The aims of this project are to use a cDNA probe to detect the presence of mRNA for insulin or insulin-like peptide in extrapancreatic tissues including the brain and to investigate their physiological role. RNAs are extracted from various tissues using liquid nitrogen pulverization followed by homogenization in the presence of GuSCN. The RNA pellets recovered from CsCl-cushion following centrifugation of the homogenate are subjected to oligo-dt columns for the purification of poly A plus minus RNAs which include mRNAs. These isolated mRNAs are electrophoresed on agarose gel followed by blotting to a nitrocellulose membrane. These immobilized mRNAs are then hybridized to a cloned cDNA fragment of proinsulin gene which has been nick-translated with 32P-dCTP. We found that the 32P-cDNA probe is hybridized to mRNA from extrapancreatic tissues under stringent conditions (i.e., high temperature and low salts). However, the molecular sizes of these hybridizable mRNA transcripts are different from that detected in the pancreas. Thus, the size of pancreatic mRNA is of 0.5 kilobase, whereas two species of mRNA transcripts detected in the gut, heart and to a lesser extent, liver have approximately 4.2 and 2.2 kilobases. We also detected these two mRNA transcripts in the brain and a cloned cell line NCB-20 (neuroblastoma x fetal hamster brain cell hybrid), suggesting a neuronal location of these transcripts. Thus, mRNA for insulin and insulin-like can be detected in extrapancreatic tissues including the brain. Ontogenetic studies and regional distribution of these mRNAs in the brain are now in progress. Their role in the CNS awaits further investigation.
