Exendin-4对游离脂肪酸诱导的小鼠胰岛β细胞凋亡的保护作用及其机制研究
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摘要
目的:探讨PI3K/Akt/Bcl-2信号通路在游离脂肪酸诱导的小鼠胰岛β细胞株MIN6细胞凋亡中的作用;在此基础上,进一步研究GLP-1受体激动剂Exendin-4对FFA诱导的MIN6细胞凋亡的作用及其可能的机制。
方法:选用小鼠胰岛β细胞株MIN6细胞为研究对象,用不同浓度(0.2-1.6mmol/L)FFA诱导刺激,模拟体外β细胞脂毒性损伤后的细胞凋亡模型,通过MTT观察FFA作用后细胞的存活率;Hoechst-PI染色荧光显微镜观测细胞凋亡形态,Annexin-Ⅴ-PI染色流式细胞技术测定细胞凋亡率,Western blot技术检测胞浆Akt、P-Akt、Bcl-2和Bax蛋白表达水平的变化。在此基础上,用不同浓度(12.5-200nmol/L)的Exendin-4预处理细胞,观察Exendin-4对FFA诱导的MIN6细胞凋亡的保护作用,并选定一个合适的浓度进一步研究Exendin-4是否通过PI3K/Akt/Bcl-2信号通路影响FFA诱导MIN6细胞发生凋亡性损伤。
结果:MTT显示FFA(0.4-1.6mmol/L)作用不同时间后可明显抑制MIN6细胞的生长,并呈现一定的量效关系;Hoechst-PI染色荧光显微镜及Annexin-Ⅴ-PI双染流式细胞检测证实不同浓度的FFA处理48小时后,随着FFA浓度的增高,其诱导MIN6细胞的凋亡作用就更强。不同浓度的FFA处理48小时后,随着FFA浓度的增加,细胞内P-Akt、Bcl-2蛋白水平降低,而Akt、Bax蛋白水平变化不明显。不同浓度的Exendin-4预处理24小时,可以抑制FFA诱导的β细胞凋亡,并呈现一定的剂量依赖性,并且Exendin-4能抑制FFA诱导的胞内P-Akt、Bcl-2蛋白水平的降低。
结论:(1)FFA可诱导胰岛β细胞发生凋亡性损伤,在此过程中伴随细胞内P-Akt、Bcl-2蛋白水平的降低;(2)PI3K/Akt/Bcl-2信号通路可能是FFA诱导胰岛β细胞发生凋亡的重要通路之一;(3)GLP-1受体激动剂Exendin-4可以剂量依赖性地抑制FFA诱导胰岛β细胞的凋亡;(4)Exendin-4可能通过激活细胞内Akt的活化、提高胞浆Bcl-2蛋白表达水平来抑制FFA诱导的β细胞凋亡。Exendin-4在胰腺β细胞保护治疗中具有潜在的应用价值。
OBJECTIVE: To investigate the effects of free fatty acid(FFA)on cell apoptosis in murine MIN6 pancreaticβ-cells and the changes in signaling pathway of Akt /Bcl-2 in this process.Further, to explore the effect and possible mechanism of Exendin-4 onβ-cells apoptosis induced by FFA.
METHODS: Murine MIN6 pancreaticβ-cells were cultured in vitro and the cell apoptosis model of lipotoxicity was established by FFA. Cells were pretreated with Exendin-4 at the concentration of 12.5 to 200nmol/L.Choose a correct concentration of Exendin-4 to continue the following study.Cell viability was measured by MTT assay.The morphological changes of cell damage was evaluated by epifluorescence microscopy after staining with Hoechst-PI.The percentage of cell apoptosis was determined by flow cytometric assay after Annexin-Ⅴ-FITC-PI staining. Protein levels of Akt、P-Akt、Bcl-2 and Bax were detected by Western blot.
RESULTS: MIN6 cells viability were significantly reduced in a dose-dependent manner after exposure of MIN6 cells to free fatty acid at range of 0.4-1.6 mmol/L for 24 and 48 hours.It was verified by Hoechst-PI and Annexin-Ⅴ-FITC-PI staining that the percentage of cell apoptosis was significantly increased after treating with FFA. Meanwhile,the protein levels of P-Akt、Bcl-2 but Akt、Bax were dose-dependently decreased.Intriguingly, cell apoptosis was dose-dependently reduced by being pretreated 24 hours with Exendin-4.Furthermore,the present study showed that Exendin-4 patently inhibited the decreased protein levels of P-Akt、Bcl-2 induced by FFA.
CONCLUSION: The cell signaling pathway of Akt/ Bcl-2 mediated FFA-induced apoptosis in MIN6 pancreaticβ-cells. Exendin-4 protectedβ-cells from FFA-induced apoptosis via up-regulation of Akt/ Bcl-2 pathway.Glucagon-like peptide-1 receptor agonist—Exendin-4 is potentially a highly effective therapeutic agent in protecting pancreaticβ-cells.
方法:选用小鼠胰岛β细胞株MIN6细胞为研究对象,用不同浓度(0.2-1.6mmol/L)FFA诱导刺激,模拟体外β细胞脂毒性损伤后的细胞凋亡模型,通过MTT观察FFA作用后细胞的存活率;Hoechst-PI染色荧光显微镜观测细胞凋亡形态,Annexin-Ⅴ-PI染色流式细胞技术测定细胞凋亡率,Western blot技术检测胞浆Akt、P-Akt、Bcl-2和Bax蛋白表达水平的变化。在此基础上,用不同浓度(12.5-200nmol/L)的Exendin-4预处理细胞,观察Exendin-4对FFA诱导的MIN6细胞凋亡的保护作用,并选定一个合适的浓度进一步研究Exendin-4是否通过PI3K/Akt/Bcl-2信号通路影响FFA诱导MIN6细胞发生凋亡性损伤。
结果:MTT显示FFA(0.4-1.6mmol/L)作用不同时间后可明显抑制MIN6细胞的生长,并呈现一定的量效关系;Hoechst-PI染色荧光显微镜及Annexin-Ⅴ-PI双染流式细胞检测证实不同浓度的FFA处理48小时后,随着FFA浓度的增高,其诱导MIN6细胞的凋亡作用就更强。不同浓度的FFA处理48小时后,随着FFA浓度的增加,细胞内P-Akt、Bcl-2蛋白水平降低,而Akt、Bax蛋白水平变化不明显。不同浓度的Exendin-4预处理24小时,可以抑制FFA诱导的β细胞凋亡,并呈现一定的剂量依赖性,并且Exendin-4能抑制FFA诱导的胞内P-Akt、Bcl-2蛋白水平的降低。
结论:(1)FFA可诱导胰岛β细胞发生凋亡性损伤,在此过程中伴随细胞内P-Akt、Bcl-2蛋白水平的降低;(2)PI3K/Akt/Bcl-2信号通路可能是FFA诱导胰岛β细胞发生凋亡的重要通路之一;(3)GLP-1受体激动剂Exendin-4可以剂量依赖性地抑制FFA诱导胰岛β细胞的凋亡;(4)Exendin-4可能通过激活细胞内Akt的活化、提高胞浆Bcl-2蛋白表达水平来抑制FFA诱导的β细胞凋亡。Exendin-4在胰腺β细胞保护治疗中具有潜在的应用价值。
OBJECTIVE: To investigate the effects of free fatty acid(FFA)on cell apoptosis in murine MIN6 pancreaticβ-cells and the changes in signaling pathway of Akt /Bcl-2 in this process.Further, to explore the effect and possible mechanism of Exendin-4 onβ-cells apoptosis induced by FFA.
METHODS: Murine MIN6 pancreaticβ-cells were cultured in vitro and the cell apoptosis model of lipotoxicity was established by FFA. Cells were pretreated with Exendin-4 at the concentration of 12.5 to 200nmol/L.Choose a correct concentration of Exendin-4 to continue the following study.Cell viability was measured by MTT assay.The morphological changes of cell damage was evaluated by epifluorescence microscopy after staining with Hoechst-PI.The percentage of cell apoptosis was determined by flow cytometric assay after Annexin-Ⅴ-FITC-PI staining. Protein levels of Akt、P-Akt、Bcl-2 and Bax were detected by Western blot.
RESULTS: MIN6 cells viability were significantly reduced in a dose-dependent manner after exposure of MIN6 cells to free fatty acid at range of 0.4-1.6 mmol/L for 24 and 48 hours.It was verified by Hoechst-PI and Annexin-Ⅴ-FITC-PI staining that the percentage of cell apoptosis was significantly increased after treating with FFA. Meanwhile,the protein levels of P-Akt、Bcl-2 but Akt、Bax were dose-dependently decreased.Intriguingly, cell apoptosis was dose-dependently reduced by being pretreated 24 hours with Exendin-4.Furthermore,the present study showed that Exendin-4 patently inhibited the decreased protein levels of P-Akt、Bcl-2 induced by FFA.
CONCLUSION: The cell signaling pathway of Akt/ Bcl-2 mediated FFA-induced apoptosis in MIN6 pancreaticβ-cells. Exendin-4 protectedβ-cells from FFA-induced apoptosis via up-regulation of Akt/ Bcl-2 pathway.Glucagon-like peptide-1 receptor agonist—Exendin-4 is potentially a highly effective therapeutic agent in protecting pancreaticβ-cells.
引文
1. Butler AE, Janson J, Bonner-Weir S, et al. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes.Diabetes, 2003,52:102-110.
2. Johnson JD, Ahmed NT, Luciani DS, et al. Increased islet apoptosis in Pdx1+/-mice . J Clin Invest, 2003, 111: 1147-1160.
3. Ye CL, Jin YL, Ye KH, et al. Effects of EGb 761 on the cell apoptosis induced by H2O2 in RIN-m beta cells.Zhong Yao Cai, 2007,30: 424-428.
4. 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 [J]. Diabetes,2003 ,52:726-733.
5. Kang ES, Han D, Park J, et al. O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells. Exp Cell Res,2008,314:2238-2248.
6. St?rling J, Binzer J, Andersson AK, et al. Nitric oxide contributes to cytokine-induced apoptosis in pancreatic beta cells via potentiation of JNK activity and inhibition of Akt. Diabetologia,2005,48:2039-2050.
7. Brown JE, Dunmore SJ. Leptin decreases apoptosis and alters BCL-2 : Bax ratio in clonal rodent pancreatic beta-cells. Diabetes Metab Res Rev,2007,23:497-502.
8. 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,2007,148:81-91.
9. Hui H, Wright C, Perfetti R. Glucagons-like peptide-1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting-cells.Diabetes, 2001, 50: 785-796.
10. Stoffers DA, Kieffer TJ, Hussain MA, et a1. 1nsulinotropic glucagons-like peptide l agonists stimulate expression of homeodomain protein IDX-l and increase islet size in mouse pancreas. Diabetes, 2000 , 49: 741-748
11. Wang Q, Brubaker PL.Glucagon like peptide l treatment delays the onset of diabetes in 8 week old db/db mice.Diabetologia, 2002, 45:1263- 1273.
12. Li Y, Hansotia T, Yusta B, et al. Glucagon-like-peptide-1 receptor signaling modulatesβcell apoptosis. J Biol Chem, 2003, 278: 471-478.
13. Li L, El-Kholy W, Rhodes CJ, et al. Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B. Diabetologia, 2005, 48: 1339-1349.
14. Farilla L, Bulotta A, Hirshberg B, et al. Glucagon-like-peptide-1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology, 2003, 144: 5149-5158.
15. Bregenholt S, M?ldrup A, Blume N, et al. The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro. Biochem Biophys Res Commun,2005,330:577-584.
16. Nogueira TC, AnhêGF, Carvalho CR, et al. Involvement of phosphatidylinositol-3 kinase/AKT/PKCzeta/lambda pathway in the effect of palmitate on glucose-induced insulin secretion. Pancreas,2008,37:309-315.
17. Peterson JM, Wang Y, Bryner RW, et al. Bax signaling regulates palmitate-mediated apoptosis in C(2)C(12) myotubes. Am J Physiol Endocrinol Metab,2008,295:E1307-1314.
18. Urahama Y, Ohsaki Y, Fujita Y, et al. Lipid droplet-associated proteins protect renal tubular cells from fatty acid-induced apoptosis. Am J Pathol,2008,173:1286-1294.
19. Chai W, Liu Z. p38 mitogen-activated protein kinase mediates palmitate-induced apoptosis but not inhibitor of nuclear factor-kappaB degradation in human coronary artery endothelial cells. Endocrinology,2007,148:1622-1628.
20. Li HL,Yu YR,Yu HL,et al. Relationship between peripheral insulin resistance and beta-cell function in obese subjects. Sichuan Da Xue Xue Bao Yi Xue Ban, 2005,36:378-381.
21. Karaskov E,Scott C,Zhang L, et al.Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress,which may contribute to INS-1 pancreaticbeta-cell apoptosis.Endocrinology,2006,147(7):3398-3407.
22. Lai E, Bikopoulos G, Wheeler MB, et al. Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic beta-cells.Am J Physiol Endocrinol Metab,2008,294:E540-550.
23. Lingohr MK,Buettner R,Rhodes CJ.Pancreatic beta-cell growth and survival-a role in obesity-linked type 2 diabetes. Trends Mol Med,2002,8:375-384.
24. Girard J. Contribution of free fatty acids to impairment of insulin secretion and action. mechanism of beta-cell lipotoxicity. Med Sci (Paris), 2005 ,21:19-25.
25. Higa M,Shimabukuro M, Shimajiri Y,et al.Protein kinase B/Akt signalling is required for palmitate-inducedβ-cell lipotoxicity. Diabetes Obes Metab,2006,8:228-233.
26. Lupi R,Dotta F,Marselli L, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated.Diabetes,2002,51:1437-1442.
27. Tuttle RL,Gill NS,Pugh W, et al.Regulation of pancreaticβ-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha.Nat Med,2001 ,7:1133-1137.
28. Wrede CE, Dickson LM, Lingohr MK, et al. Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1). J Biol Chem,2002,277:49676-49684.
29. Hui H, Nourparvar A, Zhao X, et al. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5’-adenosine monophophate-dependent protein A-and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology,2003,144:1444-1455.
30.Farilla L, Hui H, Bertolotto C, et al. Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats.Endocrinology,2002,143:4397-4408.
31. Buteau J, El-Assaad W, Rhodes CJ, et al. Glucagon-like peptide-1 prevents beta cell glucolipotoxicity. Diabetologia,2004,47:806-815.
32. Wang Q, Li L, Xu E, et al. Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells. Diabetologia,2004,47:478-487.
33. Tews D, Werner U, Eckel J. Enhanced protection against cytokine- and fatty acid-induced apoptosis in pancreatic beta cells by combined treatment with glucagon-like peptide-1 receptor agonists and insulin analogues. Horm Metab Res,2008,40:172-180.
34. Ranta F, Avram D, Berchtold S, et al. Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4. Diabetes,2006,55:1380-1390.
[1] Li HL, Yu YR, Yu HL, et al. Relationship between peripheral insulin resistance and beta-cell function in obese subjects [J]. Sichuan Da Xue Xue Bao Yi Xue Ban, 2005 May;36(3):378-381.
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[4] Karaskov E, Scott C, Zhang L, et al. Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis[J]. Endocrinology, 2006 Jul;147(7):3398-3407.
[5] Lai E, Bikopoulos G, Wheeler MB, et al. Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic beta-cells[J]. Am J Physiol Endocrinol Metab, 2008 Mar;294(3):E540-550.
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2. Johnson JD, Ahmed NT, Luciani DS, et al. Increased islet apoptosis in Pdx1+/-mice . J Clin Invest, 2003, 111: 1147-1160.
3. Ye CL, Jin YL, Ye KH, et al. Effects of EGb 761 on the cell apoptosis induced by H2O2 in RIN-m beta cells.Zhong Yao Cai, 2007,30: 424-428.
4. 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 [J]. Diabetes,2003 ,52:726-733.
5. Kang ES, Han D, Park J, et al. O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells. Exp Cell Res,2008,314:2238-2248.
6. St?rling J, Binzer J, Andersson AK, et al. Nitric oxide contributes to cytokine-induced apoptosis in pancreatic beta cells via potentiation of JNK activity and inhibition of Akt. Diabetologia,2005,48:2039-2050.
7. Brown JE, Dunmore SJ. Leptin decreases apoptosis and alters BCL-2 : Bax ratio in clonal rodent pancreatic beta-cells. Diabetes Metab Res Rev,2007,23:497-502.
8. 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,2007,148:81-91.
9. Hui H, Wright C, Perfetti R. Glucagons-like peptide-1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting-cells.Diabetes, 2001, 50: 785-796.
10. Stoffers DA, Kieffer TJ, Hussain MA, et a1. 1nsulinotropic glucagons-like peptide l agonists stimulate expression of homeodomain protein IDX-l and increase islet size in mouse pancreas. Diabetes, 2000 , 49: 741-748
11. Wang Q, Brubaker PL.Glucagon like peptide l treatment delays the onset of diabetes in 8 week old db/db mice.Diabetologia, 2002, 45:1263- 1273.
12. Li Y, Hansotia T, Yusta B, et al. Glucagon-like-peptide-1 receptor signaling modulatesβcell apoptosis. J Biol Chem, 2003, 278: 471-478.
13. Li L, El-Kholy W, Rhodes CJ, et al. Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B. Diabetologia, 2005, 48: 1339-1349.
14. Farilla L, Bulotta A, Hirshberg B, et al. Glucagon-like-peptide-1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology, 2003, 144: 5149-5158.
15. Bregenholt S, M?ldrup A, Blume N, et al. The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro. Biochem Biophys Res Commun,2005,330:577-584.
16. Nogueira TC, AnhêGF, Carvalho CR, et al. Involvement of phosphatidylinositol-3 kinase/AKT/PKCzeta/lambda pathway in the effect of palmitate on glucose-induced insulin secretion. Pancreas,2008,37:309-315.
17. Peterson JM, Wang Y, Bryner RW, et al. Bax signaling regulates palmitate-mediated apoptosis in C(2)C(12) myotubes. Am J Physiol Endocrinol Metab,2008,295:E1307-1314.
18. Urahama Y, Ohsaki Y, Fujita Y, et al. Lipid droplet-associated proteins protect renal tubular cells from fatty acid-induced apoptosis. Am J Pathol,2008,173:1286-1294.
19. Chai W, Liu Z. p38 mitogen-activated protein kinase mediates palmitate-induced apoptosis but not inhibitor of nuclear factor-kappaB degradation in human coronary artery endothelial cells. Endocrinology,2007,148:1622-1628.
20. Li HL,Yu YR,Yu HL,et al. Relationship between peripheral insulin resistance and beta-cell function in obese subjects. Sichuan Da Xue Xue Bao Yi Xue Ban, 2005,36:378-381.
21. Karaskov E,Scott C,Zhang L, et al.Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress,which may contribute to INS-1 pancreaticbeta-cell apoptosis.Endocrinology,2006,147(7):3398-3407.
22. Lai E, Bikopoulos G, Wheeler MB, et al. Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic beta-cells.Am J Physiol Endocrinol Metab,2008,294:E540-550.
23. Lingohr MK,Buettner R,Rhodes CJ.Pancreatic beta-cell growth and survival-a role in obesity-linked type 2 diabetes. Trends Mol Med,2002,8:375-384.
24. Girard J. Contribution of free fatty acids to impairment of insulin secretion and action. mechanism of beta-cell lipotoxicity. Med Sci (Paris), 2005 ,21:19-25.
25. Higa M,Shimabukuro M, Shimajiri Y,et al.Protein kinase B/Akt signalling is required for palmitate-inducedβ-cell lipotoxicity. Diabetes Obes Metab,2006,8:228-233.
26. Lupi R,Dotta F,Marselli L, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated.Diabetes,2002,51:1437-1442.
27. Tuttle RL,Gill NS,Pugh W, et al.Regulation of pancreaticβ-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha.Nat Med,2001 ,7:1133-1137.
28. Wrede CE, Dickson LM, Lingohr MK, et al. Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1). J Biol Chem,2002,277:49676-49684.
29. Hui H, Nourparvar A, Zhao X, et al. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5’-adenosine monophophate-dependent protein A-and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology,2003,144:1444-1455.
30.Farilla L, Hui H, Bertolotto C, et al. Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats.Endocrinology,2002,143:4397-4408.
31. Buteau J, El-Assaad W, Rhodes CJ, et al. Glucagon-like peptide-1 prevents beta cell glucolipotoxicity. Diabetologia,2004,47:806-815.
32. Wang Q, Li L, Xu E, et al. Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells. Diabetologia,2004,47:478-487.
33. Tews D, Werner U, Eckel J. Enhanced protection against cytokine- and fatty acid-induced apoptosis in pancreatic beta cells by combined treatment with glucagon-like peptide-1 receptor agonists and insulin analogues. Horm Metab Res,2008,40:172-180.
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