It has been suggested that insulin-like proteins may be growth factors in the nerous systems.
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. We extracted RNAs from various tissues using liquid nitrogen pulverization and by homogenization in the presence of GuSCN. The RNA pellets recovered from CsC1-cushion following centrifugation of the homogenate are subjected to oligo-dt columns for the purification of poly A plus or 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 major 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. Some minor mRNA species hybridizable to the cDNA probe were also found in the brain and other tissues. Thus, mRNA for insulin and insulin-like peptides can be detected in extrapancreatic tissues including the brain. We are current attempting to study the role of these insulin-like peptides in the growth, maturation and survival of neurons.
