Fig. 5.

Munc18b restores the deficient GSIS in GK rat islets to normal levels.

(a) Western blot analysis of Munc18b and cognate SNARE proteins in pancreatic islets of normal Wistar and GK rats. Bottom: Summary of densitometric analysis of blots from 3 separate experiments; results expressed as percentage of control values.

(b) Western blot analysis of SM and SNARE proteins of Ad-Munc18b/eGFP and Ad-eGFP-transduced GK rat islets. Rat brain and INS-1 are positive controls. Data shown is representative of 3 sets of experiments. Bottom: densitometric analysis of Munc18b overexpression compared to Ad-eGFP-transduced GK rat islets (N = 3). Other proteins showed no change after Munc18b overexpression (analysis not shown).

(c) Islet perifusion assays of Lenti-mCherry- and Lenti-Munc18b/mCherry-transduced GK rat islets, and Lenti-mCherry-transduced normal Wistar rat islets; and corresponding AUCs (right) of first-phase (10–25 min) and second-phase (25–50 min) insulin release. Data shown are from 3 sets of experiments.

(d) TIRFM images showing SG density on the PM at basal condition (i, N = 9; scale bars represent 2 μm; Bottom: Comparison of averaged SG densities) and histograms of the different fusion events after glucose-stimulation (ii–iv). First-phase (5 min after 16.7 mM glucose stimulation) and second-phase (5–16 min) in Lenti-mCherry-transduced Wistar rat β-cells (i, N = 15), and GK rat β-cells transduced with Lenti-mCherry (ii, N = 17) or Lenti-Munc18b/mCherry (iii, N = 16). Data obtained from 3 independent experiments. (v) Although (ii), (iii) and (iv) are shown in three modes of fusion (predocked, short-dock and no-dock newcomer SGs) events in first- and second-phase GSIS, the summary combined the two populations of newcomer SGs.

Data are shown as mean ± SEM, *p < 0.05; **p < 0.01, NS: no significant difference.