Microassay for glucose-induced preproinsulin mRNA expression to assess islet functional potency for islet transplantation.
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.
Omori
Keiko
K
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
Mitsuhashi
Masato
M
Todorov
Ivan
I
Rawson
Jeffrey
J
Shiang
Keh-Dong
KD
Kandeel
Fouad
F
Mullen
Yoko
Y
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*
D017931:DNA Primers; D018076:DNA, Complementary; D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; C013737:preproinsulin; D005947:Glucose