ABA induces the increase of the [cAMP]_i in hNCI-H716 cells.

<p>(A) hNCI-H716 cells were incubated for the indicated time in the absence or presence of 200 μM ABA (squares), or of 10 mM glutamine (rhombi); [cAMP]_i was then measured on cell extracts. Data are mean±SD of at least 3 different experiments; *, p<0.05 compared with untreated cells; #, p<0.05 compared to glutamine-treated cells (for the same time). (B) hNCI-H716 cells were transfected with an empty plasmid (control) or with a LANCL2-containing plasmid (LANCL2). After 48 h from transfection, cells were lysed and a Western blot analysis was performed using an anti-LANCL2 monoclonal antibody [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140588#pone.0140588.ref019" target="_blank">19</a>]; a representative blot is shown, confirming LANCL2 overexpression after transfection. LANCL2 expression was normalized on vinculin levels. (C) After 48 h from transfection, cells were stimulated for 2.5 min in the absence or presence of 200 μM ABA. [cAMP]_i was measured on cell extracts and data, expressed as fold increase over values in unstimulated cells, are expressed as mean±SD of at least 3 different experiments; basal cAMP values were not significantly different upon transfection. *, p<0.05 compared to control. (D) After 48 h from transfection, cells were incubated for 2 h in the absence or presence of 200 μM ABA. GLP-1 levels in the culture media were then estimated with an ELISA kit. Data, expressed as fold increase over values in unstimulated cells, are expressed as mean±SD of at least 3 different experiments. *, p<0.05 compared to untreated cells.</p

ABA induces GLP-1 release and transcription in hNCI-H716 cells.

<p>(A) hNCI-H716 cells were incubated for 2 h in the absence or presence of ABA (at the indicated concentrations), or of 200 mM glucose or 10 mM glutamine (gln). In some experiments, cells were pre-incubated for 10 min in the absence or presence of 20 μM 2′,3′-Dideoxyadenosine, a specific adenylyl cyclase inhibitor (grey bar) or of 1 μM of a cell permeable PKA inhibitor (protein kinase A inhibitor 14–22 amide, myristoylated, black bar), prior to stimulation with 200 μM ABA. GLP-1 levels in the culture media were then estimated with an ELISA kit. Data, expressed as fold increase over values in untreated cells, are expressed as mean±SD of at least 3 different experiments. *, p<0.05 compared to untreated cells. (B) hNCI-H716 cells were incubated for 2 h in the absence or presence of 200 μM ABA and qPCR was performed with specific primers for GLP-1 and glucagon; *, p<0.05 compared to expression in untreated cells.</p

Effect of oral ABA on plasma GLP-1, insulin and glucose levels in rats.

<p>ABA (50 mg/Kg, black squares) or vehicle alone (open squares) were orally administered to rats pre-treated with Sitagliptin (6 animals per experimental group) and blood samples were collected at 0, 20, 40 and 60 min to evaluate plasma GLP-1 (A), insulin (C) and glucose (E). The AUC corresponding to the curves of GLP-1 (B), insulin (D) and glycemia (F) were calculated. Inset to panel A: blood samples were collected from the portal vein of rats not pre-treated with Sitagliptin, 10 min after ABA or vehicle administration and GLP-1 levels were evaluated (n = 5 rats per group). *, p<0.05 and **, p<0.01 compared with the corresponding value in vehicle-treated animals; #, p<0.05 and ##, p<0.01 compared with time zero.</p