Fig. 5.
GLP-2 stimulates IGF-I mRNA transcript levels in murine ISEMF cells through a PI3K- and Akt-dependent mechanism. A, ISEMF cells were treated with 10−8 m h(Gly2)GLP-2 for 30 min, and cell lysates were analyzed by Western blotting for the levels of pAkt and tAkt; actin was used as the loading control. A representative blot is shown for two different experiments; graph shows results from densitometry analysis. *, P < 0.05 (n = 6; L2 and L3, at P10–P15). B, ISEMF cells were treated with 10−8 m h(Gly2)GLP-2 for 2 h in the absence (open bars) or presence (closed bars) of either vehicle alone, wortmannin (500 nm), or LY294002 (50 μm). Total RNA was analyzed by qRT-PCR for IGF-I mRNA transcript levels, and data are expressed relative to 18S rRNA levels. *, P < 0.05 vs. GLP-2 in the absence of inhibitor (n = 4–12; L2–L5, at P10–P15). C, ISEMF cells were infected with either Adv-GFP (control) or Adv-kinase-dead-Akt (Adv-KD) at 109 PFU/ml for 2 h. Two days later, cells were treated with 10−8 m GLP-2 for 30 min, and cell lysates were analyzed by Western blotting for the levels of pAkt and tAkt; actin was used as the loading control; a representative blot is shown, with the graph showing results from densitometry analysis. *, P < 0.05 vs. GLP-2 with Adv-GFP (n = 4; L2–L4, at P10–P15). D, Total RNA from Adv-GFP- and Adv-KD-infected ISEMF cells was analyzed by qRT-PCR for IGF-I mRNA transcript levels. *, P < 0.05 vs. GLP-2 with Adv-GFP (n = 4–7; L2–L4, at P10–P15). E, Mice were fasted overnight and then injected with vehicle (PBS) or 0.5 μg/g h(Gly2)GLP-2, and jejunal samples were collected at t = 90 min for analysis of IGF-I mRNA transcript levels in mucosal scrapes by qRT-PCR. In a separate study, animals were pretreated with wortmannin (Wm) [1.5 mg/kg in 4% (vol/vol) methanol in saline] or with vehicle (Veh) alone 30 min before administration of GLP-2 and analysis, as above. **, P < 0.01 (PBS vs. GLP-2, n = 5–6; Veh vs. Wm, n = 8–9).