Ghrelin对高糖诱导下胰岛β细胞凋亡的作用
摘要
背景与目的无论1型糖尿病还是2型糖尿病,诊断时β细胞已丧失50-80%。β细胞数量减少与细胞凋亡有关,糖尿病时胰岛β细胞凋亡加速;另外,糖尿病患者长期处于高血糖状态,使“高糖毒性”作用诱导的β细胞凋亡进一步加重,形成恶性循环。β细胞凋亡主要包括外源性(死亡受体介导的信号途径)、内源性凋亡途径(线粒体途径)以及穿孔素/颗粒酶B途径,其中bcl-2家族不仅通过改变线粒体外膜通透性而调控线粒体活化,在线粒体诱导的内源性凋亡途径中起重要作用,还起着连接β细胞三条凋亡途径的作用。动物实验研究显示生长激素释放肽(ghrelin)可阻止β细胞破坏所致的糖尿病;其他研究也表明ghrelin可抑制细胞饥饿、阿霉素及细胞因子等诱导的胰岛β细胞及其细胞株的凋亡。目前ghrelin对高糖环境下诱导的β细胞凋亡的作用尚不清楚。本研究拟ghrelin预处理高糖培养的小鼠胰岛β细胞株NIT-1细胞,检测β细胞凋亡率及其bcl-2、bax mRNA表达水平,探讨ghrelin对高糖诱导下胰岛β细胞凋亡的作用。
材料与方法(1)葡萄糖浓度为5.6mmol/L的DMEM常规培养NIT-1细胞,倒置显微镜下观察细胞培养过程中的形态学特点,生长方式。(2)Annexin V-FITC/PI双染和流式细胞仪联合测定不同处理组:对照组(葡萄糖浓度5.6mmol/L,C)、ghrelin预处理组(10-7mmol/L ghrelin,GP)、高浓度葡萄糖诱导凋亡组(葡萄糖浓度33.3mmol/L,HG)、ghrelin预处理+高浓度葡萄糖组(GP+HG)的NIT-1细胞早期凋亡率。(3)Hoechst 33258标记细胞核,在荧光显微镜下检测不同处理组浓集核和碎裂核的数目,计算各组的细胞总凋亡率。(4)RT-PCR分别测定不同处理组bcl-2、bax基因mRNA表达量的变化,电泳后进行灰度值分析,得出不同组bcl-2、bax的相对表达量。
结果(1) NIT-1细胞为多边形上皮样细胞,单层簇状生长,排列紧密。(2)流式细胞仪Annexin V-FITC/PI双染及Hoechst 33258测定细胞凋亡率显示,与对照组NIT-1细胞早期凋亡率及总凋亡率比较,Ghrelin预处理组无显著差异(P均>0.05)、高糖组增加(P均<0.01);与单纯高糖组比较,Ghrelin预处理+高糖组细胞早期凋亡率及总凋亡率降低(P<0.01,P<0.05)。(3) RT-PCR结果显示:与对照组比较,Ghrelin预处理组bax、bcl-2、bcl-2/bax水平无变化,而高糖组bcl-2、bcl-2/bax水平下降(P<0.05);与单纯高糖组比较,Ghrelin预处理+高糖组bcl-2、bcl-2/bax水平有升高趋势,但无统计学差异;各组bax表达水平无差别。
结论(1) Ghrelin对正常葡萄糖浓度培养的胰岛β细胞凋亡无影响,但可抑制高浓度葡萄糖诱导的NIT-1胰岛β细胞凋亡增加。提示高浓度的葡萄糖对β细胞有毒性作用,Ghrelin可能对高浓度葡萄糖诱导的β细胞凋亡有保护作用。(2) Ghrelin抑制β细胞凋亡的同时,Bcl-2 mRNA及bcl-2/bax表达水平有升高趋势,但无统计学差异,这可能与β细胞类型、刺激因子的不同等有关,也不排除与其他细胞凋亡途径相关因子有关。
BACKGROUND AND OBJECTIVE Regardless of type 1 or type 2 diabetes being newly diagnosed,βcell mass has lost about 50-80%.Decreasedβcell mass is associated with its apoptosis,andβcell apoptosis is accelerated in diabetes.Moreover,chronic hyperglycemia makes glucotoxicity-inducedβcell apoptosis more severe in diabetes, forming a vicious circle.βcell apoptosis effects mainly through the following three pathophysiological pathways:extrinsic apoptotic pathway (death receptors mediated signal pathway),intrinsic apoptotic pathway (mitochondrial pathway) and perforin/granzyme B pathway.Bcl-2 family not only regulates mitochondrial activation through changing mitochondrial outer membrane permeability,and plays an important role in mitochondrion induced intrinsic pathway,it also connects the three apoptotic pathways. It is found that ghrelin,also called as growth hormone release peptide,may prevent development of diabetes resulted fromβcell damage.And ghrelin can also inhibitβcell apoptosis induced by serum starvation,doxorubicin,and some cytokines.However,it is unclear about the effects of ghrelin on high glucose-inducedβcell apoptosis.This study in vitro is to pretreat high glucose cultured mouse pancreatic isletβ-cell line NIT-1 cell with ghrelin,and determine cell apoptotic rates and expression of bcl-2 and bax.Then,to investigate the effects of ghrelin on high glucose-inducedβcell apoptosis.
METHODS
(1) NIT-1cells were cultured in DMEM culture medium,which containing 5.6mmol/L glucose.To observe the morphology characteristics and its growth style under inverted microscope.(2) NIT-1 cells were divided into four groups,control group(5.6mmol/L glucose,C),ghrelin pretreatment group(10-7mmol/Lghrelin,GP),high glucose-induced apoptosis group (33.3mmol/L glucose,HG), ghrelin pretreatment +high glucose group(GP+HG).NIT-1 cell early apoptotic rates were tested by Annexin V-FITC/PI double staining and flowcytometry technique.(3) NIT-1 cell total apoptotic rates were evaluated by Hoechst 33258. NIT-1 cells were stained with Hoechst 33258 to detect apoptotic nuclei showing features of condensation and fragmentation under fluorescence microscope. (4) Expression of Bcl-2 and Bax in four groups were assayed by reverse transcription ploymerase chain reaction (RT-PCR) method. After DNA gel eletrophoresis,then to analyze the gray value and reach the relative expression of bcl-2 and bax.
RESULTS
(1) NIT-1 cells is polygon epithelial-like cells,growing monolayer-clustered and arranged densely.
(2) Both Annexin V/PI FACS and Hoechst33258 results indicated that ghrelin inhibits high glucose-induced NIT-1 cell apoptosis.The early apoptotic rates and total apoptotic rates were no significant difference in GP group(P>0.05),but increased significantly in HG group (P<0.01),as compared with C group.However,the early and total apoptotic rates were significantly lower in GP+HG group than in HG group(P<0.01,P<0.05). (3) RT-PCR results show that ghrelin had no effects on bcl-2 and bax expression in high glucose-induced NIT-1 cell. The level of bax mRNA was no significant difference in all groups. The level of bcl-2 mRNA, bcl-2/bax were decreased significantly in HG group(P<0.05), but no significant difference in GP group (P>0.05), as compared with C group. Unexpectedly, The level of bcl-2 mRNA, bcl-2/bax were no significant difference between GP+HG group and HG group,just an increasing trend was seen in the former.
CONCLUSIONS
(1) Ghrelin has no effects on pancreatic isletβcell apoptosis cultured in normal glucose,however,it can inhibit high glucose-induced NIT-1 cell apoptosis.It is indicated high glucose has toxic effects onβcells,and ghrelin may have proctective effects on high glucose- inducedβcell apoptosis.
(2) As ghrelin inhibitingβcell apoptosis,there is still an increasing trend of bcl-2 and bcl-2/bax expression inβcell,although it is no statistic significance.We speculates that ghrelin’s effects onβcell apoptosis maybe relates to theβcell types and stimulating factors.However, it could not be precluded that ghrelin inhibitingβcell apoptosis is through other apoptotic pathway associated factors.
材料与方法(1)葡萄糖浓度为5.6mmol/L的DMEM常规培养NIT-1细胞,倒置显微镜下观察细胞培养过程中的形态学特点,生长方式。(2)Annexin V-FITC/PI双染和流式细胞仪联合测定不同处理组:对照组(葡萄糖浓度5.6mmol/L,C)、ghrelin预处理组(10-7mmol/L ghrelin,GP)、高浓度葡萄糖诱导凋亡组(葡萄糖浓度33.3mmol/L,HG)、ghrelin预处理+高浓度葡萄糖组(GP+HG)的NIT-1细胞早期凋亡率。(3)Hoechst 33258标记细胞核,在荧光显微镜下检测不同处理组浓集核和碎裂核的数目,计算各组的细胞总凋亡率。(4)RT-PCR分别测定不同处理组bcl-2、bax基因mRNA表达量的变化,电泳后进行灰度值分析,得出不同组bcl-2、bax的相对表达量。
结果(1) NIT-1细胞为多边形上皮样细胞,单层簇状生长,排列紧密。(2)流式细胞仪Annexin V-FITC/PI双染及Hoechst 33258测定细胞凋亡率显示,与对照组NIT-1细胞早期凋亡率及总凋亡率比较,Ghrelin预处理组无显著差异(P均>0.05)、高糖组增加(P均<0.01);与单纯高糖组比较,Ghrelin预处理+高糖组细胞早期凋亡率及总凋亡率降低(P<0.01,P<0.05)。(3) RT-PCR结果显示:与对照组比较,Ghrelin预处理组bax、bcl-2、bcl-2/bax水平无变化,而高糖组bcl-2、bcl-2/bax水平下降(P<0.05);与单纯高糖组比较,Ghrelin预处理+高糖组bcl-2、bcl-2/bax水平有升高趋势,但无统计学差异;各组bax表达水平无差别。
结论(1) Ghrelin对正常葡萄糖浓度培养的胰岛β细胞凋亡无影响,但可抑制高浓度葡萄糖诱导的NIT-1胰岛β细胞凋亡增加。提示高浓度的葡萄糖对β细胞有毒性作用,Ghrelin可能对高浓度葡萄糖诱导的β细胞凋亡有保护作用。(2) Ghrelin抑制β细胞凋亡的同时,Bcl-2 mRNA及bcl-2/bax表达水平有升高趋势,但无统计学差异,这可能与β细胞类型、刺激因子的不同等有关,也不排除与其他细胞凋亡途径相关因子有关。
BACKGROUND AND OBJECTIVE Regardless of type 1 or type 2 diabetes being newly diagnosed,βcell mass has lost about 50-80%.Decreasedβcell mass is associated with its apoptosis,andβcell apoptosis is accelerated in diabetes.Moreover,chronic hyperglycemia makes glucotoxicity-inducedβcell apoptosis more severe in diabetes, forming a vicious circle.βcell apoptosis effects mainly through the following three pathophysiological pathways:extrinsic apoptotic pathway (death receptors mediated signal pathway),intrinsic apoptotic pathway (mitochondrial pathway) and perforin/granzyme B pathway.Bcl-2 family not only regulates mitochondrial activation through changing mitochondrial outer membrane permeability,and plays an important role in mitochondrion induced intrinsic pathway,it also connects the three apoptotic pathways. It is found that ghrelin,also called as growth hormone release peptide,may prevent development of diabetes resulted fromβcell damage.And ghrelin can also inhibitβcell apoptosis induced by serum starvation,doxorubicin,and some cytokines.However,it is unclear about the effects of ghrelin on high glucose-inducedβcell apoptosis.This study in vitro is to pretreat high glucose cultured mouse pancreatic isletβ-cell line NIT-1 cell with ghrelin,and determine cell apoptotic rates and expression of bcl-2 and bax.Then,to investigate the effects of ghrelin on high glucose-inducedβcell apoptosis.
METHODS
(1) NIT-1cells were cultured in DMEM culture medium,which containing 5.6mmol/L glucose.To observe the morphology characteristics and its growth style under inverted microscope.(2) NIT-1 cells were divided into four groups,control group(5.6mmol/L glucose,C),ghrelin pretreatment group(10-7mmol/Lghrelin,GP),high glucose-induced apoptosis group (33.3mmol/L glucose,HG), ghrelin pretreatment +high glucose group(GP+HG).NIT-1 cell early apoptotic rates were tested by Annexin V-FITC/PI double staining and flowcytometry technique.(3) NIT-1 cell total apoptotic rates were evaluated by Hoechst 33258. NIT-1 cells were stained with Hoechst 33258 to detect apoptotic nuclei showing features of condensation and fragmentation under fluorescence microscope. (4) Expression of Bcl-2 and Bax in four groups were assayed by reverse transcription ploymerase chain reaction (RT-PCR) method. After DNA gel eletrophoresis,then to analyze the gray value and reach the relative expression of bcl-2 and bax.
RESULTS
(1) NIT-1 cells is polygon epithelial-like cells,growing monolayer-clustered and arranged densely.
(2) Both Annexin V/PI FACS and Hoechst33258 results indicated that ghrelin inhibits high glucose-induced NIT-1 cell apoptosis.The early apoptotic rates and total apoptotic rates were no significant difference in GP group(P>0.05),but increased significantly in HG group (P<0.01),as compared with C group.However,the early and total apoptotic rates were significantly lower in GP+HG group than in HG group(P<0.01,P<0.05). (3) RT-PCR results show that ghrelin had no effects on bcl-2 and bax expression in high glucose-induced NIT-1 cell. The level of bax mRNA was no significant difference in all groups. The level of bcl-2 mRNA, bcl-2/bax were decreased significantly in HG group(P<0.05), but no significant difference in GP group (P>0.05), as compared with C group. Unexpectedly, The level of bcl-2 mRNA, bcl-2/bax were no significant difference between GP+HG group and HG group,just an increasing trend was seen in the former.
CONCLUSIONS
(1) Ghrelin has no effects on pancreatic isletβcell apoptosis cultured in normal glucose,however,it can inhibit high glucose-induced NIT-1 cell apoptosis.It is indicated high glucose has toxic effects onβcells,and ghrelin may have proctective effects on high glucose- inducedβcell apoptosis.
(2) As ghrelin inhibitingβcell apoptosis,there is still an increasing trend of bcl-2 and bcl-2/bax expression inβcell,although it is no statistic significance.We speculates that ghrelin’s effects onβcell apoptosis maybe relates to theβcell types and stimulating factors.However, it could not be precluded that ghrelin inhibitingβcell apoptosis is through other apoptotic pathway associated factors.
引文
[1] Kloppel G, Lohr M, Habich K, et al. Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Pathol Res,1985,4(2):110-125.
[2] Defronzo R A. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes,2009,58(4):773-795.
[3] U.K. prospective diabetes study 16. Overview of 6 years' therapy of type II diabetes: a progressive disease. U.K. Prospective Diabetes Study Group. Diabetes,1995,44(11):1249-1258.
[4] Marchetti P, Dotta F, Lauro D, et al. An overview of pancreatic beta-cell defects in human type 2 diabetes: implications for treatment. Regul Pept,2008,146(1-3):4-11.
[5] Matveyenko A V, Butler P C. Beta-cell deficit due to increased apoptosis in the human islet amyloid polypeptide transgenic (HIP) rat recapitulates the metabolic defects present in type 2 diabetes. Diabetes,2006,55(7):2106-2114.
[6] Donath M Y, Storling J, Maedler K, et al. Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes. J Mol Med,2003,81(8):455-470.
[7] Hui H, Dotta F, Di M U, et al. Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death. J Cell Physiol,2004,200(2):177-200.
[8] Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature,1999,402(6762):656-660.
[9] Ashraf A, Mick G, Meleth S, et al. Insulin treatment reduces pre-prandial plasma ghrelin concentrations in children with type 1 diabetes. Med Sci Monit,2007,13(12):533-537.
[10] Ostergard T, Hansen T K, Nyholm B, et al. Circulating ghrelin concentrations are reduced in healthy offspring of Type 2 diabetic subjects, and are increased in women independent of a family history of Type 2 diabetes. Diabetologia,2003,46(1):134-136.
[11] Poykko SM, Kellokoski E, Horkko S, et al. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes. 2003; 52:2546-53.
[12] Irako T, Akamizu T,Hosoda H,et al. Ghrelin prevents development of diabetes at adult age in streptozotocin-treated newborn rats.Diabetologia, 2006, 49(6): 1264-1273.
[13] Granata R, Settanni F, Biancone L, et al. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology,2007,148(2):512-529.
[14] Zhang Y, Ying B, Shi L, et al. Ghrelin inhibit cell apoptosis in pancreatic beta cell line HIT-T15 via mitogen-activated protein kinase/phosphoinositide 3-kinase pathway.Toxicology, 2007,237 (1-3):194-202.
[15] Prado C L, Pugh-bernard A E, Elghazi L, et al. Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci U S A,2004,101(9):2924-2929.
[16] Rhodes C J. Type 2 diabetes-a matter of beta-cell life and death?Science,2005,307(5708):380-384.
[17] Halban P A. Cellular sources of new pancreatic beta cells and therapeutic implications for regenerative medicine. Nat Cell Biol,2004,6(11):1021-1025.
[18] Dor Y, Brown J, Martinez O I, et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature,2004,429(6987):41-46.
[19] Bonner-weir S, Weir G C. New sources of pancreatic beta-cells.Nat Biotechnol,2005,23(7): 857- 861.
[20] Sakuraba H, Mizukami H, Yagihashi N, et al. Reduced beta-cell mass and expression of oxidativestress-related DNA damage in the islet of Japanese Type II diabetic patients. Diabetologia,2002,45(1): 85 -96.
[21] Eizirik D L, Mandrup-poulsen T. A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia,2001,44(12):2115-2133.
[22] Cnop M, Welsh N, Jonas J C, et al. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities.Diabetes,2005,54 Suppl 2:97-107.
[23] Qin H,Trudeau JD,Reid GS,et al.Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL.Int Immunol,2004,16(11):1657-1662.
[24] Donath MY, Storling J,Maedler K, et al.Inflammatory mediators and isletβ-cell failure: a link between type 1 and type 2 diabetes.J Mol Med ,2003(8),81:455-470.
[25] Maedler K,Spinas GA,Lehmann R,et al.Glucose induces beta-cell apoptosis via upregulation of the Fas receptor in human islets.Diabetes,2001,50(8):1683-1690.
[26] Donath M Y, Halban P A. Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications. Diabetologia,2004,47(3):581-589.
[27] Westermark P, Wilander E. The influence of amyloid deposits on the islet volume in maturity onset diabetes mellitus.Diabetologia,1978,15(5):417-421.
[28] Saito K, Iwama N, Takahashi T. Morphometrical analysis on topographical difference in size distribution, number and volume of islets in the human pancreas. Tohoku J Exp Med,1978,124(2):177-186.
[29] Gepts W, Lecompte P M. The pancreatic islets in diabetes. Am J Med,1981,70(1):105-115.
[30] Yoon K H, Ko S H, Cho J H, et al. Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrinol Metab,2003,88(5):2300-2308.
[31] Butler A E, Janson J, Bonner-weir S, et al. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes,2003,52(1):102-110.
[32] Marchetti P, Del G S, Marselli L, et al. Pancreatic islets from type 2 diabetic patients have functional defects and increased apoptosis that are ameliorated by metformin. J Clin Endocrinol Metab,2004,89(11):5535-5541.
[33] Del G S, Lupi R, Marselli L, et al. Functional and molecular defects of pancreatic islets in human type 2 diabetes. Diabetes,2005,54(3):727-735.
[34] Andreozzi F, D A C, Federici M, et al. Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells.Endocrinology,2004,145(6):2845-2857.
[35] Patane G, Anello M, Piro S, et al. Role of ATP production and uncoupling protein-2 in the insulin secretory defect induced by chronic exposure to high glucose or free fatty acids and effects of peroxisome proliferator-activated receptor-gamma inhibition. Diabetes,2002,51(9):2749-2756.
[36] Maedler K,Serqeev P,Ris F,et al.Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest,2002 Sep;110(6):851-60.
[37] Reed J C, Pellecchia M. Apoptosis-based therapies for hematologic malignancies. Blood, 2005, 106(2): 408-418.
[38] Packham G, Stevenson F K. Bodyguards and assassins: Bcl-2 family proteins and apoptosis control in chronic lymphocytic leukaemia. Immunology,2005,114(4):441-449.
[39] Cory S, Huang D C, Adams J M. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene, 2003,22(53):8590-8607.
[40] Scaffidi C, Schmitz I, Zha J, et al. Differential modulation of apoptosis sensitivity in CD95 type I and type II cells.J Biol Chem,1999,274(32):22532-22538.
[41] Mckenzie M D, Carrington E M, Kaufmann T, et al. Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells.Diabetes,2008,57(5):1284-1292.
[42] Iwahashi H, Hanafusa T, Eguchi Y, et al. Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2.Diabetologia,1996,39(5):530-536.
[43] Liu Y, Rabinovitch A, Suarez-pinzon W, et al. Expression of the bcl-2 gene from a defective HSV-1 amplicon vector protects pancreatic beta-cells from apoptosis.Hum Gene Ther,1996,7(14):1719-1726.
[44] Tran V V, Chen G, Newgard C B, et al. Discrete and complementary mechanisms of protection of beta-cells against cytokine-induced and oxidative damage achieved by bcl-2 overexpression and a cytokine selection strategy.Diabetes,2003,52(6):1423-1432.
[45] Rabinovitch A, Suarez-pinzon W, Strynadka K, et al. Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. Diabetes, 1999,48(6): 1223-1229.
[46] Ou D, Wang X, Metzger D L, et al. Synergistic inhibition of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human pancreatic beta cells by Bcl-2 and X-linked inhibitor of apoptosis.Hum Immunol,2005,66(3):274-284.
[47] Zhou Y P, Pena J C, Roe M W, et al. Overexpression of Bcl-xL in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion. Am J Physiol Endocrinol Metab,2000,278(2):340-351.
[48] Klein D, Ribeiro M M, Mendoza V, et al. Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets. Biochem Biophys Res Commun,2004,323(2):473-478.
[49] Holohan C, Szegezdi E, Ritter T, et al. Cytokine-induced beta-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL.J Cell Mol Med,2008,12(2):591-606.
[50] Lindsten T, Ross A J, King A, et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell,2000,6(6):1389-1399.
[51] Wei M C, Zong W X, Cheng E H, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science,2001,292(5517):727-730.
[52] Huang Q, Bu S, Yu Y, et al. Diazoxide prevents diabetes through inhibiting pancreatic beta-cells from apoptosis via Bcl-2/Bax rate and p38-beta mitogen-activated protein kinase. Endocrinology, 2007, 148(1): 81-91.
[53] Onoue S, Hanato J, Yamada S. Pituitary adenylate cyclase-activating polypeptide attenuates streptozotocin-induced apoptotic death of RIN-m5F cells through regulation of Bcl-2 family protein mRNA expression. FEBS J,2008,275(22):5542-5551.
[54] Buteau J, El-assaad W, Rhodes C J, et al. Glucagon-like peptide-1 prevents beta cell glucolipotoxicity. Diabetologia,2004,47(5):806-815.
[55] Van C M, Kefas B A, Ling Z, et al. Specific expression of Bax-omega in pancreatic beta-cells is down-regulated by cytokines before the onset of apoptosis. Endocrinology,2002,143(1):320-326.
[56] Federici M, Hribal M, Perego L, et al. High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program. Diabetes,2001,50(6):1290-1301.
[57] Maedler K, Oberholzer J, Bucher P, et al. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover and function. Diabetes, 2003, 52(3): 726-733.
[58] Shimabukuro M, Wang M Y, Zhou Y T, et al. Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression. Proc Natl Acad Sci U S A,1998,95(16):9558-9561.
[59] Chanoine J P, Wong A C. Ghrelin gene expression is markedly higher in fetal pancreas compared with fetal stomach: effect of maternal fasting. Endocrinology,2004,145(8):3813-3820.
[60] Mori K, Yoshimoto A, Takaya K, et al. Kidney produces a novel acylated peptide,ghrelin. FEBS Lett,2000,486(3):213-216.
[61] Broglio F, Gottero C, Prodam F, et al. Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans.J Clin Endocrinol Metab,2004,89(6):3062-3065.
[62] Barazzoni R, Zanetti M, Ferreira C, et al. Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome. J Clin Endocrinol Metab,2007,92(10):3935-3940.
[63] Gauna C, Delhanty P J, Hofland L J, et al. Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes. J Clin Endocrinol Metab,2005,90(2):1055-1060.
[64] Gauna C, Meyler F M, Janssen J A, et al. Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab,2004,89(10):5035-5042.
[65] M.o.Weickert, C.v. Loeffelholz, A.m. Arafat,et al. Euglycemic hyperinsulinemia differentially modulates circulating total and acylated-ghrelin in humans. J Endocrinol Invest,2008,31:119-124.
[66] Celi F, Bini V, Papi F, et al. Circulating acylated and total ghrelin and galanin in children with insulin-treated type 1 diabetes: relationship to insulin therapy, metabolic control and pubertal development. Clin Endocrinol (Oxf),2005,63(2):139-145.
[67] Masaoka T, Suzuki H, Hosoda H, et al. Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes.FEBS Lett,2003,541(1-3):64-68.
[68] Soriano-guillen L, Barrios V, Lechuga-sancho A, et al. Response of circulating ghrelin levels to insulin therapy in children with newly diagnosed type 1 diabetes mellitus. Pediatr Res,2004,55(5):830-835.
[69] Martos-moreno G A, Barrios V, Soriano-guillen L, et al. Relationship between adiponectin levels, acylated ghrelin levels, and short-term body mass index changes in children with diabetes mellitus type 1 at diagnosis and after insulin therapy. Eur J Endocrinol,2006,155(5):757-761.
[70] Bideci A, Camurdan M O, Cinaz P, et al. Ghrelin, IGF-I and IGFBP-3 levels in children with type 1 diabetes mellitus. J Pediatr Endocrinol Metab,2005,18(12):1433-1439.
[71] Thompson N M, Gill D A, Davies R, et al. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor.Endocrinology,2004,145(1):234-242.
[72] Rodriguez A, Gomez-ambrosi J, Catalan V, et al. Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes.Int J Obes (Lond),2009.
[73] Bacha F, Arslanian S A. Ghrelin suppression in overweight children: a manifestation of insulin resistance? J Clin Endocrinol Metab,2005,90(5):2725-2730.
[74] Mccowen K C, Maykel J A, Bistrian B R, et al. Circulating ghrelin concentrations are lowered by intravenous glucose or hyperinsulinemic euglycemic conditions in rodents. J Endocrinol,2002,175(2):7-11.
[75] Saad M F, Bernaba B, Hwu C M, et al. Insulin regulates plasma ghrelin concentration.J Clin Endocrinol Metab,2002,87(8):3997-4000.
[76] Purnell J Q, Weigle D S, Breen P, et al. Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans.J Clin Endocrinol Metab,2003,88(12):5747-5752.
[77] Broglio F, Arvat E, Benso A, et al. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab,2001,86(10):5083-5086.
[78] Zigman J M, Nakano Y, Coppari R, et al. Mice lacking ghrelin receptors resist the development of diet-induced obesity. J Clin Invest,2005,115(12):3564-3572.
[79] Granata R, Settanni F, Gallo D, et al. Obestatin promotes survival of pancreatic beta-cells and human islets and induces expression of genes involved in the regulation of beta-cell mass and function.Diabetes,2008,57(4):967-979.
[80] Kim S W, Choi O K, Jung J Y, et al. Ghrelin inhibits early osteogenic differentiation of C3H10T1/2 cells by suppressing Runx2 expression and enhancing PPARgamma and C/EBPalpha expression.J Cell Biochem,2009,106(4):626-632.
[81] Xu Z,Lin S,Wu W,et al. Ghrelin prevents doxorubicin-induced cardiotoxicity through TNF-alpha/ NF-kappaB pathways and mitochondrial protective mechanisms.Toxicology,2008,247(2-3): 133-138.
[82] Kui L, Weiwei Z, Ling L, et al. Ghrelin inhibits apoptosis induced by high glucose and sodium palmitate in adult rat cardiomyocytes through the PI3K-Akt signaling pathway. Regul Pept,2009.
[83] Hwang S, Moon M, Kim S, et al. Neuroprotective Effect of Ghrelin is Associated with Decreased Expression of Prostate Apoptosis Response-4. Endocr J,2009.
[84] Miao Y, Xia Q, Hou Z, et al. Ghrelin protects cortical neuron against focal ischemia/reperfusion in rats.Biochem Biophys Res Commun,2007,359(3):795-800.
[85] Zhao H, Liu G, Wang Q, et al. Effect of ghrelin on human endothelial cells apoptosis induced by high glucose. Biochem Biophys Res Commun,2007,362(3):677-681.
[1] Date Y, Nakazato M, Hashiguchi S, et al. Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes,2002,51(1):124-129.
[2] Volante M, Allia E, Gugliotta P, et al. Expression of ghrelin and of the GH secretagogue receptor by pancreatic islet cells and related endocrine tumors. J Clin Endocrinol Metab,2002,87(3):1300-1308.
[3] Prado C L, Pugh-bernard A E, Elghazi L, et al. Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci USA, 2004, 101 (9):2924-2929.
[4] Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature,1999,402(6762):656-660.
[5] Gormsen L C, Nielsen C, Gjedsted J, et al. Effects of free fatty acids, growth hormone and growth hormone receptor blockade on serum ghrelin levels in humans. Clin Endocrinol (Oxf),2007,66(5):641-645.
[6] Irako T, Akamizu T,Hosoda H,et al. Ghrelin prevents development of diabetes at adult age in streptozotocin-treated newborn rats.Diabetologia, 2006, 49(6): 1264-1273.
[7] Granata R, Settanni F, Biancone L, et al. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology,2007,148(2):512-529.
[8] Zhang Y, Ying B, Shi L, et al. Ghrelin inhibit cell apoptosis in pancreatic beta cell line HIT-T15 via mitogen-activated protein kinase/phosphoinositide 3-kinase pathway. Toxicology, 2007,237 (1-3):194-202.
[9] Dezaki K, Hosoda H, Kakei M, et al. Endogenous Ghrelin in pancreatic islets restricts insulin release by attenuating Ca2+ signaling in beta-cells: implication in the glycemic control in rodents. Diabetes,2004,53(12):3142-3151.
[10] Esler WP, Rudolph J, Claus TH, et al. Small-molecule Ghrelin receptor antagonists improve glucose tolerance, suppress appetite, and promote weight loss. Endocrinology, 2007,148(11): 5175-5185.
[11] Sun Y, Asnicar M, K. Saha PK,et al.Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice.Cell Metab,2006,3(5):379-386.
[12] Dezaki K, Kakei M, Yada T. Ghrelin uses Gαi2 and activates voltage-dependent K+ channels to attenuate glucose-induced Ca2+ signaling and insulin release in islet beta-cells: novel signal transduction of Ghrelin. Diabetes,2007,56(9):2319-2327.
[13] Flanagan DE,Evans ML,Monsod TP. The influence of insulin on circulating ghrelin. Am J Physiol Endocrinol Metab,2003,284(2):313-316.
[14] Weickert MO, Loeffelholz CO, Arafat AM,et al. Euglycemic hyperinsulinemia differentially modulates circulating total and acylated-ghrelin in humans. J Endocrinol Invest,2008,31(2):119-124.
[15] Ashraf A, Mick G, Meleth S, et al. Insulin treatment reduces pre-prandial plasma ghrelin concentrations in children with type 1 diabetes. Med Sci Monit,2007,13(12):533-537.
[16] Soriano-guillen L, Barrios V, Lechuga-sancho A, et al. Response of circulating ghrelin levels to insulin therapy in children with newly diagnosed type 1 diabetes mellitus. Pediatr Res,2004,55(5):830-835.
[17] Mager U, Lindi V, Lindstrom J, et al. Association of the Leu72Met polymorphism of the ghrelin gene with the risk of Type 2 diabetes in subjects with impaired glucose tolerance in the Finnish Diabetes Prevention Study. Diabet Med,2006, 23(6): 685-689.
[18] Poykko SM, Kellokoski E, Horkko S, et al. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes.Diabetes,2003,52(10): 2546 -2553.
[2] Defronzo R A. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes,2009,58(4):773-795.
[3] U.K. prospective diabetes study 16. Overview of 6 years' therapy of type II diabetes: a progressive disease. U.K. Prospective Diabetes Study Group. Diabetes,1995,44(11):1249-1258.
[4] Marchetti P, Dotta F, Lauro D, et al. An overview of pancreatic beta-cell defects in human type 2 diabetes: implications for treatment. Regul Pept,2008,146(1-3):4-11.
[5] Matveyenko A V, Butler P C. Beta-cell deficit due to increased apoptosis in the human islet amyloid polypeptide transgenic (HIP) rat recapitulates the metabolic defects present in type 2 diabetes. Diabetes,2006,55(7):2106-2114.
[6] Donath M Y, Storling J, Maedler K, et al. Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes. J Mol Med,2003,81(8):455-470.
[7] Hui H, Dotta F, Di M U, et al. Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death. J Cell Physiol,2004,200(2):177-200.
[8] Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature,1999,402(6762):656-660.
[9] Ashraf A, Mick G, Meleth S, et al. Insulin treatment reduces pre-prandial plasma ghrelin concentrations in children with type 1 diabetes. Med Sci Monit,2007,13(12):533-537.
[10] Ostergard T, Hansen T K, Nyholm B, et al. Circulating ghrelin concentrations are reduced in healthy offspring of Type 2 diabetic subjects, and are increased in women independent of a family history of Type 2 diabetes. Diabetologia,2003,46(1):134-136.
[11] Poykko SM, Kellokoski E, Horkko S, et al. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes. 2003; 52:2546-53.
[12] Irako T, Akamizu T,Hosoda H,et al. Ghrelin prevents development of diabetes at adult age in streptozotocin-treated newborn rats.Diabetologia, 2006, 49(6): 1264-1273.
[13] Granata R, Settanni F, Biancone L, et al. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology,2007,148(2):512-529.
[14] Zhang Y, Ying B, Shi L, et al. Ghrelin inhibit cell apoptosis in pancreatic beta cell line HIT-T15 via mitogen-activated protein kinase/phosphoinositide 3-kinase pathway.Toxicology, 2007,237 (1-3):194-202.
[15] Prado C L, Pugh-bernard A E, Elghazi L, et al. Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci U S A,2004,101(9):2924-2929.
[16] Rhodes C J. Type 2 diabetes-a matter of beta-cell life and death?Science,2005,307(5708):380-384.
[17] Halban P A. Cellular sources of new pancreatic beta cells and therapeutic implications for regenerative medicine. Nat Cell Biol,2004,6(11):1021-1025.
[18] Dor Y, Brown J, Martinez O I, et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature,2004,429(6987):41-46.
[19] Bonner-weir S, Weir G C. New sources of pancreatic beta-cells.Nat Biotechnol,2005,23(7): 857- 861.
[20] Sakuraba H, Mizukami H, Yagihashi N, et al. Reduced beta-cell mass and expression of oxidativestress-related DNA damage in the islet of Japanese Type II diabetic patients. Diabetologia,2002,45(1): 85 -96.
[21] Eizirik D L, Mandrup-poulsen T. A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia,2001,44(12):2115-2133.
[22] Cnop M, Welsh N, Jonas J C, et al. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities.Diabetes,2005,54 Suppl 2:97-107.
[23] Qin H,Trudeau JD,Reid GS,et al.Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL.Int Immunol,2004,16(11):1657-1662.
[24] Donath MY, Storling J,Maedler K, et al.Inflammatory mediators and isletβ-cell failure: a link between type 1 and type 2 diabetes.J Mol Med ,2003(8),81:455-470.
[25] Maedler K,Spinas GA,Lehmann R,et al.Glucose induces beta-cell apoptosis via upregulation of the Fas receptor in human islets.Diabetes,2001,50(8):1683-1690.
[26] Donath M Y, Halban P A. Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications. Diabetologia,2004,47(3):581-589.
[27] Westermark P, Wilander E. The influence of amyloid deposits on the islet volume in maturity onset diabetes mellitus.Diabetologia,1978,15(5):417-421.
[28] Saito K, Iwama N, Takahashi T. Morphometrical analysis on topographical difference in size distribution, number and volume of islets in the human pancreas. Tohoku J Exp Med,1978,124(2):177-186.
[29] Gepts W, Lecompte P M. The pancreatic islets in diabetes. Am J Med,1981,70(1):105-115.
[30] Yoon K H, Ko S H, Cho J H, et al. Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrinol Metab,2003,88(5):2300-2308.
[31] Butler A E, Janson J, Bonner-weir S, et al. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes,2003,52(1):102-110.
[32] Marchetti P, Del G S, Marselli L, et al. Pancreatic islets from type 2 diabetic patients have functional defects and increased apoptosis that are ameliorated by metformin. J Clin Endocrinol Metab,2004,89(11):5535-5541.
[33] Del G S, Lupi R, Marselli L, et al. Functional and molecular defects of pancreatic islets in human type 2 diabetes. Diabetes,2005,54(3):727-735.
[34] Andreozzi F, D A C, Federici M, et al. Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells.Endocrinology,2004,145(6):2845-2857.
[35] Patane G, Anello M, Piro S, et al. Role of ATP production and uncoupling protein-2 in the insulin secretory defect induced by chronic exposure to high glucose or free fatty acids and effects of peroxisome proliferator-activated receptor-gamma inhibition. Diabetes,2002,51(9):2749-2756.
[36] Maedler K,Serqeev P,Ris F,et al.Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest,2002 Sep;110(6):851-60.
[37] Reed J C, Pellecchia M. Apoptosis-based therapies for hematologic malignancies. Blood, 2005, 106(2): 408-418.
[38] Packham G, Stevenson F K. Bodyguards and assassins: Bcl-2 family proteins and apoptosis control in chronic lymphocytic leukaemia. Immunology,2005,114(4):441-449.
[39] Cory S, Huang D C, Adams J M. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene, 2003,22(53):8590-8607.
[40] Scaffidi C, Schmitz I, Zha J, et al. Differential modulation of apoptosis sensitivity in CD95 type I and type II cells.J Biol Chem,1999,274(32):22532-22538.
[41] Mckenzie M D, Carrington E M, Kaufmann T, et al. Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells.Diabetes,2008,57(5):1284-1292.
[42] Iwahashi H, Hanafusa T, Eguchi Y, et al. Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2.Diabetologia,1996,39(5):530-536.
[43] Liu Y, Rabinovitch A, Suarez-pinzon W, et al. Expression of the bcl-2 gene from a defective HSV-1 amplicon vector protects pancreatic beta-cells from apoptosis.Hum Gene Ther,1996,7(14):1719-1726.
[44] Tran V V, Chen G, Newgard C B, et al. Discrete and complementary mechanisms of protection of beta-cells against cytokine-induced and oxidative damage achieved by bcl-2 overexpression and a cytokine selection strategy.Diabetes,2003,52(6):1423-1432.
[45] Rabinovitch A, Suarez-pinzon W, Strynadka K, et al. Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. Diabetes, 1999,48(6): 1223-1229.
[46] Ou D, Wang X, Metzger D L, et al. Synergistic inhibition of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human pancreatic beta cells by Bcl-2 and X-linked inhibitor of apoptosis.Hum Immunol,2005,66(3):274-284.
[47] Zhou Y P, Pena J C, Roe M W, et al. Overexpression of Bcl-xL in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion. Am J Physiol Endocrinol Metab,2000,278(2):340-351.
[48] Klein D, Ribeiro M M, Mendoza V, et al. Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets. Biochem Biophys Res Commun,2004,323(2):473-478.
[49] Holohan C, Szegezdi E, Ritter T, et al. Cytokine-induced beta-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL.J Cell Mol Med,2008,12(2):591-606.
[50] Lindsten T, Ross A J, King A, et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell,2000,6(6):1389-1399.
[51] Wei M C, Zong W X, Cheng E H, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science,2001,292(5517):727-730.
[52] Huang Q, Bu S, Yu Y, et al. Diazoxide prevents diabetes through inhibiting pancreatic beta-cells from apoptosis via Bcl-2/Bax rate and p38-beta mitogen-activated protein kinase. Endocrinology, 2007, 148(1): 81-91.
[53] Onoue S, Hanato J, Yamada S. Pituitary adenylate cyclase-activating polypeptide attenuates streptozotocin-induced apoptotic death of RIN-m5F cells through regulation of Bcl-2 family protein mRNA expression. FEBS J,2008,275(22):5542-5551.
[54] Buteau J, El-assaad W, Rhodes C J, et al. Glucagon-like peptide-1 prevents beta cell glucolipotoxicity. Diabetologia,2004,47(5):806-815.
[55] Van C M, Kefas B A, Ling Z, et al. Specific expression of Bax-omega in pancreatic beta-cells is down-regulated by cytokines before the onset of apoptosis. Endocrinology,2002,143(1):320-326.
[56] Federici M, Hribal M, Perego L, et al. High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program. Diabetes,2001,50(6):1290-1301.
[57] Maedler K, Oberholzer J, Bucher P, et al. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover and function. Diabetes, 2003, 52(3): 726-733.
[58] Shimabukuro M, Wang M Y, Zhou Y T, et al. Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression. Proc Natl Acad Sci U S A,1998,95(16):9558-9561.
[59] Chanoine J P, Wong A C. Ghrelin gene expression is markedly higher in fetal pancreas compared with fetal stomach: effect of maternal fasting. Endocrinology,2004,145(8):3813-3820.
[60] Mori K, Yoshimoto A, Takaya K, et al. Kidney produces a novel acylated peptide,ghrelin. FEBS Lett,2000,486(3):213-216.
[61] Broglio F, Gottero C, Prodam F, et al. Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans.J Clin Endocrinol Metab,2004,89(6):3062-3065.
[62] Barazzoni R, Zanetti M, Ferreira C, et al. Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome. J Clin Endocrinol Metab,2007,92(10):3935-3940.
[63] Gauna C, Delhanty P J, Hofland L J, et al. Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes. J Clin Endocrinol Metab,2005,90(2):1055-1060.
[64] Gauna C, Meyler F M, Janssen J A, et al. Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab,2004,89(10):5035-5042.
[65] M.o.Weickert, C.v. Loeffelholz, A.m. Arafat,et al. Euglycemic hyperinsulinemia differentially modulates circulating total and acylated-ghrelin in humans. J Endocrinol Invest,2008,31:119-124.
[66] Celi F, Bini V, Papi F, et al. Circulating acylated and total ghrelin and galanin in children with insulin-treated type 1 diabetes: relationship to insulin therapy, metabolic control and pubertal development. Clin Endocrinol (Oxf),2005,63(2):139-145.
[67] Masaoka T, Suzuki H, Hosoda H, et al. Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes.FEBS Lett,2003,541(1-3):64-68.
[68] Soriano-guillen L, Barrios V, Lechuga-sancho A, et al. Response of circulating ghrelin levels to insulin therapy in children with newly diagnosed type 1 diabetes mellitus. Pediatr Res,2004,55(5):830-835.
[69] Martos-moreno G A, Barrios V, Soriano-guillen L, et al. Relationship between adiponectin levels, acylated ghrelin levels, and short-term body mass index changes in children with diabetes mellitus type 1 at diagnosis and after insulin therapy. Eur J Endocrinol,2006,155(5):757-761.
[70] Bideci A, Camurdan M O, Cinaz P, et al. Ghrelin, IGF-I and IGFBP-3 levels in children with type 1 diabetes mellitus. J Pediatr Endocrinol Metab,2005,18(12):1433-1439.
[71] Thompson N M, Gill D A, Davies R, et al. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor.Endocrinology,2004,145(1):234-242.
[72] Rodriguez A, Gomez-ambrosi J, Catalan V, et al. Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes.Int J Obes (Lond),2009.
[73] Bacha F, Arslanian S A. Ghrelin suppression in overweight children: a manifestation of insulin resistance? J Clin Endocrinol Metab,2005,90(5):2725-2730.
[74] Mccowen K C, Maykel J A, Bistrian B R, et al. Circulating ghrelin concentrations are lowered by intravenous glucose or hyperinsulinemic euglycemic conditions in rodents. J Endocrinol,2002,175(2):7-11.
[75] Saad M F, Bernaba B, Hwu C M, et al. Insulin regulates plasma ghrelin concentration.J Clin Endocrinol Metab,2002,87(8):3997-4000.
[76] Purnell J Q, Weigle D S, Breen P, et al. Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans.J Clin Endocrinol Metab,2003,88(12):5747-5752.
[77] Broglio F, Arvat E, Benso A, et al. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab,2001,86(10):5083-5086.
[78] Zigman J M, Nakano Y, Coppari R, et al. Mice lacking ghrelin receptors resist the development of diet-induced obesity. J Clin Invest,2005,115(12):3564-3572.
[79] Granata R, Settanni F, Gallo D, et al. Obestatin promotes survival of pancreatic beta-cells and human islets and induces expression of genes involved in the regulation of beta-cell mass and function.Diabetes,2008,57(4):967-979.
[80] Kim S W, Choi O K, Jung J Y, et al. Ghrelin inhibits early osteogenic differentiation of C3H10T1/2 cells by suppressing Runx2 expression and enhancing PPARgamma and C/EBPalpha expression.J Cell Biochem,2009,106(4):626-632.
[81] Xu Z,Lin S,Wu W,et al. Ghrelin prevents doxorubicin-induced cardiotoxicity through TNF-alpha/ NF-kappaB pathways and mitochondrial protective mechanisms.Toxicology,2008,247(2-3): 133-138.
[82] Kui L, Weiwei Z, Ling L, et al. Ghrelin inhibits apoptosis induced by high glucose and sodium palmitate in adult rat cardiomyocytes through the PI3K-Akt signaling pathway. Regul Pept,2009.
[83] Hwang S, Moon M, Kim S, et al. Neuroprotective Effect of Ghrelin is Associated with Decreased Expression of Prostate Apoptosis Response-4. Endocr J,2009.
[84] Miao Y, Xia Q, Hou Z, et al. Ghrelin protects cortical neuron against focal ischemia/reperfusion in rats.Biochem Biophys Res Commun,2007,359(3):795-800.
[85] Zhao H, Liu G, Wang Q, et al. Effect of ghrelin on human endothelial cells apoptosis induced by high glucose. Biochem Biophys Res Commun,2007,362(3):677-681.
[1] Date Y, Nakazato M, Hashiguchi S, et al. Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes,2002,51(1):124-129.
[2] Volante M, Allia E, Gugliotta P, et al. Expression of ghrelin and of the GH secretagogue receptor by pancreatic islet cells and related endocrine tumors. J Clin Endocrinol Metab,2002,87(3):1300-1308.
[3] Prado C L, Pugh-bernard A E, Elghazi L, et al. Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci USA, 2004, 101 (9):2924-2929.
[4] Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature,1999,402(6762):656-660.
[5] Gormsen L C, Nielsen C, Gjedsted J, et al. Effects of free fatty acids, growth hormone and growth hormone receptor blockade on serum ghrelin levels in humans. Clin Endocrinol (Oxf),2007,66(5):641-645.
[6] Irako T, Akamizu T,Hosoda H,et al. Ghrelin prevents development of diabetes at adult age in streptozotocin-treated newborn rats.Diabetologia, 2006, 49(6): 1264-1273.
[7] Granata R, Settanni F, Biancone L, et al. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology,2007,148(2):512-529.
[8] Zhang Y, Ying B, Shi L, et al. Ghrelin inhibit cell apoptosis in pancreatic beta cell line HIT-T15 via mitogen-activated protein kinase/phosphoinositide 3-kinase pathway. Toxicology, 2007,237 (1-3):194-202.
[9] Dezaki K, Hosoda H, Kakei M, et al. Endogenous Ghrelin in pancreatic islets restricts insulin release by attenuating Ca2+ signaling in beta-cells: implication in the glycemic control in rodents. Diabetes,2004,53(12):3142-3151.
[10] Esler WP, Rudolph J, Claus TH, et al. Small-molecule Ghrelin receptor antagonists improve glucose tolerance, suppress appetite, and promote weight loss. Endocrinology, 2007,148(11): 5175-5185.
[11] Sun Y, Asnicar M, K. Saha PK,et al.Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice.Cell Metab,2006,3(5):379-386.
[12] Dezaki K, Kakei M, Yada T. Ghrelin uses Gαi2 and activates voltage-dependent K+ channels to attenuate glucose-induced Ca2+ signaling and insulin release in islet beta-cells: novel signal transduction of Ghrelin. Diabetes,2007,56(9):2319-2327.
[13] Flanagan DE,Evans ML,Monsod TP. The influence of insulin on circulating ghrelin. Am J Physiol Endocrinol Metab,2003,284(2):313-316.
[14] Weickert MO, Loeffelholz CO, Arafat AM,et al. Euglycemic hyperinsulinemia differentially modulates circulating total and acylated-ghrelin in humans. J Endocrinol Invest,2008,31(2):119-124.
[15] Ashraf A, Mick G, Meleth S, et al. Insulin treatment reduces pre-prandial plasma ghrelin concentrations in children with type 1 diabetes. Med Sci Monit,2007,13(12):533-537.
[16] Soriano-guillen L, Barrios V, Lechuga-sancho A, et al. Response of circulating ghrelin levels to insulin therapy in children with newly diagnosed type 1 diabetes mellitus. Pediatr Res,2004,55(5):830-835.
[17] Mager U, Lindi V, Lindstrom J, et al. Association of the Leu72Met polymorphism of the ghrelin gene with the risk of Type 2 diabetes in subjects with impaired glucose tolerance in the Finnish Diabetes Prevention Study. Diabet Med,2006, 23(6): 685-689.
[18] Poykko SM, Kellokoski E, Horkko S, et al. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes.Diabetes,2003,52(10): 2546 -2553.