Fig 2: Expression of GLP-1 and DPP-4 in human islets. (A) Immunofluorescence staining showing GLP-1 expression in pancreatic islets. Normal human pancreatic slices were co-stained with anti-glucagon and anti-amidated GLP-17–36 antibodies. DAPI staining (blue) was used to mark nuclei. Shown are representative images of >20 islets. The open arrow marks an example of cells expressing both GLP-1 and glucagon; the block arrow marks one predominantly expressing glucagon; and the diamond arrow marks one predominantly expressing GLP-1. (B) Western blotting assay showing expression of DPP-4 in human islets. The assay was repeated 5 times with different batches of normal human islets. (C) Immunofluorescence staining of DPP-4 in normal human pancreatic tissue. Shown are representative images of >20 islets.

Fig 3: Immunofluorescence staining showing GLP-1 and glucagon expression following linagliptin treatment. (A) Representative images showing GLP-1 (red) and glucagon (green) expression in cultured human islets after 3 days of linagliptin treatment. (B,C) The ratio of GLP-1+ to glucagon+ cells in ND (n = 5) (B) and T2D (n = 5) (C) human islets. The numbers of GLP-1+ cells and glucagon+ cells were manually counted and the ratio was calculated for 25–30 islets in each group. The data were expressed as Mean ± SEM. *indicates p < 0.05 when compared to untreated control.

Fig 4: GLP-1R and DPP-4 expression in ND and T2D human islets. (A) Western blotting assay examining GLP-1R and DPP-4 expression in the lysates of ND and T2D islets. Beta-actin was included as loading control and reference for quantification. Shown are cropped images, and the full-length blots are included in the supplemental information. (B) Quantification of GLP-1R band signal that was normalized to ß-actin signal (n = 5 for each group). (C) Quantification of DPP-4 signal normalized to ß-actin (n = 5). The data were expressed as Mean ± SEM. *p < 0.05, and **p < 0.01.

Fig 5: Basal GLP-1, insulin and glucagon secretion in cultured human islets following linagliptin treatment. Human islets isolated from ND and T2D donors were cultured at a density of 150 IEQ/0.5 ml in the presence of 0, 5, 30, and 50 nM linagliptin for 3 days with daily media refreshing. The media was collected and used to measure GLP-1 (A,B), insulin (C,D), and glucagon (E,F) concentrations using corresponding ELISA kits. Shown are representative data obtained from the 3rd day culture. All data were normalized with the total protein of islet lysates that were collected at the end of the experiments (per ug protein), and expressed as Mean ± SEM. *p < 0.05; **p < 0.01 when compared to untreated group.