Fig 1.
(A) Fluorescein diacetate/propidium iodide staining of control islets (CTL) and of islets treated with 10 μmol/L of liraglutide (Lira) at the indicated time-points. (B) Islet viability was also represented in percentage as histograms with controls (black bars) versus Lira (grey bars) (C) Glucose stimulation test: Insulin secreted by islet incubated in conditioned medium containing 2.5 mmol/L (black bars) and 25 mmol/L (grey bars) of glucose. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.
Fig 2.
The angiogenic effects of liraglutide in vitro.
(A) Insulin intensity toward islet surface in control islets (CTL; black bar) as compared to islets treated with 10 μmol/L of liraglutide (Lira; grey bar) (B) CD31 intensity toward islet surface in CTL (black bar) versus Lira (grey bar) group (C) Immunostaining of insulin and endothelial cells after 24 h for control islets (CTL; a, b, c, d) versus islets cultured with Lira10μM (Lira; e, f, g, h). Nuclear DAPI staining is shown in blue (a, e); insulin staining in green (b, f); vessels are stained red (c, g); and these are merged in d and h. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.
Fig 3.
Evaluation of angiogenic markers.
(A) Hypoxia-inducible factor-1α (HIF-1α) expression, evaluated by qPCR after 12 h under the indicated conditions and (B) Vascular endothelial growth factor (VEGF) gene expression, determined by quantitative polymerase chain reaction (qPCR) after 12-h exposure to 10μmol/L of liraglutide (Lira) or control (CTL) treatment. (C) VEGF secretion after 12, 24 and 48 h exposure to the indicated treatments using an enzyme-linked immunosorbent assay kit. (D) Platelet-derived growth factor-α (PDGF-α) gene expression, determined by qPCR after 12 h. Black bars, control; grey bars, Lira. Results were expressed as mean ± SEM. Statistics were expressed by * or using # when it is compared respectively to control or to Control + Rapa; *; # p<0.05.
Fig 4.
Angiogenic mechanisms induced in rat pancreatic islets by 10 μmol/L of liraglutide.
Evaluation of mammalian target of rapamycin (mTOR) activation, determined by the ratio of phosphorylated mTOR/mTOR using western blotting at the indicated time-points. Black bars, control; grey bars, Lira10μM. Results were expressed as mean ± SEM. *p<0.05.
Fig 5.
Metabolic control in diabetic rats after islet transplantation.
(A) Body-weight gain versus t = 0 day in no transplanted diabetic rats called SHAM (filled diamond), in transplanted diabetic animals with untreated islets called control group (filled square) and in transplanted diabetic rats using treated islets with Lira called Lira group (filled triangle). (B) The mean body-weight gains in SHAM (white bar), control (CTL; black bar) and 10μmol/L of liraglutide (Lira; grey bar) groups over the entire experiment. (C) Fasting glycaemia in the SHAM (filled diamond), control (filled square) and Lira (filled triangle) groups at the indicated time-points. (D) Mean fasting glycaemia in SHAM (white bar), control (black bar) and Lira (grey bar) during the experiment. Results were expressed as means ± SEM. ***p<0.001; **p<0.01; *p<0.05.
Fig 6.
The angiogenic effects of liraglutide in vivo.
(A) CD31 intensity toward islet surface of rats transplanted with control islets (CTL; black bar) as compared to animals grafted with islets treated with 10 μmol/L of liraglutide (Lira; grey bar) (B) CD31 intensity surrounding transplanted islets toward analysed surface in CTL (black bars) versus Lira (grey bars) groups (C) Insulin intensity toward islet surface in CTL islets (black bars) as compared to Lira (grey bar) (C) Immunostaining of insulin and endothelial cells 30 days post implantation for control islets (CTL; a, b, c, d) versus Lira (e, f, g, h). Nuclear DAPI staining is shown in blue (a, e); insulin staining in green (b, f); vessels are stained red (c, g); and these are merged in d and h. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.