Figure 7: PC mediates the regulatory effects of MDM2 on GSIS and mitochondrial metabolism.

(a) Pancreatic islets isolated from 10-week-old male β-MDM2KO mice and its WT littermates were stimulated with non-glucose secretagogues, including α-ketoisocaproic acid (KIC, 10 mM), and methyl succinate (MS, 10 mM) for 30 min, followed by measurement of insulin levels in the conditional medium (n=5). (b) Intracellular OAA levels in response to glucose stimulation (20 mM) for 30 min (n=4). (c) MIN6 β-cells were infected with adenovirus encoding luciferase (Adv-Luci) or dominant negative form of p53 (Adv-DN-p53) at m.o.i.=50 for 24 h. The infected cells were treated with nutlin-3a (10 μg ml⁻¹) or DMSO as vehicle for 6 h. The cells were subjected to serum and glucose starvation for an hour. Intracellular OAA levels were measured after glucose (20 mM) stimulation for 30 min (n=4). (d–k) The islets isolated from 10-week-old male β-MDM2KO mice and its WT littermates were infected with adenovirus encoding luciferase (Adv-Luci) or pyruvate carboxylase (Adv-PC) at m.o.i.=100 for 24 h. (d) The infected islets were subjected to immunoblotting using an antibody against PC or β-actin as indicated. Representative immunoblot images from three independent experiments are shown. The bottom panel is the densitometric analysis for the relative abundance of PC normalized with β-actin (n=4). Static (e) or dynamic (f) GSIS (n=8). Note that insulin content of isolated islets was similar among the three groups. Intracellular levels of OAA (g) and ATP (h) were measured on glucose stimulation for 30 and 10 min, respectively. (i) NADPH levels were measured on glucose stimulation for 30 min. OCR (j) and calcium influx (k) were measured as in Fig. 5 (n=4). *P<0.05, ^$P<0.05 (WT+Adv-Luci versus KO+Adv-Luci), ^#P<0.01 (KO+Adv-Luci versus KO+Adv-PC) (Student’s t-test). All data are represented as the mean±s.e.m.