Thaliporphine 與Resveratrol 降血糖作用之研究

Abstract

近年來，糖尿病的病患有日益增加的趨勢，糖尿病也成為國人十大疾病之一。因此，為了有效控制血糖改善糖尿病症狀，因此分離或合成化學成分探討其降血糖作用為本論文之主要研究目標。本論文主要是探討白藜蘆醇 (resveratrol)、thaliporphine 及相關衍生物對正常及糖尿病大鼠之降血糖作用。由於thaliporphine被發現與血清素受體有很強結合能力，因此本論文更進一步探討血清素對大鼠血糖之調控作用及血清素受體亞型及其相關訊息路徑與thaliporphine降血糖作用之關係。 第一部份 Resveratrol在STZ誘發糖尿病大鼠降血糖作用與PI3-kinase訊息路徑之關係 白藜蘆醇 (resveratrol) 是一種多酚類結構的物質，存在於葡萄皮中，有研究報告指出紅酒對心臟血管有保護的作用。本論文利用正常及不同糖尿病模式包括STZ誘發之胰島素依賴型 (IDDM) 大鼠、Nicotinamide合併STZ誘發之非胰島素依賴型 (NIDDM) 糖尿病大鼠探討白黎蘆醇的血糖調控作用及其作用機制。研究發現口服給予白藜蘆醇在正常及NIDDM的大鼠可以產生劑量相關性的降血糖作用及增加胰島素的釋放。在第一型IDDM大鼠同樣口服給予白藜蘆醇後90分鐘可以產生有統計意義的降血糖作用。同時，利用外來的葡萄糖耐受性試驗 (oral glucose tolerance test) 顯示白藜蘆醇也可以增加正常大鼠對葡萄糖的利用率。口服給予白藜蘆醇產生降血糖的作用可以被PI3K的阻斷劑LY294002與Wortmannin所抑制。另外，以C2C12小鼠肌母細胞 (mouse myoblast cell line) 進行體外實驗發現白藜蘆醇亦能以劑量相關性增加C2C12小鼠肌母細胞對葡萄糖的吸回，此作用亦能被PI3K的阻斷劑LY294002與Wortmannin所抑制。在此阻斷劑的存在下可以抑制白藜蘆醇使IDDM糖尿病大鼠骨骼肌Akt磷酸化的作用。在第一型IDDM糖尿病大鼠以口服餵食白藜蘆醇3.0 mg/kg 每天三次治療7天發現可以改變骨骼肌葡萄糖轉運第四型蛋白質 (GLUT4) 及肝臟葡萄糖新生速率限制酵素 (PEPCK) 之表現量，白黎蘆醇可以增加GLUT4蛋白質的表現量並且使肝臟葡萄糖新生速率限制酵素 (PEPCK) 過度表現恢復正常。因此，從實驗結果發現，白藜蘆醇具降血糖作用，在非胰島素依賴型大鼠有促進胰島素釋放產生降血糖之作用，但在胰島素依賴型大鼠則可經由活化PI3K-Akt的訊息作用來增加葡萄糖的回收及利用而降血糖。 第二部份 血清素在STZ誘發糖尿病大鼠降血糖作用 血清素、血清素回收抑制劑和血清素的前趨物質，都會對人類或嚙齒動物產生降血糖作用，但血清素在胰島素依賴型的糖尿病大鼠是否有降低血糖的作用一直不是很清楚。本篇論文主要是探討血清素對此糖尿病大鼠血糖調節作用。評估血糖與胰島素、腦內啡和腎上腺素分泌量。血清素對STZ誘發糖尿病大鼠產生降血糖作用但沒有改變血漿中胰島素和腎上腺素的濃度，但是會增加血液中腦內啡的濃度。以腹腔注射方式給予血清素0.3 mg/kg 到糖尿病大鼠體內在90分鐘後可以增加骨骼肌肝醣合成量。血清素對STZ誘發糖尿病大鼠產生降血糖的作用可以被血清素接受體阻斷劑dihydroergotamine與pimozide所抑制。將STZ誘發糖尿病大鼠兩側的腎上腺摘除，發現血清素的降血糖作用也同樣被抑制。摘除糖尿病大鼠的腎上腺作離體實驗，發現血清素可以直接刺激腎上腺釋放腦內啡，同樣的離體實驗，在血清素接受體阻斷劑dihydroergotamine與pimozide的存在下血清素促進腦內啡的釋放作用受抑制。在STZ誘發糖尿病大鼠預先以腹腔注射投與鴉片受體阻斷劑naloxon 1.0 mg/kg再投與血清素，結果發現血清素的降血糖作用被抑制。上述實驗結果證明，血清素在STZ誘發糖尿病大鼠產生降血糖作用是藉由活化腎上腺血清素第七型接受器使腦內啡的釋放，並且刺激到鴉片接受體進而增加周邊組織對葡萄糖的利用率，因而達到降血糖的效果。 第三部份 Thaliporphine與相關衍生物在正常大鼠與糖尿病大鼠降血糖作用 糖尿病當今臨床常見的一種代謝性疾病，而且，隨著生活品質的提昇及物質充裕與飲食習慣改變，糖尿病的病例也日益增多。因此，新藥物的研究仍有需要。許多芳香環阿浦酚類的生物鹼存在於許多中草藥，並被發現具有抗高血壓或心臟保護作用。本實驗分別在正常大鼠、streptozotocin所誘導的第一型胰島素依賴型 (insulin-dependent DM, IDDM) 糖尿病大鼠及nicotinamide 與 streptozocin合併誘導的第二型非胰島素依賴型 (non-insulin-dependent DM, NIDDM) 糖尿病大鼠評估並比較各種阿浦酚化合物之降血糖效果。實驗上所評估的化合物包括有：thaliporphine、glaucine、boldine、N-methyl-laurotetanine 與predicentrine 及阿浦酚類衍生物有N-[2-(2-methoxyphenoxy)ethyl]norglaucine與diacetyl-N-allylsecoboldine。以靜脈注射方式給藥，注射不同劑量到正常大鼠及糖尿病大鼠，實驗結果發現，這些化合物接可以產生劑量相關性的降血糖作用。實驗過程中發現，thaliporphine具有刺激正常大鼠與NIDDM糖尿病大鼠胰臟分泌胰島素，同時，在外給的葡萄糖試驗 (glucose tolerance test) 發現thaliporphine 1mg/kg 可增加大鼠對葡萄糖的利用率。同樣的，thaliporphine對正常大鼠與糖尿病大鼠亦能促進骨骼肌肝醣合成。因此，實驗結果得知，thaliporphine及其衍生物皆具有降血糖作用，而thaliporphine降血糖機制則與促進胰島素釋放及增強葡萄糖的利用率有關。

第四部份 Thaliporphine在STZ誘發糖尿病大鼠降血糖作用 Thaliporphine在STZ誘發糖尿病大鼠可以劑量相關性的產生降血糖作用，同樣地，可以增加內生性腦內啡的釋放。Thaliporphine對STZ誘發糖尿病大鼠產生降血糖的作用可以被血清素接受體阻斷劑dihydroergotamine與pimozide所抑制。而且，在腎上腺摘除之STZ誘發糖尿病大鼠，thaliporphine的降血糖作用及刺激腦內啡的釋放之作用均消失。在STZ誘發糖尿病大鼠預先給予鴉片受體阻斷劑naloxon再投與thaliporphine，結果發現thaliporphine的降血糖作用被抑制。上述實驗結果證明，thaliporphine在STZ誘發糖尿病大鼠產生降血糖作用是藉由活化腎上腺血清素第七型接受器使腦內啡的釋放，然後刺激到鴉片接受體進而增加周邊組織對葡萄糖的利用率，因而達到降血糖的效果。 結論： 本論文證實resveratrol、thaliporphine及其相關衍生物在正常與糖尿病大鼠具有降血糖作用。Resveratrol之降血糖作用機制包括促進胰島素釋放與直接活化PI3-k-Akt signaling pathway以增加葡萄糖之利用。在thaliporphine與相關衍生物中以thaliporphine具有最好的降血糖作用。 Thaliporphine 之降血糖作用部分是因促進胰島素釋放所貢獻，但另外之作用機制是因thaliporphine活化腎上腺血清素受體而使腦內啡釋放進而產生降血糖作用，雖然thaliporphine會活化血清素受體引起降血糖作用，但增加3-10倍thaliporphine注射投與劑量並不會如注射較高劑量之血清素促進凝血作用與血管收縮反應及引起動物死亡。因此有關thaliporphine與血清素在其它藥理作用之差異與其對不同血清素受體亞型作用之差異仍需進一步研究。

Recently, the population of diabetic patients is increasing. It becomes one of the ten most common diseases in our country. Therefore, searching for chemical principles from plant or synthesis as antidiabetic agents become an important aim of this thesis. We examined and compared the antidiabetic activity of resveratrol, thaliporphine and its derivatives in normal and diabetic rat models. Since thaliporphine was found to have strong binding activity to serotonin receptors, we further investigated the pharmacological action of serotonin in plasma glucose regulation and characterize serotonin receptor subtype and the associated signaling pathway in mediating thaliporphine-induced antidiabetic action. Part One : Antihyperglycemic action of resveratrol in streptozotocin-induced diabetic rats : The role of Phosphatidylinositol-3-kinase activation. Resveratrol, a polyphenolic substance found in grape skin, is proposed to account in part for the protective effect of red wine in the cardiovascular system. The aim of the present study is to investigate the action and possible mechanisms of resveratrol-produced regulation in plasma glucose of normal and diabetic rats including the animal model of streptozotocin (STZ)-induced and nicotinamide-STZ-induced (NA-STZ), and insulin-resistance diabetic rats. Resveratrol (p.o.) produced a hypoglycemic effect in a dose-dependent manner in normal and diabetic rats, and the insulin level was increased following resveratrol treatment in normal and NA-STZ diabetic rats. In insulin-deficient STZ-diabetic rats, resveratrol significantly lowered the plasma glucose 90 min after oral treatment, and the hypoglycemic effect was abolished by PI3K inhibitors (LY294002 and wortmannin) which also inhibited resveratrol-induced Akt phosphorylation in soleus muscle of STZ-diabetic rats. The change in the protein expression level of glucose transporter subtype 4 (GLUT4) in the soleus muscle and phosphoenolpyruvate carboxykinase (PEPCK) in the liver of STZ-diabetic rats treated with resveratrol (3 mg/kg, p.o.) for 7 days were examined. Resveratrol normalized hepatic PEPCK expression and increased GLUT4 expression in the soleus muscle of STZ-diabetic rats. The results indicate that the mechanisms contributing to the hypoglycemic effect of resveratrol include insulin-dependent and insulin-independent pathway, and PI3K-Akt-signaling was involved in the latter mechanism to enhance glucose uptake in skeletal muscle. Part Two : Antihyperglycemic action of serotonin in streptozotocin-induced diabetic rats. Although serotonin, serotonin uptake inhibitor and serotonin precursor (including tryptophan or 5-hydroxytryptophan) are known to have hypoglycemic action in rodents or human, it is not clear whether serotonin has hypoglycemic effect in streptozotocin-induced diabetic rats (STZ-diabetic rats). The aim of this study was to investigate the action of serotonin in regulating the plasma glucose STZ-diabetic rats. Plasma glucose, insulin, β-endorphin (BER) and adrenaline were assessed after intraperitoneal administration with serotonin. Serotonin produced hypoglycemic effect without altering plasma insulin and adrenaline levels but increasing β-endorphin level in STZ-diabetic rats. The glycogen content in soleus muscle was increased at 90 min after applying serotonin 0.3 mg/kg in STZ-diabetic rats. Dihydroergotamine (non-selective 5-HT receptor blocker) and pimozide (5-HT7 receptor blocker) abolished the hypoglycemic effect of serotonin in STZ-diabetic rats. Serotonin-induced hypoglycemic effect in association with the increase of β-endorphin release was abolished in bilateral adrenalectomized STZ-diabetic rats. In isolated adrenal gland of STZ-diabetic rats, the increase of β-endorphin secretion in response to serotonin was reduced by either dihydroergotamine or pimozide. Naloxon (1.0 mg/kg, i.p.) pretreatment prevented serotonin-induced plasma glucose lowering effect in STZ-diabetic rat. The results demonstrated that serotonin may activate 5-HT7 receptor on rat adrenal gland to enhance of β-endorphin secretion, which then stimulated the opioid receptor to increase peripheral glucose utilization and resulted in lowering plasma glucose in STZ-diabetic rats. Part three: Antihyperglycemic effect of aporphines and their derivatives in normal and diabetic rats. The antihyperglycemic action of some aporphines and their derivatives in normal Wistar, streptozotocin (STZ)-induced diabetic (IDDM) and nicotinamide-STZ induced diabetic (NIDDM) rats were investigated and compared in this study. These compounds included the thaliporphine, glaucine, boldine, N-methyl-laurotetanine, and predicentrine and the derivatives, N-[2-(2-methoxyphenoxy)ethyl]norglaucine and diacetyl-N-allylsecoboldine. Bolus intravenous injection of these compounds decreased the plasma glucose levels dose-dependently both in normal and diabetic rats. Among them, thaliporphine was found to have the most potent antihyperglycemic effect in both NIDDM and IDDM diabetic rats. It was found that thaliporphine could stimulate the release of insulin both in normal and diabetic rats, and a dose of 1 mg per kg thaliporphine could significantly attenuate the increase of plasma glucose induced by an intravenous glucose challenge test in normal rats. Similar treatment with thaliporphine significantly increased the skeletal muscle glycogen synthesis in both normal and diabetic rats. Hence the hypoglycemic effect of thaliporphine in diabetic rats could be attributed to the stimulation of insulin release and the increase of glucose utilization. Part four: Antihyperglycemic action of thaliporphine in streptozotocin-induced diabetic rats. In STZ-diabetic rats, thaliporphine dose dependently lowered plasma glucose concentrations and increased plasma beta-endorphin-like immunoreactivity (BER). Both of these effects of thaliporphine were abolished by pretreatment of rats with dihydroergotamine (non-selective 5-HT receptor blocker) and pimozide (5-HT7 receptor blocker) at doses sufficient to block 5-HT7-serotonin receptor. Moreover, bilateral adrenalectomy in STZ-diabetic rats eliminated the activities of thaliporphine, including the plasma glucose-lowering effect and the plasma BER-elevating effect. Naloxon inhibited the plasma glucose-lowering activity of thaliporphine at doses sufficient to block opioid receptors. In conclusion, The results demonstrated that thaliporphine may activate 5-HT7 receptor on rat adrenal gland to enhance of β-endorphin secretion, which then stimulated the opioid receptor to increase peripheral glucose utilization and resulted in lowering plasma glucose in STZ-diabetic rats. In conclusion, resveratrol、thaliporphine and many of its derivative were found to have hypoglycemic actions both in normal and diabetic rats. The hypoglycemic mechanism of resveratrol involved the activation of PI3K-Akt signaling pathway as demonstrated in IDDM rats and the increase of insulin secretion as demonstrated isolated in pancreatic beta cells or NIDDM rats. Thaliporphine was found to have the most potent antihyperglycemic effect among aporphine derivatives. Part of the hypoglycemic effect of thaliporphine could be attributed to the stimulation of insulin release. Part of the other hypoglycemic action of thaliporphine is related to the stimulation of glucose uptake probably via the release of endorphin from adrenal gland through the stimulation of 5-HT7 receptor. Although the activation of serotonin receptor contributed to the hypoglycemic effect of thaliporphine, intravenous administration of 3 to 10 folds the hypoglycemic dose of thaliporphine did not enhance blood coagulation, vasoconstriction, and animal death as serotonin per se did. Therefore, the difference between thaliporphine and serotonin in the hypoglycemic action and other biological activities and their involvement of different serotonin receptor subtypes remains to be further investigated.