利用Cre/lox P系统构建胰岛β细胞特异性敲除PDHA1基因的小鼠模型
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摘要
背景:研究表明,2型糖尿病啮齿类动物模型及人体的丙酮酸脱氢酶复合体表达量降低,PDHA1基因缺陷也是导致丙酮酸脱氢酶复合体酶活性改变最常见的原因。目的:构建并鉴定胰岛β细胞特异性敲除PDHA1基因小鼠,为后续探究PDHA1基因在糖尿病发病机制方面作用提供模型基础。方法:实验方案经南方医科大学伦理委员会批准。利用Cre/lox P系统理论,将PDHA1~(loxp/loxp)小鼠与胰岛β细胞特异性表达Cre重组酶的小鼠进行数代杂交,3-4周龄小鼠通过PCR法鉴定筛选基因型,子2代中基因型为PDHA1~(loxp/loxp)Cre~(+/-)小鼠即为实验所需要构建的模型小鼠。取4周龄PDHA1~(flox/flox)Cre~(+/-)小鼠(βKO小鼠组)与Cre~(+/-)小鼠(对照组)各5只,给予相同的水和饲料,待8周龄时连续7d注射腹腔注射他莫昔芬50mg/kg诱导PDHA1基因敲除,观察3周后腹腔注射麻醉,取胰腺组织、脂肪组织、肝脏组织。采用q PCR法、Western Blot法、免疫组织化学法鉴定PDHA1基因敲除效果,观察小鼠表型。结果与结论:(1)PCR基因扩增筛选出基因型为PDHA1~(loxp/loxp)Cre~(+/-)小鼠;(2)q PCR法检验小鼠胰腺、脂肪、肝脏组织,证实敲除效果具有组织特异性,胰岛中βKO组小鼠PDHA1 mRNA的相对表达较对照组明显下降;(3)WesternBlot检测及免疫组织化学法观察显示,βKO组小鼠PDHA1蛋白表达较对照组小鼠明显降低,证实敲除PDHA1基因效果明显;(4)结果说明,利用Cre/loxP系统成功构建胰岛β细胞特异性敲除PDHA1基因的小鼠模型,为动物水平上探究糖尿病发病机制提供新的靶点。
BACKGROUND: In rodent models of type 2 diabetes mellitus and human, the expression level of pyruvate dehydrogenase complex is decreased, and PDHA1 deficiency is the most common cause of changed pyruvate dehydrogenase complex activity.OBJECTIVE: To construct and identify the inducible islet-specific PDHA1 knockout mice, so as to provide basis for the study on the role of PDHA1 in pathogenesis of diabetes mellitus.METHODS: The study was approved by the Ethics Committee of Southern Medical University. By using Cre-loxP recombination system,PDHA1~(flox/flox)Cre~(+/-)mice were generated by crossing PDHA1~(flox/flox) mice with Cre~(+/-)mice. Genotypic identification was performed by PCR at the age of 3-4 weeks and the PDHA1~(flox/flox)Cre~(+/-)mice were the required mouse model. PDHA1~(flox/flox)Cre~(+/-)mice(βKO group, n=5) and Cre~(+/-)mice(control group, n=5) at the age of 4 weeks were selected and received the same water and feed. Tamoxifen(50 mg/kg) was intraperitoneally injected at the age of 8 week to induce gene knockout. Three weeks later, pancreatic tissue, adipose tissue, and liver tissue were removed under anesthesia. qPCR, western blot and immunohistochemical staining were applied to identify the PDHA1 knockout effect and to observe the mouse phenotype.RESULTS AND CONCLUSION:(1) PCR analysis selected PDHA1~(flox/flox)Cre~(+/-)mice.(2) qPCR method was used to test the pancreas, fat and liver tissues of mice, and it was confirmed that the knockout effect had tissue specificity. The expression level of PDHA1 mRNA in islet in theβKO group was significantly lower than that in the control group.(3) The results of western blot and immunohistochemical scanning showed a significant decrease in PDHA1 protein in the βKO group compared with the control group, suggesting the PDHA1 knockout effect was obvious.(4) In summary, the β cell-specific deletion of the PDHA1 gene is successfully constructed by Cre-loxP system, which provides a novel target for studying the pathogenesis of diabetes at animal level.
BACKGROUND: In rodent models of type 2 diabetes mellitus and human, the expression level of pyruvate dehydrogenase complex is decreased, and PDHA1 deficiency is the most common cause of changed pyruvate dehydrogenase complex activity.OBJECTIVE: To construct and identify the inducible islet-specific PDHA1 knockout mice, so as to provide basis for the study on the role of PDHA1 in pathogenesis of diabetes mellitus.METHODS: The study was approved by the Ethics Committee of Southern Medical University. By using Cre-loxP recombination system,PDHA1~(flox/flox)Cre~(+/-)mice were generated by crossing PDHA1~(flox/flox) mice with Cre~(+/-)mice. Genotypic identification was performed by PCR at the age of 3-4 weeks and the PDHA1~(flox/flox)Cre~(+/-)mice were the required mouse model. PDHA1~(flox/flox)Cre~(+/-)mice(βKO group, n=5) and Cre~(+/-)mice(control group, n=5) at the age of 4 weeks were selected and received the same water and feed. Tamoxifen(50 mg/kg) was intraperitoneally injected at the age of 8 week to induce gene knockout. Three weeks later, pancreatic tissue, adipose tissue, and liver tissue were removed under anesthesia. qPCR, western blot and immunohistochemical staining were applied to identify the PDHA1 knockout effect and to observe the mouse phenotype.RESULTS AND CONCLUSION:(1) PCR analysis selected PDHA1~(flox/flox)Cre~(+/-)mice.(2) qPCR method was used to test the pancreas, fat and liver tissues of mice, and it was confirmed that the knockout effect had tissue specificity. The expression level of PDHA1 mRNA in islet in theβKO group was significantly lower than that in the control group.(3) The results of western blot and immunohistochemical scanning showed a significant decrease in PDHA1 protein in the βKO group compared with the control group, suggesting the PDHA1 knockout effect was obvious.(4) In summary, the β cell-specific deletion of the PDHA1 gene is successfully constructed by Cre-loxP system, which provides a novel target for studying the pathogenesis of diabetes at animal level.
引文
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[7]Yonashiro R,Eguchi K,Wake M,et al.Pyruvate Dehydrogenase PDH-E1βControls Tumor Progression by Altering the Metabolic Status of Cancer Cells.Cancer Res.2018;78(7):1592-1603.
[8]Rodríguez-Enríquez S,Hernández-Esquivel L,Marín-Hernández A,et al.Mitochondrial free fatty acidβ-oxidation supports oxidative phosphorylation and proliferation in cancer cells.Int J Biochem Cell Biol.2015;65:209-221.
[9]Kaufman BA,Li C,Soleimanpour SA.Mitochondrial regulation ofβ-cell function:maintaining the momentum for insulin release.Mol Aspects Med.2015;42:91-104.
[10]Park S,Jeon JH,Min BK,et al.Role of the Pyruvate Dehydrogenase Complex in Metabolic Remodeling:Differential Pyruvate Dehydrogenase Complex Functions in Metabolism.Diabetes Metab J.2018;42(4):270-281.
[11]Talchai C,Xuan S,Lin HV,Sussel L,Accili D.Pancreaticβcell dedifferentiation as a mechanism of diabeticβcell failure.Cell.2012;150(6):1223-1234.
[12]Requejo-Aguilar R,Lopez-Fabuel I,Fernandez E,et al.PINK1deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1.Nat Commun.2014;5:4514.
[13]Whitley MJ,Arjunan P,Nemeria NS,et al.Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in theαV138M variant of human pyruvate dehydrogenase.J Biol Chem.2018;293(34):13204-13213.
[14]Zhang S,Hulver MW,McMillan RP,et al.The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility.Nutr Metab(Lond).2014;11(1):10.
[15]Liu YQ,Moibi JA,Leahy JL.Chronic high glucose lowers pyruvate dehydrogenase activity in islets through enhanced production of long chain acyl-CoA:prevention of impaired glucose oxidation by enhanced pyruvate recycling through the malate-pyruvate shuttle.J Biol Chem.2004;279(9):7470-7475.
[16]Yang F,Liu C,Chen D,et al.CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.Mol Ther Nucleic Acids.2017;7:378-386.
[17]Ma Y,Yu L,Pan S,et al.CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre-loxP-mediated lineage tracing.FEBS J.2017;284(19):3262-3277.
[18]Mulder H.Transcribingβ-cell mitochondria in health and disease.Mol Metab.2017;6(9):1040-1051.
[19]Sun W,Quan N,Wang L,et al.Cardiac-Specific Deletion of the Pdha1 Gene Sensitizes Heart to Toxicological Actions of Ischemic Stress.Toxicol Sci.2016;151(1):193-203.
[20]Lewis AJ,Neubauer S,Tyler DJ,et al.Pyruvate dehydrogenase as a therapeutic target for obesity cardiomyopathy.Expert Opin Ther Targets.2016;20(6):755-766.
[21]Ussher JR,Wang W,Gandhi M,et al.Stimulation of glucose oxidation protects against acute myocardial infarction and reperfusion injury.Cardiovasc Res.2012;94(2):359-369.
[22]Gudiksen A,Bertholdt L,Vingborg MB,et al.Muscle interleukin-6 and fasting-induced PDH regulation in mouse skeletal muscle.Am J Physiol Endocrinol Metab.2017;312(3):E204-E214.
[23]Harrison BC,Bell ML,Allen DL,et al.Skeletal muscle adaptations in response to voluntary wheel running in myosin heavy chain null mice.J Appl Physiol(1985).2002;92(1):313-322.
[24]Krus U,Kotova O,Spégel P,et al.Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells.Biochem J.2010;429(1):205-213.
[25]Bortesi L,Zhu C,Zischewski J,et al.Patterns of CRISPR/Cas9 activity in plants,animals and microbes.Plant Biotechnol J.2016;14(12):2203-2216.
[26]Valny M,Honsa P,Kirdajova D,et al.Tamoxifen in the Mouse Brain:Implications for Fate-Mapping Studies Using the Tamoxifen-Inducible Cre-loxP System.Front Cell Neurosci.2016;10:243.
[27]Aguirre AJ,Meyers RM,Weir BA,et al.Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.Cancer Discov.2016;6(8):914-929.
[28]Cerniglia GJ,Dey S,Gallagher-Colombo SM,et al.The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase(PDH)-E1αPhosphorylation.Mol Cancer Ther.2015;14(8):1928-1938.
[2]Cho NH,Shaw JE,Karuranga S,et al.IDF Diabetes Atlas:Global estimates of diabetes prevalence for 2017 and projections for 2045.Diabetes Res Clin Pract.2018;138:271-281.
[3]Wang L,Gao P,Zhang M,et al.Prevalence and Ethnic Pattern of Diabetes and Prediabetes in China in 2013.JAMA.2017;317(24):2515-2523.
[4]中华医学会糖尿病学分会,国家基层糖尿病防治管理办公室.国家基层糖尿病防治管理指南(2018)[J].中华内科杂志,2018,57(12):885-893.
[5]Morrow RM,Picard M,Derbeneva O,et al.Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity.Proc Natl Acad Sci U S A.2017;114(10):2705-2710.
[6]Kim-Muller JY,Zhao S,Srivastava S,et al.Metabolic inflexibility impairs insulin secretion and results in MODY-like diabetes in triple FoxO-deficient mice.Cell Metab.2014;20(4):593-602.
[7]Yonashiro R,Eguchi K,Wake M,et al.Pyruvate Dehydrogenase PDH-E1βControls Tumor Progression by Altering the Metabolic Status of Cancer Cells.Cancer Res.2018;78(7):1592-1603.
[8]Rodríguez-Enríquez S,Hernández-Esquivel L,Marín-Hernández A,et al.Mitochondrial free fatty acidβ-oxidation supports oxidative phosphorylation and proliferation in cancer cells.Int J Biochem Cell Biol.2015;65:209-221.
[9]Kaufman BA,Li C,Soleimanpour SA.Mitochondrial regulation ofβ-cell function:maintaining the momentum for insulin release.Mol Aspects Med.2015;42:91-104.
[10]Park S,Jeon JH,Min BK,et al.Role of the Pyruvate Dehydrogenase Complex in Metabolic Remodeling:Differential Pyruvate Dehydrogenase Complex Functions in Metabolism.Diabetes Metab J.2018;42(4):270-281.
[11]Talchai C,Xuan S,Lin HV,Sussel L,Accili D.Pancreaticβcell dedifferentiation as a mechanism of diabeticβcell failure.Cell.2012;150(6):1223-1234.
[12]Requejo-Aguilar R,Lopez-Fabuel I,Fernandez E,et al.PINK1deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1.Nat Commun.2014;5:4514.
[13]Whitley MJ,Arjunan P,Nemeria NS,et al.Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in theαV138M variant of human pyruvate dehydrogenase.J Biol Chem.2018;293(34):13204-13213.
[14]Zhang S,Hulver MW,McMillan RP,et al.The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility.Nutr Metab(Lond).2014;11(1):10.
[15]Liu YQ,Moibi JA,Leahy JL.Chronic high glucose lowers pyruvate dehydrogenase activity in islets through enhanced production of long chain acyl-CoA:prevention of impaired glucose oxidation by enhanced pyruvate recycling through the malate-pyruvate shuttle.J Biol Chem.2004;279(9):7470-7475.
[16]Yang F,Liu C,Chen D,et al.CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.Mol Ther Nucleic Acids.2017;7:378-386.
[17]Ma Y,Yu L,Pan S,et al.CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre-loxP-mediated lineage tracing.FEBS J.2017;284(19):3262-3277.
[18]Mulder H.Transcribingβ-cell mitochondria in health and disease.Mol Metab.2017;6(9):1040-1051.
[19]Sun W,Quan N,Wang L,et al.Cardiac-Specific Deletion of the Pdha1 Gene Sensitizes Heart to Toxicological Actions of Ischemic Stress.Toxicol Sci.2016;151(1):193-203.
[20]Lewis AJ,Neubauer S,Tyler DJ,et al.Pyruvate dehydrogenase as a therapeutic target for obesity cardiomyopathy.Expert Opin Ther Targets.2016;20(6):755-766.
[21]Ussher JR,Wang W,Gandhi M,et al.Stimulation of glucose oxidation protects against acute myocardial infarction and reperfusion injury.Cardiovasc Res.2012;94(2):359-369.
[22]Gudiksen A,Bertholdt L,Vingborg MB,et al.Muscle interleukin-6 and fasting-induced PDH regulation in mouse skeletal muscle.Am J Physiol Endocrinol Metab.2017;312(3):E204-E214.
[23]Harrison BC,Bell ML,Allen DL,et al.Skeletal muscle adaptations in response to voluntary wheel running in myosin heavy chain null mice.J Appl Physiol(1985).2002;92(1):313-322.
[24]Krus U,Kotova O,Spégel P,et al.Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells.Biochem J.2010;429(1):205-213.
[25]Bortesi L,Zhu C,Zischewski J,et al.Patterns of CRISPR/Cas9 activity in plants,animals and microbes.Plant Biotechnol J.2016;14(12):2203-2216.
[26]Valny M,Honsa P,Kirdajova D,et al.Tamoxifen in the Mouse Brain:Implications for Fate-Mapping Studies Using the Tamoxifen-Inducible Cre-loxP System.Front Cell Neurosci.2016;10:243.
[27]Aguirre AJ,Meyers RM,Weir BA,et al.Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.Cancer Discov.2016;6(8):914-929.
[28]Cerniglia GJ,Dey S,Gallagher-Colombo SM,et al.The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase(PDH)-E1αPhosphorylation.Mol Cancer Ther.2015;14(8):1928-1938.