Changes in beta cell function occur in prediabetes and early disease in the Lepr db mouse model of diabetes
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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.
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.
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.
Beta cell function is altered in prediabetes and there are further changes in the progression to early disease.
KeywordsBeta cell Compound exocytosis Exocytosis Insulin granules Islets Prediabetes Progression
Fasting blood glucose
Glucose tolerance test
Impaired fasting glucose
Impaired glucose tolerance
Quantitative real-time PCR
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor
Type 2 diabetes is a chronic, progressive disease characterised by loss of glucose homeostasis and leading to morbidity and premature mortality [1, 2, 3, 4]. A key feature of the later disease stages is the failure of insulin secretion from beta cells to meet the demand of increased peripheral insulin resistance [1, 5]. However, in early disease there can be an increase in insulin secretion that might be explained by an increase in beta cell mass; nevertheless, it is unknown whether beta cell function also changes. Here, we have used the Lepr db mouse model of diabetes to investigate beta cell function in early disease.
Prior to overt type 2 diabetes, a prediabetic state can be identified by impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) after a glucose tolerance test (GTT). A study of prediabetic individuals (identified by IFG) showed increased insulin secretion in response to an OGTT . Some studies have also shown that individuals with IGT have enhanced insulin secretion [6, 7], but it is not known whether this is due to upregulation of beta cell secretion or an increased number of beta cells .
There is indirect evidence that compensatory increases in beta cell number can occur in obese humans [8, 9] and in animal models such as prediabetic Zucker diabetic fatty rats , TALLYHO diabetic mice , ob/ob mice  and animals fed a high-fat diet [13, 14, 15].
There is no direct evidence that beta cell function changes in prediabetes or early disease. However, it is known to occur in other contexts. For example beta cell secretory function is enhanced in pregnancy to cope with the increased demand from the developing fetus . Moreover, in the ob/ob model of obesity, in addition to increased numbers of beta cells, there is increased secretion per cell .
In this paper, we describe experiments using the Lepr db mouse model of type 2 diabetes  aimed at understanding beta cell function during prediabetes and early disease. Our results show beta cells do undergo functional changes during prediabetes and their exocytic capacity is upregulated in response to glucose. We conclude that pathological changes are already occurring in beta cells during prediabetes.
Glucose tolerance test
We performed i.p. glucose tolerance tests (1 g/kg body weight) for wild-type (WT, +/+) and all db/db mice (see electronic supplementary material [ESM] Methods for details).
Mice of both sexes and aged 13–18 weeks were used, except where otherwise stated (BKS.Cg-Dock7m+/+Lepr db /J, The Jackson Laboratory, Bar Harbor, ME, USA) were humanely killed according to local animal ethics procedures (approved by the University of Queensland Anatomical Biosciences Ethics Committee). Islets were prepared by enzymatic digestion of the pancreas from WT, stage 1 and stage 2 db/db mice for experiments (except the insulin assay, which was performed in WT and db/db mice of all stages; see ESM Methods for details).
Two-photon imaging, insulin assays and calcium measurements were performed in a sodium-rich extracellular solution (see ESM Methods for details).
Isolated islets were first cultured for 2–3 days and then prior to imaging were bathed in an extracellular solution containing 3 mmol/l glucose for 30 min (37°C, 95%/5% air/CO2). Islets were then transferred to an extracellular solution containing 15 mmol/l glucose and 0.8 mmol/l sulforhodamine B (SRB). Two-photon imaging was performed at 34°C, with exocytic events recorded as entry of the SRB extracellular dye (excitation 950 nm, detection 550–650 nm) into each fused granule (see ESM Methods for details).
Homogeneous time resolved fluorescence with the HTRF Insulin Kit (no. 62INSPEB, Cisbio Bioassays, Brisbane, QLD, Australia) was used to measure islet insulin secretion (see ESM Methods for details).
For intracellular calcium measurement, we used the ratiometric calcium indicator Fura2-AM (see ESM Methods for details).
Freshly isolated islets were fixed in 4% paraformaldehyde, permeabilised with Triton X-100 and blocked with donkey serum containing BSA for 1 h prior to incubation with primary antibodies (see ESM Methods for details).
Quantitative real-time PCR
Total RNA was isolated from islets using RNeasy plus Micro kit (Qiagen). cDNA was synthesised from 100 ng total RNA using the SuperScript III reverse transcriptase (Invitrogen, Mt Waverley, VIC, Australia; see ESM Methods for details).
Serial block-face scanning electron microscopy
Islets cultured for 2 days were stimulated with 15 mmol/l glucose for 7 min and then fixed with 2.5% glutaraldehyde and processing for imaging (see ESM Methods for details).
All numerical data are presented as the mean ± SEM. Statistical analysis was performed using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism version 6 (La Jolla, CA, USA). Datasets containing just two groups were subjected to a two-tailed, unpaired Student’s t test with statistical significance identified at p < 0.05. Islets from at least three animals were used in each experiment. Statistical significance is indicated in the figures as *p < 0.05, **p < 0.01, and ***p < 0.001.
We next tested islet function to determine whether insulin secretion changed according to the stage of disease severity. Isolated islets were stimulated for 20 min with 3 or 15 mmol/l glucose. At stage 1, db/db islets showed a significant increase in insulin secretion at both 3 and 15 mmol/l glucose compared with WT (Fig. 1c; 24 WT and 25 db/db mice). At stage 2, insulin secretion decreased to levels resembling those of WT islets; at stage 3, there was a further decrease in insulin secretion; and at stage 4, insulin secretion was significantly lower than in WT islets (Fig. 1c).
We used this classification to subdivide the animals’ phenotype. At stage 1, db/db animals were heavier than WT (Fig. 1d), with no significant difference in the GTT (Fig. 1e) and a small, but significant, increase in fasting blood glucose (FBG) (Fig. 1f). By stage 2, the body weight, GTT and FBG of db/db animals all differed significantly from those of WT animals and were indicative of frank diabetes (Fig. 1d–f). These changes approximate to the progression of human type 2 diabetes.
Islet size and insulin content increase in prediabetes
Insulin secretion during stage 1 shows upregulation of the exocytic capacity
To study secretory function within individual beta cells in intact islets, we used a two-photon, granule fusion assay that we previously used to characterise the fusion of individual insulin granules from beta cells within intact islets . When recording from the islet core, where most cells are beta cells (see Fig. 2g), this method detects the fusion of granules that are the same size as insulin granules, colocalise with insulin and, in terms of fusion numbers, fully account for insulin secretion from the islets .
The increased exocytic response at stage 1 reflects both an apparent increase in the numbers of cells responding to glucose and an increase in the number of fusing granules per cell (Fig. 3a; nine WT and 21 db/db mice). This shows that individual beta cells have become more responsive to a glucose stimulus.
Phenotype of 6-week-old WT and stage 1 db/db mice
AUC (mmol l−1 min−1)
916 ± 43
1,076 ± 108
6.3 ± 0.5
7.6 ± 1.0
Islet diameter (μm)
145 ± 7
132 ± 11
Islet insulin content (ng/islet)
40.2 ± 10.3
43.2 ± 8.7
Islet insulin secretionb (% of insulin content)
1.23 ± 0.43
1.43 ± 0.41
Our data also indicate the mechanism responsible for decreased insulin secretion at stage 2. Stage 2 islets showed continued upregulation of the mRNA for SNAREs (as measured by qPCR; Fig. 4d), an enhanced high extracellular potassium response and an intact potassium-induced calcium response compared with WT islets (Fig. 4b). However, their glucose-induced calcium response was much lower (Fig. 4a). Although we haven’t explored this in detail, the reduced mRNA for Slc2a2 (which encodes glucose transporter 2 [GLUT2]; Fig. 4d) suggests that beta cells at stage 2 are still secretory competent but are defective in glucose sensing.
Granule fusion behaviour in stage 1 prediabetic islets
We used GTTs to classify disease severity in db/db animals and demonstrated that changes in beta cell function do occur in prediabetes. In stage 1 db/db mice, the GTT is the same but FBG is elevated compared with WT mice; however, we observe an increase in islet size and insulin content, which supports previous work demonstrating beta cell expansion in early disease. Our new finding is that function is also altered, including an increase in basal insulin secretion and an increase in the number of glucose-induced granule fusion events, the latter due to upregulation of compound exocytosis.
Beta cell function in prediabetes
Our evidence demonstrates significant changes in the secretory function of islets in prediabetes.
First, we observed an increase in basal (i.e. at 3 mmol/l glucose) insulin secretion. This increase is maintained in stage 2 islets, consistent with previous work on the db/db model [20, 22, 23]. The increased secretion could be driven by the elevated basal intracellular calcium concentration that was previously observed at stage 2  and which we now show also occurs in stage 1 islets (Fig. 4a), possibly through calcium-dependent granule mobilisation step .
Second, after stimulation, we showed that stage 1 islets have more glucose-induced insulin granule fusion events compared with WT islets (Fig. 3a), but with no change in the calcium response (Fig. 4a). Since the high potassium-induced response is also bigger, again with no increase in the calcium response, these data are consistent with upregulation of the terminal stages of the stimulus secretion pathway that are downstream of calcium entry. This conclusion is supported by the increase in mRNA expression of some of the SNAREs (Fig. 4d).
A direct comparison of insulin secretion from islets (Figs 1c, 2f) shows that the fold increase in secretion is decreased at all disease stages in db/db islets. This largely reflects the reduced capacity of the beta cells to respond to glucose at stages 2–4. However, at stage 1 this is mostly due to increased basal secretion: glucose-stimulated secretion increases when measured per islet (Fig. 1c) but remains the same when measured per insulin content (Fig. 2f).
Given the similarities in islet insulin secretion between WT and stage 1 db/db islets, the large increase in insulin granule fusion numbers was surprising: the explanation for the discrepancy between increased fusion events but comparable secretory output is not clear. One possibility is heterogeneity in the beta cell content and function, which has been observed in late-stage disease , possibly due to cell dedifferentiation . Heterogeneity might give rise to some cells that contain insulin but are non-responsive, therefore explaining the increase in granule fusion events in the other cells. An alternative possibility is that insulin granule fusion at stage 1, in particular via compound exocytosis, is less efficient. This idea is difficult to test, but we might expect that multiple granules releasing content through a common, single, fusion pore would at least have slower kinetics of insulin release compared with normal fusion, in which each granule releases its content through its own fusion pore.
Compound exocytosis is rarely observed in WT cells after glucose stimulation but is specifically upregulated, for example with glucagon-like peptide 1 (GLP-1) stimulation . Although it is possible that stage 1 db/db islets selectively enhance pathways that normally regulate compound exocytosis, we suggest that this is more likely to result from non-specific upregulation of the exocytic machinery. Given the critical importance of the balanced expression of SNARES for regulating granule fusion , it is possible that an imbalance might lead to an increased prevalence of compound exocytosis.
Comparison with disease progression in humans
Our phenotypic data showing disease progression in db/db mice are consistent with previous work  and with the progression of type 2 diabetes in humans . In humans, compensatory increases in the insulin response occur in stage 1 disease [18, 29] and are associated with increased islet size and insulin content [8, 9]. As the disease progresses, insulin content decreases  and, as we observed in the mouse, glucose-induced insulin secretion is reduced [31, 32].
Humans with relatively poorer insulin sensitivity tend to have a higher insulin secretion . The product of insulin sensitivity and pancreatic responsivity (the disposition index), has a hyperbolic relationship  that does not normally change over time. However, evidence, from obese humans that are developing insulin resistance, shows that insulin secretion can increase according to the demand . Another human study showed increasing insulin secretion up to a maximum at a FBG of 5.6 mmol/l, followed by a decline . Longitudinal studies show that individuals who progressed to diabetes did not have compensatory increases in insulin secretion; in contrast, those that did, did not progress [18, 35]. Consistent with this, animal models of diabetes also show increased insulin secretion in early disease [11, 36, 37].
Our data represent a step towards understanding the mechanisms that underlie increased insulin secretion in prediabetes and show that, in addition to an expansion in cell numbers, functional changes are occurring in the beta cell.
Summary of disease progression to diabetes
We conclude that altered beta cell function is intimately associated with prediabetes and progression to diabetes.
Serial block-face imaging was performed by Robyn Webb and Rick Webb, in the Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Australia.
All authors designed the experiments; OHD performed the experiments; all authors drafted and revised the manuscript and approved this version. PT is the guarantor of this work.
This work was supported by the National Health and Medical Research Council (APP1002520 and APP1059426) to PT and HYG and by a Diabetes Australia grant (Y15G-THOP) to PT. OHD was supported by a Prime Minister’s Australia Asia Endeavour Award.
Duality of interest statement
The authors declare that there is no duality of interest associated with this manuscript.
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