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1996 Fiscal Year Final Research Report Summary

Gene therapy of streptozotocin-induced diabetic rats with a regulatable insulin expression vector

Research Project

Project/Area Number 06557051
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section試験
Research Field 内分泌・代謝学
Research InstitutionGunma University, Institute for Molecular and Cellular Regulation

Principal Investigator

TAKEUCHI Toshiyuki  Gunma Univ.Inst.for Mol.& Cell.Regulation Professor, 生体調節研究所, 教授 (00109977)

Co-Investigator(Kenkyū-buntansha) KONDA Yoshitaka  Gunma Univ.Inst.for Mol.& Cell.Regulation Associate Professor, 生体調節研究所・遺伝子調節部門, 講師 (90261867)
KAWAZU Shoji  Gunma Univ.School of Medicine.Dept.of Internal Medicine Associate Professor, 医学部, 助教授 (30134547)
Project Period (FY) 1994 – 1996
KeywordsInsulin / Gene therapy / PEPCK / Adenovirus vector
Research Abstract

For engineering insulin-producing surrogate cells for diabetes gene therapy, two types of cells are available : neuroendocrine and non-neuro-endocrine cells. Neuroendocrine cells are equipped with a processing mechanism that converts a propeptide into a mature bioactive peptide, as well as a regulatory secretion system by which a mature peptide is secreted in response to extracellular stimuli. Thus, when neuroendocrine cells such as anterior pituitary corticotroph-derived endocrine cells AtT20 are used for diabetes gene therapy, the primary focus has been oriented to engineer an insulin secretion mechanism that responds to a physiological range of glucose stimuli. On the other hand, when non-neuroendocrine cells are used as insulin-producing cells, the cells need to be engineered with a processing mechanism and a regulatory secretion system.
In pancreatic beta cells, proinsulin is processed to mature insulin by prohormone convertases PC2 and PC3 (also named PC1) and carboxypeptidase H ( … More CPH). These two proteolytic reactions appeared specific to secretory granule-containing neuroendocrine cells. However, when we replaced the processing sites of proinsulin with those cleavable by a yeast Kex2 family endoprotease furin, the non-neuroendocrine cell lines including COS-7, HepG2, CHO,and NIH3T3 produced insulin with the same size as synthetic human insulin. Next, the furin-cleaved proinsulin required the removal of basic residues by carboxypeptidases for its maturation. Although non-neuroendocrine cells expressed different quantities of carboxypeptidase HmRNA,these cells contained considerable levels of carboxypeptidase activity. The insulins resulting from these cell lines were eluted as a single peak at the mature insulin position on a cation-exchange chromatography column. Thus, non-neuroendocrine cells are able to produce correctly processed insulin if proinsulin is first mutated to possess furin-cleavable processing sites.
For developing an insulin expression system that can be regulated by extracellular stimili, most desirable system is to use glucose as a stimulator. Alternative regulator may be insulin itself for down-regulating the expression. Since non-neuroendocrine cells do not carry secretory granules and secrete proteins and peptides through a constitutive pathway without their retention in the cytoplasm, a regulatory step for the production of insulin is limited to a gene transcription level. Many genes are known to be regulated their expression by glucose. For the regulatory expression of these genes target cells are required to equip with both glucose transporter type 2 and glucokinase for responding to a physiological range of glucose consentrations. For this criterion the primarily cultured hepatocytes are the cell of choice for assessing the expression of these genes. However, hepatocytes are relatively inefficient for DNA transfer by conventional gene transfer methods. Furthermore, hepatocyte culture cell lines with glucose transporter type 2 and glucokinase are not avalable at the present time. Thus, we attempted to generate a regulatory expression system by insulin itself using a hepatoma cell line and a phosphoenolpyruvate carboxykinase (PEPCK) promoter. The PEPCK is expressed in hepatocytes and catalyzes oxaloacetate to phosphoenolpyruvate by taking phosphate from GTP.This enzyme acts as a key step for hepatic glucose production. In diabetic subjects this enzyme is highly activated by the lack of insulin. The PEPCK promoter is known to be up-regulated by cAMP,glucocorticoids, retinoic acid (RA), and down-regulated by insulin and phorbol ester. We examined the insulin production using a PEPCK promoter plus insulin DNA construct in H4Ell cells.
The H4llE cells secreted immunoreactive insulin (IRI) constantly at a level of 2-3 fmol/10^6 cells/h. IRI was increased about 2-fold upon stimulation with 0.5 mM cAMP,further 3-fold with cAMP-dependent phosphodiesterase inhibitor IBMX.IRI was elevated only 2-fold by 5 to 700 nM dexamethasone, but 10-12-fold by the further addition of cAMP and IBMX.Retinoic acid induced IRI by 4-fold. Addition of exogenous insulin to the culture medium decreased insulin mRNA expresion strikingly on Northern blot. However, together with cAMP,IBMX,and dexamethasone, the inhibitory effect of exogenous insulin became weakened. In the insulin-producing H4llE cells the production was augmented by the addition of wortmannin, a phosphatidylinositol (PI) -3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. When the cells were perifused, insulin secretion was elevated 2h after the stimulation. Thus, the secretory pattern from the H4llE cells may correspond to the action mode of intermediate-acting insulin.
We then introduced the insulin expression unit into an adenoviral vector Adex. Using a recombinant adenoviral vector, we confirmed the production of mature insulin from primarily cultured rat hepatocytes. The insulin secretion from hepatocytes was also well regulated by dbcAMP,IBMX,glucagon, and dexamethasone. We moved onto an in vivo experiment using streptozotocin (STZ) -induced diabetic rats. After injecting STZ at 100 or 150mg/Kg body weight, rats exhibited hyperglycemia of 400-600mg/dl blood glucose. We administered the recombinant adenovirus from a rat tail vein, through where most of viruses are thought to accumulate in the liver. Insulin level went up to 2-3ng/ml around 10 days after the injection, and blood glucose went down to 300-400mg/dl. Although we could not attain normoglycemia in the recombinant virus-injected rats, we think that this insulin expression system improves diabetes mildly. We are currently determining a best virul titer and an appropriate method of virus administration. Less

  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] T.Kayo,Y.Sawada,Y.Suzuki,et al.: "Proprotein-processing endoprotease furin decreases regulated secretory pathway-specific proteins in the pancreatic β cell line MIN6" J.Biol.Chem.271. 10731-10737 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Nishigori,M.Yanagita,T.Takeuchi.: "Proinsulin cleaved by furin is processed to chromatographically mature insulin by carboxypeptidases in non-neuroendocrine cells." Peptides. 17. 789-796 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Kayo,Y.Konda,S.Tanaka,et al.: "Developmental expression of proprotein-processing endoprotease furin in rat pancreatic islets." Endocrinology. 137. 5126-5134 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] D.Lu,H.Hoshino,T.Takeuchi.: "Regulatable production of mature insulin from a hepatocyte cell line : insulin production is up-regulated by cAMP and glucocorticoids,and down-regulated by insulin" FEBS Lett.399. 37-42 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Takahashi,Y.Liu,N.Hayashi,et al.: "Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO." Peptides. (印刷中). (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Kayo,Y.Sawada,M.Suda,et al.: "Proprotein-processing endoproease furin conrols pancreatic β cells." Diabetes. (印刷中). (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 竹内 利行: "分子糖尿病学の進歩:プロインスリンのプロセシング" 金原出版社, 9 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 竹内 利行: "インスリン分泌機構:調節性分泌と構成性分泌" 科学評論社, 14 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] N.Hayashi, T.Kayo, K.Sugano and T.Takeuchi: "Production of bioactive gastrin from the non-endocrine cell lines CHO and COS-7." FEBS Lett. 337. 27-32 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Takahashi, and T.Takeuchi: "Production of bioactive enkephalin from the non-endocrine cell lines COS-7, NIH3T3, Ltk^-, and C2C12." Peptides. 16. 933-938 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Shibata, M.Kanzaki, T.Takeuchi, J-i.Miyazaki and I.Kojima: "Two distinct signaling pathways activated by activin A in glucose responsive pancreatic beta-cell lines." J.Mol.Endocrinol. 16. 249-258 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Kayo, Y.Sawada, Y.Suzuki, M.Suda, S.Tanaka, Y.Konda, J-i.Miyazaki, and T.Takeuchi: "Proprotein-processing endoprotease furin decreases regulated secretorey pathway-specific proteins in the pancreatic beta cell line MIN6" J.Biol.Chem.271. 10731-10737 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Nishigori, M.Yanagita and T.Takeuchi: "Proinsulin cleaved by furin is processed to chromatographically mature insulin by carboxy-peptidases in non-neuroendocrine cells." Peptides. 17. 789-796 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Kayo, Y.Konda, S.Tanaka, K.Takata, A.Koizumi, T.Takeuchi: "Developmental expression of proprotein-processing endoprotease furin in rat pancreatic islets." Endocrinology. 137. 5126-5134 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] D.Lu, H.Hoshino, T.Takeuchi: "Regulateble production of mature insulin from a hepatocyte cell line : insulin production is up-regulated by cAMP and glucocorticoids, and down-regulated by Insulin." FEBS Lett.399. 37-42 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Sawada, M.Inoue, T.Kanda, T.Sakamaki, S.Tanaka, N.Minamino, R.Nagai, T.Takeuchi: "Co-elevation of brain type natriuretic peptide and proprotein-processing endoprotease furin after myocardial infarction in rats." FEBS Lett.400. 177-182 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Takahashi, Y.Liu, N.Hayashi, F.Goto, M.Kato, H.Kawashima, T.Takeuchi: "Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO." Peptides. (in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Konda, H.Yokota, T Kayo, T.Horiuchi, N.Sugiyama, S.Tanaka, K.Takata, T.Takeuchi: " Proprotein-processing endoprotease furin controls the growth and differentiation of gastric surface mucous cells." J.Clin.Invest.(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Kayo, Y.Sawada, M.Suda, Y.Konda, T.Izumi, S.Tanaka, H.Shibata, T.Takeuchi: "Proteolytic activity of proprotein-processing endoprotease furin controls growth of pancreatic beta cells." Diabetes. (in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Sawada, H.Yokoyama, M.Inoue, T.Kanda, T.Sakamaki, R.Nagai, T.Takeuchi: "Stretch-induced hypertrophic growth of cardiocytes and processing of brain-type natriuretic peptide are controlled by proprotein-processing endoprotease furin." J.Biol.Chem.(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-09  

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