Figure 5: Ciliary/basal body disruption impairs insulin receptor-mediated signalling. | Nature Communications

Figure 5: Ciliary/basal body disruption impairs insulin receptor-mediated signalling.

From: Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents

Figure 5

(a) Immunoblotting of basal and insulin-stimulated levels of phosphorylated p55 (pTyr199), phospho-Akt (pSer473), pan-Akt and γ-tubulin in lysates of MIN6m9 cells transduced with adenovirus targeting Ofd1 (shOfd1), Bbs4 (shBbs4) alongside scrambled RNA (shNC). (b) pSer473 Akt to pan-Akt ratio in the presence or absence of Bbs4 and Ofd1 in basal (INS-) and stimulated (INS +) MIN6m9 cells (*P=0.035 for insulin-stimulated levels, one-way ANOVA). (c) pSer472 Akt to pan-Akt ratio in insulin-stimulated Bbs4- or Ofd1-depleted murine islets (*P=0.042, one-way ANOVA). (d) Phosphorylated p85 (pTyr458) in insulin-stimulated murine islets transduced with adenovirus targeting Bbs4 and scrambled RNA control. (e) pSer473 Akt is reduced in islets from three Bbs4−/− animals compared with wild-type controls. (f) Phospho Akt/Akt ratio for Bbs4−/− and wild-type animals. (g) Syntaxin1A levels are reduced in Ofd1- or Bbs4-depleted islets compared with controls. (h) Relative expression levels of Snap25 and Stx1a are reduced in islets of Bbs4−/− animals compared with wild-type controls (RNA pooled from three animals each). (i) Re-introduction of STX1A- into Bbs4-depleted MIN6m9 cells partially restores the disposition index. **P(control versus shBbs4)=0.007, P(shNC versus shBbs4+1,600 ng STX1A)=0.417, P(shNC versus shBbs4+2,400 ng STX1A)=1.00, one-way ANOVA (Tukey’s). Red bars indicate the mean. (j) Under basal conditions (open bars), insulin content is similar to controls (shNC, black) in MIN6m9 cells treated with adenoviral shOfd1 (blue) or shBbs4 (red). After 30 min of 16.7 mM glucose and another 15 min 25 mM KCl, the total insulin content (solid bars) in Ofd1- (blue) or Bbs4- (red) depleted cells is significantly decreased compared with control cells (n=8, P=0.002, (one-way ANOVA)). (k) One hour after insulin stimulation, Ins mRNA is reduced by up to 40%, whereas βGck mRNA is slightly but not significantly upregulated in Ofd1-depleted MIN6m9cells (n=3). Error bars: st. dev.

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