糖尿病形成之環境毒素--汞之研究及濱當歸萃取物對血糖調節作用之探討

Abstract

第一部份: 目前關於氧化性壓力和phosphoinositide 3-kinase (PI3K)訊號傳遞的互動，和對於胰臟胰島細胞分泌胰島素功能之影響仍不清楚。而汞已知是一種可導致氧化性傷害的毒性金屬。低濃度（submicromolar-concentration）的氯化汞 (mercuric chloride, HgCl2)或是甲基汞（methylmercury, MeHg）在胰島細胞衍生出的HIT-T15細胞株和由小鼠胰臟分離出的胰島細胞（islet cells），會引發自由基（reactive oxygen species, ROS）的增加以及降低胰島素的分泌，汞化合物會增加phosphoinositide 3-kinase (PI3K)的活性以及其下游AKT蛋白質磷酸化的表現。抗氧化劑N-acetyl-L-cysteine (NAC)可以避免汞所導致的胰島素分泌抑制和AKT磷酸化的表現，但不能降低PI3K活性增加的情形。利用PI3K的抑制劑LY294002或是經由表現dominant-negative p85 or Akt蛋白質體（plasmid）可預防汞化合物所導致的胰島素分泌抑制，但卻不能降低汞促進ROS的產生。實驗結果顯示，細胞實驗中，在汞化合物的刺激下，PI3K以及ROS都會獨自去調控AKT的磷酸化，進而影響胰島素分泌抑制的情形。動物實驗的結果顯示，小鼠經由口服餵食2至4週低劑量的汞化合物，發現其存在於血漿中的胰島素明顯有降低的現象，並且血糖提高和增加血漿中脂質過氧化作用。將餵食汞一定時間的小鼠胰島細胞分離出來，發現AKT磷酸化表現增加，而在合併處理NAC的小鼠，其胰島細胞無AKT磷酸化表現增加的情形。而小鼠在停止餵食汞之後，汞在小鼠體內所誘發的效應和增加高血糖的現象都可被回復。以上實驗結果顯示，低劑量的汞化合物會誘導氧化性壓力的產生，並且活化PI3K-AKT的路徑而引發胰臟胰島細胞的功能性損傷。 第二部份: 汞已知為重要的毒性物質，並且會誘導氧化性傷害的產生。由之前的文獻中顯示胰臟的胰島細胞對於氧化性傷害非常的敏感，然而汞在胰臟的胰島細胞所產生病理效應並不清楚。我們希望藉此研究探討有機汞化合物--甲基汞（methylmercury, MeHg）在引發氧化性傷害之後對於胰臟的胰島細胞的存活率及功能上的影響。 結果發現汞化合物會導致胰臟胰島細胞的死亡而導致其分泌胰島素的功能喪失。甲基汞（1-20uM）處理胰臟胰島細胞細胞株HIT-T15 24 小時，細胞存活率呈現劑量效應的減少，而ROS也有劑量效應與時間效應增加的現象。甲基汞 (2及5uM)處理24小時發現在HIT-T15細胞株和由小鼠胰臟分離出的胰島細胞都會有胰島素分泌能力降低的情形，此外，甲基汞會促進細胞的sub-G1以及annexin-V結合力的增加，這表示甲基汞會促進細胞凋亡的發生。HIT-T15 細胞株處理甲基汞後也發現其粒腺體膜電位（mitochondrial transmembrane potential）會有降低的現象，而粒腺體遭到破壞後會釋放出cytochrome c至細胞質中，進一步活化caspase-3。而上述結果均可被自由基拮抗劑N-acetylcysteine有效反轉。 我們認為甲基汞主要是透過促使胰臟胰島細胞細胞凋亡而使其分泌胰島素的能力喪失。 第三部份: 汞已知為重要的毒性物質，並且會誘導氧化姓傷害的產生。由之前的文獻中顯示胰臟的胰島細胞對於氧化性傷害非常的敏感，然而汞在胰臟的胰島細胞所產生病理效應並不清楚。我們希望藉此研究探討無機汞化合物--氯化汞（mercuric chloride, HgCl2）在引發氧化性傷害之後對於胰臟的胰島細胞的存活率及功能上的影響。 結果發現汞化合物會導致胰臟胰島細胞的死亡而導致其分泌胰島素的功能喪失。氯化汞（2-20uM）處理胰臟胰島細胞細胞株HIT-T15 24 小時，細胞存活率呈現劑量效應的減少，而ROS也有劑量效應與時間效應增加的現象。氯化汞處理24小時發現在HIT-T15細胞株和由小鼠胰臟分離出的胰島細胞都會有胰島素分泌能力降低的情形，此外，氯化汞會促進細胞的sub-G1以及annexin-V結合力的增加，並且降低粒腺體膜電位，繼而使cytochrome c釋放到細胞質中。所以氯化汞會促進細胞凋亡的發生。HIT-T15 細胞株處理氯化汞後也會增加AO (acridine orange)和EtBr (ethidium bromide) 的雙向染色和降低ATP濃度，而使細胞壞死。餵食小鼠氯化汞5 mg/kg明顯的導致血漿中胰島素減少，增加血糖和血漿中脂質過氧化作用，全血中也偵測到有大量汞的堆積現象。 我們認為氯化汞主要是透過促使胰臟胰島細胞細胞凋亡（apoptosis）和細胞壞死（necrosis）而使其分泌胰島素的能力喪失。 第四部份: 本篇主要是要研究利用甲醇萃取的濱當歸萃取物（Angelica hirsutiflora）是否有降低血糖的作用，藉以研發抗糖尿病的藥物。 實驗結果發現濱當歸萃取物能在在胰島細胞衍生出的HIT-T15細胞株和由小鼠胰臟分離出的胰島細胞（islet cells）中刺激胰島素的釋放，並且發現，腹腔注射濱當歸萃取物在空腹12小時的正常小鼠，可以降低因餵食澱粉之後的血糖增加，並且可以增加在血漿中的胰島素濃度。而在高脂飲食誘導的糖尿病鼠中，也發現濱當歸萃取物可以改善糖尿病鼠的葡萄糖不耐受性和胰島素不耐受性。在HIT-T15細胞和人類初代培養的胰島細胞都發現濱當歸萃取物會促進細胞內鈣離子的增加。此外在HIT-T15細胞也發現濱當歸萃取物會增加AKT和ERK1/2（extracellular signal-regulated protein kinases）的磷酸化作用，並且也發現濱當歸萃取物和胰島素在骨骼肌細胞衍生出的C2C12細胞株都可以促進葡萄糖的利用。我們也進一步發現分離自濱當歸萃取物的幾種純化物中具有促進HIT-T15胰島素的分泌的作用。 根據以上的實驗結果，我們推測濱當歸可以促進胰島素的分泌並且具有類似胰島素作用的效應，而降低在糖尿病動物體內的高血糖情形。

Part1: The relationship between oxidation stress and phosphoinositide 3-kinase (PI3K) signaling in pancreatic beta-cell dysfunction remains unclear. Mercury is a well-known toxic metal that induces oxidative stress. Submicromolar-concentration HgCl2 or methylmercury triggered reactive oxygen species (ROS) production and decreased insulin secretion in beta-cell–derived HIT-T15 cells and isolated mouse islets. Mercury increased PI3K activity and its downstream effector Akt phosphorylation. Antioxidant N-acetyl-L-cysteine (NAC) prevented mercury-induced insulin secretion inhibition and Akt phosphorylation but not increased PI3K activity. Inhibition of PI3K/Akt activity with PI3K inhibitor or by expressing the dominant-negative p85 or Akt prevented mercury-induced insulin secretion inhibition but not ROS production. These results indicate that both PI3K and ROS independently regulated Akt signaling–related, mercury-induced insulin secretion inhibition. We next observed that 2- or 4-week oral exposure to low-dose mercury to mice significantly caused the decrease in plasma insulin and displayed the elevation of blood glucose and plasma lipid peroxidation and glucose intolerance. Akt phosphorylation was shown in islets isolated from mercury-exposed mice. NAC effectively antagonized mercury-induced responses. Mercury-induced in vivo effects and increased blood mercury were reversed after mercury exposure was terminated. These results demonstrate that low-dose mercury–induced oxidative stress and PI3K activation cause Akt signaling–related pancreatic beta-cell dysfunction. Part2: Mercury is a well-known toxic metal, which induces oxidative stress. Pancreatic beta-cells are vulnerable to oxidative stress. The pathophysiological effect of mercury on the function of pancreatic beta-cells remains unclear. The present study was designed to investigate the effects of methylmercury (MeHg)-induced oxidative stress on the cell viability and function of pancreatic beta -cells. The number of viable cells was reduced 24 h after MeHg treatment in a dose-dependent manner with a range from 1 to 20 uM. 2',7'-Dichlorofluorescein fluorescence as an indicator of reactive oxygen species (ROS) formation after exposure of HIT-T15 cells or isolated mouse pancreatic islets to MeHg significantly increased ROS levels. MeHg could also suppress insulin secretion in HIT-T15 cells and isolated mouse pancreatic islets. After 24 h of exposure to MeHg, HIT-T15 cells had a significant increase in mercury levels with a dose-dependent manner. Moreover, MeHg displayed several features of cell apoptosis including an increase of the sub-G1 population and annexin-V binding. Treatment of HIT-T15 cells with MeHg resulted in disruption of the mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytosol and activation of caspase-3. Antioxidant N-acetylcysteine effectively reversed the MeHg-induced cellular responses. Altogether, our data clearly indicate that MeHg-induced oxidative stress causes pancreatic beta -cell apoptosis and dysfunction. Part3: Mercury is a well-known toxic metal, which induces oxidative stress. Pancreatic beta-cells are vulnerable to oxidative stress. The pathophysiological effect of inorganic mercury on the function of pancreatic beta-cells remains unclear. The present study was designed to investigate the effects of mercuric chloride (HgCl2)-induced oxidative stress on the cell viability and function of pancreatic beta -cells. The number of viable cells was reduced 24 h after HgCl2 treatment in a dose-dependent manner with a range from 2 to 20 uM. 2’,7’- Dichlorofluorescein fluorescence as an indicator of reactive oxygen species (ROS) formation after exposure of HIT-T15 cells or isolated mouse pancreatic islets to HgCl2 significantly increased ROS levels. HgCl2 could also suppress insulin secretion in HIT-T15 cells and isolated mouse pancreatic islets. After 24 h of exposure to HgCl2, HIT-T15 cells had a significant increase in mercury levels with a dose-dependent manner. Moreover, HgCl2 displayed several features of cell apoptosis including an increase of the sub- G1 population and annexin-V binding. Treatment of HIT-T15 cells with HgCl2 resulted in disruption of the mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytosol. Antioxidant N-acetylcysteine effectively reversed the HgCl2-induced cellular responses. Besides, HgCl2 displayed several features of cell necrosis including an increase of the AO (acridine orange) and EtBr (ethidium bromide) dual-staining and decrease ATP levels. We next observed that 2- or 4-week oral exposure to HgCl2 5 mg/kg to mice significantly caused the decrease in plasma insulin and displayed the elevation of blood glucose and plasma lipid peroxidation and whole blood mercury accumulation. Altogether, our data clearly indicate that HgCl2-induced oxidative stress causes pancreatic beta-cell apoptosis, necrosis and dysfunction. Part4: The methanolic extract of Angelica hirsutiflora was studied for its hypoglycemic activity. A. hirsutiflora extract potently stimulated the release of insulin from cultured pancreatic beta-cells (HIT-T15 cells) and isolated mouse and human islets. When A. hirsutiflora extract was peritoneally administrated to the fasted mice, it decreased the increase in blood glucose level after starch loading. The plasma insulin level was also increased by A. hirsutiflora extract treatment. In high fat diet-induced diabetic mice, A. hirsutiflora extract markedly improved the oral glucose intolerance as compared with the vehicle control. A. hirsutiflora extract could also effectively improve the insulin intolerance in high fat diet-induced diabetic mice. Besides, the A. hirsutiflora extract enhances calcium concentration in HIT-T15 cells and primary human islet cells. Moreover, both insulin and A. hirsutiflora extract were capable of increasing the phosphorylation of Akt and extracellular signal-regulated protein kinases (ERK) 1/2 proteins in HIT-T15 beta-cells. Both insulin and A. hirsutiflora extract could also increase the glucose consumption in differentiated C2C12 skeletal muscle cells. We had further found that pure compounds of A.hirsutiflora, including of Isopimpinellin, Bergapten, Byakanglicin, and Isobyakangelicin, were capable of increasing insulin secretion in HIT-T15 cells. These results suggest that the A. hirsutiflora may exert both insulin secretagogue and insulinomimetic activities to lower blood glucose concentrations in vivo.