行政院國家科學委員會專題研究計畫成果報告
Sor bitol 在糖尿病老鼠膀胱病變所扮演之角色
The Role of Sor bitol in Diabetic Cystopathy of the Rat
NSC89-2314-B-006-143
執行期限: 2000-8-1 至 2001-7-31
主持人：唐一清 Yat-Ching Tong
國立成功大學
醫學系
泌尿科
Depar tment of Ur ology, National Cheng Kung Univer sity
Medical College, Tainan
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Abstr act
This study investigated the role of sorbitol, a metabolic product of glucose, in the pathogenesis of rat diabetic cystopathy. Three-month-old male Wistar rats were
divided into 4 groups: 1)normal controls; 2) rats rendered diabetic by streptozotocin; 3) rats fed with glucose; and 4) rats injected with sorbitol. The M2 muscarinic receptor (M2-mAChR) protein and mRNA densities of the bladder tissue were measured by Western immunoblotting and Northern blotting respectively. The densities of M2-mAChR protein and mRNA in the bladder tissue were significantly increased in diabetic rats, and rats given either glucose or sorbitol (increases in receptor protein: 27.3 ± 3.3 %, 19.8 ± 2.3 % and 18.0 ± 2.1 %; increases in mRNA: 39.6 ± 3.7 %, 33.1 ± 2.9% and 20.2 ± 2.2% respectively). The findings suggest that sorbitol plays a role in the pathogenesis of diabetic cystopathy in rats rendered diabetic by streptozotocin. Keywords: Diabetes mellitus, urinary bladder, autonomic nerves, muscarinic
receptors.
摘要
本研究主要目的探討 sorbitol 在老鼠糖尿病性膀胱病變之角色。三月大之雄 性 Wistar 大白鼠分成四組: (1) 正常對照組; (2) 兩週糖尿病組。糖尿病乃以 streptozotocin 60mg/kg i.v. 誘發；(3) 餵食 glucose 組; (4) 注射 sorbitol 組。膀胱 中 M2-副交感神經受體 protein 含量以 Western immuno-bolting 測定；M2-副交 感神經受體 m-RNA 以 Northern bolting 測定。結果發現兩者在後三組皆有明顯 上升，M2-副交感神經受體 protein 分別上升: 27.3 ± 3.3 %, 19.8 ± 2.3 % and 18.0 ± 2.1 %; M2-副交感神經受體 mRNA 分別上升: 39.6 ± 3.7 %, 33.1 ± 2.9% and 20.2 ± 2.2% (p<0.05, n=8)。因此可見糖尿病性膀胱病變時 sorbitol 扮演之可能角色。 Keywords: 糖尿病, 膀胱, 副交感神經, 神經受體
緣由與目的
Since Ruth van Heyningen [1959] first demonstrated an excess of sorbitol in the lenses of diabetic rats, substantial accumulation of evidence has supported the role of the sorbitol pathway as a major contributor in some late complications of diabetes [Frank, 1994]. Sorbitol accumulation in peripheral nerves accompanied with impaired nerve conduction has been demonstrated in diabetic animals [Gabbay et al., 1972]. Both diabetic cataract and diabetic neuropathy can be delayed or partially corrected by inhibitors of aldose reductase, the initial enzyme reducing glucose to sorbitol [Kinoshita, 1974; Sarges et al., 1993]. Diabetes induced by STZ treatment in rats causes profound morphological, biochemical, and pharmacological changes in the urinary bladder. Increase in the density of diabetic bladder muscarinic receptors has been reported [Latipour et al., 1989]. We recently demonstrated up-regulations of both bladder M2 receptor (M2-mAChR) protein and mRNA in 2-week STZ-induced
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role of sorbitol in the pathogenesis of diabetic cystopathy using the alterations in M2 receptor protein and mRNA as indicators.
方法
Three-month-old male Wistar rats were kept in a temperature controlled room (25oC) with a 12-h to 12-h dark and light cycle. The rats were divided into 4 groups: 1)normal controls; 2) rats fed with glucose 3 g/kg 4 times/day for 7 days; 3) rats intraperitoneally injected with sorbitol 12 g/kg/day for 7 days; and 4) rats rendered diabetic by streptozotocin (STZ). Diabetes was induced by single intravenous
injection of 60mg/kg STZ dissolved in 0.1M citrate buffer (pH4.5) via the lateral tail vein. The control rats were injected with the same volume of citrate buffer. Overnight 8-hour fasting blood glucose levels were determined 72 h after STZ administration to confirm the presence of diabetes (blood glucose>300mg/dl). The amount of M2 receptor protein and mRNA in the bladder tissue of the four groups of rats were measured and compared by Western immunoblotting and Northern blotting
techniques respectively. The results were expressed as the mean ±standard error of the mean (SEM). Statistical analysis was performed using analysis of variance followed by the Students t test for comparisons between two groups. A probability level of <0.05 was required for statistical significance.
結果與討論
When compared to the control, the Western blotting for urinary bladder
M2-mAChR protein density was significantly increased in the STZ-induced diabetic rat, as well as in rats given either glucose or sorbitol. Bladder M2-mAChR protein increased 27.3 ±3.3 %, 19.8 ± 2.3 % and 18.0 ± 2.1 % respectively in the latter three groups of rats (n=8, p<0.05 for each comparison to the control group). However, no significant differences were noted among these three groups. The Northern blotting for urinary bladder M2-mAChR mRNA density was also significantly increased in the STZ-induced diabetic rat, as well as in rats administered with either glucose or
sorbitol (39.6 ± 3.7 %, 33.1 ± 2.9% and 20.2 ± 2.2% increments respectively, n=8, p<0.05 for each comparison to the control). The present study in the rat provides evidence for the role of sorbitol in the pathogenesis of diabetic cystopathy. The polyol pathway is one of the possible biochemical mechanisms by which diabetes could impair the function and structure of organ systems affected by diabetic complications. Hypotheses of the pathogenesis include the polyol osmotic theory, alteration in myo-inositol and sodium metabolism, intermediary metabolites, abnormal changes of the redox state and an abnormality of kinase C dependent protein phosphorylation. Recently, increasing evidence suggests that glycation and oxidative stress may have a cross-link with polyol pathway, contributing to the development of diabetic
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hyperactivity has an important role in the etiology of late-onset diabetic complications, the inhibition of aldose reductase, the rate-limiting enzyme of the pathway, can be utilized in the treatment and prevention of diabetic complications. From the results obtained mainly in animal models of diabetes, it is well recognized that aldose reductase inhibitors have a positive inhibitory effect on neuropathy, retinopathy, nephropathy, keratopathy, cataract-formation, possibly infection and atherosclerosis [Frank, 1994; Hotta, 1997]. The possible beneficial effect of aldose reductase inhibitors in preventing diabetic cystopathy will be the research theme of our further effort.
成果自評
Our study results have provided evidence for the role of sorbitol in the pathogenesis of diabetic cystopathy. This will open the development of new pharmacological treatment for this disease.
References
1. Frank RN. 1994. The aldose reductase controversy. Diabetes 43:169-72.
2. Gabbay KH, Snider JJ. 1972. Nerve conduction defect in galactose-fed rats. Diabetes 21:295-300.
3. Hotta N. 1997. New concepts and insights on pathogenesis and treatment of diabetic complications: polyol pathway and its inhibition. Nagoga J Med Sci 60: 89-100.
4. Kinoshita JH. 1974. Mechanisms initiating cataract formation. Invest. Ophthalmol Vis Sci 13:713-24.
5. Latifpour J, Gousse A, Kondo S, Morita T, Weiss RM. 1989. Effects of experimental diabetes on biochemical and functional characteristics of bladder muscarinic receptors, J Pharmacol Exp Ther 248: 81-8.
6. Sarges R, Oates PJ. 1993. Aldose reductase inhibitors: recent developments. Progr Drug Res 40:99-161
7. Tong YC, Chin WT, Cheng JT. 1999. Alterations in M2-muscarinic receptor protein and mRNA in diabetic rat urinary bladders. Neurosci Lett 277: 173-6. 8. Van Heyningen R. 1959. Formation of polyols by the lens of the rat with sugar
