since treatment with lOOmg/kg of FR145237 for 2 weeks had no toxic effects in normal rats (data not shown), the reduction of food intake induced by FR145237 was probably not due to its side effects. It is possible that the reduction of cholesterol levels induced by FR145237 treatment may improve hyperphagia, and resulting in the decrease of plasma glucose levels and body weight. In summary, cholesterol feeding for 1 week induced a remarkable increase in plasma cholesterol levels in STZ rats. An ACAT inhibitor, FR145237, dose-dependently reduced this rise and normalized it at a dose of 1 mg/kg/day. These results suggest that ACAT is involved in the hyperresponse to dietary cholesterol in the diabetic state.
References 1. 2. 3. 4.
B.V. HOWARD, J. Lipid Res.28 613-628 (1987). G. STEINER, Drugs 3 22-26 (1988). M-R. TASKINEN, Baillieres Clin. Endocrinol. Metab. 4 743-755 (1990). M.L. J.UUSITUPA, L.K. NISKANEN, 0. SIITONEN, E. VOUTILAINEN and K. PYORALA, Circulation u 27-36 (1990). 5. P.H. WINOCOUR, P.N. DURRINGTON, D. BHATNAGAR, M. ISHOLA and M. MACKNESS, Arterioscler. Thromb. 12 920-928 (1992). 6. F.O. NERVI, A. GONZALEZ and V.D. VALDIVIESO, Metabolism 23 495-503 (1974).
356
ACAT in Diabetic Hyperchoksterolemia
Vol. 60, No. 6, 1997
7. R. SADAHIRO, N. TAKEUCHI, A. KUMAGAI and Y. YAMAMURA, Endocrinol. Jpn. fl225-232 (1970). 8. N.L. YOUNG, D.R. LOPEZ and D.J. MCNAMARA, Diabetes 37 151-156 (1988). 9. J.R. COLCA, C.F. DAILEY, B.J. PALAZUK, R.M. HILLMAN, D.M. DINH, G.W. MELCHIOR and C.H. SPILMAN, Diabetes & 1669-1674 (1991). 10. C.M. ARBEENY, D. EDELSTEIN, S.R. FREEDMAN and H.A. EDER, Diabetes 2j 774-777 (1980). 11. Y. MARUHAMA, Metabolism 14 78-87 (1965). 12. S. JIAO, Y. MATSUZAWA, K. MATSUBARA, S. KIHARA, S. NAKAMURA, K. TOKUNAGA, M. KUBO and S. TARUI, Diabetes 21342-346 (1988). 13. K.E. SUCKLING and E.F. STANGE, J. Lipid Res.26 647-671 (1985). 14. R. HAUGEN and K.R. NORUM, Stand. J. Gastroenterol.LL 615-621 (1976). 15. M. MATSUO, M. AKETA, R. OZAKI, M. TOM01 and K. SHIMOMURA, Drug Dev. Res. x 186-192 (1996). 16. M. MATSUO, F. ITO, A. KONTO, M. AKETA, M. TOM01 and K. SHIMOMURA, Biochim. Biophys. Acta 1259 254-260 (1995). 17. H.P. SCHEDL and H.D. WILSON, J. Exp. Zool. 176 487-496 (1971). 18. N.T.P. KAM, E. ALBRIGHT, S.N. MATHUR and F. J. FIELD, J. Lipid Res.O 371-377 (1989). 19. C.S. BENNETT and A.M. TERCYAK, J. Lipid Res.25 148-159 (1984). 20. M. TANAKA, H. JINGAMI, H. OTANI, M. CHO, Y. UEDA, H. ARAI, Y. NAGANO, T. DOI, M. YOKODE and T. KITA, J. Biol. Chem.a 12713-12718 (1993). 21. N. DASHTI, J. Biol. Chem.267 7160-7169 (1992). 22. B. LEWIS, A. CHAIT, I.D.P. WOOTON, C.M. OAKELYA and D.M. KRIKLER, Lancet 1 141-146 (1974).
PI1 SOO24-3205(96)00458-3
EFFECTS OF FR145237, AN Acyl-CoA:CHOLESTEROL ACYLTRANSFERASE INHIBITOR, ON DIET-INDUCED HYPERCHOLESTEROLEMIA IN DIABETIC RATS Yuri Sakuma, Hiroyuki Hagihara, Akira Nagayoshi, Kazuhiko Ohne, Seitaro Mutoh*, Yoshikuni Ito, Kunio Nakahara, Yoshitada Notsu and Masakuni Okuhara Exploratory Research Laboratories, Fujisawa Pharmaceutical 5-2-3 Tokodai, Tsukuba, Ibaraki 300-26, Japan
Co., Ltd.
(Received in find form November l&19%)
Summary Recent studies have shown that acyl-CoA:cholesterol acyltransferase (ACAT) plays an important role in the initiation of diabetes-associated hypercholesterolemia. To confirm this hypothesis, effects of a potent ACAT inhibitor, FR145237, on diet-induced hypercholesterolemia were examined in streptozotocin (STZ)-induced diabetic rats. One-week feeding of 1 % cholesterol and 0.5 % cholic acid to normal rats and STZ-induced diabetic rats increased plasma cholesterol levels in both groups, and the response was more remarkable in the STZ rats than in the normal ones (1266f476 mg/dl and 146*7 mg/dl, respectively). FR145237 dose-dependently reduced the rise in plasma cholesterol levels in the STZ rats and the levels were almost normalized by treatment with 1 mg/kg/day of the compound. These results suggest that hyperresponse to dietary cholesterol was induced in the STZ rats and that ACAT is involved in the hyperresponse. The effects of FR145237 on other plasma lipids such as high density lipoprotein (HDL) cholesterol and triglyceride (TG) levels were also examined. Key Words: acyI-CoAxholesterol acykransferase,ACAT inhibitor,diabetes, atherosderosis
Hypercholesterolemia is one of complications frequently observed in patients with diabetes mellitus (l-5) and certainly contributes to the development of atherosclerosis in these patients. To clarify the mechanism by which the diabetic state induces hypercholesterolemia, many studies have been performed using diabetic animal models such as streptozotocin (STZ)-induced diabetic rats (6,7), and recent studies have shown an increased cholesterol absorption in STZ rats (8-l 1). Recently, Acyl-CoA:cholesterol acytransferase (ACAT, EC 2.3.1.26), the rate-limiting enzyme for cholesterol absorption, was reported to be increased in STZ rats (12). In addition, Maechler et al. clearly demonstrated that ACAT plays a major role in the initiation of hypercholesterolemia in STZ rats (13,14). In their studies, CL-277082, an ACAT inhibitor, was used to see the involvement of intestinal ACAT in the hypercholesterolemia, and the inhibitor completely improved it in the dose of 5Omg/kg/day. To confirm that ACAT is involved in the development of diet-induced hypercholesterolemia the diabetic state, we examined the effects of a potent ACAT inhibitor, FR145237, on
in
352
ACAT in Diabetic Hypercholesterolemia
Vol. 60, No. 6, 1997
hypercholesterolemia induced by cholesterol feeding to STZ rats. Additionally, we examined the effects of the compound on the changes of plasma triglyceride and HDL cholesterol levels in the cholesterol-fed STZ rats. FR145237 has potent inhibitory activities on ACAT activities in the intestine, aorta and liver (15). The ICso values are around 1x10-*M. In addition, the compound attenuated aortic cholesterol depositions in cholesterol-fed rabbits and Watanabe heritable hyperlipidemic (WHHL) rabbits (16).
Methods Chemicals FR145237 (N-Benzyl-N-[3-(4-chorophenyl)-5-methyl-2-benzofuranyl] trifluorophenyl) urea) was synthesized at Exploratory Research Pharmaceutical Co., Ltd. (Ibaraki, Japan).
methyl-N’-(2,4,6Laboratories, Fujisawa
Animals Male Sprague-Dawley rats (180-220 g body weight, 6 weeks, Clea, Tokyo, Japan) were used. Rats were acclimated for 1 week at a controlled temperature of 22-25 “C under illumination from 0800 to 2000 h with free access to standard chow (Clea, Tokyo, Japan) and tap water. After 16 hours fasting, diabetes was induced by injection to the tail vein of streptozotocin (Sigma, St.Louis, MO, USA) dissolved in 5 mM citrate buffer, 0.1 M NaCl, pH 4.5 (60 mg/kg). Nondiabetic control rats were injected with the buffer only. One week after the injection, about 100 pl of blood was taken from the tail vein and glucose was measured to ensure the induction of diabetes. The rats were then fed for 1 week a high-fat diet prepared with the standard chow to which 1 % cholesterol and 0.5 % cholic acid (w/w) were added. The total amount of food intake of each cage (5 rats) was measured everyday for 1 week starting from cholesterol feeding, and the average of daily food intake was calculated on the basis of the 1 week observation. The rats were killed by collecting blood from the abdominal aorta under ether anesthesia. All animal procedures were carried out as approved by the Animal Care and Use Committee at Fujisawa Pharmaceutical Co., Ltd. Drum Treatment FR145237 was dissolved in ethanol and mixed with the high-fat diet at final concentrations of 0.00001 %, 0.0001 % and 0.001 % (w/w) in order to obtain daily doses of approximately 0.01 mg/kg, 0.1 mg/kg and 1 mg/kg, respectively. Biochemical Analvsis of Serum Plasma glucose, cholesterol, triglyceride (TG) and free fatty acid were determined enzymatically using kits purchased, respectively, from Wako Pure Chemicals (Osaka, Japan). High density lipoprotein (HDL) cholesterol was measured after removal of very low density lipoprotein (VLDL) and LDL cholesterol by heparin-manganese precipitation. Statistical Analvsis Data were expressed as mean&SEM. Statistical differences between two groups were evaluated by Student’s t-test. For evaluation of effects of FR145237, analysis of variance (ANOVA) followed by Dunnett’s t-test was used. P values less than 0.05 were considered significant.
Results Hyperresuonse to high cholesterol diet in diabetic rats As shown in Table 1, plasma glucose levels were higher than 600 mg/dl in the STZ rats, indicating a diabetic state. Compared to the normal rats, the food intake was increased in the STZ
Vol. 60, No. 6, 1997
ACAT in Diabetic Hypercholesterolemia
353
rats, but the body weight was significantly reduced. One-week feeding of cholesterol had no effect on plasma glucose levels, food intake or body weight in either the normal or the STZ rats. One-week feeding of a high cholesterol diet to the normal rats significantly increased plasma cholesterol levels compared to those in the normal rats fed the standard chow (Fig. 1A) The same treatment markedly increased plasma cholesterol levels in the STZ rats and the level was about 8.7 fold higher than that of the cholesterol-fed normal rats (1266&476 mg/dl and 146f7 mg/dl, respectively). Plasma TG levels were not affected significantly by cholesterol feeding to the STZ rats, but a significant decrease in plasma HDL cholesterol levels was induced (Fig.lB,lC). TABLE
I
Effect of a High Cholesterol Diet on Plasma Glucose, Food Intake and Body Weight of Normal and Diabetic rats Rat
Normal Normal STZ STZ STZ
Diet
Plasma Glucose (mg/dU
Food Intake (g/day/rat)
Final Body Weight
141f3 131s 625+32** 614&39**
25 26 41 39
327&l 1 328f6 271+8** 271f17**
607f25 572k43 514*41
38 36 30
274f7 27M3 263tlO
Standard Cholesterol Standard Cholesterol Cholesterol + FR145237 O.Olmg/kg 0.1 1
Data are expressed as mea&SEM
Cholesterol
-
+
Normal rak
-
+
STZ rats
(8)
(n=5). **PcO.Ol versus respective normal controls.
Cholesterol
-
+
Normal rats
-
+
STZ rak
Cholesterol
-
+
Normal rak
-
+
STZ rak
Fig.1 Cholesterol, TG and HDL cholesterol levels in the plasma of cholesterol-fed normal and diabetic rats. Data represent mea&SEM (n=5). The rats were fed a high cholesterol diet or a standard chow for 1 week. *Pc0.05,***Pc0.001 versus the standard chow-fed normal rats. ##PcO.Ol versus the standard chow-fed STZ rats.
Vol. 60, No. 6, l!N7
ACAT in Diabetic Hypercholesterolemia
354
Hvpocholesterolemic acti ‘ty of FR145237 in cholesterol-fed diabetic rats As shown in Table 1, ‘Al45237 dose-dependently decreased plasma glucose concentrations, the food intake and the final body weight in the STZ rats fed a high cholesterol diet, but those values were not significant. As mentioned above, a marked increase in plasma cholesterol levels was observed in diabetic rats fed a high cholesterol diet for 1 week (Fig. 1A). FR145237 dose-dependently reduced this increase and at a dose of 1 mg/kg/day the levels were almost the same as those of the standard chow-fed control (Fig.2A). Treatment of the diabetic rats with FR145237 did not affect significantly the changes of plasma TG (Fig.2B) and HDL cholesterol (Fig.2C) levels although the tendency for those values to return towards normal was observed.
Cholesterol FR145237
0
+
+
+
0
0.01
0.1
+
Cholesterol-
1
FR145237 0
(wWday)
CholesterolFR145237 0
+
+
+
+
0
0.01
0.1
1
bg/Wday)
+ 0
+
+
0.01
0.1
+ 1
(mg/k%day)
Fig.2 Effects of FR145237 on plasma cholesterol, TG and HDL cholesterol levels in cholesterol-fed diabetic rats. Data represent meamtSEM (n=5). The rats were fed a high cholesterol diet for 1 week and FR145237 was administered as a dietary admixture. **P
Vol. 60, No. 6, 1997
ACAT in Diabetic Hypereholesterolemia
355
hyperresponse to dietary cholesterol in the STZ rats is due to the increase in food intake because the hyperresponse was too great. This speculation is supported by previous reports that hyperresponse to dietary cholesterol in STZ rats is induced independently of hyperphagia and that ACAT plays an important role in the initiation of the condition (13,14). Since intestinal ACAT is involved in cholesterol absorption, the remarkable increase in plasma cholesterol levels in the STZ rats suggests that the diabetic state may enhance the response of intestinal ACAT to cholesterol feeding. The involvement of ACAT in the hyperresponse was further supported by our finding that FR145237 effectively normalized hypercholesterolemia in the STZ rats. These results coincided well with those reported by Maechler et al. who used CL-277082 as an ACAT inhibitor (13,14). Since FR145237 is a specific inhibitor of ACAT, there is no doubt that the reduction of plasma cholesterol levels was due to inhibition of ACAT. The mechanism of the enhancement of ACAT activity in STZ rats is unknown. It is reported that intestinal hypertrophy is induced in STZ rats and it is assumable that the hypertrophy contributes to the enhancement of ACAT activity (17). However, it was reported that cholesterol hyperabsorption due to ACAT activation occurs in the absence of intestinal hypertrophy in STZ rats (13,14). Further studies are needed to clarify this point. FR145237 did not show clear effects on plasma TG and HDL cholesterol levels in comparison with those on plasma cholesterol levels in the cholesterol-fed STZ, but additional studies are suggested by the tendency for values of TG and HDL cholesterol to return towards normal, even if the changes were not statistically significant in this limited study. TG is incorporated into chylomicrons in the intestine together with cholesterol esters, phospholipids and apolipoproteins, and esterification of cholesterol is critical for the assembly of chylomicrons (18-2 1). Therefore, inhibition of cholesterol ester formation by ACAT inhibitors might interfere with the assembly of chylomicrons, and possibly result in the reduction of plasma TG levels. It is known that circulating concentrations of TG and HDL cholesterol are inversely related (22). This inverse relationship was also observed in the diabetic rats treated with lmg/kg of FR145237 although the precise mechanism is unknown. FR145237 dose-dependently reduced the food intake and body weight in the STZ rats. This reduction is associated with the slight decrease in plasma glucose levels. Since treatment with lOOmg/kg of FR145237 for 2 weeks had no toxic effects in normal rats (data not shown), the reduction of food intake induced by FR145237 was probably not due to its side effects. It is possible that the reduction of cholesterol levels induced by FR145237 treatment may improve hyperphagia, and resulting in the decrease of plasma glucose levels and body weight. In summary, cholesterol feeding for 1 week induced a remarkable increase in plasma cholesterol levels in STZ rats. An ACAT inhibitor, FR145237, dose-dependently reduced this rise and normalized it at a dose of 1 mg/kg/day. These results suggest that ACAT is involved in the hyperresponse to dietary cholesterol in the diabetic state.
References 1. 2. 3. 4.
B.V. HOWARD, J. Lipid Res.28 613-628 (1987). G. STEINER, Drugs 3 22-26 (1988). M-R. TASKINEN, Baillieres Clin. Endocrinol. Metab. 4 743-755 (1990). M.L. J.UUSITUPA, L.K. NISKANEN, 0. SIITONEN, E. VOUTILAINEN and K. PYORALA, Circulation u 27-36 (1990). 5. P.H. WINOCOUR, P.N. DURRINGTON, D. BHATNAGAR, M. ISHOLA and M. MACKNESS, Arterioscler. Thromb. 12 920-928 (1992). 6. F.O. NERVI, A. GONZALEZ and V.D. VALDIVIESO, Metabolism 23 495-503 (1974).
356
ACAT in Diabetic Hyperchoksterolemia
Vol. 60, No. 6, 1997
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