Double-stranded RNA-dependent protein kinase (PKR) is shown to be a key regulator of the inflammasome; PKR is central for caspase-1 activation and the release of interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1) in response to a diverse range of stimuli. The inflammasome, an intracellular oligomeric protein complex involved in innate immunity, has been linked to the pathogenesis of obesity, type 2 diabetes, atherosclerosis, gout, sepsis and colitis. This paper identifies the double-stranded RNA-dependent protein kinase (PKR) as an important regulator of inflammasome activation. PKR autophosphorylation is required for its interaction with the Nod-like receptor NLRP3, for caspase 1 activation and for the release of the cytokines interleukin (IL)-1β, IL-18 and HMGB1 in response to a diverse set of stimuli. This finding raises the possibility of designing therapeutics to inhibit inflammasome activity in inflammatory conditions without broadly impairing immunity. The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1)1,2,3,4,5. By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1β, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.

Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKR_WT) or PKR with a point mutation in each dsRNA-binding motif (PKR_RM). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV–cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKR_WT samples after PA treatment, but not in the PKR_RM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5′-triphosphate enhanced PKR activity compared with the activity with a 5′-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress. Significance Animals respond to stress in many ways, including initiating cell death to eliminate damaged cells. Protein kinase RNA-activated (PKR) is a protein that senses stress, and it promotes cell death by phosphorylating eIF2α to block protein synthesis in damaged cells. PKR is activated by metabolic stress, such as that associated with obesity, and this activation depends on its RNA-binding domain. Here we investigated whether endogenous RNA triggers PKR activation in response to lipid exposure. Our results indicate that a noncoding RNA, the small nucleolar RNA (snoRNA), binds PKR during cellular metabolic stress, and multiple experiments suggest snoRNAs also activate PKR during metabolic stress. snoRNAs have established roles in RNA modification, and our studies suggest they have additional roles in metabolic stress.

Obesity is a life‐threatening factor and is often associated with dysregulation of gene expression. Here, we show that the CNOT3 subunit of the CCR4–NOT deadenylase complex is critical to metabolic regulation. Cnot3^+/− mice are lean with hepatic and adipose tissues containing reduced levels of lipids, and show increased metabolic rates and enhanced glucose tolerance. Cnot3^+/− mice remain lean and sensitive to insulin even on a high‐fat diet. Furthermore, introduction of Cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs is not altered in Cnot3^+/− vis‐à‐vis wild‐type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism‐related PDK4 and IGFBP1, are increased in Cnot3^+/− hepatocytes, having poly(A) tails that are longer than those seen in control cells. We provide evidence that CNOT3 is involved in recruitment of the CCR4–NOT deadenylase to the 3′ end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decrease upon fasting, we propose that CNOT3 responds to feeding conditions to regulate deadenylation‐specific mRNAs and energy metabolism.

Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.

Childhood obesity continues to be a major public health concern. Obesity causes various metabolic complications, including insulin resistance, type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), dyslipidemia, and cardiovascular disease. However, currently, we have a limited understanding of the pathophysiology in the development of these processes. Extracellular vesicles (EVs) are nano-sized vesicles secreted by different cell types that travel to various organ systems carrying molecular and genetic information. These vesicles have been proposed as a novel intercellular communication mode in systemic metabolic regulation and in several pathophysiologic processes. In particular, recent studies indicate that EVs play a critical role in the pathogenesis of obesity and its metabolic complications. In this study, we reviewed the current literature that supports the role of EVs in the regulation of metabolic homeostasis and pathogenesis of obesity and its associated metabolic complications, with a short discussion about future directions in the EV research field.