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). CONCLUSION: 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.
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 humaninsulin 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). CONCLUSION: 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 diabeticpatients.
Authors: C Ricordi; D W Gray; B J Hering; D B Kaufman; G L Warnock; N M Kneteman; S P Lake; N J London; C Socci; R Alejandro Journal: Acta Diabetol Lat Date: 1990 Jul-Sep
Authors: B Leibiger; T Moede; T Schwarz; G R Brown; M Köhler; I B Leibiger; P O Berggren Journal: Proc Natl Acad Sci U S A Date: 1998-08-04 Impact factor: 11.205
Authors: J Wang; L Shen; H Najafi; J Kolberg; F M Matschinsky; M Urdea; M German Journal: Proc Natl Acad Sci U S A Date: 1997-04-29 Impact factor: 11.205
Authors: Mauro Giuliani; Wolfgang Moritz; Elvira Bodmer; Daniel Dindo; Patrick Kugelmeier; Roger Lehmann; Max Gassmann; Peter Groscurth; Markus Weber Journal: Cell Transplant Date: 2005 Impact factor: 4.064
Authors: K Omori; M Mitsuhashi; K Ishiyama; I Nair; J Rawson; I Todorov; F Kandeel; Y Mullen Journal: Diabetologia Date: 2011-05-13 Impact factor: 10.122
Authors: Orison O Woolcott; Richard N Bergman; Joyce M Richey; Erlinda L Kirkman; L Nicole Harrison; Viorica Ionut; Maya Lottati; Dan Zheng; Isabel R Hsu; Darko Stefanovski; Morvarid Kabir; Stella P Kim; Karyn J Catalano; Jenny D Chiu; Robert H Chow Journal: Pancreas Date: 2012-01 Impact factor: 3.327