Serum-free Medium Increases and Insulin Decreases RDH10mRNA Stability—We determined the elimination half-life (t1⁄2)of RDH10 mRNA in cells cultured 16 h in serum-free medium,or maintained in growth medium, and then exposed to ActD for24 h (Fig. 5A). RDH10 mRNA had a t1⁄2 of 35 h in the absence ofserum and insulin. In the absence of serum, but in the presenceof insulin, the t1⁄2 decreased to 19 h. In the presence of serum andabsence of insulin (growth medium), RDH10 mRNA had abiphasic t1⁄2. The mRNA was decreased 50% by 8 h; after 8 h themRNAwas stabilized with a t1⁄2 of 118 h. Adding serum for 8 h tocells exposed to serum-free medium for 16 h also decreased theamount of RDH10 mRNA by 50%. Because RDH10 expressionis elevated 2–3-fold in serum-free medium, the amounts ofmessage in cells with serum-free medium, with or without insulin,continued to exceed those of cells maintained in growthmedium. Destabilization of RDH10 mRNA by insulin wasattenuated by inhibition of PI3K or expression of dnFoxO1, andcompletely prevented by inhibition of Akt (Fig. 5B). In theabsence of ActD, RDH10 expression in cells exposed to serumfreemedium 16 h, then treated 4 h with insulin, was notchanged significantly (Fig. 5C). In contrast, treatment withserum or serum and insulin reduced RDH10 mRNA to theamount in growth medium. These data show that mRNAinduced by long-term exposure to serum-free medium is moresensitive to serum than insulin. In cells exposed to serum-freemedium for 16 h, then treated 8 h with CHX and ActD versusActD alone, maximum RDH10 mRNA stability required translation(Fig. 5D).