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. 2014 Feb;63(2):526-34.
doi: 10.2337/db13-1019. Epub 2013 Oct 22.

Small-molecule inhibitors of PKR improve glucose homeostasis in obese diabetic mice

Takahisa Nakamura  1 , Alessandro Arduini, Brenna Baccaro, Masato Furuhashi, Gökhan S Hotamisligil
Affiliations

Small-molecule inhibitors of PKR improve glucose homeostasis in obese diabetic mice

Takahisa Nakamura et al. Diabetes. 2014 Feb.

Abstract

Obesity and metabolic diseases appear as clusters, often featuring high risk for insulin resistance and type 2 diabetes, and constitute a major global health problem with limited treatment options. Previous studies have shown that double-stranded RNA-dependent kinase, PKR, plays an important role in the nutrient/pathogen-sensing interface, and acts as a key modulator of chronic metabolic inflammation, insulin sensitivity, and glucose homeostasis in obesity. Recently, pathological PKR activation was also demonstrated in obese humans, strengthening its prospects as a potential drug target. Here, we investigate the use of two structurally distinct small-molecule inhibitors of PKR in the treatment of insulin resistance and type 2 diabetes in cells and in a mouse model of severe obesity and insulin resistance. Inhibition of PKR reduced stress-induced Jun NH2-terminal kinase activation and insulin receptor substrate 1 serine phosphorylation in vitro and in vivo. In addition, treatment with both PKR inhibitors reduced adipose tissue inflammation, improved insulin sensitivity, and improved glucose intolerance in mice after the establishment of obesity and insulin resistance. Our findings suggest that pharmacologically targeting PKR may be an effective therapeutic strategy for the treatment of insulin resistance and type 2 diabetes.

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Figures

Figure 1
Figure 1
Imoxin inhibits PKR activity in vitro and in vivo. PKR activity was assessed by in vitro kinase assay of PKR with IRS1 (A) or eIF2α (B) as substrates in the presence or absence of 16.7 μmol/L imoxin as indicated. An oxindole compound was used as a negative control for PKR inhibitor. PKR, IRS1, and eIF2α protein levels were examined by immunoblotting. C: TNF-α–induced IRS1 phosphorylation in wild-type MEFs. Cells were pretreated with 1 μmol/L imoxin before the addition of 10 ng/mL TNF-α for 3 h. IRS1 immunoprecipitates and cell lysates were analyzed by Western blot. D: Effect of PKR inhibitor (imoxin) on ER stress–induced PERK phosphorylation in wild-type MEFs. Cells were pretreated with imoxin (0.2, 0.5, or 1 μmol/L) before adding 300 nmol/L thapsigargin (TG) for 3 h. Cell lysates were analyzed by Western blot. PKR activity was assessed by the autophosphorylation level of PKR using ATP[g-32P]. E: PKR activity and expression and JNK1 activity in WAT of ob/ob mice after 30 days of treatment with vehicle or PKR inhibitor (imoxin). PKR and JNK activities were examined by kinase assay. In vitro assay of JNK1, JNK2, and JNK3 (F); p38 (G); and IKKβ (H) kinase activity with substrates in the presence or absence of PKR inhibitor are as indicated. p, phospho; IB, immunoblot; IP, immunoprecipitate; MBP, myelin basic protein; IκB, inhibitor of the nuclear factor κB.
Figure 2
Figure 2
Imoxin improves glucose homeostasis and reduces inflammation in genetically obese mice. A: GTTs performed after 9 days of treatment in ob/ob mice with vehicle (Veh) (n = 6) or imoxin (n = 6). B: ITTs performed after 16 days of treatment in ob/ob mice with vehicle (n = 6) or imoxin (n = 6). C: ALT/AST ratio and blood urea nitrogen (BUN) levels were measured in the serum of ob/ob mice treated with vehicle (n = 7) or imoxin (n = 8). D: GTT was performed on male C57BL/6J mice after 24 days of treatment with vehicle or imoxin. E: Hematoxylin-eosin staining of WAT from ob/ob mice treated with vehicle or imoxin for 35 days. Scale bar, 200 µm. F: Expression of Tnfa and Il6 in WAT of ob/ob mice treated with vehicle (n = 5) or imoxin (n = 5). G: Insulin-stimulated IRβ tyrosine 1162/1163 and Akt serine 473 phosphorylation in WAT of ob/ob mice treated with vehicle or imoxin. Quantification of the Western blot band intensity is shown in the lower panels. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01. The statistical difference between groups was calculated by two-way ANOVA. p, phospho.
Figure 3
Figure 3
The PKR inhibitor 2-AP improves glucose homeostasis in ob/ob mice. A: GTTs performed after 7 days of treatment in ob/ob mice with vehicle (Veh) (n = 8) or 2-AP (n = 8). B: ITTs performed after 14 days of treatment in ob/ob mice with vehicle (n = 8) or 2-AP (n = 8). C: Expression of Tnfa, Il6, F4/80, and Mcp-1 in WAT of ob/ob mice treated with vehicle (n = 8) or 2-AP (n = 8) for 21 days. D and E: Serum ALT and AST levels in ob/ob mice after 21 days of treatment with vehicle (n = 9) or 2-AP (n = 9). Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01. The statistical difference between groups was calculated by two-way ANOVA.
Figure 4
Figure 4
Imoxin treatment improves systemic insulin sensitivity without altering metabolic rate. Metabolic cage studies were performed on ob/ob mice treated with vehicle (Veh) (n = 10) or imoxin (n = 10). A: Rate of Vo2. B: Heat production. Hyperinsulinemic–euglycemic clamp studies were performed in ob/ob mice after 18 days of treatment with vehicle (n = 8) or imoxin (n = 5). C: Glucose infusion rates (GIRs) during the clamp procedure. D: Average (GIR). E: Whole-body glucose disposal rates (Rd). F: HGP during the clamp. G: Basal HGP. H: Tissue glucose uptake in gastrocnemius muscle. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01.
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References

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