Figure 7. SIK2 is essential for beta cells to meet insulin demand in models of metabolic syndrome.

(a) (Top) Western blot analysis of SIK2 protein levels in islets from control C57BL/6J (B6) and B6.V-Lep^ob/J (ob/ob) mice. (Bottom) Histogram showing relative intensity of SIK2 protein levels normalized to beta actin loading control. Data are mean ± s.d. from n=2 mice from a single experiment, and are representative of three independent experiments with consistent results. (b) Western blot analysis for SIK2, p35 and Lkb1 levels in islets of high fat diet-fed (20 weeks) and age matched control (normal chow diet) mice. (c) Western blot analysis of SIK2 protein levels in islets from B6 and ob/ob mice infected with non-targeting control or SIK2 shRNA lentivirus. (d) Static GSIS assay using islets from B6 and ob/ob mice infected with non-targeting control or SIK2 shRNA lentivirus (n=3 mice for each condition). Error bars represent s.d. (e) Western blot anaylsis of SIK2 levels. MIN6 cells were cultured for 15 h in 0, 5 or 25 mM glucose in the presence or absence of 2DG (25 mM). (f) (Top) Western blot anaylsis of SIK2 levels. MIN6 cells were pretreated with 100 nM rapamycin or EtOH (control) for 3h, then cultured for 15 h in 0, 5 or 25 mM glucose in the presence or absence of 100 nM rapamycin. (Bottom) Western blot anaylsis of SIK2 levels. MIN6 cells and mouse islets were pre-treated with MG132 (20 μM) or DMSO vehicle for 3h, then cultured for 15 h in 0, 5 or 25 mM glucose in the presence or absence of MG132. (g) In vitro kinase assay using GST-p35 WT and SIK2 purified from MIN6 cells cultured for 15 h in 0, 5 or 25 mM glucose together with or without 20 μM MG132. (h) Top left: Blood glucose levels of random-fed BKS.Cg-Dock7 (BKS) and BKS.Cg-Dock7m +/+ Leprdb/J (db/db) mice (n=6 for both strains). Error bars represent s.e.m. Top right: Percentage of insulin secretion from islets of BKS and db/db mice (n=3 mice for both strains). Error bars represent s.d. (Bottom) Western blot analysis of SIK2 protein levels in islets from BKS and db/db mice. Statistical significance for all data was determined using two-tailed unpaired Student’s t-test (* p < 0.01, ** p < 0.05). All western blot data are representative of two or three independent experiments with consistent results. The statistics source data for (d and h) are provided in Supplementary Table 1.