Figure 6. Chronic hyperglycaemia induces glucagon expression in β-cells.

(a) Schematic illustrating how Rip-CreER^+/+ (i), Rosa^RFP/− (ii) and Rosa^V59M/− (iii) were used to generate Rosa^RFP/V59M mice (βV59M-RFP mice). β-Cells were selectively and irreversibly labelled following tamoxifen injection, by crossing an inducible rat insulin promoter Cre line (i; β) with a floxed tdRFP reporter line in which tdRFP expression was driven by the endogenous ROSA promoter (ii; RFP). These β-RFP control mice were then crossed with an inducible Kir6.2-V59M line (iii; V59M) to create βV59M-RFP mice (iv). (b) Representative examples of immunofluorescence staining for insulin (green), glucagon (pink) and RFP (red) in control (top panel, β-RFP) and 4-week-diabetic βV59M-RFP (middle and bottom panels) isolated islet cells. White arrows, RFP⁺/glu⁺ cells. White arrowhead, RFP⁺/glu⁺/ins⁺ cell. Scale bar, 10 μm. (c,d) Islet cells from β-RFP and 4-week-diabetic βV59M-RFP mice were FAC-sorted into RFP⁺ and RFP⁻ populations, and analysed for preproglucagon mRNA by qPCR (c) and glucagon protein (d); n=4 mice per genotype. Data are mean values±s.e.m. *P<0.05; Mann–Whitney test.