Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis

Abstract

As chronic inflammation is a hallmark of obesity, pathways that integrate nutrient- and pathogen sensing pathways are of great interest in understanding the mechanisms of insulin resistance, type 2 diabetes, and other chronic metabolic pathologies. Here, we provide evidence that double-stranded RNA-dependent protein kinase (PKR) can respond to nutrient signals as well as endoplasmic reticulum (ER) stress and coordinate the activity of other critical inflammatory kinases such as the c-Jun N-terminal kinase (JNK) to regulate insulin action and metabolism. PKR also directly targets and modifies insulin receptor substrate and hence integrates nutrients and insulin action with a defined pathogen response system. Dietary and genetic obesity features marked activation of PKR in adipose and liver tissues and absence of PKR alleviates metabolic deterioration due to nutrient or energy excess in mice. These findings demonstrate PKR as a critical component of an inflammatory complex that responds to nutrients and organelle dysfunction.

Figure 1. Regulation of PKR activity in obesity and lipid exposure

(A) A genetic mouse model of obesity (ob/ob) was used to examine PKR activity by a kinase assay using immunopurified PKR and ATP ^[γ-32P] in white adipose tissue (WAT) and liver compared with age- and sex- matched lean controls. Fatty acid synthase (FAS) and β-tubulin proteins are shown as controls. (B) PKR activity was examined in white adipose tissue (WAT) and liver of the male wild type mice kept either on regular diet (RD) or high fat diet (HFD) for 20 weeks. (C) PKR activity in liver of 10 week-old male wild type mice, which were infused with lipid or saline for 5 hours. Total S6 protein is shown as control. (D) PKR activity in primary MEFs. Cells were cultured in the absence or presence of 0.5 mM palmitic acid for 2 hours. See also Supplemental Figure S1.

Figure 2. PKR regulates JNK activity resulting in inhibition of insulin signaling

(A) Primary Pkr^+/+ and Pkr^−/− MEFs were treated with 0.5 mM palmitic acid for 2 hours. JNK activity was assessed by a kinase assay using recombinant c-Jun protein as substrate. (B) Induction of JNK phosphorylation after 300 nM thapsigargin treatment for 1 hour in primary Pkr^+/+ and Pkr^−/− MEFs. Phosphorylation level of JNK was examined with anti-phospho-JNK (Thr183/Tyr185) antibody. (C and D) Induction of IRS-1 phosphorylation after 0.5 mM palmitic acid (C) or 300 nM thapsigargin (D) treatment for 2 hours in Pkr^+/+ and Pkr^−/− MEFs. Phosphorylation level of IRS-1 on Ser307 was examined with anti-phospho-IRS-1 (serine 307) antibody. (E) Induction of IRS-1 phosphorylation in retrovirally PKR-reconstituted Pkr^−/− MEFs. The cells were serum-starved for 14 hours followed by western blot analysis with anti-phospho-IRS-1 (serine 307) antibody. (F) The PKR-reconstituted Pkr^−/− MEFs were stimulated with 10 nM Insulin for 3 minutes. The cell lysates were immunoprecipitated with anti-IRS-1 antibody followed by western blot analysis with anti-phospho-tyrosine and anti-PI3K (p85 subunit) antibodies. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM. *P<0.05. (G and H) Induction of palmitic acid- and thapsigargin-induced PKR activity requires intact RNA binding domain of PKR. Pkr^−/− MEFs were reconstituted with vector, flag-tagged wild type (WT), RNA binding domain mutant (K64E), or kinase dead mutant (K296R) of PKR by retrovirus-mediated gene transfer. These cells were maintained in serum-free DMEM containing 0.5% BSA for 14 hours followed by treatment with 0.5 mM palmitic acid for 90 minutes (G) or 300 nM thapsigardin for 1 hour (H). The cell lysates were immunoprecipitated with anti-Flag antibody followed by PKR kinase assay and western blot analysis with anti-Flag antibody. See also Supplemental Figure S2.

Figure 3. PKR directly regulates IRS-1 phosphorylation

(A) Induction of interaction between IRS-1 and PKR after TNFα treatment in Pkr^+/+ and Pkr^−/− MEFs. IRS-1 and PKR protein level were examined either with immunoprecipitation (IP) followed by immunoblotting (IB) or by direct immunobloting in cells treated with 5 ng/ml TNFα treatment for 3 hours. (B) Physical interaction between IRS-1 and PKR in TNFα-treated MEF cells. Cell lysates were prepared from Pkr^+/+ or Pkr^−/− MEFs treated with 5 ng/ml TNFα for 3 hour followed by immunoprecipitation with anti-PKR antibody and western blot analysis with anti-IRS-1 antibody. (C) Physical interaction between IRS-1 and PKR in a pull-down assay in vitro using recombinant IRS-1 and PKR proteins. (D) Direct phosphorylation of IRS-1 by PKR in kinase assay in vitro using recombinant IRS-1 and PKR proteins. Phosphorylation level of IRS-1 was assessed by autoradiography or western blot analysis with anti-phospho-IRS-1 (serine 307) antibody. (E and F) In vitro PKR kinase assay. IRS-1 phosphorylation by immunopurified PKR prepared from 5 ng/ml TNFα (E)- or 300 nM thapsigargin (F)-treated MEFs and analyzed by autoradiography or western blot analysis with anti-phospho-IRS-1 (serine 307) antibody. (G) Effects of exogenous expression of PKR on IRS-1 serine phosphorylation in primary Jnk1^+/+ and Jnk1^−/− MEFs. Flag-tagged human PKR was introduced to primary Jnk1^+/+ and Jnk1^−/− MEFs by adenovirus-mediated gene transfer. Phosphorylation level of IRS-1 on serine 307 was examined with anti-phospho-IRS-1 (serine 307) antibody. Both exogenous and endogenous PKR expression was detected by anti-PKR antibody. The graph on the right shows the quantification of the data. See also Supplemental Figure S3.

Figure 4. Glucose metabolism and insulin sensitivity in Pkr^−/− mice

(A) Total body weight on regular (RD) or high fat (HFD) diet. Obesity is induced by HFD starting immediately after weaning at 3 weeks of age. (B) Analysis of body fat by dual energy X-ray absorptiometry (DEXA). (C and D) Serum leptin (C) and adiponectin (D) levels after 6 hours daytime food withdrawal in Pkr^+/+ (n = 5) and Pkr^−/− (n = 6) mice on HFD for 15 weeks. (E and F) Blood glucose (E) and serum insulin (F) levels after 6 hours daytime food withdrawal in Pkr^+/+ (n = 6) and Pkr^−/− (n = 6) mice on HFD for 8 weeks. (G) Glucose tolerance tests were performed on Pkr^+/+ (n = 6) and Pkr^−/− mice (n = 6) on RD and HFD for 6 weeks. See also Supplemental Figure S4.

Figure 5. Biochemical and molecular alterations in Pkr^−/− tissues

(A and B) Phosphorylation level of Akt on serine 473 in WAT (A) and liver (B) of Pkr^+/+ and Pkr^−/− mice on HFD for 20 weeks. The graphs on the right of each blot show the quantification of the results. Data are shown as the mean ± SEM. *P<0.05, **P<0.01. AU: Arbitrary unit. (C and D) Phosphorylation level of eIF2α on serine 52 and JNK1 kinase activity, which was detected by a kinase assay using immunopurified JNK1, ATP ^[γ-32P] and recombinant c-Jun protein as substrate, in WAT (C) and liver (D) of Pkr^+/+ and Pkr^−/− mice on HFD for 20 weeks. β-Tubulin is shown as a control. (E) Gene expression in WAT including proinflammatory cytokine levels in Pkr^+/+ and Pkr^−/− mice on HFD for 20 weeks. (F and G) Haematoxylin and eosin staining of WAT (F) and liver (G) sections of Pkr^+/+ and Pkr^−/− mice, respectively. Scale bar, 200 μm. (H) Triglyceride contents in liver of Pkr^+/+ (n = 6) and Pkr^−/− (n = 6) mice on HFD for 20 weeks. (I) Serum alanine aminotransferase level after 6 hours daytime food withdrawal in Pkr^+/+ (n = 6) and Pkr^−/− (n = 6) mice on HFD for 15 weeks. Data are shown as the mean ± SEM. See also Supplemental Figure S5.

Figure 6. PKR mediates lipid-induced insulin resistance

(A–F) Hyperinsulinaemic–euglycaemic clamp studies performed in Pkr^+/+ (n = 5) and Pkr^−/− mice (n = 5) infused with lipid for 5 hours. Glucose infusion rates (GIR) throughout the clamp procedure (A). Average GIR (B). Whole body glucose disposal rates (Rd) (C). Hepatic glucose production (HGP) during the clamp (D). Tissue glucose uptake in gastrocnemius muscle (E) and epididymal fat (F) tissues of Pkr^+/+ and Pkr^−/− mice. Data are shown as the mean ± SEM. See also Supplemental Figure S6.

Figure 7. Regulation of systemic metabolic responses by PKR

PKR senses and responds to obesity, ER stress, and pathogen-related stress in concert with JNK, leading to metabolic disease under diverse physiological and pathological conditions. In this capacity, PKR not only integrates immune and metabolic response systems but also links endoplasmic reticulum homeostasis and unfolded protein response (UPR), to translational regulation through eIF2α and insulin signaling through IRS-1. Finally, the kinases, IRS-1 and eIF2α may represent a “metabolic inflammasome” complex assembled and regulated by PKR.