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Transplantation. 2010 Jan 27;89(2):146-54. doi: 10.1097/TP.0b013e3181c4218d.

Microassay for glucose-induced preproinsulin mRNA expression to assess islet functional potency for islet transplantation.

Author information

  • 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

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).

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.

PMID:
20098276
[PubMed - indexed for MEDLINE]
PMCID:
PMC2852640
Free PMC Article

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