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Long-term effects of bezafibrate on in vivo VLDL-triglyceride production in the rat
Diabetes Research and Clinical Practice, 9 (1990)
163-168
163
Elsevier DIABET
00392
Long-term effects of bezafibrate on in vivo VLDL-triglyceride production in the rat Tsutomu Kazumi ‘, Gen Yoshino2, Masahide Iwai2, Kohji Matsuba2, Masayuki Matsushita 2, Ippei Iwatani2, Toshio Kasama ‘, Muneyoshi Yoshida ’ and Shigeaki Baba2 ’ Division of Endocrinology and Metabolirm, Department of Medicine, Hyogo Medical Center for Adults, Akashi, and 2 Second Department of Internal Medicine, Kobe University School of Medicine, Kobe. Japan
(Received 1 August 1989) (Revision received 20 December 1989) (Accepted 29 December 1989)
Summary Long-term effects of bezafibrate on in vivo production of VLDL-triglyceride were studied in the rat. Bezafibrate given at a daily dose of 30 mg/kg body weight for 14 days produced a decrease not only in triglyceride by 51 y0 but in cholesterol by 28% and phospholipid by 18%. Despite a marked reduction in plasma triglyceride concentrations, there was no significant change in the rate of VLDL-triglyceride secretion from the liver into the circulation between beztibrate-treated and control animals (1113 k 58 and 1234 + 63 pg/min, respectively). In addition, bezafibrate produced no change in lipid composition in VLDL. These results suggest that bezafibrate enhances triglyceride removal from the circulation, which leads to reduction in plasma triglyceride. Key words: Bezafibrate;
VLDL-triglyceride
production;
Introduction Bezafibrate has been shown to reduce increased levels of very low density lipoproteins (VLDL) in patients with hypertriglyceridemia [l-3]. These studies demonstrated that bezaflbrate produced an increase in lipoprotein lipase activity. This Address for correspondence: Dr. Tsutomu Kazumi, Division of Endocrinology and Metabolism, Department of Medicine, Hyogo Medical Center for Adults, 13-70, Kitaohji-cho, Akashi 673, Japan. 0168~8227/90/$03.50 0 1990 Elsevier Science Publishers
Rat plasma
could result in an increased removal of circulating triglyceride-rich proteins. In addition, a recent in vitro study has revealed that bezafibrate inhibited the secretion of VLDL from cultured human hepatocytes into the medium [4], indicating that the fibric acid derivative lowers plasma VLDL concentrations by reducing the secretion of VLDL from the liver into the circulation. There are, however, many differences between in vivo and in vitro studies. The most striking difference is in the time during which cells in culture or organs in the body, especially the liver, were
B.V. (Biomedical
Division)
164
exposed to bezafibrate. Hypertriglyceridemic patients had received bezaflbrate for 4 or 8 weeks [l-3]. In contrast, cultured human hepatocytes had been exposed to bezafibrate for as little as 18 hours [4]. Therefore, the present study was undertaken to see the long-term effects of bezaflbrate on the rate of VLDL-triglyceride secretion in the rat. In addition, lipid composition of circulating VLDL was also examined.
Materials and methods Male Wistar rats weighing 290-330 g were used in the present study. They were kept on rat chow (Oriental MF, Oriental Yeast Co., Tokyo, Japan) for 7 days prior to the study. Then, they were given powdered rat chow (Oriental Yeast Co.) or powdered rat chow containing bezafibrate at the concentration of 360 mg/kg of rat chow until the end of the study. Fourteen days later, the rate of VLDL-triglyceride secretion from the liver into the circulation was measured by the Triton method [ 51. The batch of Triton WR 1339 (Nakarai Chemicals, Kyoto, Japan) used was demonstrated to completely block the removal of triglyceride from the circulation (Fig. 1) and to produce a linear increase in plasma triglyceride concentrations for at least 90 min. The Triton was dissolved in distilled water (300 mg/ml) and injected into the caudal veins under light ether anesthesia (600 mg/kg body weight). Cholesterol and phospholipid as well as triglyceride were measured in samples taken before, at 45 and 90 min after Triton injection. Since the removal of VLDL from the plasma has been shown to be blocked during this time period, and since more than 90% of triglyceride in plasma taken 90 min after Triton injection was recovered in VLDL fraction after ultracentrifugal separation of plasma lipoproteins, it is reasonable to consider that an increase in lipid concentrations per unit time after Triton injection is due to an increase in VLDL-lipid concentrations. Therefore, we subtracted plasma concentrations of cholesterol, phospholipid or triglyceride before Triton injec-
““L
=
Tritp
(-I-)
OOV
.
90 Time
(mtn)
Fig. 1. Effect of Triton WR 1339 on disappearance of triglyceride radioactivity in plasma of two normal rats after injection of biologically 3H-labeled rat VLDL-triglyceride. The biological labeling of VLDL-triglyceride was done by injecting [2-3H]glycerol into a donor rat, exanguinating it 30 min later, and isolating the VLDL from plasma. VLDLtriglyceride was then injected into recipient rats 10 min after one had received Triton (600 mg/kg body weight, + Triton) and the other had received an equal volume of distilled water ( - Triton).
tion from those at 45 min after Triton injection and calculated the lipid composition (weight %) of VLDL. The same calculations were also done using data at 90 min after Triton injection. The rat chow consumed was measured every day in each rat, and the daily dose of bezatibrate calculated was 30 mg/kg of body weight. This dosage was reported to effectively reduce plasma concentrations of triglyceride and cholesterol in rats [6]. Plasma concentrations of bezafibrate were measured by high-performance liquid chromatography [ 71 and averaged 5.60 k 0.59 pg/ml in samples taken 90 min after Triton injection. Triglyceride [ 81, cholesterol [9] and phospholipid [lo] were measured by enzymatic methods, respectively. Plasma glucose and insulin were measured by the glucose oxidase method and RIA [ 111, respectively. High-density lipoproteins were measured by the precipitation method [ 121. Data are expressed as the mean f SE and statistical comparison was made using Student’s non-paired t-test.
165
There was no change in body weight, food consumption and the plasma levels of glucose and insulin between bezafibrate-treated and control rats (Table 1). As shown in Fig. 2, bezafibrate given at a daily dose of 30 mg/kg of body weight for 14 days produced a decrease in plasma cholesterol by 28% (51.5 k 2.7 vs. 36.9 f 1.7 mg/dl) and phospholipid by 18% (116 + 5 vs. 95 k 4 mg/dl), the former being associated with a decrease in HDL-cholesterol by 48 % (40.0 k 1.9 vs. 21.3 + 2.3 mg/dl). As described in Materials and methods, a single intravenous injection of Triton was followed by a linear increase in plasma triglyceride concentrations, not only in control
TABLE
ATG
TG
Results
Wde)
ha) 800
r
T
8oor
600 -
1
Body weights, chow consumption, plasma glucose insulin levels in rats given bezatibrate and controls
Starting body weights (g) Final body weights (g) Chow consumed (g/day) Plasma glucose (mg/dl) Plasma insulin (pU/ml)
and Time(min)
Bezalibrate-treated
Controls
309 f. 3 316 k 6 21.1 + 1.2 168+4 12.9 f 1.4
309 + 315 * 21.5 f 164k 13.5 +
5 9 1.2 5 2.3
Fig. 3. Responses tions to a single weight of Triton (TG, right panel) rats
after
Triton
Injection
of plasma triglyceride (TG) concentraintravenous injection of 600 mg/kg body (left panel) and increments of triglyceride in bezafibrate (BF)-treated (0) and control (0). Mean k SE of eight rats.
Mean + SE of eight rats in each group.
TG
TGSR
(ma/dN
(mglmd
80Total
Cholesterol
HDL-Cholesterol
1.4 -
Phosphollpid (W/d8)
L
IOO-
1.2 60-
t
1.0 -
40 -
*
0.8 -
400.6 20 -
20-
0.4 0.2 -
O-
-
Fig. 2. Long-term (14 days) effects of bezafibrate on plasma levels of cholesterol, HDL-cholesterol and phospholipids in the rat. Hatched bar, bezafibrate-treated rats; open bar, control rats. Mean + SE of eight rats in each group. Significance of differences: ** P < 0.0 1, ***P < 0.00 1.
o-
O-
Fig. 4. Plasma triglyceride (TG) concentrations and the rate of triglyceride secretion (TGSR) in bezafibrate-treated (shaded colums) and control rats (open colums). Mean + SE of eight rats in each group. Significance of differences, * P < 0.05.
166 TABLE
2
Lipid composition
(weight percent) of very-low-density
lipoproteins
in bezafibrate-treated
45 min after Triton
Cholesterol Triglyceride Phospholipid
and control rats
90 min after Triton
Bezafibrate-treated
Controls
Bezatibrate-treated
Controls
7.1 f 1.3 77.8 f 3.5 14.4 f 2.1
7.4 + 1.1 77.1 f 2.8 15.5 f 1.6
4.9 k 0.6 84.5 +- 1.6 10.7 f 1.0
5.9 + 0.7 81.6 + 1.5 12.5 f 0.8
Mean + SE of 8 rats in each group. Plasma levels of three lipids were measured in samples taken before, at 45 and 90 min after Triton injection. Since the removal of VLDL-triglyceride was demonstrated to be completely blocked at least for 90 min after Triton injection, and since more than 90% of triglyceride in plasma taken 90 min after Triton injection were recovered in VLDL fraction after ultracentrifugal separation of plasma lipoproteins, it seems reasonable to infer that increments of lipid concentrations during unit time periods are those of VLDL-lipids. Therefore, we calculated the lipid composition in VLDL using increments of triglyceride, cholesterol and phospholipid during 45-min and 90-min periods, respectively.
rats but also in bezafibrate-treated animals (Fig. 3). Although plasma triglyceride levels at all three time points were significantly lower (P < 0.05, respectively) in bezafibrate-treated rats than in controls, there were no significant differences in the increments of plasma triglyceride between the two groups. Plasma triglyceride fell by 5 1% from 70 k 15 mg/dl to 34 k 6 mg/dl (Fig. 4). Despite this, there was no change in the rate of triglyceride secretion between bezaflbratetreated and control rats (Fig. 4). However, the production rate tended to be lower in the bezafibrate-treated rats (1113 k 58 pg/min) than in controls (1234 + 63 pg/min). Furthermore, bezafibrate produced no change in lipid composition of VLDL (Table 2) as well as lipid mass of VLDL (421 + 33 and 393 + 20 mg/dl in bezafibratetreated and control rats), which was the sum of the increments of the three lipids during the first 45 min after Triton injection.
Discussion The present study has revealed that bezatibrate given as 30 mg per kg of body weight daily for 14 days had no signiticant effects on the rate of triglyceride secretion in the rat, despite a 50 % reduc-
tion in plasma triglyceride concentrations. Plasma triglyceride concentrations are the reflection of the balance between the secretion into and the removal from the circulation. In order to see the rate of triglyceride secretion, we completely blocked the triglyceride removal in the present study. Therefore, we could not directly measure the removal rate in the same rat. However, since we measured initial plasma triglyceride levels as well in each rat, we could make inferences about the rates of triglyceride removal, assuming that the volume of distribution did not change. The current results suggest that bezafIbrate stimulates the removal of VLDL-triglyceride from the circulation and this is consistent with the previous observations that the fibric acid derivative reduced the VLDL-triglyceride concentration by stimulating lipoprotein lipase activities [ l-31. They, however, were not in accordance with a recent study [4] using human hepatocytes that incubation in the presence of bezafibrate produced a decrease in both total VLDL neutral lipids and apolipoprotein B secreted into the medium. Although the reasons for the discrepancy between our in vivo and the in vitro study are not clear, the most striking difference between the two is in the time period when hepatocytes were exposed to bezaflbrate: 14 days in the current in vivo study and 18 hours in the in
167
vitro study. However, the present study does not exclude the possibility that fibric acid reduces the production rate of VLDL-triglyceride, since the production rate in bezatibrate-treated rats tended to be lower than that of controls in the present study. We have previously shown in hypertriglyceridemic patients that bezafibrate decreased plasma VLDL concentrations without modifying its structure [ 131. This may be consistent with the present finding that the fibrate produced no significant changes in lipid composition of VLDL which was calculated using increments of triglyceride, cholesterol and phospholipid after Triton. Since the removal of VLDLtriglyceride was demonstrated to be completely blocked for at least 90 min after Triton injection, and since more than 90% of the triglycerides in plasma taken 90 min after the Triton injection were recovered in the VLDL fraction, after ultracentrifugal separation of plasma lipoproteins, it seems reasonable to infer that the increments in lipid concentration during unit time periods are those of the VLDL-lipids, although they are likely to represent an accumulation of different classes of lipoproteins. In patients with type III hyperlipoproteinemia, therapy with bezafibrate not only increased the fractional clearance of VLDL apolipoprotein B (flotation constant of 60 to 400) but reduced the production of this fraction of VLDL apolipoprotein B [ 141. In a study in hypertriglyceridemic subjects by Shepherd et al. [ 151, bezafibrate therapy produced a decrease in VLDL apolipoprotein B levels associated with an increase in the fractional clearance rate of VLDL apolipoprotein B (flotation constant of 100 to 400). A reduction in the rate of VLDL apolipoprotein B production was found in four out of six patients, although the difference between baseline and post-treatment values was not statistically significant. There were, as far as we know, no data available concerning the effects of bezaflbrate on VLDL production in experimental hyperlipoproteinemia. In the present study, treatment with bezafibrate for 14 days resulted in a decrease in HDLcholesterol in the rat. The same decrease has been
shown in normal rats [ 161. In humans, bezafibrate raised HDL-cholesterol not only in hyperlipoproteinemics [l-3,14,15] but also in normal subjects [ 171. The reason for the discrepancy in the effects of bezalibrate on the HDL-cholesterol concentrations between humans and rats is obscure and we have no explanation for this matter.
Acknowledgement The authors are indebted to Kissei Pharmaceutical Co. for their kind supply of bezatibrate.
References 1 Eisenberg, S., Gavish, D., Oschry, Y., Fainaru M. and Deckelbaum, R. J. (1984) Abnormalities in very-low, low and high-density lipoproteins in hypertriglyceridemia. Reversal toward normal with bezafibrate treatment. J. Clin. Invest. 74, 470-482. 2 Vessby, B., Lithell, H., Hellsina,-. K., Ostlund-Lindavist. A.-M., Gustafsson, I.-B., Boberg, J and Ledermann, H. (1980) Effects of bezafibrate on the serum lipoprotein lipid and apolipoprotein composition, lipoprotein triglyceride removal capacity and the fatty acid composition of the plasma lipid esters. Atherosclerosis, 37, 257-269. Vessby, B., Lithell, H. and Ledermann, H. (1982) Elevated lipoprotein lipase activity in skeletal muscle tissue during treatment of hypertriglyceridaemic patients with bezafibrate. Atherosclerosis 44, 113-l 18. Kosykh, V. A., Podrez, E. A., Novikov, D. K., Victorov, A. V., Dolbin, A. G., Repin, V. S. and Smirnov, V. N. (1987) Effect of bezaflbrate on lipoprotein secretion by cultured human hepatocytes. Atherosclerosis, 68,67-76. Steiner, G., Haynes, F. J., Yoshino, G. and Vranic, M. (1984) Hyperinsulinemia and in vivo very-low-density lipoprotein-triglyceride. Am. J. Physiol. 246, E187-E192. Berndt, J., Gaumert, R. and Still, J. (1978) Mode of action of the lipid-lowering agents, clofibrate and BM 15075, on cholesterol biosynthesis in rat liver, Atherosclerosis 30, 147-152. Castoldi, D., Monzani, V., Tofanetti, 0. (1985) Determination of bezafibrate in human plasma and urine by highperformance liquid chromatography. J. Chromatogr. 344, 259-265. Sullivan, D. R., Kruijswijk, Z., West, C. E., Kohlmeier, M. and Katan, M. B. (1985) Determination of serum triglyceride by an accurate enzymatic method not affected by free glycerol. Clin. Chem. 31, 1227-1228. 1
I
168 9 Richmond, W. (1973) Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin. Chem. 19, 1350-1356. 10 Takayama, M., Itoh, S., Nagasaki, T. and Tanimizu, I. (1977) A new enzymatic method for determination of serum choline-containing phospholipid. Clin. Chem. Acta 79, 93-98. 11 Morgan, C. R. and Lazarow, A. (1963) Immunoassay of insulin: two antibody system; plasma insulin level of normal, subdiabetic and diabetic rats. Diabetes 12, 115-126. 12 Noma, A., Okabe, H., Netsu-Nakayama, K., Ueno, Y. and Shinohara, H. (1979) Improved method for simultaneous determination of cholesterol in high- and lowdensity lipoproteins. Clin. Chem. 25, 1480-1481. 13 Kazumi, T., Yoshino, G., Matsuba, K., Iwai, M., Iwatani, I., Kasama, T., Yoshida, M. and Baba, S. (1989) Bezafibrate lowers VLDL concentrations without modifying its structure. Horm. Metabol. Res. 21, 341-342.
14 Packard, C. J., Clegg, R. J., Dominiczak, M. H., Lorimer, A. R. and Shepherd, J. (1986) Effects of bezafibrate on apolipoprotein B metabolism in type III hyperlipoproteinemic subjects. J. Lipid Res. 27, 930-938. 15 Shephered, J., Packard, C. J., Stewart, J. M., Atmeh, R. F., Clark, R. S., Boag, D. E., Carr, K., Lorimer, A. R., Ballantyne, D., Morgan, H. G. and Lawrie, T. D. V. (1984) Apolipoprotein A and B (Sf 100-400) metabolism during bezafibrate therapy in hypertriglyceridemic subjects. J. Clin. Invest. 74, 2164-2177. 16 Catapano, A. L., Trezzi, E. and Roma, P. (1982) Effects of bezafibrate on experimental hyperlipoproteinemias induced in the rat, In: G. Crepaldi, H. Greten, G. Schettler and G. Baggio (Eds.), Lipoprotein Metabolism and Therapy of Lipid Disorders, pp. 83-86, Excerpta Medica, Amsterdam. 17 Moulin, P., Bourdillon, M.-C., De Parscan, L., Perrot, L., Ponsin, G. and Berthezene, F. (1987) High-density lipoprotein alternations induced by bezafibrate in healthy male volunteers. Atherosclerosis 67, 17-22.
163-168
163
Elsevier DIABET
00392
Long-term effects of bezafibrate on in vivo VLDL-triglyceride production in the rat Tsutomu Kazumi ‘, Gen Yoshino2, Masahide Iwai2, Kohji Matsuba2, Masayuki Matsushita 2, Ippei Iwatani2, Toshio Kasama ‘, Muneyoshi Yoshida ’ and Shigeaki Baba2 ’ Division of Endocrinology and Metabolirm, Department of Medicine, Hyogo Medical Center for Adults, Akashi, and 2 Second Department of Internal Medicine, Kobe University School of Medicine, Kobe. Japan
(Received 1 August 1989) (Revision received 20 December 1989) (Accepted 29 December 1989)
Summary Long-term effects of bezafibrate on in vivo production of VLDL-triglyceride were studied in the rat. Bezafibrate given at a daily dose of 30 mg/kg body weight for 14 days produced a decrease not only in triglyceride by 51 y0 but in cholesterol by 28% and phospholipid by 18%. Despite a marked reduction in plasma triglyceride concentrations, there was no significant change in the rate of VLDL-triglyceride secretion from the liver into the circulation between beztibrate-treated and control animals (1113 k 58 and 1234 + 63 pg/min, respectively). In addition, bezafibrate produced no change in lipid composition in VLDL. These results suggest that bezafibrate enhances triglyceride removal from the circulation, which leads to reduction in plasma triglyceride. Key words: Bezafibrate;
VLDL-triglyceride
production;
Introduction Bezafibrate has been shown to reduce increased levels of very low density lipoproteins (VLDL) in patients with hypertriglyceridemia [l-3]. These studies demonstrated that bezaflbrate produced an increase in lipoprotein lipase activity. This Address for correspondence: Dr. Tsutomu Kazumi, Division of Endocrinology and Metabolism, Department of Medicine, Hyogo Medical Center for Adults, 13-70, Kitaohji-cho, Akashi 673, Japan. 0168~8227/90/$03.50 0 1990 Elsevier Science Publishers
Rat plasma
could result in an increased removal of circulating triglyceride-rich proteins. In addition, a recent in vitro study has revealed that bezafibrate inhibited the secretion of VLDL from cultured human hepatocytes into the medium [4], indicating that the fibric acid derivative lowers plasma VLDL concentrations by reducing the secretion of VLDL from the liver into the circulation. There are, however, many differences between in vivo and in vitro studies. The most striking difference is in the time during which cells in culture or organs in the body, especially the liver, were
B.V. (Biomedical
Division)
164
exposed to bezafibrate. Hypertriglyceridemic patients had received bezaflbrate for 4 or 8 weeks [l-3]. In contrast, cultured human hepatocytes had been exposed to bezafibrate for as little as 18 hours [4]. Therefore, the present study was undertaken to see the long-term effects of bezaflbrate on the rate of VLDL-triglyceride secretion in the rat. In addition, lipid composition of circulating VLDL was also examined.
Materials and methods Male Wistar rats weighing 290-330 g were used in the present study. They were kept on rat chow (Oriental MF, Oriental Yeast Co., Tokyo, Japan) for 7 days prior to the study. Then, they were given powdered rat chow (Oriental Yeast Co.) or powdered rat chow containing bezafibrate at the concentration of 360 mg/kg of rat chow until the end of the study. Fourteen days later, the rate of VLDL-triglyceride secretion from the liver into the circulation was measured by the Triton method [ 51. The batch of Triton WR 1339 (Nakarai Chemicals, Kyoto, Japan) used was demonstrated to completely block the removal of triglyceride from the circulation (Fig. 1) and to produce a linear increase in plasma triglyceride concentrations for at least 90 min. The Triton was dissolved in distilled water (300 mg/ml) and injected into the caudal veins under light ether anesthesia (600 mg/kg body weight). Cholesterol and phospholipid as well as triglyceride were measured in samples taken before, at 45 and 90 min after Triton injection. Since the removal of VLDL from the plasma has been shown to be blocked during this time period, and since more than 90% of triglyceride in plasma taken 90 min after Triton injection was recovered in VLDL fraction after ultracentrifugal separation of plasma lipoproteins, it is reasonable to consider that an increase in lipid concentrations per unit time after Triton injection is due to an increase in VLDL-lipid concentrations. Therefore, we subtracted plasma concentrations of cholesterol, phospholipid or triglyceride before Triton injec-
““L
=
Tritp
(-I-)
OOV
.
90 Time
(mtn)
Fig. 1. Effect of Triton WR 1339 on disappearance of triglyceride radioactivity in plasma of two normal rats after injection of biologically 3H-labeled rat VLDL-triglyceride. The biological labeling of VLDL-triglyceride was done by injecting [2-3H]glycerol into a donor rat, exanguinating it 30 min later, and isolating the VLDL from plasma. VLDLtriglyceride was then injected into recipient rats 10 min after one had received Triton (600 mg/kg body weight, + Triton) and the other had received an equal volume of distilled water ( - Triton).
tion from those at 45 min after Triton injection and calculated the lipid composition (weight %) of VLDL. The same calculations were also done using data at 90 min after Triton injection. The rat chow consumed was measured every day in each rat, and the daily dose of bezatibrate calculated was 30 mg/kg of body weight. This dosage was reported to effectively reduce plasma concentrations of triglyceride and cholesterol in rats [6]. Plasma concentrations of bezafibrate were measured by high-performance liquid chromatography [ 71 and averaged 5.60 k 0.59 pg/ml in samples taken 90 min after Triton injection. Triglyceride [ 81, cholesterol [9] and phospholipid [lo] were measured by enzymatic methods, respectively. Plasma glucose and insulin were measured by the glucose oxidase method and RIA [ 111, respectively. High-density lipoproteins were measured by the precipitation method [ 121. Data are expressed as the mean f SE and statistical comparison was made using Student’s non-paired t-test.
165
There was no change in body weight, food consumption and the plasma levels of glucose and insulin between bezafibrate-treated and control rats (Table 1). As shown in Fig. 2, bezafibrate given at a daily dose of 30 mg/kg of body weight for 14 days produced a decrease in plasma cholesterol by 28% (51.5 k 2.7 vs. 36.9 f 1.7 mg/dl) and phospholipid by 18% (116 + 5 vs. 95 k 4 mg/dl), the former being associated with a decrease in HDL-cholesterol by 48 % (40.0 k 1.9 vs. 21.3 + 2.3 mg/dl). As described in Materials and methods, a single intravenous injection of Triton was followed by a linear increase in plasma triglyceride concentrations, not only in control
TABLE
ATG
TG
Results
Wde)
ha) 800
r
T
8oor
600 -
1
Body weights, chow consumption, plasma glucose insulin levels in rats given bezatibrate and controls
Starting body weights (g) Final body weights (g) Chow consumed (g/day) Plasma glucose (mg/dl) Plasma insulin (pU/ml)
and Time(min)
Bezalibrate-treated
Controls
309 f. 3 316 k 6 21.1 + 1.2 168+4 12.9 f 1.4
309 + 315 * 21.5 f 164k 13.5 +
5 9 1.2 5 2.3
Fig. 3. Responses tions to a single weight of Triton (TG, right panel) rats
after
Triton
Injection
of plasma triglyceride (TG) concentraintravenous injection of 600 mg/kg body (left panel) and increments of triglyceride in bezafibrate (BF)-treated (0) and control (0). Mean k SE of eight rats.
Mean + SE of eight rats in each group.
TG
TGSR
(ma/dN
(mglmd
80Total
Cholesterol
HDL-Cholesterol
1.4 -
Phosphollpid (W/d8)
L
IOO-
1.2 60-
t
1.0 -
40 -
*
0.8 -
400.6 20 -
20-
0.4 0.2 -
O-
-
Fig. 2. Long-term (14 days) effects of bezafibrate on plasma levels of cholesterol, HDL-cholesterol and phospholipids in the rat. Hatched bar, bezafibrate-treated rats; open bar, control rats. Mean + SE of eight rats in each group. Significance of differences: ** P < 0.0 1, ***P < 0.00 1.
o-
O-
Fig. 4. Plasma triglyceride (TG) concentrations and the rate of triglyceride secretion (TGSR) in bezafibrate-treated (shaded colums) and control rats (open colums). Mean + SE of eight rats in each group. Significance of differences, * P < 0.05.
166 TABLE
2
Lipid composition
(weight percent) of very-low-density
lipoproteins
in bezafibrate-treated
45 min after Triton
Cholesterol Triglyceride Phospholipid
and control rats
90 min after Triton
Bezafibrate-treated
Controls
Bezatibrate-treated
Controls
7.1 f 1.3 77.8 f 3.5 14.4 f 2.1
7.4 + 1.1 77.1 f 2.8 15.5 f 1.6
4.9 k 0.6 84.5 +- 1.6 10.7 f 1.0
5.9 + 0.7 81.6 + 1.5 12.5 f 0.8
Mean + SE of 8 rats in each group. Plasma levels of three lipids were measured in samples taken before, at 45 and 90 min after Triton injection. Since the removal of VLDL-triglyceride was demonstrated to be completely blocked at least for 90 min after Triton injection, and since more than 90% of triglyceride in plasma taken 90 min after Triton injection were recovered in VLDL fraction after ultracentrifugal separation of plasma lipoproteins, it seems reasonable to infer that increments of lipid concentrations during unit time periods are those of VLDL-lipids. Therefore, we calculated the lipid composition in VLDL using increments of triglyceride, cholesterol and phospholipid during 45-min and 90-min periods, respectively.
rats but also in bezafibrate-treated animals (Fig. 3). Although plasma triglyceride levels at all three time points were significantly lower (P < 0.05, respectively) in bezafibrate-treated rats than in controls, there were no significant differences in the increments of plasma triglyceride between the two groups. Plasma triglyceride fell by 5 1% from 70 k 15 mg/dl to 34 k 6 mg/dl (Fig. 4). Despite this, there was no change in the rate of triglyceride secretion between bezaflbratetreated and control rats (Fig. 4). However, the production rate tended to be lower in the bezafibrate-treated rats (1113 k 58 pg/min) than in controls (1234 + 63 pg/min). Furthermore, bezafibrate produced no change in lipid composition of VLDL (Table 2) as well as lipid mass of VLDL (421 + 33 and 393 + 20 mg/dl in bezafibratetreated and control rats), which was the sum of the increments of the three lipids during the first 45 min after Triton injection.
Discussion The present study has revealed that bezatibrate given as 30 mg per kg of body weight daily for 14 days had no signiticant effects on the rate of triglyceride secretion in the rat, despite a 50 % reduc-
tion in plasma triglyceride concentrations. Plasma triglyceride concentrations are the reflection of the balance between the secretion into and the removal from the circulation. In order to see the rate of triglyceride secretion, we completely blocked the triglyceride removal in the present study. Therefore, we could not directly measure the removal rate in the same rat. However, since we measured initial plasma triglyceride levels as well in each rat, we could make inferences about the rates of triglyceride removal, assuming that the volume of distribution did not change. The current results suggest that bezafIbrate stimulates the removal of VLDL-triglyceride from the circulation and this is consistent with the previous observations that the fibric acid derivative reduced the VLDL-triglyceride concentration by stimulating lipoprotein lipase activities [ l-31. They, however, were not in accordance with a recent study [4] using human hepatocytes that incubation in the presence of bezafibrate produced a decrease in both total VLDL neutral lipids and apolipoprotein B secreted into the medium. Although the reasons for the discrepancy between our in vivo and the in vitro study are not clear, the most striking difference between the two is in the time period when hepatocytes were exposed to bezaflbrate: 14 days in the current in vivo study and 18 hours in the in
167
vitro study. However, the present study does not exclude the possibility that fibric acid reduces the production rate of VLDL-triglyceride, since the production rate in bezatibrate-treated rats tended to be lower than that of controls in the present study. We have previously shown in hypertriglyceridemic patients that bezafibrate decreased plasma VLDL concentrations without modifying its structure [ 131. This may be consistent with the present finding that the fibrate produced no significant changes in lipid composition of VLDL which was calculated using increments of triglyceride, cholesterol and phospholipid after Triton. Since the removal of VLDLtriglyceride was demonstrated to be completely blocked for at least 90 min after Triton injection, and since more than 90% of the triglycerides in plasma taken 90 min after the Triton injection were recovered in the VLDL fraction, after ultracentrifugal separation of plasma lipoproteins, it seems reasonable to infer that the increments in lipid concentration during unit time periods are those of the VLDL-lipids, although they are likely to represent an accumulation of different classes of lipoproteins. In patients with type III hyperlipoproteinemia, therapy with bezafibrate not only increased the fractional clearance of VLDL apolipoprotein B (flotation constant of 60 to 400) but reduced the production of this fraction of VLDL apolipoprotein B [ 141. In a study in hypertriglyceridemic subjects by Shepherd et al. [ 151, bezafibrate therapy produced a decrease in VLDL apolipoprotein B levels associated with an increase in the fractional clearance rate of VLDL apolipoprotein B (flotation constant of 100 to 400). A reduction in the rate of VLDL apolipoprotein B production was found in four out of six patients, although the difference between baseline and post-treatment values was not statistically significant. There were, as far as we know, no data available concerning the effects of bezaflbrate on VLDL production in experimental hyperlipoproteinemia. In the present study, treatment with bezafibrate for 14 days resulted in a decrease in HDLcholesterol in the rat. The same decrease has been
shown in normal rats [ 161. In humans, bezafibrate raised HDL-cholesterol not only in hyperlipoproteinemics [l-3,14,15] but also in normal subjects [ 171. The reason for the discrepancy in the effects of bezalibrate on the HDL-cholesterol concentrations between humans and rats is obscure and we have no explanation for this matter.
Acknowledgement The authors are indebted to Kissei Pharmaceutical Co. for their kind supply of bezatibrate.
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