Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling, Insulin Secretion, and Glucose Tolerance (original) (raw)
Figure 1
Detection of CaMKII-mediated phosphorylation on RyR2 and determination of its sensitivity to glucose in INS-1 cells.
(A) Quantification of qRTPCR analyses showing mRNA transcript levels of RyR1, RyR2 and RyR3 in islets from WT mice (represented in arbitrary units (A.U.)). (B) Representative Western blots for RyR2 expression and phosphorylated RyR2-S2814 in lysates of skeletal muscles (SM), heart (Ht), and brain (Br) from WT mice and rat insulinoma INS-1 cells (IS). β-actin was used to normalize protein concentrations. Observation indicated that RyR2 was present and could be phosphorylated in IS cells, comparable to the Ht. (C) Representative Western blots for RyR2 and its phosphorylation at S2814 from immunoprecipitates obtained from IS lysates using anti-RyR2 antibody. The lysates were prepared from INS-1 cells incubated with 2.8 mM (−) glucose or stimulated with 25 mM (+) glucose with and without pre-treatment with 10 µM KN-93. (D) Representative Western blots for CaMKII and its autophosphorylation at T287 from IS lysates generated in the above experiments. (E) Quantification revealed increased S2814 phosphorylation (pS2814) normalized to total RyR2 level upon glucose (25 mM) stimulation. Pre-treatment with 10 µM KN-93 blunted this increase. (F) Quantification also revealed increased autophosphorylation of CaMKII at T287 (pT287) normalized to total CaMKII level upon stimulation with 25 mM glucose but not in the presence of KN-93. Data (N = 3–6 experiments) are represented as average ± SEM. *P<0.05, vs. 2.8 mM glucose; #P<0.05 vs. 25 mM glucose.