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Published on: June 25, 2012
Microassay for glucose-induced preproinsulin mRNA expression to assess islet functional potency for islet
Keiko Omori1, Masato Mitsuhashi, Ivan Todorov
1Department 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
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.
