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// Ha Ram Bae 1,* , Dae Hyun Kim 1,* , Min Hi Park 1 , Bonggi Lee 1 , Min Jo Kim 1 , Eun Kyeong Lee 1 , Ki Wung Chung 1 , Seong Min Kim 1 , Dong Soon Im 1 and Hae Young Chung 1 1 Molecular Inflammation Research Center for Aging Intervention, College of Pharmacy, Pusan National University, Geumjeong-gu, Busan, Republic of Korea * These authors have contributed equally to this work Correspondence to: Hae Young Chung, email: // Keywords : β-hydroxybutyrate; endoplasmic reticulum; aging; inflammasome; AMPK; Gerotarget Received : May 10, 2016 Accepted : August 26, 2016 Published : September 19, 2016 Abstract β-Hydroxybutyrate, a ketone body that is used as an energy source in organs such as the brain, muscle, and heart when blood glucose is low, is produced by fatty acid oxidation in the liver under the fasting state. Endoplasmic reticulum (ER) stress is linked with the generation of intracellular reactive oxygen species and the accumulation of misfolded protein in the ER. ER stress is known to induce the NOD-like receptor protein 3 inflammasome, which mediates activation of the proinflammatory cytokine interleukin-1β, whose maturation is caspase-1-dependent. We investigated whether β-hydroxybutyrate modulates ER stress, inflammasome formation, and insulin signaling. Sprague Dawley rats (6 and 24 months of age) that were starved for 3 d and rats treated with β-hydroxybutyrate (200 mg·kg -1 ·d -1 i.p., for 5 d) were used for in vivo investigations, whereas human hepatoma HepG2 cells were used for in vitro studies. Overexpression of AMPK in cultured cells was performed to elucidate the molecular mechanism. The starvation resulted in increased serum β-hydroxybutyrate levels with decreased ER stress (PERK, IRE1, and ATF6α) and inflammasome (ASC, caspase-1, and NLRP3) formation compared with non-fasted 24-month-old rats. In addition, β-hydroxybutyrate suppressed the increase of ER stress- and inflammasome-related marker proteins. Furthermore, β-hydroxybutyrate treatment increased the expression of manganese superoxide dismutase and catalase via the AMP-activated protein kinase-forkhead box protein O3α transcription factor pathway both in vivo and in vitro . The significance of the current study was the discovery of the potential therapeutic role of β-hydroxybutyrate in suppressing ER-stress-induced inflammasome formation.

// Daeui Park 1,2,3 , Byoung-Chul Kim 1,2 , Chul-Hong Kim 4 , Yeon Ja Choi 1 , Hyoung Oh Jeong 1 , Mi Eun Kim 5 , Jun Sik Lee 5 , Min Hi Park 1 , Ki Wung Chung 1 , Dae Hyun Kim 1 , Jaewon Lee 1 , Dong-Soon Im 1 , Seokjoo Yoon 2,3 , Sunghoon Lee 6 , Byung Pal Yu 7 , Jong Bhak 6 and Hae Young Chung 1 1 Molecular Inflammation Research Center for Aging Intervention, Pusan National University, Busan, Korea 2 Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, Korea 3 Human and Environmental Toxicology, School of Engineering, University of Science and Technology, Daejeon, Korea 4 GenomicTree Inc., Yuseong-gu, Daejeon, Korea 5 Department of Biology, College of Natural Sciences, Chosun University, Gwangju, Korea 6 Personal Genomics Institute,Genome Research Foundation, Suwon, Korea 7 Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Correspondence to: Hae Young Chung, email: // Jong Bhak, email: // Keywords : aging; inflammation; RNA-Seq; differentially expressed genes; novel genes; alternative splicing, Gerotarget Received : October 05, 2015 Accepted : April 18, 2016 Published : May 03, 2016 Abstract Age-related dysregulated inflammation plays an essential role as a major risk factor underlying the pathophysiological aging process. To better understand how inflammatory processes are related to aging at the molecular level, we sequenced the transcriptome of young and aged rat kidney using RNA-Seq to detect known genes, novel genes, and alternative splicing events that are differentially expressed. By comparing young (6 months of age) and old (25 months of age) rats, we detected 722 up-regulated genes and 111 down-regulated genes. In the aged rats, we found 32 novel genes and 107 alternatively spliced genes. Notably, 6.6% of the up-regulated genes were related to inflammation ( P < 2.2 × 10 -16 , Fisher exact t-test); 15.6% were novel genes with functional protein domains ( P = 1.4 × 10 -5 ); and 6.5% were genes showing alternative splicing events ( P = 3.3 × 10 -4 ). Based on the results of pathway analysis, we detected the involvement of inflammation-related pathways such as cytokines ( P = 4.4 × 10 -16 ), which were found up-regulated in the aged rats. Furthermore, an up-regulated inflammatory gene analysis identified the involvement of transcription factors, such as STAT4, EGR1, and FOSL1, which regulate cancer as well as inflammation in aging processes. Thus, RNA changes in these pathways support their involvement in the pro-inflammatory status during aging. We propose that whole RNA-Seq is a useful tool to identify novel genes and alternative splicing events by documenting broadly implicated inflammation-related genes involved in aging processes.

The beneficial role of FoxO during aging has been proposed for its promotion of resistance to oxidative stress and inhibition of pro-inflammatory mediators. On the other hand, NF-κB is a pro-inflammatory transcription factor which is a key mediator of inflammatory cytokine generation. However, the correlation between FoxO6 and NF-κB during aging has not fully been explored. The main purpose of the present study was to elucidate mechanisms underlying the protective role of FoxO6 in the maintenance of cellular homeostasis under potent pro-inflammatory conditions induced by LPS. Initial experimentation revealed that reduced FoxO6 activity during aging was caused by its phosphorylation, which suppressed its transcriptional activity in aged livers. Transfection with FoxO6-wt virus and FoxO6-siRNA in HepG2 cells revealed that FoxO6 phosphorylation by LPS leads to NF-κB activation via Akt and Pak1 pathways. Furthermore, Pak1 activity was increased in a phosphatidylinositol 3-kinase independent manner, and LPS-induced FoxO6 phosphorylation and FoxO6 inactivation were Pak1-dependent in nuclear fractions of cells. Further revealed Pak1 phosphorylation by LPS permitted interaction between FoxO6 and Akt. Current study suggests FoxO6 phosphorylation facilitates the nuclear translocation of NF-κB via Akt and Pak1 pathways induced by LPS in aged rats.

Recently, agonists targeting multiple peroxisome proliferator-activated receptors (PPARs) have been developed to improve metabolic disorders and minimize the side effects of selective PPAR agonists such as weight gain and dyslipidemia. We newly synthesized six 2-methyl-2-(o-tolyloxy)propanoic acid derivatives based on the structure of a well-known PPAR pan agonist, bezafibrate. Of six compounds, MHY2013 was screened as the strongest activator of three PPAR subtypes based on protein docking simulation and luciferase assays. When treated orally in db/db mice, MHY2013 ameliorated obesity-induced insulin resistance, dyslipidemia, and hepatic steatosis without changes of the body weight and levels of liver and kidney injury markers. MHY2013 decreased the serum triglyceride and fatty acid levels, which is associated with an increase in fatty acid oxidation signaling in the liver and thermogenic signaling on white adipose tissue, respectively. Furthermore, MHY2013 markedly increased serum levels of insulin-sensitizing hormones including fibroblast growth factor 21 (FGF21) and adiponectin. In conclusion, this study suggests that, MHY2013 is a novel PPAR pan agonist that improves obesity-induced insulin resistance, dyslipidemia and hepatic steatosis and elevates insulin-sensitizing hormones in the blood.