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. Author manuscript; available in PMC: 2010 Sep 16.
Published in final edited form as: Sci Signal. 2010 Jan 26;3(106):ra7. doi: 10.1126/scisignal.2000514

Fig. 5. RACK1 antagonizes IRE1α-dependent upregulation of insulin production in β-cells.

Fig. 5

(A) Overexpression of RACK1 in β-cells attenuates the IRE1α phosphorylation-dependent increase in the intracellular insulin content. INS-1 cells maintained in 11.1 mM glucose were infected with control adenovirus Ad-GFP (at an MOI of 20), or co-infected at a total MOI of 20 with Ad-GFP (at an MOI of 10) plus viruses expressing the indicated forms of IRE1α, Ad-RACK1, or Ad-IRE1α-WT with Ad-RACK1. Cell lysates were analyzed by immunoblotting at 48 hours post-infection using the indicated antibodies. Intracellular insulin content was measured by radioactive immunoassay (RIA) and values are shown as means ± SEM (n=3 independent experiments). *P<0.05 compared to Ad-GFP-infected control cells, and #P<0.05 compared to cells infected with Ad-IRE1α by one-way ANOVA. (B) RACK1 knockdown increases IRE1α phosphorylation and insulin content in β-cells. INS-1 cells maintained in 11.1 mM glucose were infected at an MOI of 40 with Ad-shNC, Ad-shRACK1 #1, or Ad-shRACK1 #2. Intracellular insulin content was determined and is shown as means ± SEM (n=3 independent experiments). *P<0.05 compared to the Ad-shNC control by one-way ANOVA. (C) Adenoviral RACK1 overexpression reduces IRE1α phosphorylation and insulin content in mouse pancreatic islets. Primary islets isolated from 6–8 male C57BL/6 mice were pooled and infected at an MOI of 100 (with the assumption of 2500 cells/islet) by Ad-GFP or Ad-RACK1 for 48 hours. After pre-culture in 5 mM glucose for 18 hours, islets were incubated in 25 mM glucose for 3 hours prior to immunoblotting analysis. Insulin content was measured and are shown as means ± SEM (n=3 independent experiments). *P<0.05 by unpaired two-tailed t-test. (D) Restored expression of RACK1 reduces IRE1α phosphorylation and decreases insulin content in the islets of db/db mice. Islets isolated and pooled from 6–7 C57BL/6 db/db mice at 14–16 weeks of age were infected at an MOI of 100 with Ad-GFP or Ad-RACK1 for 48 hours or left uninfected. Uninfected islets from wild-type littermates were used as the control. Immunoblotting and RIA insulin measurement were performed, and insulin contents are shown as means ± SEM (n=3 independent experiments). *P<0.05 by unpaired two-tailed t-test.