Changes in beta cell function occur in prediabetes and early disease in the Lepr (db) mouse model of diabetes

Abstract

Aims/hypothesis: Type 2 diabetes is a progressive disease that increases morbidity and the risk of premature death. Glucose dysregulation, such as elevated fasting blood glucose, is observed prior to diabetes onset. A decline in beta cell insulin secretion contributes to the later stages of diabetes, but it is not known what, if any, functional beta cell changes occur in prediabetes and early disease.

Methods: The Lepr (db) mouse (age 13-18 weeks) was used as a model of type 2 diabetes and a two-photon granule fusion assay was used to characterise the secretory response of pancreatic beta cells.

Results: We identified a prediabetic state in db/db mice where the animals responded normally to a glucose challenge but have elevated fasting blood glucose. Isolated islets from prediabetic animals secreted more and were bigger. Insulin secretion, normalised to insulin content, was similar to wild type but basal insulin secretion was elevated. There was increased glucose-induced granule fusion with a high prevalence of granule-granule fusion. The glucose-induced calcium response was not changed but there was altered expression of the exocytic machinery. db/db animals at the next stage of disease had overt glucose intolerance. Isolated islets from these animals had reduced insulin secretion, reduced glucose-induced granule fusion events and decreased calcium responses to glucose.

Conclusions/interpretation: Beta cell function is altered in prediabetes and there are further changes in the progression to early disease.

Keywords: Beta cell; Compound exocytosis; Exocytosis; Insulin granules; Islets; Prediabetes; Progression.

Fig. 1

Classification of db/db animals into separate stages of disease. (a) The overall mean GTT was significantly impaired in db/db (triangles) compared with WT (+/+; circles) and +/− mice (squares). (b) db/db mice (black bars) are classified into four stages based on the AUC for the GTT: stage 1, <1,600 mmol/l × min; stage 2, 1,600–2,200 mmol/l × min; stage 3, 2,200–2,800 mmol/l × min; stage 4, >2,800 mmol/l × min. White bars, WT. (c) Glucose-induced islet insulin secretion is increased at stage 1 and reduced for the later stages of disease. (d) Stage 1 animals are significantly heavier than WT (+/+) animals. (e) GTT is comparable in stage 1 db/db (dotted line) and WT (+/+) islets (solid line) but significantly increases in stage 2 db/db islets (dashed line). (f) Stage 1 db/db mice have a small but significant increase in FBG. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT

Fig. 2

Islet morphology changes in db/db islets. Stage 1 and stage 2 db/db islets have (a–d) a larger size (scale bar 200 μm) and (e) increased islet insulin content compared with WT (+/+) islets. (f) Insulin secretion, expressed as percentage insulin content for WT (+/+) and stage 1 and stage 2 db/db islets in response to 3 and 15 mmol/l glucose. (g) Immunostaining shows a selective expansion in beta cells compared with alpha cells in stage 1 and 2 db/db islets compared with WT. Scale bar 50 μm. *p < 0.05; **p < 0.01; ***p < 0.001. Gluc, glucagon

Fig. 3

Glucose-induced insulin granule fusion is substantially altered in db/db islets. (a) A two-photon granule fusion assay identified the sites of granule fusion events (yellow circles) over a 20 min period in response to 15 mmol/l glucose. Compared with WT (+/+), many more fusion events are seen in stage 1 db/db islets, with greater spatial clustering of fusion events. In contrast, stage 2 db/db islets show a dramatic decrease in fusion events. Histograms show changes in granule exocytosis in terms of the numbers of responding cells and responses per cell within the two-photon volume. (b) At 6 weeks of age, the number of glucose-induced exocytic events is comparable with that of age-matched WT (+/+) islets. Scale bars 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT

Fig. 4

Characterisation of beta cell changes in stage 1 and 2 db/db islets. (a) Glucose-induced islet calcium responses are comparable between WT (solid line) and db/db stage 1 islets (grey dotted line) but reduced in db/db stage 2 islets (black dashed line). Scale bar 50 μm. (b) Potassium-induced islet calcium responses are comparable among WT (solid line), db/db stage 1 (grey dotted line) and db/db stage 2 (black dashed line) islets. (c) Responses to 50 mmol/l KCl show enhanced insulin secretion at stage 1 and 2 in db/db compared with WT (+/+) beta cells. (d) qPCR shows elevations in SNARE mRNA expression at stage 1 (light grey bars) and 2 (dark grey bars) but a reduction in Slc2a2 mRNA at stage 2 in db/db compared with WT (white bars) beta cells. *p < 0.05, ***p < 0.001 vs WT

Fig. 5

Compound exocytosis is increased in stage 1 db/db beta cells. (a) Analysis of a 4 μm² region around each exocytic event shows that the frequency of observing more than seven events is much lower in WT (dashed line, circles) and stage 2 db/db (dotted line, squares) islets but not stage 1 db/db islets (solid line, triangles). (b) Evidence for multiple granule fusion events occurring within the same region of interest. (c) This is shown as an image series with the numbers (i), (ii), and (iii) indicating the peaks of fluorescence as each granule fuses. Scale bar 1 μm. (d) Histograms of primary (under seven events within a 4 μm² region) exocytosis and compound exocytosis show increases in stage 1 and decreases in stage 2 db/db compared with WT (+/+) beta cells. **p < 0.01, ***p < 0.001. 2P, two-photon; AU, arbitrary units; ROI, region of interest

Fig. 6

Electron microscopy evidence for compound exocytosis. (a, b) Serial block-face electron micrographs through two typical examples of multigranular compound exocytosis in db/db islets stimulated with 15 mmol/l glucose. Low magnification images show the area that is enlarged and shown as serial sections (i–iv) 50 nm apart taken through a compound exocytic event composed of multiple fused granules. The reconstructed models show the spatial relationships between each fused granule (in colour) and also identify the adjacent cell plasma membrane (white mesh). (c) In some cases, granules within the cell were fused together but made no contact with the cell surface, as shown in the model. Scale bar 2 μm

Fig. 7

Spider graph showing changes in db/db islet and beta cell secretion at different stages of disease. Each line is centred on the mean for each variable and the width represents the SEM. Note, for clarity, the AUC is inverted and shown as a fraction of 5,000 mmol l⁻¹ × min. Green, WT; yellow, db/db stage 1; orange, db/db stage 2. 2P, two-photon