新型AMPK活化劑Nstpbp168對於胰島分泌細胞的保護作用

全文

(1)國立臺灣師範大學生命科學系碩士論文. 新型AMPK活化劑Nstpbp168對於胰島分泌細胞的保護作用 The protective effect of a novel AMPK activator Nstpbp168 on insulin secreting cells. 研究生：邱富嶼 Fu-Yu Chiu 指導教授：林炎壽博士 Yenshou Lin, Ph.D. 共同指導教授：謝博軒博士 Po-Shiuan Hsieh, Ph.D. 徐鳳麟博士 Feng-Lin Hsu, Ph.D. 中華民國 102 年一月.

(2) 致謝兩年半碩班生涯即將劃下句點，回想這一切真的波折不斷但也因為如此所學到的東西相對是非常多，從一開始在師大林老師那學了分生生化的技術再來去了北醫在徐老師 Lab 學了化學分離純化的方法，最後到了國防謝老師那邊完成論文外也學習到如何做動物實驗，整個碩班生活或許看似離奇但也豐富了我人生，這真的要感謝林老師給我的逆境，如果沒有他我無法有周遊各校的機會也不會學到研究該有懷疑與解決方法的能力，而在生藥研究這方面也很感謝北醫徐老師與國防謝老師的全力的幫助，沒有這兩位老師支持論文無法順利完成。再來是 C308 的伙伴仁華學姐與柏強很感謝你們，雖然只有一年的時間跟你們在 Lab 打拼，那一年真的過得很開心真的很希望還有那樣機會，而仁華學姐妳真的給我許多的協助與幫忙，在我無助的時候給予實驗上面許多的建議；北醫的伙伴景鴻、雅植、哲良及宗儒這一年半來很謝謝你們也把我當成生藥所的一員讓我這段時間不覺得孤獨，約吃飯都不會忘了我的份，最後是國防的伙伴呂醫師、晨婷、炫寰、梓姮、宏哲、飯友筱婷也很感謝你們這一年來的幫忙，特別是炫寰沒有你的協助共儀使用上面無法那麼的順心，炫寰謝謝你這一年半來這麼挺我這學長，還有就是我的父母與大學的好友謝謝你們一路對.

(3) 我的支持。或許這篇論文不比別人來的華麗有著各式各樣的技術證明實驗的結果，但這是以最有限的資源盡最大的使用所得來的成果，是集結了各位給我的支持所完成的論文，最後還是要說謝謝這些在我研究所中的貴人。邱富嶼謹誌於國立臺灣師範大學生命科學系(所) 中華民國 102 年二月一號.

(4) Contents. Abbreviations………………………………………………………...1 Chinese abstract……………………………………………………...2. Abstract ………………………………………………………………4. Chapter1. Introduction………………………………………………5 1.1 Current status of diabetes. 6. 1.2 β-Cell and the development of diabetes. 6. 1.3 Reactive oxygen species (ROS) and β-Cell dysfunction. 8. 1.4 AMP-activated protein kinase (AMPK) pathway. 9. 1.5 AMPK activation and diabetes. 11. 1.6 AMPK activator Nstpbp168. 13. Chapter 2. Materials and Methods…………………………………16 2.1 Reagents. 16. 2.2 Cell Culture. 16. 2.3 Palmitate/BSA complex preparation. 17. 2.4 Cell viability assay. 17. 2.5 Western blotting. 18. 2.6 Determination of reactive oxygen species (ROS). 19. 2.7 Evaluation of ROS production by DCFH-DA. 19. 2.8 Glucose stimulated insulin secretion (GSIS). 20. 2.9 Statistical Analysis. 20 I. .

(5) Chapter 3. Results…………………………………………………….21 3.1 The effects of Nstpbp168 on cell survival. 21. 3.2 Scavenging capacity of Nstpbp168 on H2O2-induced ROS 22 3.3 The anti-oxidant protective ability of Nstpbp168 is through activating AMPK. 22. 3.4 Nstpbp168 prevented Palmitate-induced lipotoxicity. 24. 3.5 Inhibition of palmitate-induced ROS production by Nstpbp168. 26. 3.6 Effects of Nstpbp168 on glucose- stimulated insulin secretion in mouse islets.. 27. Chapter 4. Discussion…………………………………………………. 28 4.1 Nstpbp168 has antioxidant potential. 28. 4.2 Activated by Nstpbp168 can reduce the oxidative-induced cellular damage. 29. 4.3 Different level of palmitate-induced AMPK activation and cellular damage in pancreatic cells and liver cells. 31. 4.4 AMPK can be activated under H2O2/Palmitate stimulation. 33. 4.5 Activation of AMPK is essential in early onset of diabetes. 34. 4.6 Conclusion. 35. References………………………………………………………………36. II. .

(6) Figures………………………………………………………………….46 Figure 1. Nstpbp168 could suppress H2O2-induced cytotoxicity in a dose-dependent manner.. 46. Figure 2. Suppressive effect of Nstpbp168 on the production of H2O2-induced ROS.. 48. Figure 3. The protective effect of Nstpbp168 on H2O2-induced cell damage through activation of AMPK.. 49. Figure 4. The effect of Nstpbp168 on palmitate-induced lipotoxicity in RINm5F.. 51. Figure 5. The effect of Nstpbp168 on AMPK activation in palmitate treated RINm5F.. 53. Figure 6. The protective effect of Nstpbp168 on HepG2 cells with or without palmitate treatment.. 54. Figure 7. The effect of Nstpbp168 on AMPK expression in palmitate-treated HepG2 cells.. 55. Figure 8. Imagination of DCFH-DA stained ROS in palmitate treated RINm5f cells with or without drug co-administration.. 56. Figure 9. Effects of Nstpbp168 on intracellular ROS scavenging in HepG2.. 58. Figure 10. Effects of Nstpbp168 on glucose-stimulated insulin secretion in mouse islets. III. . 60.

(7) Abbreviations. AMPK. AMP-activated protein kinase. AICAR. 5-Aminoimidazole-4-carboxamide ribonucleotide. BSA. Bovine serum albumin. DCFH-DA. 2′,7′-Dichlorofluorescin diacetate. DMSO. Dimethyl sulfoxide. DMEM. Dulbecco’s modified Eagle’s medium. FBS. Fetal bovine serun. GSIS. Glucose stimulated insulin secretion. H2O2. Hydrogen peroxide. NBT. Nitrotetrazolium blue chloride. Metformin. N,N-Dimethylimidodicarbonimidic diamide. PBS. Phosphate-buffered saline. ROS. Reactive oxygen species. RPMI-1640. Roswell Park Memorial Institute 1640. 1. .

(8) 摘要 Nstpbp168 是由植物分離出來的純化合物，在我們先前的研究發現它是一種新穎的 AMPK 活化劑。而本次研究目主要是探討 Nstpbp168 在氧化壓力及脂質毒性下對胰島素分泌細胞的活存率與活性氧化物產生是否是有正面影響。在氧化壓力的刺激下，胰島素分泌細胞 RINm5F 曝露於含有 40 μM 過氧化氫溶液，而後投予 100 μM Nstpbp168 培養十八小時，計數細胞的存活率與活性氧化物生成量。此外，在脂質毒性刺激方面，則是將 RINm5F 及肝臟細胞 HepG2 培養於含有 100 μM Nstpbp168 及 palmitate/BSA 混合物的培養液，進而以 MTT 檢測其細胞存活率及 DCFH-DA 染劑觀察細胞內超氧物質含量的螢光圖像。研究的結果顯示，Nstpbp168 可以在過氧化氫誘導之氧化壓力下呈現劑量依存的方式防止細胞死亡。此外，在 NBT 檢測的結果發現細胞經由 Nstpbp168 處理後能有效抑制過氧化氫所誘導出活性氧化物，並證明 Nstpbp168 對於 AMPK 的活化呈現劑量依存的關係。AMPK 的拮抗劑 Compound C 可明顯阻止 Nstpbp168 的保護作用在過氧化氫對於胰島素分泌細胞產生的傷害。此外，Nstpbp168 具有防止 palmitae 引起之細胞死亡，推論是藉由促進 AMPK 活化以減少活性氧的產生。綜以上所述，Nstpbp168 對於細胞具有保護作用，可有效防止氧化性壓力及脂質累積之毒性所造成的傷害，而這些保護 2. .

(9) 的功能可能均是藉由促進 AMPK 活化的機制所達成。. 關鍵字：β-cell、Reactive oxygen species (ROS)、AMP-activated protein kinase (AMPK)、diabetes、oxidative stress、lipotoxicity. 3. .

(10) Abstract Nstpbp168, a pure compound isolated from natural product has been shown to be a novel AMPK activator in our previous study. The aim of this study was to assess the possible beneficial effect of Nstpbp168 on cell survival and ROS production in insulin secreting cell under oxidative stress/lipotoxicity. In the part of oxidative stress stimulation, RINm5F was first exposed to 40 μM hydrogen peroxide and then incubated in medium w/o 100 μM Nstpbp168 for following cell viability, ROS level and related signal transduction measurement, respectively. Besides, in the part of lipotoxicity, RINm5F and the liver cell, HepG2, were treated w/o 100 μM Nstpbp168 and exposed to palmitate/BSA mixture to employ the MTT/DCFH-DA assessment. The present results showed that Nstpbp168 could prevent cell death from H2O2-induced oxidative stress dose-dependently along with lowering H2O2-induced ROS production. Meanwhile, Nstpbp168 treatment also activated AMPK dose-dependently. Compound C, a selective AMPK antagonist, could significantly block the protective effect of Nstpbp168 on H2O2-induced damage in insulin secreting cell. Moreover, Nstpbp168 had the potential to prevent cell death from palmitae-induced injury, which might be through promoting AMPK activity as well as reduced the ROS production. Taken together, it is suggested that Nstpbp168 might have a potential protective effect on ROS/lipotoxicity-induced cell damage through AMPK-mediated pathway in insulin secreting cells. Key word: β-cell、Reactive oxygen species (ROS)、AMP-activated protein kinase (AMPK)、diabetes、oxidative stress、lipotoxicity 4. .

(11) Ch hapter 1. 1 Introduction n 1.1 Current status of diabetes onic metabbolic disorrder which h affects oover 150 Diabetes is a chro milllion people in the world, w andd several reesearches predict thhat over 36 65 milllion people will sufffer from ddiabetes until u the yeear 2030 (W Wild et all., 20004a). Due to diet fav vorite channging and d excessivee obesity, the poppulation off diabetes worldwidde is expan nding in an amazingg rate. Diaabetes has been reco ognized ass a global public heaalth conceern currenttly. Diabetes can be distinguish d hed in two o types. Ty ype 1 diabeetes is also o refeerred to ass immune--mediatedd diabetes which w resu ults from the autooimmune destructio on of insullin secreting pancreeatic β-cellls. Type 2 diabbetes was named ass adult-onsset diabetees or non-insulin-deependent diabbetes prevviously, wh hich is asccribable to o high blood glucos e caused by b insuulin resisttance and relative r innsulin defiiciency. It is the mosst common form m of diabeetes aboutt 90% – 955% of the case diagn nosed. Thhe typical diabbetes is chharacterizeed in chroonic hyperg glycemia owing to dyssfunctionaal insulin receptors, r irregular insulin i seccretion or abnormall posst-receptorr events in nfluencingg metaboliism of carb bohydratee, fats, and d prootein. Therrefore, thee long-term m damage and failurre of differrent organ ns, succh as cardiiovascularr disease, bblindness,, kidney co omplicatioons, and ampputations are also caused by cchronic hy yperglycem mia (Wildd et al., 20 004b, Ugaz and Reesnick, 200 08). Obesityy has been known asssociated with w a con ndition of cchronic lo ow level inflamm mation. It was also ddiscovered d that the generation g n of reactiive 5. .

(12) oxygen species (ROS) is elevated in obesity, which may further invoke the inflammatory pathways. Current diabetes medicine administrated along cannot completely improve long-term hyperglycemia, but it is necessary to combine with other directions or drugs to cure diabetes (Mohler et al., 2009). So far, clinical pharmacy of type 2 diabetes can be classified into five major aspects. These include stimulating insulin secretion, decreasing gluconeogenesis, postponing the digestion and absorption of intestinal carbohydrate, increasing glucose utilization rate or enhancing insulin sensitivity of peripheral tissue (Krentz and Bailey, 2005, Mohler et al., 2009). The most representative anti-diabetic compounds are metformin and thiazolidinediones (TZD), which exert effects through activating AMPK. Metformin could not induce insulin secretion but reversed insulin resistance in peripheral tissues via decreasing hepatic glucose output and enlarging peripheral glucose uptake (Kim et al., 2010).. 1.2 β-Cell and the development of diabetes The major insulin secreting cells are β-cells on the inside of pancreatic islets. They play a central role on mammalian systemic regulating glucose metabolism. Glut2 is one type of glucose transporter, expressing on β-cells to enhance glucose uptaking efficacy. Glucose influx provides substrates for hexokinases to generate energy in the form of ATP. High ratio of ATP/AMP sequentially stimulate ATP-sensitive KATP channels closure followed by increasing depolarized membrane potential, 6. .

(13) resulting in the opening of L-type voltage-depending calcium channels to lead numerous Ca2+ influxes and then inducing vesicles exocytosis to release insulin. Hence, mitochondria as the main organelle for energy maintenance as well as energy/ATP producing are indeed critical for sustaining β-cell physiological normality, particularly for insulin secretion. Many factors have been identified to cause mitochondria dysfunction. These include well known high glucose content, reactive oxygen species (ROS) and free fatty acids-induced lipotoxicity. Catabolic pathway generated electrons from sugar, fatty acid, and amino acids accumulate in the electron carrier to form ROS, the byproduct of mitochondrial respiration. Increased free-radical often occurred when ATP exceeds cellular energy demand. ROS attack results in mitochondrial inactivation to interrupt insulin secreting signaling. Besides, glucose and fatty acid depend on mitochondria to process metabolism. Low glucose could activate carnitine palmitoyltransferase I (CPT1) to transport long chain fatty acid into mitochondria for β oxidation to produce NADH and FADH2 and promote ATP production consequently. On the other hand, high glucose stimulation switch this pathway to glucose oxidation, causing lipid accumulate in cytosol. Chronically exposing to low glucose enables constitutive activated CPTI to inhibit GSIS, while ER stress would be elevated under long-term high glucose stimulation to activate mitochondrial apoptosis and impairs GSIS.(Supale et al., 2012). 7. .

(14) Invvolvementt of oxida ative stres s in β-Celll dysfuncction foun nd in diaabetes.(Kaaneto et al., a 2005). 1.3 Reactivee oxygen species s (R ROS) and β-Cell dy ysfunction n It is noted that ox xidative strress damages severaal cellularr functionss gy of differrent diseaases. In typ pe 1 andd has majoor roles in the pathopphysiolog diabbetes, RO OS is produ uced by m macrophagees and attrributed to apoptosiss or neccrosis of thhe insulin--secreting cells (Xio ong et al., 2006). Instead, the progrression of type 2 diaabetes is generally g ddescribed in i and dysfuunctional pancreatic botth insulin resistance r p c β-cell. β--cell com mpensationn against insulin i ressistance iss through increasing i g insulin secretion or β-cell β masss, while ffailure com mpensation n causes thhe onset of o gluucose intollerance (K Kaneto et aal., 2005). Chronicc hypergly ycemia eliccit the form mation off ROS maiinly from the t glyycation reaaction in various v tisssues (Num mazawa et al., 2008)). These prooduction of ROS inccluding, hyydroxyl raadicals (•O OH), hydroogen perroxide (H2O2), superroxide aniion (•O2), and the concomitannt formation 8. .

(15) of nitric oxide (NO) (Dröge, 2002), have been associated with β-cell dysfunction and led cell death both in type 1 and type 2 diabetes (Shimabukuro et al., 1997). In addition, due to β-cells possess low level of antioxidant enzyme such as Cu/Zn superoxide dismutase (SOD), Mn SOD, catalase, and glutathione peroxide dismutase, they are more sensitive to the damage caused by oxidative stress than other tissues (Tiedge et al., 1997, Tiedge et al., 1998). Several signal pathways are the target of oxidative stresses, including the p38 MAPK, namely, the stress-activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK) and MAPK pathways (Hou et al., 2008). Chronic exposure to long-chain saturated fatty acid is also the other major inducer of type 2 diabetes. Accelerated free fatty acid (FFA) will promote oxidative process in mitochondria which may enhance ROS production as well. Moreover, with irregular protein synthetic rate, ER (endoplasmic reticulum) is accumulated with increasing unfolded protein in its lumen and is always encountered with abnormal oxidation. Aggregated numerous misfolding protein may in turn cause excess ROS that will induce sequential gradual progression of apoptosis to led pancreatic β-cell die (Fonseca et al., 2011).. 1.4 AMP-activated protein kinase (AMPK) pathway AMPK is indicated as cellular energy sensor by switching AMP/ATP status. It is comprised of three subunits, catalytic α (α1or α2) subunit, regulatory β (β1, β2 or β3) and γ (γ1, γ2 or γ3) subunits (Hardie and Carling, 1997, Hardie et al., 1998). With various splicing, 12 9. .

(16) heterotrmeric combinations mainly distribute in particular tissues. For instance, α1 subunit is abundant in the cytoplasm of kidney pancreatic β-cell, the lung and the adipose tissue, while α2 subunit is in the heart and skeletal muscles; most of β1 subunit is in the liver and β2 in the skeletal muscle (Birk and Wojtaszewski, 2006, Treebak et al., 2007). The N-terminal of α subunit contains serine/threonine protein kinase domain which can be phosphorylated on threonine172 within the activation loop (T-loop) by upstream kinases so that AMPK can be activated (Hardie et al., 2003), while γ subunit is the main site for AMP or ATP by binding on its specific four cystathionine-β synthase (CBS) domains (Xiao et al., 2007). AMP binding on AMPK would promote allosteric activation which is predominantly phosphorylated by LKB1/STRAD/MO25 complex, whereas recent studies indicated that another alternative CaMKK β was capable to activate AMPK via increasing calcium without raising amount of AMP (Woods et al., 2003, Momcilovic et al., 2006, Towler and Hardie, 2007). Several environmental stresses, such as hypoxia and hyperglycemia, are capable to enhance the non-metabolic activation of AMPK.. 10. .

(17) Strructure and a regulaation of AMPK.(Vi A iollet et all., 2009). 1.5 AMPK activation a and diab betes Active AMPK A can n target too multiple protein un nder varioous mulation. These T targ get proteinns include several biiosynthetiic enzymes stim succh as acetyyl-CoA carrboxylase (ACC) an nd glycogeen synthasse, which resuults in fattty acid oxiidation annd glucosee uptake reespectivelyy (Higa ett al., 19999, El-Asssaad et al.,, 2003). Thherefore, its i main fu unction inn liver cells is to aaugment fatty f acid oxidation o so as to prrevent lipo ogenesis, w whereas th he defficient AM MPK activiity would cause sterrol regulattory elemeent binding prootein-1 (SR REBP-1) over-funct o tion to lead dislipideermia in tyype 2 diabbetes. The other noticeaable one iss that AMP PK activity y could reecover the pheenomenonn of compeensatory inn pancreattic β-cell by b inhibitiing the mT TOC1 pathhway throu ugh activaating TSC2 2 (Bartolo omé et al., 2010). Th he connsequentiaal decreaseed S6K acctivity wou uld reducee protein trranslation n to preevent over amount of o unfoldinng protein in the lum men of ER R which may m resuult in loweering the lipotoxicit l ty or hypeerglycemiaa-induced ER stress. 11. .

(18) Althouggh promoting AMPK K activatio on is confi firmed to bbe beneficial in m most of tisssues, the role of AM MPK remaained conttroversial in the reggulating off the physiiology of ββ-cells (K Kim et al., 2007, 2 Ribboulet-Chaavey et al.., 2008). D During thee progressiion of typee 2 diabetes, varrious degreees of insu ulin secrettion are in nvolved in pathologiical β-cells. Thee energy status s is sw witching w with time which w also o concomiitants diveerse effeects of thee fuel sensor AMPK K. For instaance, in prrediabetic states, hig gh gluucose intakke for incrreasing inssulin secreetion makees β-cells present in n higgh energy status s thatt would deecrease thee activity of AMPK K. Hence, it i is takiing into acccount thaat enhancinng AMPK K activity can c suppreess insulin n secretion to prevent p the exhaustiion of β-cells (Eto et e al., 20022). On the othher hand, im β-cells aft mpaired functional fu fter chronic compen sation would deccline insulin release followed by AMPK K activatio on, which may pottentiate glyycolipotox xicity-induuced cell death d (Ricchards et aal., 2005). Colllectively, AMPK pathway is importantt in regulaating glucoose hom meostasis and is also o a major target for the therap py of Typee 2 diabetees.. Roole of AM MPK in inssulin secrretion(Fu et al., 20113) 12. .

(19) 1.6 AMPK activator Nstpbp168. Since AMPK involves in multiple cellular and physiological interactions, its activators are applied in the therapy of diabetes or other metabolic diseases. One common anti-diabetic drug is metformin which promote AMPK activity through binding to the γ unit of AMPK. Metformin can’t induce LKB1 phosphorylation and fails to increase calcium influx in skeletal muscles, even in pancreatic β-cells, but the effects is reverse in its main target organ, liver. The other pharmaceutical compound, AICAR can be metabolized to AMP analog ZMP (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranotide) to activate AMPK by interacting with γ subunit(Corton et al., 1995). Similarly, ZMP can inhibit glucogenesis via blocking fructose-1,6-biphosphatase in heptocytes, whereas it could increase glucose uptake in skeletal muscle. Therefore, it was concerned as a potent drug for increasing insulin sensitivity in peripheral tissues. However, it can also interact with other enzymes, such as glycogen phosphorylase, meaning that it lack a specificity for AMPK(Gruzman et al., 2009). Traditional Chinese herbs have been found from many potential resources for the production of drugs to treat diabetes. Nstpbp168, a pure compound purified from methanol extracts of a fern plant Hypolepis punctata (Thunb.) Mett. Its chemical structure was identified as a small molecular weight natural product, pterosin A. Previous studies found that streptozotocin-induced diabetic mice (Type-1 diabetes) treated with Nstpbp168 could against glucose intolerance. In differentiated C2C12 13. .

(20) cellls, it also significan s tly promooted the glu ucose uptaake abilityy, improveed the insulin seensitivity, and enhannced AMP PK phosph horylationn level, wh hich in tturn regulaated carbo ohydrate an and fatty accid metabolism. In vvivo and in i vitrro studies indicated that Nstpbbp168 cou uld effectively decreeased PEP PCK mRN NA expresssion and ssignifican ntly improv ve the intrracellular glyycogen conntents in hepatotic h ccells. Furth hermore, db/db d diabbetic mousse isleet hypertroophy could d be improoved by trreated with h Nstpbp1168 and in n culttured β-ceells also sh howed thaat effectiveely attenuaated STZ--induced cell c deaath and NO O productiion, and innterleukin n-1β–increased NO prooduction(H Hsu et al., 2013). Thherefore, we w proposee that Nstppbp168 may m playy a protecctive role in i pancreaatic β-cell.. Ch hemical sttructure of o pterosin nA. Currentlly, using AMPK A agoonist or its stimulato or, combinned with exeercise and diet contrrol is the cclinical strrategy for diabetes th therapy (Krrentz and Bailey, B 20 005). Receently, seveeral natural herbs beeing able to o actiivate AMP PK are alsso alternattive option ns. As men ntioned abbove, we have h gott the nature compou und, Nstpbbp168, which have been b validdated to bee able to activaate AMPK K in the caase of in viivo or in vitro. This intrigues us to iinvestigatee its role in n metabollic related pancreatic and liveer cells and d to studdy whetheer it possess protectiive effectss under miimic oxidaative 14. .

(21) stress/lipotoxicity caused by H2O2/palmitate. We hope this study can provide more information of Nstbp168 in regards of its protective effects on pancreatic β-cell/liver cell in the development of diabetes.. 15. .

(22) Chapter 2. Materials and Methods. 2.1 Reagents The materials/reagents were purchased from companies indicated in the parentheses. RPMI-1640 and Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL-Life Technologies, Grand Island, NY, USA); Fetal bovine serum (Thermo Scientific, South Logan, Utah, USA); 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), hydrogen peroxide (H2O2), nitrotetrazolium blue chloride (NBT) and 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) (Sigma Chemical Co, St. Louis, MO, USA); 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyr imidine dihydrochloride (Compound C) (Tocris Bioscience, Bristal, UK); Anti-phospho AMPK alpha (Thr172) antibody (Cell Signaling Technology); Horseradish peroxidase anti-rabbit secondary antibody (Jackson, West Grove, PA, USA). Nstpbp168 (provided from Dr. Feng-Lin Hsu, Taipei Medical University, Taipei, Taiwan). 2.2 Cell Culture Rat pancreatic insulin secreting cell line, RINm5F, were obtained from the American Type Culture Collection (Rockville, MD). RINm5F were maintained in RPMI-1640 medium containing 10% fetal bovine serum, 1mM sodium pyruvate, 2 mM glutamine, 11.1 mM glucose, 24 mM NaHCO3, 2.5 g/l HEPES, 10,000 units/l of penicillin and 100 mg/l of streptomycin at 37°C in an incubator with a humidified atmosphere of 5% 16. .

(23) CO2. The growth rate of RINm5F was slow that the suspension would be able to attach onto culture dishes for 3 days after passage and it was necessary to maintain for additional 3 days to reach the confluence. Therefore, when performing pharmaceutical treatment, it is batter to let cell grow for at least 4 days so that the cell numbers could be enough to be applied with various concentrations of drugs. Human hepatocellular liver carcinoma cell line, HepG2 cells were purchased from American Type Culture Collection (Rockville, MD) and grown in DMEM supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, 100 lg/mL of streptomycin at 37°C under a humidified, 5% CO2 atmosphere.. 2.3 Palmitate/BSA complex preparation A stock palmitate solution was dissolved in absolute alcohol and 1% FFA-free BSA solutions were prepared in serum free medium. To prepare 250 μM palmitate /1% BSA stock solutions, 10 μl of 25 mM palmitate stock solution was added to 990 μl of 1% FFA-free BSA solution and then mixed by vortexing for 30 seconds followed by additional 1~2 hours of incubation at room temperature . All control conditions contained a solution of vehicle (absolute alcohol) mixed with FFA-free BSA as the abovementioned ratio. This mixture was so called palmitate/BSA solution.. 2.4 Cell viability assay Cell viability was measured by MTT assay. MTT was yellow 17. .

(24) tetrazole which could be reduced to insoluble purple formazon dye crystals by mitochondrial succinate dehydrogenase in living cells. Briefly, the RINm5F cell was seeded in a 24-well plat at a density of 2×105 cell/0.5 ml. After adhering for three days, cells began to expose to different treatment for variant time. Then, 50 μl MTT solution (1 mg/ml in phosphate-buffered saline (PBS)) was added to each well. The plate was further incubated for another 2 hours at 37 °C. The supernatant was removed and then 200 μl dimethyl sulphoxide (DMSO) was added to thoroughly dissolve the dark blue crystal formazan. The absorbance of the royal purple solution at 570 nm was detected with a spectrophotometer.. 2.5 Western blotting Cells were seeded in a 6-well plate at a density of 5×105 cells/2 ml and incubated for 6 days w/o drug treatment when the cell confluence was reaching 70-80%. After cells were harvested, 30μl of lysis buffer (20 mM Tris base, pH 7.9, 20 mM NaCl, 1 mM EDTA, 5 mM EGTA, 20 mM β-glycerolphosphate, 1 mM DTT, 1 mM PMSF in isopropanol, protease inhibitor, 25nM Calyculin A, 05.% TritonX-100) was added. Lysates were centrifuged at 13,000 rpm for 15 minutes. Aliquots of samples containing equal amounts of protein, measured by Bradford assay (Bio-Rad, Hercules, CA, USA), was resolved by SDS-PAGE for separation and transferred to PVDF membrane for immunoblotting. The membrane were blocked with TBST including 5% skim milk at room temperature for 1 h and then incubated with primary antibodies for AMPK alpha, phospho 18. .

(25) AMPK alpha(Thr172) in TBST at 4℃overnight. After washing with TBST, blots were visualized using horseradish peroxidase-conjugated secondary antibody followed by ECL detection (Millipore Corporation, Billerica, MA, USA).. 2.6 Determination of reactive oxygen species (ROS) The generation of ROS was examined by NBT assay. NBT is membrane permeable yellow-colored nitroblue tetrazolium (Choi et al., 2006). After absorbed by cells, NBT would interact with O2- to be reduced to blue formazon. Cells were seeded in a 24-well plate at a density of 2×105 cell/0.5 ml and were grown for three days. Cells were begun to expose to various drugs under variant time. After treatment, the plate was further incubated for 90 minutes at incubator. The supernatant was withdrawn and the pellet was washed with PBS one more time. The dark blue crystal formazan was solved in 400 μl dimethylsulphoxide (DMSO) and 600μl KOH (1N). The absorbance of the bluish green solution at 630 nm was detected with a spectrophotometer.. 2.7 Evaluation of ROS production by DCFH-DA A fluorometric assay, 2′, 7′-Dichlorofluorescin diacetate (DCFH-DA) is performed to detected intracellular oxidation. DCFH-DA crosses the cell membrane and then undergoes deacetylation by cellular esterases to non-fluorescent DCFH, which is quickly oxidized to highly fluorescent DCF by ROS (Rosenkranz et al., 1992). Cells were seeded on a cover-slip-loaded 12-well plate at a density of 3×105 cell/ ml. After 19. .

(26) adhering for three days, cells began to expose to different treatment for variant time. The cells were added with 10 μM DCFH-DA in no serum medium at incubator for 20 min. After washing the cells with PBS twice, digital images of DCF fluorescence were observed by a fluorescence microscope system at an excitation wavelength of 488 nm (argon laser) and a 515-nm long-pass emission filter.. 2.8 Glucose stimulated insulin secretion (GSIS) Isolated islets were incubated w/o Nstpbp168 in RPMI-1640 without glucose for 2 hours. Islets were washed once in RPMI-1640 without glucose and washed twice in RPMI-1640 containing 2.8 mM glucose, and then they were pre-incubated in the same medium for 30 min at 37°C. This buffer was then replaced with RPMI-1648 containing 2.8 mM or 16.7 mM glucose as indicated for a further 60 min at 37°C. Supernatants was then collected for analysis of insulin secretion by ELISA.. 2.9 Statistical Analysis Data are presented as mean ± SEM. Treatment effects were evaluated by using a two-tailed Student’s t test. A p value < 0.05 was considered to be statistically significant.. 20. .

(27) Chapter 3. Results. 3.1 The effects of Nstpbp168 on cell survival In previous study, we have found that Nstpbp168 could enhance fatty acid oxidation and glycolysis in animal model. Even more, it could reduce pancreatic cell hyperplasia in diabetic mouse model. This implied that Nstpbp168 may play a protective role in β-cell dysfunction during the progression of type 2 diabetes. Thereby, we intended to investigate the toxicity of Nstpbp168 on RINm5F by utilizing cell viability assay at first. After treated with Nstpbp168, 1 μM, 10 μM, 30 μM, 100 μM and DMSO for 18 hours, cells were applied to MTT assay. As shown in Fig 1A, Nstpbp168 did not display any cellular toxicity even up to 100 μM. Thus, the concentrations from 0 to 100 μM of Nstpbp168 would be further employed in the present study. H2O2 was indicated as an ideal stimulator for oxidative stress instead of β-cell-specific toxins, such as alloxan and streptozotocin because H2O2 is the prevailing production in most oxidative stress processes (Szkudelski, 2001). For determining the appropriate dose of H2O2, we exerted the prior study to test the dose-dependent viability losses caused by H2O2 on RINm5F cells. After treated with different doses of H2O2 (25– 100 μM) for 2 hours, cell survival was estimated by MTT assay. As indicated in Fig 1B, H2O2 dose-dependently induced cell death, with a maximum effect at 100 μM. Since 40 μM H2O2 caused 50% cell impairment, it was identified as efficient dose in the following experiments. 21. .

(28) 3.2 Scavenging capacity of Nstpbp168 on H2O2-induced ROS In advance, cells treated with various concentrations of Nstpbp168 in the absence or presence of 40 μM H2O2 were measured by MTT assay as indicated in Fig 1C. The survival rate of Nstpbp168 combined with 40 μM H2O2 was elevated in a little tendency compared to H2O2 treatment, implied that Nstpbp168 may have mild protective effects on H2O2-induced cell death, just by sustaining but not promoting cell viability. Therefore, we tried to raise Nstpbp168 up to 200 and 600 μM. Interestingly, efficient increasing survival rate was obtained in this range of higher concentration. Although the protective effect of 100 μM Nstpbp168 was mild in MTT assessment, we are still interested in analyzing the change of cellular ROS under these conditions. Intracellular ROS levels in H2O2-treated RINm5f β-cell were determined by using NBT assay which was shown in Fig 2. The ROS scavenging capacity of Nstpbp168 was presented in a dose dependent manner, especially at 100 μM. Hence, these data provided evidence that Nstpbp168 might exert as an anti-oxidant drug to reduce oxidative stress- induced cell death in β-cell.. 3.3 The anti-oxidant protective ability of Nstpbp168 is through activating AMPK In our previous research, we found that Nstpbp168 was a novel AMPK activator. Some reports had mentioned that phosphorylated AMPK would suppress nitric oxide induced apoptosis on β-cell (Nyblom et al., 2008). Therefore, in order to determine whether the cytoprotection 22. .

(29) of Nstpbp168 was due to activate AMPK, RINm5F cells were pretreated with the AMPK inhibitor, Compound C for 2 hours before applied with Nstpbp168 or H2O2 as presented in Fig 3C. Consequently, 20 μM Compound C attenuated the protective effect of Nstpbp168 on H2O2-induced oxidative stress, implying that the cytoprotective effect of Nstpbp168 might be partly mediated through AMPK activation. In order to further clarify the association, we tried to detect the AMPK phosphorylated level under different doses of Nstpbp168 in a short-term or long-term manner. Regardless short-term or long-term treatment, the pattern was similar as shown in Fig 3A. Phosphorylated AMPK was apparently increased under 75~100 μM Nstpbp168 treatment for 3 or18 hours, revealing that Nstpbp168 could activate AMPK in a short time incubation and the effect could prolong for an extend time scale. Because 100 μM Nstpbp168 performed well in activating AMPK no matter in 3hr or 18hr treatment, we subsequently chose this concentration to investigate its influence on H2O2-induced damage. Markedly, as indicated in Fig 3B, additional Nstpbp168 introduced into cells with 40 μM H2O2 pretreatment could promote higher AMPK activation than that of treated with Nstpbp168 or H2O2 alone. Owing to the protective role of 100 μM Nstpbp168 in preventing H2O2-inducing cell death (Fig 1C), preliminary data might also provide the insight that Nstpbp168 could regulate cellular physiological status through activating AMPK, even under oxidative stress.. 23. .

(30) 3.4 Nstpbp168 prevented Palmitate-induced lipotoxicity From the protective effects of Nstpbp168 under H2O2 -induced oxidative stress on pancreatic β cells, it is suggested that Nstpbp168 might behave as an anti-oxidant drug as well as an activator of AMPK. However, H2O2 is the production of oxidative stress which may be resulted from excess high glucose or lipid intake. Therefore, we were interested in studying how Nstpbp168 regulated cellular physiology or signal transduction under the stress of fatty acid overload. Here, we treated cells with various doses of palmitate to create an environment with over-loaded lipid. As indicated in Fig. 4A, the MTT assay revealed that palmitate treatment for 24 hours would cause cell death significantly in a dose-dependent manner from 100 to 500 μM and 250 μM palmitate led 50% leaithality efficiently. Therefore, we chose 250 μM palmitate for further studying its lipotoxic effects on pancreatic/liver cells. Notably, in Fig 4B and 4C, the MTT results showed that the cytotoxic effect of palmitate treatment would be reversed by administrated with Nstpbp168, particularly at 600 μM. While pretreated with Compound C before palmitate plus Nstpbp168 treatment, cells would display decreased survival rate implying that Nstpbp168 might protect cells from lipotoxicity through activating AMPK (Fig. 4D). Furthermore, in Fig 5, we used western blotting analysis to prove that phosphorylated level of AMPK would increase under different doses (50, 100, 250, 500 μM) of palmitate treatment for 24 hours. Nevertheless, the increased phosphorylated AMPK level would be further promoted in presence of palmitate-induced injury after exposured to Nstpbp168 for at 24. .

(31) least 24 hours as indicated in Fig 5B. According to the results, it is suggested that Nstpbp168 might have a protective role in reducing lipotoxicity- induced cell death in β-cell through activated AMPK. In advance, liver cell is also an important organ for lipid metabolism. In order to assess the protective effects of Nstpbp168 against the lipotoxicity on liver cell, HepG2, MTT assay was conducted. As shown in Fig. 6A, cells were treated with different dosage of palmitate for 24 hours and cell viability was declined respectively. The correspondent viability loss was achieved 50% under 350 μM palmitate treatment in HepG2. Pre-treated with Nstpbp168 at 10, 50 or 100 μM for 48 hours could present the protective potential to sustain cell viability from palmitate injury, although there was no significant variation when compared to the result of control as presented in Fig 6B. Fig 5A showed that the activity of AMPK was enhanced by palmitate treatment on RINm5F cells. Thus, we would like to verify whether it also employed the same effects on HepG2. Cells were applied with different doses of palmitate for 24 hours and compared the following results with that on RINm5F. However, from Fig 7A, the pattern of AMPK phosphorylation was not similar with that in RINm5F. Instead, 250μM palmitate could induce higher AMPK activity significantly, but not 350μM palmitate. As abovementioned, we knew that Nstpbp168 was an AMPK activator. Previous studies also indicated that a phenylpropanoid dibenzylbutyrolactone lignan from Arctium lappa L, Arctigenin could efficiently suppress HepG2 cells from palmitate-induced cell death via 25. .

(32) activating AMPK (Gu et al., 2012). These intrigued us to further investigate whether Nstpbp168 also exerted its suppressive effects on palmitate-induced damage through activating AMPK in HepG2. As shown in Fig. 7B, 10 μM and 50 μM Nstpbp168 pretreatment could enhance the palmitate-inducied phosphorylation of AMPK but not in 100 μM Nstpbp168 pretreatment , which is not consistent to that in RINm5F cells. This could only provide the possibility that Nstpbp168 might activate AMPK to prevent cell death from palmitate in HepG2 which is needed more evidence to be proved.. 3.5 Inhibition of palmitate-induced ROS production by Nstpbp168 Palmitate is recognized as a lipototoxic fatty acid which can elevate oxidative stress and ROS production to cause insulin resistance in hepatocytes (Shimano et al., 1999). Recent studies indicate that Luteolin could reduce lipid accumulation in HepG2 cells which was related to the activation of AMPK and mitigation of oxidative stress (Liu et al., 2011). Accordingly, the effects of Nstpbp168 on palmitate-induced ROS production would be detected by using DCFH-DA. Here we utilized fluorescence microscopy to observe the distribution of luminescence resulting from DCF formation. As illustrated in Fig. 8 & 9, treated RINm5F and HepG2 cells with palmitate could enhance the ROS level at the 24th hour apparently. However, from 10 to 100 μM Nstpbp168 attenuated the ROS generation in the palmitate-stimulated RINm5F and HepG2 cells dose dependently. AICAR was known as an AMPK activator also could diminish the palmitate-induced ROS production. 26. .

(33) Thus, corresponding to previous observation, we proposed that the ameliorated effect of palmitate-induced lipotoxicity by Nstpbp168 might through regulating AMPK activation to reduce ROS generation.. 3.6 Effects of Nstpbp168 on glucose-stimulated insulin secretion in mouse islets. Since Nstpbp168 could protect cells from H2O2 or palmitate-induced cell death or ROS injury and activate AMPK, we would like to evaluate whether Nstpbp168 stimulated rapid GSIS from mouse islets and whether the phenomenon is similar to that of AMPK activato, AICAR treatment. As shown in Fig. 10, after mouse islets were pre-incubated with 100 μM Nstpbp168 or AICAR for 2 hours, the response of islets to high glucose significantly decreased in the last 1 h incubation without Nstpbp168. This apparently indicated the inhibition effects of Nstpbp168 on insulin secretion like the result of AICAR.. 27. .

(34) Chapter 4. Discussion. Previous study has shown that Nstpbp168 employed as an AMPK activator is beneficial in promoting the ability of glucose uptake and improving insulin sensitivity in vivo and also in C2C12 muscular cell line. In present study, we further examined the role of Nstpbp168 on insulin-secreting pancreatic β-cells. The present result demonstrated that Nstpbp168 could significantly reduce H2O2/palmitate-induced oxidative stress and reduce ROS generation in insulin secreting cell line RINm5F. Additionally, the protective effect of Nstpbp168 on H2O2-induced cell death was significantly suppressed by AMPK inhibitor Compound C. It could enhance AMPK activity in RINm5F cells both in short-term and long-term treatment. Taken together, it is suggested that Nstpbp168 might have a protective potential on ROS-induced cell damage through mediating AMPK signaling pathway in insulin secreting cells.. 4.1 Nstpbp168 has antioxidant potential In our study, we utilized H2O2 as the source of oxidative stress but not oxidant toxin, such as streptozotocin. Although H2O2 is only by-production from oxidative stress, it is proposed to be good for studying oxidative issues due to its derived from oxygen-derived intermediate. Pancreatic abnormal glucose metabolism and long-term treatment of free fatty acid (FFAs) can elicit deficient mitochondrial function to raise H2O2 production gradually (Carlsson et al., 1999, Evans et al., 2003, Wang et al., 2004). Hence, H2O2 is supposed to be more 28. .

(35) closed to the real physiological status to mimic the oxidative situation directly. In previous literature, Nstpbp168 has been shown to attenuate NO production so it was also considered as an anti-NOS reagent. However, according to Fig 1C and Fig 2, we found that low-dose of Nstpbp168 such as 100 μM could not reverse the cell survival rate but show the ability of significant reducing ROS generation. This un-consistence perhaps is due to the deficient anti-oxidant enzymes in pancreatic β-cells so that low dose of Nstpbp168 could not protect β-cells well from oxidative stress. In spite of abovementioned phenomenon, it is notable that the remaining survived cells co-incubated under palmitate/H2O2 plus Nstpbp168 would generate apparently lower ROS than those with palmitate/H2O2 treatment alone via DCFH-DA/NBT analysis (Fig 2, Fig 8 and Fig 9). This implies that Nstpbp168 may behave like some Chinese herbs by which cellular anti-oxidant enzymes such as NADPH oxidase 4 (NOX4) can be activated to reduce ROS-caused damage though it can only sustain viability of β-cells in oxidative status (Xiong et al., 2006). Thus, we would like to take more efforts on exploring the activity of antioxidant enzymes under Nstpbp168 treatment in the future.. 4.2 Activated AMPK by Nstpbp168 can reduce oxidant-induced cellular damage How is active AMPK link to oxidative stress? When cells are under lower energy state, phosphorylation of AMPK can be triggered so as to enhance glucose utilization and fatty acid oxidation, and further to 29. .

(36) conduct cells away from lipogenesis during which superoxide can be accumulated to promote ROS generation (Zhang and Kim, 1995, Winder and Hardie, 1999, Zhou, 2001, Yamauchi et al., 2002). Based on such point of view, we propose that the AMPK activator Nstpbp168 might decrease ROS generation through activated AMPK directly or indirectly. Although we can’t clearly indicate these association in present data, we still can confirm that Nstpbp168 protecting cells from oxidative stress or lipotoxicity induced damage was via activating AMPK. This is due to compound C co-treatment not only attenuate AMPK activation but also destroyed the protective effect of Nstpbp168 on cell viability under H2O2/palmitate-inducing cell injury. (Fig 3C, 4C) However, whether active AMPK can attenuate ROS generation is still not very clear. So far according to previous investigations, AMPK activation would have positive effects against the functional impairment and cell mass of β-cells from glucotoxicity (Nyblom et al., 2008). Although the role of AMPK on β-cells is controversial, it was indicated that TSC2, the downstream of AMPK, coordinated signals from various pathways to regulate cell size, translation, and apoptosis which can protect cell death from an unfavorable growth environment (Inoki et al., 2003). In advance, AMPK activity may also be beneficial for promoting the physiological function of β-cells; for instance, the allosteric activator of AMPK such as AICAR can stimulate insulin release both in vitro and in vivo (Malaisse et al., 1994). Therefore, in our preliminary data, the activated AMPK induced by Nstpbp168 protecting cells from oxidative damage may be through abovementioned 30. .

(37) mecchanisms,, which is needed too be furtheer evidenced. Howevver, proolonged ovver activattion of AM MPK by AICAR or the t anti-diiabetic dru ug Meetformin has been in ndicated too lead β-ceells apopto osis (Kefaas et al., 20004).. AM MPK/TSC C2/mTOR R-signalingg pathwa ay (Bartolomé et al.., 2010). 4.3 Differentt level of palmitate p e-induced d AMPK activation a n and celllular dam mage in pa ancreatic cells and liver cells In Fig 4 and Fig 6, 6 pancreaatic cells or liver cells exposedd to high conncentrationn of palmiitate resultted in apparent cell death, imp mplying thaat it hadd evoked lipotoxicitty under suuch condittions. Thereby, we ppropose th hat oveer-loaded FFA F migh ht enhancee the proceess of β ox xidation annd corrrespondedd with the promotioon of ROS level to im mpair celll viability. 31. .

(38) Moreover, the following long-term accumulation of palmitoylated protein would cause the formation of ER stress which damage energy status and induce AMPK activation. The sequential phosphorylated AMPK may play an essential protective role to prevent lipogenesis, to reduce protein translation, to retard the apoptosis progress etc (Borradaile et al., 2006). In our study, we found that the palmitate-induced AMPK phosphorylation was dose-dependent in RINm5F but the phenomenon was not apparent in HepG2 as indicated in Fig 5 and Fig 7, which was consistent with those evidence in other studies(Sun et al., 2008). That may be related to the lower anti-oxidative enzymes presented in RINm5F so that palmitate treatment would stimulate more ROS production than that in HepG2. In addition, from Fig 4 and Fig 6, we found that the efficient cytotoxic dose of palmitate in HepG2 was 1.5-fold higher than that in β-cell. We supposed that two possibilities might related to such difference. First, palmitate treated time might also direct different expression level of AMPK. Other studies found that chronic palmitate exposure of β-cell for 48 hours would decrease phospho-AMPK, total AMPK protein levels as well as GSIS (Sun et al., 2008). Therefore, palmitate treatment for 24 hour might be as a chronic stimulated timing for HepG2, which have evoked overload damage, such as ER stress to inhibit AMPK expression. Second, as AMPKα can be divided to α1 and α2 predominantly distributing in different tissues, this may also direct different AMPK response. AMPKα1 is located in cytosol and AMPKα2 is in nucleus respectively, which two conduct different functions (da Silva 32. .

(39) Xavier et al., 2003). AMPKα1 is more abundant than AMPKα2 in β-cell, while it is converse in HepG2. Based on this point view, we suggest that the different results of palmitate treatment displayed in these two cells may be due to the variation of AMPKα1/α2 distribution which also relies on more efforts to prove.. 4.4 AMPK can be activated under H2O2/Palmitate stimulation Fig. 3, 5 and 7 showed that 40 µM H2O2, 250 µM or 350 µM palmitate could enhance AMPK Thr172 phosphorylation, while the phenomenon would be more obvious after plus Nstpbp168 treatment. Since AMPK plays as a cellular energy sensor to activate catabolic process and inhibit biosynthetic pathways, it is concerned as a protector when cells are facing stress-inducing damage, such as hypoxia. Markedly, two well-known signaling pathways, LKB and CAMKKβ, are involved in activating AMPK, respectively, whereas stress-induced calcium triggering CAMKKβ pathway may promote this effect without an increasing of AMP/ATP ratio. It is suggested that CAMKKβ signaling activated AMPK mediates a greater protection before promoted AMP/ATP ratio increasing AMPK phosphorylation. Accordingly, instant promotion in glucose uptaking or ATP producing derived from AMPK activation is indeed an important survival strategy in a lethal bioenergetics crisis (Emerling et al., 2009, Mungai et al., 2011). This is also corresponded to our observation. It is reported that before palmitae-induced lipotoxicity or H2O2-stimulated oxidative damage reach the point of cell lethality, AMPK would respond 33. .

(40) to the pro-oxidant conditions; however, chronic incubation of such stresses may consume ATP and evoke a cascade of ER stress, autophagy or apoptosis (Mayer and Belsham, 2010, Cardaci et al., 2012). Here, we used a short-term model to investigate the effects of abovementioned stresses, with which the exposure timing might be prolonged to study the variation of AMPK activation. Moreover, it is notable that Nstpbp168 treatment increased the pro-oxidant stimulated AMPK actions, which confirm the works by Fediuc et al. (Fediuc et al., 2006). As described in the former paper, we suggest Nstpbp168 might elicit fatty acid oxidation to provide cellular energy under palmitate treatment at 250 µM on RINm5f cells or 350 µM on HepG2 through activating AMPK. On the other hand, the effects may be abolished when the concentration of palmitate is raised to higher level that may augment the lethal toxicity. In the future, it is relied on more experiments to clarify the regulated mechanisms involved in such process.. 4.5 Activation of AMPK is essential in early onset of diabetes As mentioned in introduction, AMPK activation leads to inhibit the exocytosis of insulin, which is proposed to be beneficial for suppressing high glucose or high fat induced β-cell hyperplasia during the early onset of diabetes (Kim et al., 2007). This point of view is consistent with our data as shown in Fig. 10 indicated that Nstpbp168 and AICAR could inhibit glucose- stimulated insulin secretion (GSIS) as compared to that of control. However, some studies found that short-term AICAR treatment could enhance the GSIS, whereas long-term berberin treatment 34. .

(41) wouuld suppreess insulin n secretionn (Zhou ett al., 2008)). Therefoore, it is neeeded to eluucidate thee GSIS unnder palmiitate or H2O2 co-treaated with Nsttpbp168 inn advance.. 4.6 Conclusiion It is sugggested thaat Nstpbp1168 could protect in nsulin secrreting cells m ROS annd palmitaate-induceed cell dam mage. Therrefore, it m might be a from novvel therapeeutic drug g to prevennt β-cells failure f durring the deevelopmen nt of ttype 2 diabbetes.. 35. .

(42) References. Bartolomé A, Guillén C, Benito M (2010) Role of the TSC1-TSC2 Complex in the Integration of Insulin and Glucose Signaling Involved in Pancreatic β-Cell Proliferation. Endocrinology 151:3084-3094. Birk JB, Wojtaszewski JFP (2006) Predominant α2/β2/γ3 AMPK activation during exercise in human skeletal muscle. The Journal of physiology 577:1021-1032. Borradaile NM, Han X, Harp JD, Gale SE, Ory DS, Schaffer JE (2006) Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death. Journal of Lipid Research 47:2726-2737. Cardaci S, Filomeni G, Ciriolo MR (2012) Redox implications of AMPK-mediated signal transduction beyond energetic clues. J Cell Sci 125:2115-2125. Carlsson C, Håkan Borg LA, Welsh N (1999) Sodium Palmitate Induces Partial Mitochondrial Uncoupling and Reactive Oxygen Species in Rat Pancreatic Islets in Vitro. Endocrinology 140:3422-3428. Choi HS, Kim JW, Cha YN, Kim C (2006) A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. Journal of immunoassay & immunochemistry 27:31-44. Corton JM, Gillespie JG, Hawley SA, Hardie DG (1995) 5-Aminoimidazole-4-Carboxamide Ribonucleoside. European Journal of Biochemistry 229:558-565. 36. .

(43) da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, Rutter GA (2003) Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression. Biochem J 371:761-774. Dröge W (2002) Free Radicals in the Physiological Control of Cell Function. Physiological Reviews 82:47-95. El-Assaad W, Buteau J, Peyot M-L, Nolan C, Roduit R, Hardy S, Joly E, Dbaibo G, Rosenberg L, Prentki M (2003) Saturated Fatty Acids Synergize with Elevated Glucose to Cause Pancreatic β-Cell Death. Endocrinology 144:4154-4163. Emerling BM, Weinberg F, Snyder C, Burgess Z, Mutlu GM, Viollet B, Budinger GR, Chandel NS (2009) Hypoxic activation of AMPK is dependent on mitochondrial ROS but independent of an increase in AMP/ATP ratio. Free Radic Biol Med 46:1386-1391. Eto K, Yamashita T, Matsui J, Terauchi Y, Noda M, Kadowaki T (2002) Genetic Manipulations of Fatty Acid Metabolism in β-Cells Are Associated With Dysregulated Insulin Secretion. Diabetes 51:S414-S420. Evans JL, Goldfine ID, Maddux BA, Grodsky GM (2003) Are Oxidative Stress−Activated Signaling Pathways Mediators of Insulin Resistance and β-Cell Dysfunction? Diabetes 52:1-8. Fediuc S, Gaidhu MP, Ceddia RB (2006) Regulation of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation by palmitate in skeletal muscle cells. J Lipid Res 47:412-420. Fonseca SG, Gromada J, Urano F (2011) Endoplasmic reticulum stress 37. .

(44) and pancreatic ²-cell death. Trends in endocrinology and metabolism: TEM 22:266-274. Fu A, Eberhard CE, Screaton RA (2013) Role of AMPK in pancreatic beta cell function. Molecular and Cellular Endocrinology 366:127-134. Gruzman A, Babai G, Sasson S (2009) Adenosine Monophosphate-Activated Protein Kinase (AMPK) as a New Target for Antidiabetic Drugs: A Review on Metabolic, Pharmacological and Chemical Considerations. The review of diabetic studies : RDS 6:13-36. Gu Y, Sun X-x, Ye J-m, He L, Yan S-s, Zhang H-h, Hu L-h, Yuan J-y, Yu Q (2012) Arctigenin alleviates ER stress via activating AMPK. Acta Pharmacol Sin 33:941-952. Hardie DG, Carling D (1997) The AMP-Activated Protein Kinase. European Journal of Biochemistry 246:259-273. Hardie DG, Carling D, Carlson M (1998) The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67:821-855. Hardie DG, Scott JW, Pan DA, Hudson ER (2003) Management of cellular energy by the AMP-activated protein kinase system. FEBS Letters 546:113-120. Higa M, Zhou Y-T, Ravazzola M, Baetens D, Orci L, Unger RH (1999) Troglitazone prevents mitochondrial alterations, β cell destruction, and diabetes in obese prediabetic rats. Proceedings of the National Academy of Sciences 96:11513-11518. 38. .

(45) Hou N, Torii S, Saito N, Hosaka M, Takeuchi T (2008) Reactive Oxygen Species-Mediated Pancreatic β-Cell Death Is Regulated by Interactions between Stress-Activated Protein Kinases, p38 and c-Jun N-Terminal Kinase, and Mitogen-Activated Protein Kinase Phosphatases. Endocrinology 149:1654-1665. Hsu F-L, Huang C-F, Chen Y-W, Yen Y-P, Wu C-T, Uang B-J, Yang R-S, Liu S-H (2013) Antidiabetic Effects of Pterosin A, a Small-Molecular-Weight Natural Product, on Diabetic Mouse Models. Diabetes 62:628-638. Inoki K, Zhu T, Guan K-L (2003) TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival. Cell 115:577-590. Kaneto H, Kawamori D, Matsuoka T-a, Kajimoto Y, Yamasaki Y (2005) Oxidative Stress and Pancreatic [beta]-Cell Dysfunction. American Journal of Therapeutics 12:529-533. Kefas BA, Cai Y, Kerckhofs K, Ling Z, Martens G, Heimberg H, Pipeleers D, Casteele MVd (2004) Metformin-induced stimulation of AMP-activated protein kinase in β-cells impairs their glucose responsiveness and can lead to apoptosis. Biochemical Pharmacology 68:409-416. Kim D-S, Jeong S-K, Kim H-R, Kim D-S, Chae S-W, Chae H-J (2010) Metformin regulates palmitate-induced apoptosis and ER stress response in HepG2 liver cells. Immunopharmacology and Immunotoxicology 32:251-257. Kim W-H, Lee JW, Suh YH, Lee HJ, Lee SH, Oh YK, Gao B, Jung MH (2007) AICAR potentiates ROS production induced by chronic 39. .

(46) high glucose: Roles of AMPK in pancreatic β-cell apoptosis. Cellular Signalling 19:791-805. Krentz AJ, Bailey CJ (2005) Oral Antidiabetic Agents: Current Role in Type 2 Diabetes Mellitus. Drugs 65:385-411. Liu J-F, Ma Y, Wang Y, Du Z-Y, Shen J-K, Peng H-L (2011) Reduction of lipid accumulation in HepG2 Cells by luteolin is associated with activation of AMPK and Mitigation of oxidative stress. Phytotherapy Research 25:588-596. Malaisse WJ, Conget I, Sener A, Rorsman P (1994) Insulinotropic action of AICA riboside. II. Secretory, metabolic and cationic aspects. Diabetes Res 25:25-37. Mayer CM, Belsham DD (2010) Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation. Endocrinology 151:576-585. Mohler ML, He Y, Wu Z, Hwang DJ, Miller DD (2009) Recent and emerging anti-diabetes targets. Medicinal research reviews 29:125-195. Momcilovic M, Hong S-P, Carlson M (2006) Mammalian TAK1 Activates Snf1 Protein Kinase in Yeast and Phosphorylates AMP-activated Protein Kinase in Vitro. Journal of Biological Chemistry 281:25336-25343. Mungai PT, Waypa GB, Jairaman A, Prakriya M, Dokic D, Ball MK, Schumacker PT (2011) Hypoxia triggers AMPK activation through 40. .

(47) reactive oxygen species-mediated activation of calcium release-activated calcium channels. Mol Cell Biol 31:3531-3545. Numazawa S, Sakaguchi H, Aoki R, Taira T, Yoshida T (2008) Regulation of the susceptibility to oxidative stress by cysteine availability in pancreatic β-cells. American Journal of Physiology Cell Physiology 295:C468-C474. Nyblom HK, Sargsyan E, Bergsten P (2008) AMP-activated protein kinase agonist dose dependently improves function and reduces apoptosis in glucotoxic beta-cells without changing triglyceride levels. Journal of molecular endocrinology 41:187-194. Riboulet-Chavey A, Diraison F, Siew LK, Wong FS, Rutter GA (2008) Inhibition of AMP-Activated Protein Kinase Protects Pancreatic β-Cells From Cytokine-Mediated Apoptosis and CD8+ T-Cell– Induced Cytotoxicity. Diabetes 57:415-423. Richards SK, Parton LE, Leclerc I, Rutter GA, Smith RM (2005) Over-expression of AMP-activated protein kinase impairs pancreatic {beta}-cell function in vivo. The Journal of endocrinology 187:225-235. Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W, Zlabinger GJ (1992) A microplate assay for the detection of oxidative products using 2′,7′-dichlorofluorescin-diacetate. Journal of Immunological Methods 156:39-45. Shimabukuro M, Ohneda M, Lee Y, Unger RH (1997) Role of nitric oxide in obesity-induced beta cell disease. The Journal of Clinical Investigation 100:290-295. 41. .

(48) Shimano H, Yahagi N, Amemiya-Kudo M, Hasty AH, Osuga J-i, Tamura Y, Shionoiri F, Iizuka Y, Ohashi K, Harada K, Gotoda T, Ishibashi S, Yamada N (1999) Sterol Regulatory Element-binding Protein-1 as a Key Transcription Factor for Nutritional Induction of Lipogenic Enzyme Genes. Journal of Biological Chemistry 274:35832-35839. Sun Y, Ren M, Gao G-q, Gong B, Xin W, Guo H, Zhang X-j, Gao L, Zhao J-j (2008) Chronic palmitate exposure inhibits AMPK[alpha] and decreases glucose-stimulated insulin secretion from [beta]-cells: modulation by fenofibrate. Acta Pharmacol Sin 29:443-450. Supale S, Li N, Brun T, Maechler P (2012) Mitochondrial dysfunction in pancreatic ² cells. Trends in endocrinology and metabolism: TEM 23:477-487. Szkudelski T (2001) The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 50:537-546. Tiedge M, Lortz S, Drinkgern J, Lenzen S (1997) Relation Between Antioxidant Enzyme Gene Expression and Antioxidative Defense Status of Insulin-Producing Cells. Diabetes 46:1733-1742. Tiedge M, Lortz S, Munday R, Lenzen S (1998) Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species. Diabetes 47:1578-1585. Towler MC, Hardie DG (2007) AMP-Activated Protein Kinase in Metabolic Control and Insulin Signaling. Circulation Research 42. .

(49) 100:328-341. Treebak JT, Birk JB, Rose AJ, Kiens B, Richter EA, Wojtaszewski JFP (2007) AS160 phosphorylation is associated with activation of α2β2γ1- but not α2β2γ3-AMPK trimeric complex in skeletal muscle during exercise in humans. American Journal of Physiology - Endocrinology And Metabolism 292:E715-E722. Ugaz AG, Resnick T (2008) Assessing print and electronic use of reference/core medical textbooks. Journal of the Medical Library Association : JMLA 96:145-147. Viollet B, Lantier L, Devin-Leclerc J, Hebrard S, Amouyal C, Mounier R, Foretz M, Andreelli F (2009) Targeting the AMPK pathway for the treatment of Type 2 diabetes. Front Biosci 14:3380-3400. Wang X, Li H, De Leo D, Guo W, Koshkin V, Fantus IG, Giacca A, Chan CB, Der S, Wheeler MB (2004) Gene and Protein Kinase Expression Profiling of Reactive Oxygen Species-Associated Lipotoxicity in the Pancreatic β-Cell Line MIN6. Diabetes 53:129-140. Wild S, Roglic G, Green A, Sicree R, King H (2004a) Global Prevalence of Diabetes. Diabetes care 27:1047-1053. Wild S, Roglic G, Green A, Sicree R, King H (2004b) Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes care 27:1047-1053. Winder WW, Hardie DG (1999) AMP-activated protein kinase, a metabolic master switch: possible roles in Type 2 diabetes. American Journal of Physiology - Endocrinology And Metabolism 43. .

(50) 277:E1-E10. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LGD, Neumann D, Schlattner U, Wallimann T, Carlson M, Carling D (2003) LKB1 Is the Upstream Kinase in the AMP-Activated Protein Kinase Cascade. Current Biology 13:2004-2008. Xiao B, Heath R, Saiu P, Leiper FC, Leone P, Jing C, Walker PA, Haire L, Eccleston JF, Davis CT, Martin SR, Carling D, Gamblin SJ (2007) Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature 449:496-500. Xiong FL, Sun XH, Gan L, Yang XL, Xu HB (2006) Puerarin protects rat pancreatic islets from damage by hydrogen peroxide. European journal of pharmacology 529:1-7. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T (2002) Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 8:1288-1295. Zhang S, Kim K-H (1995) Glucose activation of acetyl-CoA carboxylase in association with insulin secretion in a pancreatic β-cell line. Journal of Endocrinology 147:33-41. Zhou G (2001) Role of AMP-activated protein kinase in mechanism of metformin action. Journal of Clinical Investigation 108:1167-1174. Zhou L, Wang X, Shao L, Yang Y, Shang W, Yuan G, Jiang B, Li F, Tang J, Jing H, Chen M (2008) Berberine acutely inhibits insulin 44. .

(51) secretion from beta-cells through 3',5'-cyclic adenosine 5'-monophosphate signaling pathway. Endocrinology 149:4510-4518.. 45. .

(52) Figgure 1.. A.. B.. C.. D... 46. .

(53) Figure 1. Nstpbp168 could suppress H2O2-induced cytotoxicity in a dose-dependent manner. (A) RINm5f cells were exposed to different dosage of Nstpbp168, 1 μM, 10 μM, 30 μM, 100 μM and DMSO for 18 hours. DMSO was used as a solvent control. (B) Cells were starved for half hour in Krebs-Ringer bicarbonate buffer (KRBH), and then exposed to 25 μM, 30 μM, 40 μM, 50 μM, 100μM H2O2 induced oxidative stress for 2 hours. (C, D) After starvation in KRBH buffer for half hour, RINm5f was treated with 40 μM H2O2 for 2 hours. Followed by H2O2 withdraw, cells were incubated in medium w/o variant dose of Nstpbp168 for additional 18 hours. Cell viability was measured by MTT Assay as Material and Methods described. Data was shown as mean ± SEM (n=4). * P< 0.05 VS. CTL .. 47. .

(54) Figgure 2.. uppressivee effect off Nstpbp1 168 on thee productiion of Figgure 2. Su H2O2-induceed ROS. After A co-inncubated 40 4 μM H2O2, cells w were then treaated with different d doses d of N Nstpbp168 for 18 ho ours. The R ROS levells werre identified by usin ng NBT asssay as described. Data D was sshown as mean ± SEM M (n=4). * P< 0.05 V VS. The grroup with 40 μM H2 O2 treaatment.. 48. .

(55) Figgure 3. A.. B.. C.. 49. .

(56) Figure 3. The protective effect of Nstpbp168 on H2O2-induced cell damage through activation of AMPK. (A) RINm5f cells were seeded on 6 well plates for treatment with different doses of Nstpbp168, 30 μM, 50 μM, 75 μM, 100 μM for 3 hours and 18 hours. (B) RINm5f cells were seeded on a 6 well plate incubated with 40 μM of H2O2 in the absence or presence of Nstpbp168 for 3 hours. (C) After treated with AMPK inhibitor (Compound C) for 2 hours, the cells were incubated with H2O2 for 2 hours and then exposed to Nstpbp168 for 18h. Metformin and no serum treatment were included as positive controls for the primary antibody of phospho-T172 AMPK. Cell viability was measured using the MTT assay as described. Data was shown as mean ± SEM (n=4). * P< 0.05 VS. The group of 40 μM H2O2 treatment.. 50. .

(57) Figgure 4. A.. . B.. C. C. D.. 51. .

(58) Figure 4. The effect of Nstpbp168 on palmitate-induced lipotoxicity in RINm5F. (A) RINm5f was exposed to palmitate 25 μM, 100 μM, 250 μM, 500 μM for 24 hours. (B, C) Cells were co-treated with increasing concentrations of Nstpbp168 and 250 μM palmitate for 24 hours. (D) Cells were pre-treated with 20 μM Compound C for 2 hours followed by 250 μM palmitate w/o Nstpbp168 co-treatment for 24 hours. (B, C, D) Cell viability was determined by MTT assay. EtOH was included as a solvent control. AICAR was as positive control. Data was shown as mean ± SEM (n=4). * P< 0.05 , *** P< 0.005. 52. .

(59) Figgure 5. A.. B.. he effect of o Nstpbp 168 on AM MPK actiivation paalmitate Figgure 5. Th treaated RIN Nm5F. (A, B) The treeatment procedures were sim milar as desscribed in Fig. 4 A, B and AM MPK was detected d by westernn blotting assay.. 53. .

(60) Figgure 6. A.. B.. he protecttive effectt of Nstpb bp168 on HepG2 H ceells with or o Figgure 6. Th witthout palm mitate treeatment. ((A) HepG2 2 cells weere treatedd under diffferent conncentration ns of palm mitate 100 μM, 250 μM, μ 350 μμM, 500 μM for 24 hours to induce injury. (B B) Cells weere pre-treeat with N Nstpbp168 for 18 hours andd then co-incubated with 350 μM palmiitate for annother 24 houurs. Data was w shown n as meann ± SEM (n n=4). * P< < 0.05, * **P< 0.01. 54. .

(61) Figgure 7. A... B.. Figgure 7. Th he effect of o Nstpbp 168 on AM MPK exp pression in n pallmitate-trreated Hep pG2 cellss. (A) Western blottiing analyssis for the phoosphorylattion of AM MPKα andd total AM MPK underr the differrent conncentrationn of palmiitate treateed for 24 hours. h (B) The cells were incuubated wiith 10, 50 and 100 μ μM Nstpbp p168 for th he indicate ted times in i the absence or o presencce of palm mitate at 35 50 μM for 24 hours. AICAR treaatment waas as positiive controol. 55. .

(62) Figgure 8. Palm mitate. C CTL. Palm mitate + Nstp pbp168 10 μM M. DCF FH-DA. . . . . . . Hochest. Pallmitate + Nsstpbp168 50 μM. Pallmitate + Nstpbp168 100 0 μM. Paalmitate + AICAR A 100 μM. DCF FH-DA. . . . . . . Hoch hest. 56. .

(63) Figure 8. Imagination of DCFH-DA stained ROS in palmitate treated RINm5f cells with or without Nstpbp168/AICAR co-administration. Cells were exposure to various concentrations of Nstpbp168 in the presence of palmitate (250 μM). Cells were stained with Hoechst for 1 hour and then loaded with 10 μM DCFH-DA for 20 minutes. After cell was washed twice with PBS, the intracellular ROS production determined by DCF formation was observed by fluorescent images (original magnification 200×). The green staining is DCFH-DA and the blue staining is Hoechst. AICAR was applied as positive control. Data are selected from three separate experiments per group.. 57. .

(64) Figgure 9. Palmita ate. CTL. Palm mitate + Nstp pbp168 10 μM μ. DCFH--DA. . . . . . Hochesst. Palmittate + Nstpb bp168 50 μM M. Palmitaate + Nstpbp p168 100 μM M. . Palmit itate + AICA AR 100 μM. DCFH-D DA. . . . . . . Hochestt. 58. .

(65) Figure 9. Effects of Nstpbp168 on intracellular ROS scavenging in HepG2. Cells were treated with 350 μM palmitate in the absence or presence of Nstpbp168 (10, 50 and 100 μM). Cells were stained with DCFH-DA and Hochest as described in Figure 6. Also, the ROS production was detected by visualizable fluorescent images (200×). Data are selected from three separate experiments per group.. 59. .

(66) Figgure 10.. ＃. *. ＃. *. E of Nstpbp16 N 68 on gluccose-stimu ulated inssulin Figgure 10. Effects seccretion in mouse isllets. Isletss (25 isletss/well) were incubatted in RPM MI-1640 with100 w μM μ AICAR R or Nstpb bp 168 in the presennce or abssence of 2..8 mM or 16.7 mM glucose for fo 1 hour at 37°C. IInsulin secreted into the superrnatants w was measurred by ELIISA. Valuues were exppressed as mean ± SEM S (n=3)). #, p < 0.05 vs. 2.8 8 mM gluccose-treateed isleets ; *, p <0.05 < vs. 16.7 mM gglucose alo one-treated islets.. 60. .

(67)