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，而這些保護的功能可能均是藉由促進AMPK活化的機制所達成。

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.

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