Recent evidence indicates that aniino acids <AA) influence gene expression in a number of mammalian sy stems. Since AA stimulate glucagon release from pancreatic cells, the purpose of this study was to determine it' individual AA influence glucagon gene expression. Mouse α-TC6 cells, uhich lack preproinsulin mRNA and secrete only glucagon, were maintained in DMHM containing 25 m M glucose. 15% horse serum, and 2.5% fetal call serum, fells were seeded at 200.000 cells/ml into either 6 cm dishes or 2-4-\vell plates. At 70% confluence, cells were incubated in serum-free maintenance media for 24 hr, then in serum-free treatment media containing 12.5 m M glucose and either 0 or 20 AA or 20 AA minus phe. met. leu. 01 trp for another 24 hr. Northern anal)sis was performed using a 516 bp mouse preproglucagon eDNA produced by RT-PCR from rat preproglucagon ei)NA primers. We found that 20 AA and minus leu enhanced glucagon mRNA abundance 3- and 5-fold, respective!}, compared to 0 AA. Similarly, immunoreactive glucagon secretion was enhanced 4- and 5-told for 20 AA and minus leu. respectively, versus U AA. A 24-hr lime course study using a nbonuclease protection assay to detect glucagon mRNA abundance m [he 0 AA. 2(1 AA. and minus leu treatments indicated that glucagon me.ssaue IcveK decreased o\er time in all groups, and lhat levels were ! .7 and 2.5-fbld greater in the 20 AA and minus leu, respectively, versus 0 AA alter 24 lit We conclude that leu may be imolved in the regulation of gtucagon mRKA leveU in α-TC6 cells.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology