REGULATION OF INSULIN PRERNA SPLICING BY GLUCOSE

Glucose tightly regulates the synthesis and secretion of insulin by be ta cells in the pancreatic islets of Langerhans, To investigate whethe r glucose regulates insulin synthesis at the level of insulin RNA spli cing, we developed a method to detect and quantify a small amount of R NA by using the branched DNA (bDNA) signal-amplification technique, Th is assay is both sensitive and highly specific: mouse insulin II mRNA can he detected from a single beta cell (beta TC3 cells or mouse islet s), whereas 1 million non-insulin-producing alpha cells (alpha TC1.6 c ells) give no signal, By using intron and er;on sequences, oligonucleo tide probes were designed to distinguish the various unspliced and par tially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA s plicing rates were estimated from the rate of disappearance of insulin preRNA signal from beta cells treated with actinomycin D to block tra nscription, We found that the two introns in mouse insulin II are not spliced with the same efficiency, Intron 2 is spliced out more efficie ntly than intron 1, As a result, some mRNA retaining intron 1 enters t he cytoplasm, making up approximate to 2-10% of insulin mRNA in the ce ll, This partially spliced cytoplasmic mRNA is quite stable, with a ha lf-life similar to the completely spliced form, When islets grown in h igh glucose are shifted to low glucose medium, the level of insulin pr eRNA and the rate of splicing fall significantly, We conclude that glu cose stimulates insulin gene transcription and insulin preRNA splicing . Previous estimates of insulin transcription rates based on insulin p reRNA levels that did not consider the rate of splicing may have under estimated the effect of glucose on insulin gene transcription.