INSULIN GENE-EXPRESSION AND INSULIN SYNTHESIS IN MAMMALIAN NEURONAL CELLS

To demonstrate the presence of de novo synthesis in central mammalian neurons, we cloned and sequenced a rabbit insulin cDNA from pancreas a nd used it to define sequences encoding insulin mRNA from postnatal ra bbit brain. We observed transcription/elongation of nascent insulin tr anscripts, characterized the size of these transcripts, and localized them to specific neurons in certain catecholaminergic-rich areas of th e central nervous system. RNase protection assays using a rabbit probe spanning a region from 14 bases 5' to the translation start site thro ugh all but 18 bases of the sequence encoding the A-chain of insulin s howed two bands in rabbit brain RNA and only one band in pancreas. The larger band in brain was the same size as that in pancreatic RNA; the other was approximately 10 bases shorter. Because the sequence of a r everse transcription-polymerase chain reaction product from brain RNA was identical to pancreatic RNA sequence in the region corresponding t o the 3' region of the probe, the smaller band in brain is most consis tent with a sequence mismatch in some brain mRNA in the region corresp onding to the 5'-end of the probe, In situ hybridization localized ins ulin mRNA to anatomical regions involved with olfaction and higher ass ociation of the limbic system. High performance liquid chromatography, radioimmunoassay, and [S-35]cysteine metabolic labeling of cultured n euronal and glial cells indicated extracellular secretion of immunopre cipitable insulin by neurons only. Presence of insulin transcripts wit hin specific neurons with extracellular secretion of the peptide sugge sts a specialized biological role.