The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells

Abstract

Aims/hypothesis: We used the db/db mouse to determine the nature of the secretory defect in intact islets.

Methods: Glucose tolerance was compared in db/db and wild-type (WT) mice. Isolated islets were used: to measure insulin secretion and calcium in a two-photon assay of single-insulin-granule fusion; and for immunofluorescence of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs).

Results: The 13-18-week-old db/db mice showed a diabetic phenotype. Isolated db/db islets showed a 77% reduction in insulin secretion induced by 15 mmol/l glucose and reductions in the amplitude and rise-time of the calcium response to glucose. Ionomycin-induced insulin secretion in WT but not db/db islets. Immunofluorescence showed an increase in the levels of the SNAREs synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) in db/db islets, but reduced syntaxin-1A. Therefore, db/db islets have both a compromised calcium response to glucose and a compromised secretory response to calcium. Two-photon microscopy of isolated islets determined the number and distribution of insulin granule exocytic events. Compared with WT, db/db islets showed far fewer exocytic events (an 83% decline at 15 mmol/l glucose). This decline was due to a 73% loss of responding cells and, in the remaining responsive cells, a 50% loss of exocytic responses per cell. An assay measuring granule re-acidification showed evidence for more recaptured granules in db/db islets compared with WT.

Conclusions/interpretation: We showed that db/db islets had a reduced calcium response to glucose and a reduction in syntaxin-1A. Within the db/db islets, changes were manifest as both a reduction in responding cells and a reduction in fusing insulin granules per cell.

Fig. 1

Insulin secretion in db/db islets is decreased. (a) A static insulin secretion assay, performed over 20 min, shows that the glucose-dependent increase in insulin secretion in WT islets (white bars) is almost completely lost in db/db islets (black bars), n = 10–12 mice. (b) The calcium response to glucose is smaller and slower in the db/db islets (dotted line) compared with WT islets (solid line) (NB individual data were aligned to the rising phase). For both WT and db/db islets, the calcium response is relatively uniform across individual islets, n = 20–30 islets. Scale bar 50 μm. ^** p < 0.01

Fig. 2

Ionomycin fails to induce insulin secretion in db/db islets. (a) The calcium response to ionomycin, measured as calibrated Fura 2-AM fluorescence, in WT (solid line) and db/db islets (dotted line) have similar kinetics, but db/db islets have a bigger magnitude. (b) A static insulin assay, performed over 20 min, shows WT islets (white bars) respond to ionomycin with a 1.6-fold increase in insulin secretion whereas the db/db islets (black bars) show no response, n = 11–12 mice. ^* p < 0.05

Fig. 3

SNARE protein distribution and expression are altered in db/db islets. Immunofluorescence of fixed islets shows insulin staining (red) SNAP25, VAMP2 and syntaxin-1A in beta cells. The histograms show average fluorescence intensity changes in db/db islets normalised to WT and % of fluorescence intensity in cytosol regions compared with plasma membrane regions in db/db and WT islets, n > 4 mice, 15–19 islets. Scale bar 10 μm. ^*** p < 0.001. Syn1A, syntaxin-1A

Fig. 4

Within intact islets from db/db mice, beta cells have fewer insulin granule fusion events. (a) Typical two-photon cross sections taken across intact islets include many cells. Each granule fusion event over a 20 min time period is identified (as shown in ESM Fig. 2) and its position marked as a yellow circle. Scale bar 10 μm. (b) Quantification of the numbers of granule fusion events (in the two-photon volume, over 20 min), across a range of glucose concentrations, shows a significant reduction in granule fusion in db/db beta cells. The reduction in fusion events observed occurs because of both (c) a decrease in the number of responding cells and (d) a decrease in the number of fusion events per responding cell. (e) Cumulative plots of insulin granule fusion events over time, in response to 15 mmol/l glucose. n = 4–9 mice and 175 islets; p values from ANOVA analysis: ^** p < 0.01 and ^*** p < 0.001. In b–d: white bars, WT; hatched bars, db/+; black bars, db/db. In e: circles, WT; squares, db/+; and triangles, db/db

Fig. 5

Insulin granule fusion lifetimes are not changed in db/db cells. Examples of (a) short and (b) longer granule lifetimes as identified by the average fluorescence signal from regions of interest (ROIs) placed over each individual fusion event. Arrows show the start and end of the event. (c, d) Frequency graphs of the binned granule lifetimes; WT (c) and db/db islets (d) have similar distributions; n = 4–9 mice, total of 1,057 events

Fig. 6

The granule fluorescence plateaus are not changed in db/db cells. Two examples of single-granule-fusion events: (a) a low fluorescence plateau (fluorescence returns to background levels) (plateau = 0.09); and (b) a high fluorescence plateau (plateau = 0.87). In each case the mean fluorescence changes within a region of interest placed over the granule fusion site is shown, as are images taken at 1 s intervals over the duration of the fusion event. Scale bar 1 μm. (c) Graphs of frequency plotted against fluorescence (SRB) plateau (normalised to the peak fluorescence) are similar for WT (circles), db/+ (squares) and db/db islets (triangles)

Fig. 7

‘Kiss-and-run’ granule recapture is increased in the db/db cells. Two extracellular dyes, SRB (pH insensitive) (solid lines) and HPTS (dashed lines) both enter each fusing granule. (a) For most granule fusion events the average fluorescence intensity changes are similar for both dyes. (b) In contrast, some granule fusion events show a selective decrease in the HPTS signal where it is quenched by acidification. Scale bars 1 μm; n = 4 WT and n = 4 db/db mice; total of 580 events