p53 is required for chloroquine-induced atheroprotection but not insulin sensitization

Abstract

An intact genotoxic stress response appears to be atheroprotective and insulin sensitizing. ATM, mutated in ataxia telangiectasia, is critical for the genotoxic stress response, and its deficiency is associated with accelerated atherosclerosis and insulin resistance in humans and mice. The antimalarial drug chloroquine activates ATM signaling and improves metabolic phenotypes in mice. p53 is a major effector of ATM signaling, but it is unknown if p53 is required for the beneficial effects of chloroquine. We tested the hypothesis that the cardiometabolic effects of chloroquine are p53-dependent. ApoE-null mice with or without p53 were treated with low-dose chloroquine or saline in the setting of a Western diet. After 8 weeks, there was no p53-dependent or chloroquine-specific effect on serum lipids or body weight. Chloroquine reduced plaque burden in mice wild-type for p53, but it did not decrease lesion extent in p53-null mice. However, chloroquine improved glucose tolerance, enhanced insulin sensitivity, and increased hepatic Akt signaling regardless of the p53 genotype. These results indicate that atheroprotection induced by chloroquine is p53-dependent but the insulin-sensitizing effects of this agent are not. Discrete components of the genotoxic stress response might be targeted to treat lipid-driven disorders, such as diabetes and atherosclerosis.

Trial registration: ClinicalTrials.gov NCT00455325 NCT00455403.

Fig. 1.

Assessment of JNK signaling in the presence and absence of p53. Western blot analyses of phospho-JNK and total-JNK were performed using thioglycollate-elicited peritoneal macrophages derived from p53^+/+ and p53^−/− (on apoE^−/− background) mice. JNK activation was achieved by treatment with (A) LPS (10 ng/ml) or (B) TNFα (10 ng/ml) for the indicated times. Densitometric analyses of Western blots normalized to total JNK are shown in the right panels for the indicated times (*P < 0.05). The discrepancy between the number of bands seen on phospho-JNK blots and the total JNK blots likely reflects the variability in the relative abundance of the three JNK subtypes (JNK1, 2, and 3) and their phosphorylation status. JNK, Jun N-terminal kinase; KO, knockout; LPS, lipopolysaccharide; TNF, tumor necrosis factor; WT, wild type.

Fig. 2.

Schematic of the 2 × 2 experimental design. p53^+/+ apoE^−/− mice (n = 29) and p53^−/− apoE^−/− mice (n = 23) were placed on Western diet (42% calories from fat) at 6–8 weeks of age. They were divided into chloroquine (loading injection of 7 mg/kg, then biweekly injections of 3.5 mg/kg) versus saline injections. Interventions were performed at 1- and 2-month intervals as indicated. GTT, glucose tolerance test; ITT, insulin tolerance test.

Fig. 3.

Effects of p53 deficiency and chloroquine treatment on common serum metabolites. Measurements of serum metabolites for (A) cholesterol, (B) triglycerides, (C) glucose, and (D) free fatty acids were performed at baseline then after 1 and 2 months of Western-diet feeding. Diet-induced increases for these parameters were not significantly different among the four groups. Chq, chloroquine; FFA, free fatty acids; KO, knockout; WT, wild type.

Fig. 4.

Effects of chloroquine on insulin sensitivity in the presence and absence of p53. p53^+/+ and p53^−/− (on apoE^−/− background) are denoted as WT and KO, respectively. (A and B) Glucose tolerance tests (GTT) for WT and KO mice. AUC is the incremental area under the glucose curve. (C and D) Insulin tolerance tests (ITT) for WT and KO mice. Glucose values are expressed as percent of the baseline (time 0) value. AUC is the incremental area under the glucose% curve. (E and F) Western blot analyses of phospho-Akt (Ser-473), total-Akt, phospho-GSK3β (Ser-9), and total-GSK3β from liver tissue homogenates of WT and KO mice after 2 months of chloroquine versus saline injections. *P < 0.05 for AUC data. All results are presented as mean ± SEM. AUC, area under the curve; CHQ, chloroquine; KO, knockout; WT, wild type.

Fig. 5.

Effects of chloroquine on atherosclerosis in the presence and absence of p53. Atherosclerotic area involvement was determined by computer image analysis of (A) en face pinned aortas for the total aorta (p53 WT, upper-left panel; p53 KO, lower-left panel), and regions of the aorta (aortic arch, thoracic aorta, and abdominal aorta, right panels), and (B) Oil-Red-O–stained sections of the aortic roots (p53 WT, left panel; p53 KO, right panel). Representative aortic root sections are shown below the data sets. Horizontal lines within the data sets represent medians. *P < 0.05. CHQ, chloroquine; KO, knockout; NS, not significant; WT, wild type.

Fig. 6.

Model depicting the potential roles of the ATM-p53 genotoxic stress response in insulin resistance and atherosclerosis. ATM, ataxia telangiectasia mutated.