Biosynthetic regulation of endogenous hamster insulin and exogenous rat insulin II in transfected HIT cells.
To investigate mechanisms underlying biosynthetic regulation of an insulin gene, the rat insulin II gene was introduced into hamster beta-cells (HIT) by cotransfection with the neomycin phosphotransferase-selectable marker. The insulin gene fragment was 2.2 kilobases (kb) in length and contained all exons, introns, and approximately 700 base pairs (bp) of 5'-flanking DNA and 300 bp of 3'-flanking DNA. The HIT cell was known to have endogenous hamster insulin production under regulation by glucose and dexamethasone. In a pool of stably transfected cells (HIT M62pR2), rat insulin II and hamster insulin were produced at comparable rates. Glucose (20 mM) stimulated cellular [3H]leucine labeling of both hamster insulin and rat insulin II by approximately twofold. Addition of 10(-6) M dexamethasone to media containing 11.1 mM glucose inhibited biosynthesis of both hamster insulin and rat insulin II by greater than 90%. Thus, with both positive and negative biosynthetic regulation, changes in the cellular labeling of exogenous rat insulin II were qualitatively and quantitatively similar to those of the endogenous hamster insulin. These data suggest that the 2.2-kb rat insulin II gene fragment contained sufficient information for both expression and apparently "normal" biosynthetic regulation of exogenous rat insulin II (when compared with endogenous hamster insulin) in response to glucose and dexamethasone.
D000818:Animals; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D011384:Proinsulin / Q000096:biosynthesis; D051381:Rats; D014162:Transfection
D007328:Insulin; D003907:Dexamethasone; D004247:DNA; D011384:Proinsulin; D005947:Glucose