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Effects of melatonin on genetic hypercholesterolemia in rats
Atherosclerosis, 69 (1988) 269-212 Elsevier Scientific Publishers Ireland,
269 Ltd.
ATH 04066
Effects of melatonin on genetic hypercholesterolemia Hiromu Aoyama ‘, Natsuko Mori 2 and Wataru Mori
in rats
1
I Department of Pathology, and ’ Third Department of Internal Medicine, Faculty of Medicine,University of Tokyo, Tokyo (Japan) (Revised,
(Received 13 March, 1987) received 17 August and 28 September, (Accepted 28 September, 1987)
1987)
Summary
Administration of the pineal hormone melatonin to genetically hypercholesterolemic rats resulted in a decrease in plasma cholesterol levels and in an improvement of fatty changes of the liver. Thus, the antihyperlipemic effect of melatonin, which was first discovered in hypercholesterolemia produced by short- or long-term administration of glucocorticoids, has now been proved to be rather universal and not simply anti-glucocorticoidal. The mechanism of the decrease of plasma cholesterol levels remains unknown. It was also found that the pathogenesis of this so-called genetic hypercholesterolemia in rats involved biochemical nephrotic changes and histopathological changes in the kidney.
Key words: Melatonin; Genetically hypercholesterolemic
Introduction
Melatonin was first isolated from the pineal gland in 1958 [l]. Our recent work has revealed that melatonin can protect rats from some of the injurious effects of glucocorticoids, such as hyperlipemia [2-41. Recently, we found that melatonin can also suppress the elevation of plasma cholesterol levels in dietary-produced hypercholesterolemic rats [5]. In order to find out whether the antihyper-
Correspondence to: Prof. W. Mori, Department thology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. 0021-9150/88/$03.50
0 1988 Elsevier Scientific
of PaHongo,
Publishers
Ireland,
rat; Antihypercholesterolemic
effect
lipemic effect of melatonin is a general phenomenon, we planned another experiment of similar nature but based on a different pathogenesis, and examined the influence of melatonin on hypercholesterolemia in genetically hypercholesterolemic rats. Materials and methods
Two 15-week-old genetically hypercholesterolemic male rats [6] of the F@ generation, which were originally separated from Sprague-Dawley JCL strain rats at the Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., were mated with female rats of the same age obtained from the same line of the same strain. Each pregnant female rat was isoLtd.
270 lated, allowed to deliver and to suckle her offspring for 3 weeks. In this subsequent generation (F,, ), a total of 14 weanling male rats were obtained, all of whom were healthy. These were used in the present experiment. The rats were kept in an animal house at constant temperature (22 k 2°C) and constant humidity (50 * 5%), but without a rigid lighting schedule (this experiment was carried out in winter). All the rats were maintained on an ordinary commercial diet (MF Oriental Yeast Co.). The animals were divided into 2 groups each containing 7 rats. Intraperitoneal injection of melatonin or saline solution was started at the beginning of the 3rd week after birth. Group I was the control; every morning, the rats were given an intraperitoneal injection of 2 ml saline solution. Rats of group II, the melatonin group, received an intraperitoneal injection of 4.0 mg melatonin daily in the morning. In this case, melatonin (N-acetyl5-methoxytryptamine; Sigma Chemical Co., U.S.A.) was first dissolved in ethanol (100 mg/ml), and this solution was diluted 50 times with physiological saline. Each rat received 2 ml of this melatonin solution intraperitoneally. Blood was collected from the tail vein at 1, 2, 3, and 4 months after birth and used for measuring total cholesterol in plasma. At the end of the experiment, 5 months after birth, all rats were anesthetized with sodium pentobarbital (50 mg/kg body weight), and blood was collected as completely as possible from the abdominal aorta. Plasma was separated, kept in tubes containing 1 mg EDTA per ml blood, and subjected to chemical analysis. Total cholesterol [7], triglyceride [8] and phospholipid [9] were measured enzymatically according to cited methods. Protein was measured by the method of Lowry et al. [lo] using bovine serum albumin as standard. Glucose concentration was determined with a FUJI DRI CHEM 2000 analyzer (Fuji Photo Film Co.) [ll]. After the animals had been killed by bleeding as described above, urine was collected from the urinary bladder, and protein, sugar and specific gravity were measured. All organs, including liver and kidneys, were preserved at autopsy and fixed in formalin solution for histological examination.
Results
No significant differences were found between the 2 groups with regard to food consumption, body weight or growth pattern. Fig. 1 shows the changes in plasma total cholesterol levels during the experiment. All the rats in both groups showed normal levels of total cholesterol until 2 months of age. After 3 months, however, the cholesterol level increased markedly in group I rats, while those in group II rats did not. At the age of 5 months (Table l), the increase of total cholesterol was reduced significantly by melatonin administration. The levels of triglyceride and phospholipid also showed some improvement, though not statistically significant, following melatonin injection. No significant differences were found between rats from groups 1 and II with regard to glucose, total protein and renal function tests. A marked elevation of protein was found in the urine of rats from both groups at the age of 5 months, but there was no significant difference between the 2 groups.
I
I
,,'
CONTROLGROUP
MELATONINGROUP
0
1
2
3
t
4
5
AGE (months) Fig. 1. Changes in the total cholesterol during the experiment.
level in plasma
of rats
271 TABLE 1 LABORATORYDATAONBLOODPLASMA Values are the mean f SD of 7 rats.
Total cholesterol (mg/dl) Triglyceride (mg/dl) Phospholipid (mg/dl) Total protein (g/dl) Albumin/globulin ratio Glucose (mg/dl) Blood urea nitrogen (mg/dl) Uric acid (mg/dl) Creatinine (mg/dl)
Group I
Group II
317 +74 221 *74 405 *70 6.4 f 0.3 0.46* 0.05 219 zt32 49.5 f 14.0 1.3 f 0.3 1.1 f 0.3
207 f 39 * 172 *lo2 312 f 65 6.1 f 0.2 0.53* 0.07 249 f 87 37.3 + 3.3 1.5 + 1.5 1.0 f 0.1
* Statistically significant protective effect of melatonin (P < 0.01).
Fig. 3. Histopathology of the kidney. Hyalinization of the glomerular tuft, fibrous thickening of the mesa&urn and capsule, adhesions and partial obliteration of Bowman’s space of glomeruli are apparent. These changes were common in rats of both groups in this experiment (HE stain, X 200).
The livers of rats of group I showed severe and diffuse fatty metamorphosis, consisting histologically of micro- to macrodroplets of fat (Fig. 2a). However, in group II there was only a small number of microdroplets of fat, limited to the periportal areas of the liver (Fig. 2b). The histopathology of the kidneys was also interesting. We found epithelial cell hyperplasia resulting in thickening of Bowman’s capsule, adhesions and partial obliteration of Bowman’s space, marked fibrous mesangial thickening, marked thickening of the basement membrane, and hyalinization of the glomerular tuft, which were observed quite diffusely in the glomeruli. However, no marked differences were seen between the 2 groups (Fig. 3). No significant differences were found between the 2 groups with regard to other organs, including the lungs, heart, thymus, spleen and adrenals. Discussion
Fig. 2. (a) Histopathology of the liver of a rat in the control group. Note severe and diffuse fatty metamorphosis, with micro- to macrodroplets of fat (HE stain, ~75). (b) Histopathology of the liver of a rat in the melatonin group. Note microdroplet fatty metamorphosis of slight degree, limited to the periportal area (HE stain, x 75).
In our previous reports [2-41, we showed for the first time that melatonin has a preventive effect against the injuries caused by glucocorticoids, and we mentioned that melatonin could. protect rats from hyperlipemia. In order to confirm and extend this observation, we wished to examine whether melatonin has a general antihyperlipemic effect. Thus, we recently found that
272 melatonin could suppress the elevation of plasma total cholesterol levels caused by a high cholesterol diet [5]. In this study, we have examined the antihypercholesterolemic effect of melatonin in genetically hypercholesterolemic rats [6]. It was demonstrated that melatonin could ameliorate, partially but definitely, the increase in total cholesterol levels in the plasma of these genetically hypercholesterolemic rats. Similar effects on triglyceride and phospholipid levels have been seen, although the differences were not statistically significant. Moreover, melatonin greatly reduced the fatty development in the liver. These results are quite similar to those of a previous experiment in which dietary-induced hypercholesterolemic rats were adopted as the animal model [5]. Thus, it appears that the antihypercholesterolemic effect of melatonin is a general one. The mechanism of this effect has, however, not yet been elucidated, although it does appear to be quantitative, not qualitative [5]. Some adverse effects of melatonin have been reported, for example disturbance of daily rhythm, loss of appetite, and hormonal and metabolic changes. In a preliminary experiment we checked these points very carefully and confirmed that melatonin administration caused no changes in plasma lipids or in some hormones when rats were fed a normal diet. Thus, we consider that the data reported here are reliable, and that the antihypercholesterolemic effect of melatonin is a general phenomenon. However, further research still seems necessary to reach a final conclusion concerning the biological activities of melatonin. The mechanism of hypercholesterolemia in genetically hypercholesterolemic rats is not known. Nephrosis has been proposed as a possibility [6], and, in fact, we found very severe damage to the kidneys in the present experiment. However, melatonin was not able to protect renal function, either biochemically or histopathologically, possibly because of the severity of the renal damage. Acknowledgements
The authors wish to express their gratitude to Dr. T. Murase, Third Department of Internal
Medicine, University of Tokyo, for his advice and assistance during the present study. Thanks are also due to Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., for providing the genetically hypercholesterolemic rats used in this experiment. This study was supported in part by a grant-inaid from the Ministry of Education, Science and Culture, Japan. References 1 Lemer, A.B., Case, J.D., Takahashi, Y., Lee, T.H. and Mori, W., Isolation of melatonin, the pineal gland factor that lightens melanocytes, J. Am. Chem. Sot., 80 (1958) 2587. 2 Mori, W., Aoyama, H. and Mori, N., Melatonin protects rats from injurious effects of a glucocorticoid, dexamethasane, Jap. J. Exp. Med., 54 (1984) 255. 3 Aoyama, H., Mori, W. and Mori, N., Anti-glucocorticoid effects of melatonin in young rats. Acta Pathol. Jpn., 36 (1986) 423. 4 Aoyama, H., Mori, N. and Mori, W., Anti-glucocorticoid effects of melatonin on adult rats, Acta Pathol. Jpn., 37 (1987) 1143. 5 Mori, N., Aoyama, H., Murase, T. and Mori, W., Effects of melatonin on dietary hypercholesterolemic rats, (in preparation). 6 Imai, Y. and Matsumura, H., Genetic studies on induced and spontaneous hypercholesterolemia in rats, Atherosclerosis, 18 (1973) 59. I Allain, C.C., Pron, L.S., Chan, C.S.S., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. 8 Bucolo, G. and David, H., Quantitative determination of serum triglycerides by the use of enzymes, Clin. Chem., 19 (1973) 476. 9 Takayama, M., Ito, S., Nagasawa, T. and Tanimizu, I., A new method for determination of serum choline containing phospholipids, Clin. Chim. Acta, 79 (1977) 93. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 11 Ohkubo, A., Kamei, S., Yamanaka, M., Arai, F., Kitajima, M. and Kondo, A., PIasma glucose concentration of whole blood, as determined with a multilayer-film analytical element, Clin. Chem., 27 (1981) 1287.
269 Ltd.
ATH 04066
Effects of melatonin on genetic hypercholesterolemia Hiromu Aoyama ‘, Natsuko Mori 2 and Wataru Mori
in rats
1
I Department of Pathology, and ’ Third Department of Internal Medicine, Faculty of Medicine,University of Tokyo, Tokyo (Japan) (Revised,
(Received 13 March, 1987) received 17 August and 28 September, (Accepted 28 September, 1987)
1987)
Summary
Administration of the pineal hormone melatonin to genetically hypercholesterolemic rats resulted in a decrease in plasma cholesterol levels and in an improvement of fatty changes of the liver. Thus, the antihyperlipemic effect of melatonin, which was first discovered in hypercholesterolemia produced by short- or long-term administration of glucocorticoids, has now been proved to be rather universal and not simply anti-glucocorticoidal. The mechanism of the decrease of plasma cholesterol levels remains unknown. It was also found that the pathogenesis of this so-called genetic hypercholesterolemia in rats involved biochemical nephrotic changes and histopathological changes in the kidney.
Key words: Melatonin; Genetically hypercholesterolemic
Introduction
Melatonin was first isolated from the pineal gland in 1958 [l]. Our recent work has revealed that melatonin can protect rats from some of the injurious effects of glucocorticoids, such as hyperlipemia [2-41. Recently, we found that melatonin can also suppress the elevation of plasma cholesterol levels in dietary-produced hypercholesterolemic rats [5]. In order to find out whether the antihyper-
Correspondence to: Prof. W. Mori, Department thology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. 0021-9150/88/$03.50
0 1988 Elsevier Scientific
of PaHongo,
Publishers
Ireland,
rat; Antihypercholesterolemic
effect
lipemic effect of melatonin is a general phenomenon, we planned another experiment of similar nature but based on a different pathogenesis, and examined the influence of melatonin on hypercholesterolemia in genetically hypercholesterolemic rats. Materials and methods
Two 15-week-old genetically hypercholesterolemic male rats [6] of the F@ generation, which were originally separated from Sprague-Dawley JCL strain rats at the Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., were mated with female rats of the same age obtained from the same line of the same strain. Each pregnant female rat was isoLtd.
270 lated, allowed to deliver and to suckle her offspring for 3 weeks. In this subsequent generation (F,, ), a total of 14 weanling male rats were obtained, all of whom were healthy. These were used in the present experiment. The rats were kept in an animal house at constant temperature (22 k 2°C) and constant humidity (50 * 5%), but without a rigid lighting schedule (this experiment was carried out in winter). All the rats were maintained on an ordinary commercial diet (MF Oriental Yeast Co.). The animals were divided into 2 groups each containing 7 rats. Intraperitoneal injection of melatonin or saline solution was started at the beginning of the 3rd week after birth. Group I was the control; every morning, the rats were given an intraperitoneal injection of 2 ml saline solution. Rats of group II, the melatonin group, received an intraperitoneal injection of 4.0 mg melatonin daily in the morning. In this case, melatonin (N-acetyl5-methoxytryptamine; Sigma Chemical Co., U.S.A.) was first dissolved in ethanol (100 mg/ml), and this solution was diluted 50 times with physiological saline. Each rat received 2 ml of this melatonin solution intraperitoneally. Blood was collected from the tail vein at 1, 2, 3, and 4 months after birth and used for measuring total cholesterol in plasma. At the end of the experiment, 5 months after birth, all rats were anesthetized with sodium pentobarbital (50 mg/kg body weight), and blood was collected as completely as possible from the abdominal aorta. Plasma was separated, kept in tubes containing 1 mg EDTA per ml blood, and subjected to chemical analysis. Total cholesterol [7], triglyceride [8] and phospholipid [9] were measured enzymatically according to cited methods. Protein was measured by the method of Lowry et al. [lo] using bovine serum albumin as standard. Glucose concentration was determined with a FUJI DRI CHEM 2000 analyzer (Fuji Photo Film Co.) [ll]. After the animals had been killed by bleeding as described above, urine was collected from the urinary bladder, and protein, sugar and specific gravity were measured. All organs, including liver and kidneys, were preserved at autopsy and fixed in formalin solution for histological examination.
Results
No significant differences were found between the 2 groups with regard to food consumption, body weight or growth pattern. Fig. 1 shows the changes in plasma total cholesterol levels during the experiment. All the rats in both groups showed normal levels of total cholesterol until 2 months of age. After 3 months, however, the cholesterol level increased markedly in group I rats, while those in group II rats did not. At the age of 5 months (Table l), the increase of total cholesterol was reduced significantly by melatonin administration. The levels of triglyceride and phospholipid also showed some improvement, though not statistically significant, following melatonin injection. No significant differences were found between rats from groups 1 and II with regard to glucose, total protein and renal function tests. A marked elevation of protein was found in the urine of rats from both groups at the age of 5 months, but there was no significant difference between the 2 groups.
I
I
,,'
CONTROLGROUP
MELATONINGROUP
0
1
2
3
t
4
5
AGE (months) Fig. 1. Changes in the total cholesterol during the experiment.
level in plasma
of rats
271 TABLE 1 LABORATORYDATAONBLOODPLASMA Values are the mean f SD of 7 rats.
Total cholesterol (mg/dl) Triglyceride (mg/dl) Phospholipid (mg/dl) Total protein (g/dl) Albumin/globulin ratio Glucose (mg/dl) Blood urea nitrogen (mg/dl) Uric acid (mg/dl) Creatinine (mg/dl)
Group I
Group II
317 +74 221 *74 405 *70 6.4 f 0.3 0.46* 0.05 219 zt32 49.5 f 14.0 1.3 f 0.3 1.1 f 0.3
207 f 39 * 172 *lo2 312 f 65 6.1 f 0.2 0.53* 0.07 249 f 87 37.3 + 3.3 1.5 + 1.5 1.0 f 0.1
* Statistically significant protective effect of melatonin (P < 0.01).
Fig. 3. Histopathology of the kidney. Hyalinization of the glomerular tuft, fibrous thickening of the mesa&urn and capsule, adhesions and partial obliteration of Bowman’s space of glomeruli are apparent. These changes were common in rats of both groups in this experiment (HE stain, X 200).
The livers of rats of group I showed severe and diffuse fatty metamorphosis, consisting histologically of micro- to macrodroplets of fat (Fig. 2a). However, in group II there was only a small number of microdroplets of fat, limited to the periportal areas of the liver (Fig. 2b). The histopathology of the kidneys was also interesting. We found epithelial cell hyperplasia resulting in thickening of Bowman’s capsule, adhesions and partial obliteration of Bowman’s space, marked fibrous mesangial thickening, marked thickening of the basement membrane, and hyalinization of the glomerular tuft, which were observed quite diffusely in the glomeruli. However, no marked differences were seen between the 2 groups (Fig. 3). No significant differences were found between the 2 groups with regard to other organs, including the lungs, heart, thymus, spleen and adrenals. Discussion
Fig. 2. (a) Histopathology of the liver of a rat in the control group. Note severe and diffuse fatty metamorphosis, with micro- to macrodroplets of fat (HE stain, ~75). (b) Histopathology of the liver of a rat in the melatonin group. Note microdroplet fatty metamorphosis of slight degree, limited to the periportal area (HE stain, x 75).
In our previous reports [2-41, we showed for the first time that melatonin has a preventive effect against the injuries caused by glucocorticoids, and we mentioned that melatonin could. protect rats from hyperlipemia. In order to confirm and extend this observation, we wished to examine whether melatonin has a general antihyperlipemic effect. Thus, we recently found that
272 melatonin could suppress the elevation of plasma total cholesterol levels caused by a high cholesterol diet [5]. In this study, we have examined the antihypercholesterolemic effect of melatonin in genetically hypercholesterolemic rats [6]. It was demonstrated that melatonin could ameliorate, partially but definitely, the increase in total cholesterol levels in the plasma of these genetically hypercholesterolemic rats. Similar effects on triglyceride and phospholipid levels have been seen, although the differences were not statistically significant. Moreover, melatonin greatly reduced the fatty development in the liver. These results are quite similar to those of a previous experiment in which dietary-induced hypercholesterolemic rats were adopted as the animal model [5]. Thus, it appears that the antihypercholesterolemic effect of melatonin is a general one. The mechanism of this effect has, however, not yet been elucidated, although it does appear to be quantitative, not qualitative [5]. Some adverse effects of melatonin have been reported, for example disturbance of daily rhythm, loss of appetite, and hormonal and metabolic changes. In a preliminary experiment we checked these points very carefully and confirmed that melatonin administration caused no changes in plasma lipids or in some hormones when rats were fed a normal diet. Thus, we consider that the data reported here are reliable, and that the antihypercholesterolemic effect of melatonin is a general phenomenon. However, further research still seems necessary to reach a final conclusion concerning the biological activities of melatonin. The mechanism of hypercholesterolemia in genetically hypercholesterolemic rats is not known. Nephrosis has been proposed as a possibility [6], and, in fact, we found very severe damage to the kidneys in the present experiment. However, melatonin was not able to protect renal function, either biochemically or histopathologically, possibly because of the severity of the renal damage. Acknowledgements
The authors wish to express their gratitude to Dr. T. Murase, Third Department of Internal
Medicine, University of Tokyo, for his advice and assistance during the present study. Thanks are also due to Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., for providing the genetically hypercholesterolemic rats used in this experiment. This study was supported in part by a grant-inaid from the Ministry of Education, Science and Culture, Japan. References 1 Lemer, A.B., Case, J.D., Takahashi, Y., Lee, T.H. and Mori, W., Isolation of melatonin, the pineal gland factor that lightens melanocytes, J. Am. Chem. Sot., 80 (1958) 2587. 2 Mori, W., Aoyama, H. and Mori, N., Melatonin protects rats from injurious effects of a glucocorticoid, dexamethasane, Jap. J. Exp. Med., 54 (1984) 255. 3 Aoyama, H., Mori, W. and Mori, N., Anti-glucocorticoid effects of melatonin in young rats. Acta Pathol. Jpn., 36 (1986) 423. 4 Aoyama, H., Mori, N. and Mori, W., Anti-glucocorticoid effects of melatonin on adult rats, Acta Pathol. Jpn., 37 (1987) 1143. 5 Mori, N., Aoyama, H., Murase, T. and Mori, W., Effects of melatonin on dietary hypercholesterolemic rats, (in preparation). 6 Imai, Y. and Matsumura, H., Genetic studies on induced and spontaneous hypercholesterolemia in rats, Atherosclerosis, 18 (1973) 59. I Allain, C.C., Pron, L.S., Chan, C.S.S., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. 8 Bucolo, G. and David, H., Quantitative determination of serum triglycerides by the use of enzymes, Clin. Chem., 19 (1973) 476. 9 Takayama, M., Ito, S., Nagasawa, T. and Tanimizu, I., A new method for determination of serum choline containing phospholipids, Clin. Chim. Acta, 79 (1977) 93. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 11 Ohkubo, A., Kamei, S., Yamanaka, M., Arai, F., Kitajima, M. and Kondo, A., PIasma glucose concentration of whole blood, as determined with a multilayer-film analytical element, Clin. Chem., 27 (1981) 1287.