title: Co-transfection of GK and mhPINS genes into HepG2 cells confers glucose-stimulated insulin secretion.
abstract: BACKGROUND
  The purpose of this study was to construct an 'artificial beta cell' that can exhibit physiologic glucose-stimulated insulin secretion for the treatment of type 1 diabetes.
  
  
  METHODS
  Retroviral vector containing the glucokinase (GK) gene and mutated human proinsulin (mhPINS) gene was constructed. HepG2 cells were first infected with recombinant retrovirus carrying the GK and mhPINS genes, then selectively cultured with G418 to obtain the positive clones. GK and mhPINS gene transcription and expression were identified by radioimmunity, Western blot and RT-PCR techniques. Finally, the dose-response effect of glucose on insulin secretion from those HepG2 cells that expressed both GK and mhPINS genes was tested with HepG2 cells that only expressed the mhPINS gene as a control.
  
  
  RESULTS
  HepG2 cells with transferred GK and mhPINS genes were selectively cultured with G418 and the positive clones were obtained in 3 weeks. Four clones with GK and mhPINS gene expression were selected from 20 positive clones by radioimmunity and Western blot. We picked up one clone with a strong GK and mhPINS gene expression and named it clone Beta. In clone Beta, differences in insulin secretion at 0.5 and 0.75 mmol/L glucose concentrations were not significant (P>0.05) and differences in insulin secretion at 2.0, 3.0, 4.0, 5.0 and 6.0 mmol/L glucose concentrations were not significant (P>0.05), while there were significant differences in insulin secretion at other glucose concentrations(P<0.05). The artificial beta cell, clone Beta, obtained a glucose-stimulated insulin secretion with maximal insulin secretion at 1.75-2.00 mmol/L glucose concentrations.
  
  
  CONCLUSIONS
  An artificial beta cell that exhibits glucose-stimulated insulin secretion can be constructed successfully.
authors.affiliation: Department of Endocrinology, First Affiliated HospitalChongqing University of Medical Sciences, Chongqing, PR China.
authors.forename: H T
authors.initials: HT
authors.lastname: Zheng
authors.forename: H C
authors.initials: HC
authors.lastname: Deng
authors.forename: C J
authors.initials: CJ
authors.lastname: Huang
authors.forename: N Z
authors.initials: NZ
authors.lastname: Lan
authors.forename: F
authors.initials: F
authors.lastname: Fang
authors.forename: R
authors.initials: R
authors.lastname: Jian
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D000078790:Insulin Secretion; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism*; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012190:Retroviridae / Q000502:physiology; D017422:Sequence Analysis, DNA; D014162:Transfection; D019065:Virus Assembly / Q000187:drug effects
chemical_list: D018076:DNA, Complementary; D007328:Insulin; D050505:Mutant Proteins; D012333:RNA, Messenger; D005941:Glucokinase; D005947:Glucose