- Email: [email protected]
Protective effect of probucol on alloxan diabetes in rats
Diabetes Research and Clinical Practice, I (1989) 3 13-3 16 Elsevier
313
DIABET 00323
Protective effect of probucol on alloxan diabetes in rats Masayuki Matsushita, Gen Yoshino, Masahide Iwai, Kohji Matsuba, Munetaka Morita, Ippei Iwatani, Muneyoshi Yoshida ‘, Tsutomu Kazumi ’ and Shigeaki Baba Second Department of Internal Medicine, Kobe UniversitySchool of Medicine, Chuo-ku, Kobe 6S0, Japan and ‘Divirion of Endocrinology and Metabolism. Hyogo Medical Center for Adults, Kitaohji-cho,Akashi 673, Japan (Received 3 April 1989) (Revision received 1 June 1989) (Accepted 15 June 1989)
Summary The diabetogenic action of alloxan is known to be attenuated by several oxygen radical scavengers. The present study was conducted to see if probucol, a drug with strong free radical scavenger action, can reduce pancreatic B-cell damage induced by alloxan in male Wistar rats. After 2 weeks of a 1 y0 probucol diet, 50 mg/kg alloxan was intravenously injected in rats (group PA, n = 34). Urine glucose of most of the injected rats not pretreated with probucol (group A, n = 22) was positive, while more than half of the rats of group PA failed to show urine glucose. The blood glucose level in group PA was significantly lower than that in group A (326 + 25 vs. 487 + 28 mg/dl, P < 0.001). Histological examination revealed that most of the pancreatic islets of group A were degranulated, whereas a lot of islets remained unaffected in group PA. Thus, the in vivo diabetogenic action of alloxan was reduced by pretreatment with probucol, although the effect was incomplete. This effect can be explained by probucol’s strong free radical scavenger action. Since accumulation of free radicals can be an initial step of B-cell damage in animal models of type 1 (insulin-dependent) diabetes, the drug can be useful for the prevention of type 1 diabetes with its long-term clinical history of safety. Key words: Probucol;
Alloxan diabetes;
Free radical; Pancreatic
Introduction Alloxan induces diabetes mellitus in experimental animals by destroying pancreatic B-cells [ 11. The precise mechanism by which the drug exerts its
Address for correspondence: Gen Yoshino, MD, Second Department of Internal Medicine, Kobe University School of Medicine. Kusunoki-cho. 7-chome. Chuo-ku. Kobe 650. Japan. 0168-8227/89/$03.50
0 1989 Elsevier Science Publishers
islet
effect is still controversial. However, several investigators have reported that the initial event in alloxan-induced B-cell damage is the generation of active oxygen species : superoxide radicals, hydrogen peroxide and hydroxy radicals [ 21. On the other hand, probucol, which is an established hypocholesterolemic drug [ 31, has been shown to possess potent antioxidant properties [4]. Therefore it is possible that probucol can protect against pancreatic B-cell damage induced by
B.V. (Biomedical
Division)
314
alloxan in experimental animals. Thus, the present study was conducted to see if pretreatment with probucol can prevent the development of alloxan diabetes in rats.
Materials and methods Male Wistar rats (Shizuoka Experimental Animal Farm Co., Shizuoka, Japan) weighing 180-200 g were used in this study. Four experimental groups were prepared; pretreatment with probucol plus alloxan injection (group PA), alloxan injection without pretreatment with probucol (group A), pretreatment with probucol plus vehicle injection (group P) and vehicle injection without pretreatment (control, group C). The rats of group A and C were fed standard powder diet (Oriental M, Oriental Yeast Co., Tokyo). The rats of group P and PA were fed the same diet containing 1 y0 probucol. After 2 weeks of each diet, alloxan monohydrate (Sigma, St. Louis, MO) dissolved in sterile phosphate-buffered saline (50 mg/ml/kg) was injected into the tail vein under light ether anesthesia (groups A and PA) without fasting. The same amount of vehicle alone was injected into the rats of groups P and C. Twenty-four hours after the injection, blood was taken from the tail vein of conscious, non-fasted rats for the
measurement of blood glucose. Urine glucose was also examined. Two weeks later, animals from each group (n = 3-4) were killed under pentobarbital anesthesia and samples of pancreatic tissue were collected for light microscopy. Tissue sections of the pancreas were stained with aldehyde-fuchsin [ 51. Statistical analyses were performed by analysis of variance and then P values were calculated by Bonferroni’s multiple comparison procedure [6] or by chi-square test.
Results Body weight gain and chow intake of the four experimental groups were all identical during the 2-week pretreatment period (Table 1). Body weight gain after alloxan injection in group A was significantly lower than in the other three groups. Urine glucose was positive in 21 of 22 rats in group A, whereas half of the rats in group PA failed to show urine glucose. The blood glucose level in group PA was signif&tntly lower than that in group A. Furthermore, the mean blood glucose level of the rats in group PA without urine glucose (214 + 12 mg/dl, n = 17) was much lower than that in group A. The difference was highly signifrcant (P < 0.001). On histological examination, most of the islets
TABLE 1 Body weight (BW) gain, urine glucose and blood glucose levels of the four experimental
Group Group Group Group
A (n = 22) PA (n = 34) P (n = 12) C (n = 8)
ABW”
ABWb
(8)
(g)
16 + 16* 79 f 76 f
2 1 5 5
61 85 87 82
groups
Urine glucose
Blood glucose (mg/dl)
f + f f
6* 2 3 2
negative
positive
1 17** 12 8
21 17 0 0
Results are expressed as means + SE. a Body weight gain (2 weeks) before alloxan or vehicle injection. b Body weight gain (2 weeks) after alloxan or vehicle injection. * Significantly lower than the other three groups (P < 0.01) by multiple comparison procedure [6]. ** Significantly higher than the group A value (P < 0.01) by chi-square test. *** Significantly lower than the group A value (P < 0.001) by multiple comparison procedure [6].
481 + 326 f 184+ 188k
28 25*** 7 7
315
Fig. 1. Photomicrograph
of rat pancreatic islets of group PA (left panel) and group A (middle panel). A normal control islet is shown in the right panel. Aldehyde-fkhsin staining [S]. x 25.
of group A rats proved to be small in size and showed remarkable degranulation (Fig. 1, middle panel). On the other hand, in group PA a lot of islets remained unaffected (Fig. 1, left panel).
Discussion It is evident from the present data that the in vivo diabetogenic action of alloxan can be reduced by pretreatment with probucol, although the effect was incomplete. The precise mechanisms by which probucol reduces the B-cell toxicity induced by alloxan are still unclear. However, the most likely explanation is that probucol, a strong free radical scavenger [4], prevents free radical accumulation in the B-cells induced by alloxan [2], thus exerting its protective effect against B-cell destruction. It is generally accepted that free radical toxicity can be an initial step in pancreatic B-cell destruction in spontaneously diabetic animals [ 71, a model of human type 1 (insulin-dependent) diabetes. Therefore, a strong radical scavenger can be a hopeful drug for the prevention of type 1 diabetes. Drash et al. [8] reported that probucol can lower the frequency and slow the rate of develop-
ment of diabetes in BB rats. Thus, our present findings support their proposal that the drug may be an alternative to cyclosporin A [9] for the prevention of type 1 diabetes. Since probucol, unlike cyclosporin A, is safe and an established hypocholesterolemic drug [3] with unique antiatherogenic potential [4], the long-term administration of probucol may prevent not only the development of macroangiopathy but also further B-cell damage in diabetic patients with hypercholesterolemia. Although an extremely high dose (1% of stock diet) of probucol was administered in rats compared to the clinical dose, the plasma level of probucol was much lower in rats than in humans (3.9 + 0.2 vs. 11.9 f 1.4 pg/ml; the latter was from unpublished data of Otsuka Pharm. Co., Tokyo, where 750 mg/day of probucol was administered to healthy volunteers for 10 days). This may also justify the clinical application of probuco1 for the prevention of type 1 diabetes.
Acknowledgement We are indebted to Otsuka Pharm. Co., Tokyo, for providing the probucol.
316
References Rerup, C. C. (1970) Drugs producing diabetes through damage of the insulin secreting cells. Pharmacol. Rev. 22, 485-518. Deamer, D. W., Heikkila, R. E., Panganamala, R. V., Cohen, G. and Corwell, D. G. (1971) The alloxan-dialuric acid cycle and generation of hydrogen peroxide. Physiol. Chem. Phys. 3.426-430. Miettinen, T. A. (1972) Mode of action of a new hypercholesterolemic drug (DH-581) in familial hypercholesterolemia. Atherosclerosis 15, 163-176. Parthasarathy, S., Young, S. G., Witztum, J., Pittman, R. C. and Steinberg, D. (1986) Probucol inhibits oxidative
modification of low density lipoprotein. J. Clin. Invest. 77, 641-644. Gomori, G. (1950) Aldehyde-fuchsin: new stain for elastic tissue. Am. J. Clin. Pathol. 20, 665-666. Granz, S. A. (1981) Primer of Biostatistics. McGraw- Hill, New York, NY. Sandler, S., Anderson, A. and Hellerstrbm, C. (1987) Inhibitory effects of interleukin I on insulin secretion, insulin biosynthesis and oxidative metabolism of isolated rat pancreatic islets. Endocrinology 121, 1424-1431. Drash, A. L., Rudert, W. A., Borquaye, S., Wang, R. and Lieberman, I. (1988) Effect of probucol on development of diabetes mellitus in BB rats. Am. J. Cardiol. 62,27B-30B. Rossini, A. A. (1988) Immunotherapy for insulin-dependent diabetics? N. Engl. J. Med. 308, 333-334.
313
DIABET 00323
Protective effect of probucol on alloxan diabetes in rats Masayuki Matsushita, Gen Yoshino, Masahide Iwai, Kohji Matsuba, Munetaka Morita, Ippei Iwatani, Muneyoshi Yoshida ‘, Tsutomu Kazumi ’ and Shigeaki Baba Second Department of Internal Medicine, Kobe UniversitySchool of Medicine, Chuo-ku, Kobe 6S0, Japan and ‘Divirion of Endocrinology and Metabolism. Hyogo Medical Center for Adults, Kitaohji-cho,Akashi 673, Japan (Received 3 April 1989) (Revision received 1 June 1989) (Accepted 15 June 1989)
Summary The diabetogenic action of alloxan is known to be attenuated by several oxygen radical scavengers. The present study was conducted to see if probucol, a drug with strong free radical scavenger action, can reduce pancreatic B-cell damage induced by alloxan in male Wistar rats. After 2 weeks of a 1 y0 probucol diet, 50 mg/kg alloxan was intravenously injected in rats (group PA, n = 34). Urine glucose of most of the injected rats not pretreated with probucol (group A, n = 22) was positive, while more than half of the rats of group PA failed to show urine glucose. The blood glucose level in group PA was significantly lower than that in group A (326 + 25 vs. 487 + 28 mg/dl, P < 0.001). Histological examination revealed that most of the pancreatic islets of group A were degranulated, whereas a lot of islets remained unaffected in group PA. Thus, the in vivo diabetogenic action of alloxan was reduced by pretreatment with probucol, although the effect was incomplete. This effect can be explained by probucol’s strong free radical scavenger action. Since accumulation of free radicals can be an initial step of B-cell damage in animal models of type 1 (insulin-dependent) diabetes, the drug can be useful for the prevention of type 1 diabetes with its long-term clinical history of safety. Key words: Probucol;
Alloxan diabetes;
Free radical; Pancreatic
Introduction Alloxan induces diabetes mellitus in experimental animals by destroying pancreatic B-cells [ 11. The precise mechanism by which the drug exerts its
Address for correspondence: Gen Yoshino, MD, Second Department of Internal Medicine, Kobe University School of Medicine. Kusunoki-cho. 7-chome. Chuo-ku. Kobe 650. Japan. 0168-8227/89/$03.50
0 1989 Elsevier Science Publishers
islet
effect is still controversial. However, several investigators have reported that the initial event in alloxan-induced B-cell damage is the generation of active oxygen species : superoxide radicals, hydrogen peroxide and hydroxy radicals [ 21. On the other hand, probucol, which is an established hypocholesterolemic drug [ 31, has been shown to possess potent antioxidant properties [4]. Therefore it is possible that probucol can protect against pancreatic B-cell damage induced by
B.V. (Biomedical
Division)
314
alloxan in experimental animals. Thus, the present study was conducted to see if pretreatment with probucol can prevent the development of alloxan diabetes in rats.
Materials and methods Male Wistar rats (Shizuoka Experimental Animal Farm Co., Shizuoka, Japan) weighing 180-200 g were used in this study. Four experimental groups were prepared; pretreatment with probucol plus alloxan injection (group PA), alloxan injection without pretreatment with probucol (group A), pretreatment with probucol plus vehicle injection (group P) and vehicle injection without pretreatment (control, group C). The rats of group A and C were fed standard powder diet (Oriental M, Oriental Yeast Co., Tokyo). The rats of group P and PA were fed the same diet containing 1 y0 probucol. After 2 weeks of each diet, alloxan monohydrate (Sigma, St. Louis, MO) dissolved in sterile phosphate-buffered saline (50 mg/ml/kg) was injected into the tail vein under light ether anesthesia (groups A and PA) without fasting. The same amount of vehicle alone was injected into the rats of groups P and C. Twenty-four hours after the injection, blood was taken from the tail vein of conscious, non-fasted rats for the
measurement of blood glucose. Urine glucose was also examined. Two weeks later, animals from each group (n = 3-4) were killed under pentobarbital anesthesia and samples of pancreatic tissue were collected for light microscopy. Tissue sections of the pancreas were stained with aldehyde-fuchsin [ 51. Statistical analyses were performed by analysis of variance and then P values were calculated by Bonferroni’s multiple comparison procedure [6] or by chi-square test.
Results Body weight gain and chow intake of the four experimental groups were all identical during the 2-week pretreatment period (Table 1). Body weight gain after alloxan injection in group A was significantly lower than in the other three groups. Urine glucose was positive in 21 of 22 rats in group A, whereas half of the rats in group PA failed to show urine glucose. The blood glucose level in group PA was signif&tntly lower than that in group A. Furthermore, the mean blood glucose level of the rats in group PA without urine glucose (214 + 12 mg/dl, n = 17) was much lower than that in group A. The difference was highly signifrcant (P < 0.001). On histological examination, most of the islets
TABLE 1 Body weight (BW) gain, urine glucose and blood glucose levels of the four experimental
Group Group Group Group
A (n = 22) PA (n = 34) P (n = 12) C (n = 8)
ABW”
ABWb
(8)
(g)
16 + 16* 79 f 76 f
2 1 5 5
61 85 87 82
groups
Urine glucose
Blood glucose (mg/dl)
f + f f
6* 2 3 2
negative
positive
1 17** 12 8
21 17 0 0
Results are expressed as means + SE. a Body weight gain (2 weeks) before alloxan or vehicle injection. b Body weight gain (2 weeks) after alloxan or vehicle injection. * Significantly lower than the other three groups (P < 0.01) by multiple comparison procedure [6]. ** Significantly higher than the group A value (P < 0.01) by chi-square test. *** Significantly lower than the group A value (P < 0.001) by multiple comparison procedure [6].
481 + 326 f 184+ 188k
28 25*** 7 7
315
Fig. 1. Photomicrograph
of rat pancreatic islets of group PA (left panel) and group A (middle panel). A normal control islet is shown in the right panel. Aldehyde-fkhsin staining [S]. x 25.
of group A rats proved to be small in size and showed remarkable degranulation (Fig. 1, middle panel). On the other hand, in group PA a lot of islets remained unaffected (Fig. 1, left panel).
Discussion It is evident from the present data that the in vivo diabetogenic action of alloxan can be reduced by pretreatment with probucol, although the effect was incomplete. The precise mechanisms by which probucol reduces the B-cell toxicity induced by alloxan are still unclear. However, the most likely explanation is that probucol, a strong free radical scavenger [4], prevents free radical accumulation in the B-cells induced by alloxan [2], thus exerting its protective effect against B-cell destruction. It is generally accepted that free radical toxicity can be an initial step in pancreatic B-cell destruction in spontaneously diabetic animals [ 71, a model of human type 1 (insulin-dependent) diabetes. Therefore, a strong radical scavenger can be a hopeful drug for the prevention of type 1 diabetes. Drash et al. [8] reported that probucol can lower the frequency and slow the rate of develop-
ment of diabetes in BB rats. Thus, our present findings support their proposal that the drug may be an alternative to cyclosporin A [9] for the prevention of type 1 diabetes. Since probucol, unlike cyclosporin A, is safe and an established hypocholesterolemic drug [3] with unique antiatherogenic potential [4], the long-term administration of probucol may prevent not only the development of macroangiopathy but also further B-cell damage in diabetic patients with hypercholesterolemia. Although an extremely high dose (1% of stock diet) of probucol was administered in rats compared to the clinical dose, the plasma level of probucol was much lower in rats than in humans (3.9 + 0.2 vs. 11.9 f 1.4 pg/ml; the latter was from unpublished data of Otsuka Pharm. Co., Tokyo, where 750 mg/day of probucol was administered to healthy volunteers for 10 days). This may also justify the clinical application of probuco1 for the prevention of type 1 diabetes.
Acknowledgement We are indebted to Otsuka Pharm. Co., Tokyo, for providing the probucol.
316
References Rerup, C. C. (1970) Drugs producing diabetes through damage of the insulin secreting cells. Pharmacol. Rev. 22, 485-518. Deamer, D. W., Heikkila, R. E., Panganamala, R. V., Cohen, G. and Corwell, D. G. (1971) The alloxan-dialuric acid cycle and generation of hydrogen peroxide. Physiol. Chem. Phys. 3.426-430. Miettinen, T. A. (1972) Mode of action of a new hypercholesterolemic drug (DH-581) in familial hypercholesterolemia. Atherosclerosis 15, 163-176. Parthasarathy, S., Young, S. G., Witztum, J., Pittman, R. C. and Steinberg, D. (1986) Probucol inhibits oxidative
modification of low density lipoprotein. J. Clin. Invest. 77, 641-644. Gomori, G. (1950) Aldehyde-fuchsin: new stain for elastic tissue. Am. J. Clin. Pathol. 20, 665-666. Granz, S. A. (1981) Primer of Biostatistics. McGraw- Hill, New York, NY. Sandler, S., Anderson, A. and Hellerstrbm, C. (1987) Inhibitory effects of interleukin I on insulin secretion, insulin biosynthesis and oxidative metabolism of isolated rat pancreatic islets. Endocrinology 121, 1424-1431. Drash, A. L., Rudert, W. A., Borquaye, S., Wang, R. and Lieberman, I. (1988) Effect of probucol on development of diabetes mellitus in BB rats. Am. J. Cardiol. 62,27B-30B. Rossini, A. A. (1988) Immunotherapy for insulin-dependent diabetics? N. Engl. J. Med. 308, 333-334.