Abscisic Acid Stimulates Glucagon-Like Peptide-1 Secretion from L-Cells and Its Oral Administration Increases Plasma Glucagon-Like Peptide-1 Levels in Rats

Abstract

In recent years, Abscisic Acid (ABA) has been demonstrated to be involved in the regulation of glucose homeostasis in mammals as an endogenous hormone, by stimulating both insulin release and peripheral glucose uptake. In addition, ABA is released by glucose- or GLP-1-stimulated β-pancreatic cells. Here we investigated whether ABA can stimulate GLP-1 release. The human enteroendocrine L cell line hNCI-H716 was used to explore whether ABA stimulates in vitro GLP-1 secretion and/or transcription. ABA induced GLP-1 release in hNCI-H716 cells, through a cAMP/PKA-dependent mechanism. ABA also enhanced GLP-1 transcription. In addition, oral administration of ABA significantly increased plasma GLP-1 and insulin levels in rats. In conclusion, ABA can stimulate GLP-1 release: this result and the previous observation that GLP-1 stimulates ABA release from β -cells, suggest a positive feed-back mechanism between ABA and GLP-1, regulating glucose homeostasis. Type 2 diabetes treatments targeting the GLP-1 axis by either inhibiting its rapid clearance by dipeptidyl-peptidase IV or using GLP-1 mimetics are currently used. Moreover, the development of treatments aimed at stimulating GLP-1 release from L cells has been considered as an alternative approach. Accordingly, our finding that ABA increases GLP-1 release in vitro and in vivo may suggest ABA and/or ABA analogs as potential anti-diabetic treatments.

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

(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.

Fig 2. ABA induces the increase of the [cAMP]_i in hNCI-H716 cells.

(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 [19]; 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.

Fig 3. Effect of oral ABA on plasma GLP-1, insulin and glucose levels in rats.

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.