title: Microassay for glucose-induced preproinsulin mRNA expression to assess islet functional potency for islet transplantation.
abstract: BACKGROUND
  The capacity for insulin synthesis in islets is important for islet transplantation to succeed. We developed a microassay that evaluates the potency of human islets by measuring changes in glucose-induced human insulin gene (INS) expression using a single islet in octuplicate samples.
  
  
  METHODS
  Poly (A) messenger RNA (mRNA) was purified from a set of single handpicked human islets. Glucose-induced mature (postspliced) and premature (prespliced) insulin mRNA were quantified by reverse-transcriptase polymerase chain reaction using several insulin mRNA primers designed at different locations including, intron, exon, and an exon-intron junction.
  
  
  RESULTS
  The synthesis of premature INS mRNA was significantly increased in islets exposed to high glucose for 16 vs. 4 hr (P<0.01), whereas mature INS mRNA showed no difference. Glucose-induced premature INS mRNA synthesis was attenuated in heat-damaged islets. Stimulation index (SI) calculated by normalizing premature by mature INS mRNA (SI_INS mRNA) positively correlated with SI of insulin release (SI_16h insulin) from the same set of islets during 16-hr incubation in high or low glucose media, and SI of glucose-mediated insulin release obtained from the same islet lot in a perifusion system (n=12). Furthermore, linear multiple regression analysis using SI_INS mRNA and SI_16h insulin predicted islet transplantation outcome in nonobese diabetic (NOD) scid mice (n=8).
  
  
  CONCLUSIONS
  The measurement of glucose-induced premature INS mRNA normalized by mature INS mRNA can be used to assess the functional quality of human islets and may predict islet function after transplantation in type 1 diabetic patients.
authors.affiliation: Department of Diabetes, Endocrinology and Metabolism, Southern California Islet Cell Resources Center, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. komori@coh.org
authors.forename: Keiko
authors.initials: K
authors.lastname: Omori
authors.forename: Masato
authors.initials: M
authors.lastname: Mitsuhashi
authors.forename: Ivan
authors.initials: I
authors.lastname: Todorov
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Rawson
authors.forename: Keh-Dong
authors.initials: KD
authors.lastname: Shiang
authors.forename: Fouad
authors.initials: F
authors.lastname: Kandeel
authors.forename: Yoko
authors.initials: Y
authors.lastname: Mullen
mesh_terms: D017209:Apoptosis; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D005784:Gene Amplification; D005786:Gene Expression Regulation / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000502:physiology; D007438:Introns; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D016381:Islets of Langerhans Transplantation / Q000379:methods*; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; C013737:preproinsulin; D005947:Glucose