Evaluation of rat insulin messenger RNA in pancreatic and extrapancreatic tissues.
The purpose of these studies was to determine whether insulin detected immunochemically in extrapancreatic tissues of the adult rat is synthesized in situ by quantitating mRNA in these tissues. A blot hybridization assay was utilized with cloned 32P-proinsulin cDNA. The lower limit of detection was estimated to be 3pg. Proinsulin mRNA concentration was found to be 1000-1500 micrograms in isolated pancreatic islets and was easily detected in total pancreatic RNA at 10-15 pg/micrograms. Proinsulin mRNA was quantitated in rat insulinoma cells adapted to culture at levels 1:50 those in normal islets. Samples of RNA (20-50 micrograms) enriched about 50-fold for mRNA sequences by repeated oligo-deoxythymidylate chromatography were assayed. No insulin mRNA was detected in 50 micrograms samples of RNA from brain or in 20 micrograms samples from subsections of brain or other extrapancreatic tissues. RNA samples were undegraded as assessed by ability to stimulate protein synthesis in a cell-free system. Proinsulin mRNA from pancreas as added to brain homogenates and recovered intact. Brain RNA samples with insulin mRNA levels 1:1000 that of pancreas would be predicted to have 50-75 pg proinsulin mRNA/50 micrograms sample assayed if present. Because none was found, brain must have a concentration less than 1:6,000 that of pancreas. These findings suggest that immunoassayable insulin detected in extrapancreatic tissues of the adult rat is synthesized by the pancreas.
Giddings
S J
SJ
Chirgwin
J
J
Permutt
M A
MA
D000818:Animals; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA; D007340:Insulinoma / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism; D008297:Male; D009693:Nucleic Acid Hybridization; D010179:Pancreas / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D011384:Proinsulin / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D011384:Proinsulin