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. 2020 Jun 19;10:9976. doi: 10.1038/s41598-020-65828-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Bihormonal cells are detected in human pancreata from Non-DM and DM subjects. Pg is present in β-cells and in a low % of α-cells as well as bihormonal cells. (A) Representative result from IF microscopy showing the presence of bihormonal cells in human (Non-DM) post-mortem samples. White arrows indicate bihormonal cells. Scale bar represents 25 μm. Insets show glucagon+, insulin+, DAPI stained and merged images. Yellow: glucagon, Green: insulin, Blue: nuclei. Representative of N = 13 Non-DM samples. For separate channel images of 40×, see Fig. S4. (B) Bihormonal cells detected in pancreata of human DM subjects. White arrows point to bihormonal cells. Insets show glucagon+, insulin+, DAPI stained and merged images. Red: glucagon, Green: insulin, Blue: nuclei. Representative of N = 12. Scale bar represents 25 μm. (C) The percentage of bihormonal cells is significantly higher in DM compared with Non-DM subjects (***p < 0.001). % bihormonal cells was determined by counting the number of cells positive for insulin and glucagon divided by the number of cells positive for glucagon. A total of 429 and 469 α-cells were counted in Non-DM and DM subjects respectively. Two fields of 400 × 300 μm rectangle area were counted per subject. Data show means ± S.D. (D) 3-D images from confocal microscopy of glucagon+, insulin+ bihormonal cells (white arrows). Scale bar represents 5 μm. (E) Orthogonal analysis of bihormonal cell shown in (D). a & b represent orthogonal projections. Yellow: glucagon, Green: insulin, Blue: nuclei. (F) Representative image of bihormonal cells positive for Pg/gingipain. Yellow: glucagon, Green: insulin, Red: Pg/gingipain, Blue: nuclei. (G) Percentage of Pg positive α-, β- and bihormonal cells. Y-axis: % cells positive for Pg, X-axis: cell types. 347 α-cells, 444 β-cells and 51 bihormonal cells were counted. N = 5 DM subjects. Data are presented as mean + S.D. ***p < 0.001.