1748 Diabetes Volume 67, September 2018 Prostaglandin F2a Facilitates Hepatic Glucose Production Through CaMKIIg/p38/FOXO1 Signaling Pathway in Fasting and Obesity Yuanyang Wang,1 Shuai Yan,2 Bing Xiao,2,3 Shengkai Zuo,2 Qianqian Zhang,2 Guilin Chen,1 Yu Yu,2,4 Di Chen,2,5 Qian Liu,1 Yi Liu,2 Yujun Shen,1 and Ying Yu1,2 Diabetes 2018;67:1748–1760 | https://doi.org/10.2337/db17-1521 Gluconeogenesis is drastically increased in patients Type 2 diabetes constitutes a major worldwide public health with type 2 diabetes and accounts for increased fasting burden and is expected to affect .642 million adults by plasma glucose concentrations. Circulating levels of 2040 (1). Type 2 diabetes is characterized by hyperglycemia, prostaglandin (PG) F2a are also markedly elevated in insulin resistance, and b-cell dysfunction, and often is diabetes; however, whether and how PGF2a regulates associated with low-grade chronic inflammation (2). Blood hepatic glucose metabolism remain unknown. Here, we glucose homeostasis is maintained by the balance between demonstrated that PGF2a receptor (F-prostanoid receptor hepatic glucose production (HGP) (glycogenolysis and glu- [FP]) was upregulated in the livers of mice upon fasting- coneogenesis) and glucose utilization by peripheral tissues. and diabetic stress. Hepatic deletion of the FP receptor In patients with type 2 diabetes, hepatic gluconeogenesis suppressed fasting-induced hepatic gluconeogenesis, is considerably elevated and contributes to both fasting whereas FP overexpression enhanced hepatic gluconeo- and postprandial hyperglycemia, and suppressing hepatic genesis in mice. FP activation promoted the expression gluconeogenesis improves insulin sensitivity and glucose METABOLISM of gluconeogenic enzymes (PEPCK and glucose-6- homeostasis, making it an attractive target for the treatment phosphatase) in hepatocytes in a FOXO1-dependent man- of diabetes (3). Hepatic gluconeogenesis is tightly controlled ner. Additionally, FP coupled with Gq in hepatocytes to elicit Ca2+ release, which activated Ca2+/calmodulin- by rate-limiting gluconeogenic enzymes, including PEPCK activated protein kinase IIg (CaMKIIg)toincreaseFOXO1 (PCK1), glucose-6-phosphatase (G6Pase), and fructose-1,6- phosphorylation and subsequently accelerate its nuclear bisphosphatase. The quantity and activity of these essential translocation. Blockage of p38 disrupted CaMKIIg- enzymes are mainly regulated by the pancreatic hormones insu- induced FOXO1 nuclear translocation and abrogated FP- lin and glucagon (4). In addition to hormonal control, hepatic mediated hepatic gluconeogenesis in mice. Moreover, gluconeogenesis is also directly regulated by inflammatory medi- knockdown of hepatic FP receptor improved insulin sensi- ators, such as cytokines (5) and phospholipid derivatives (6,7). tivity and glucose homeostasis in ob/ob mice. FP-mediated Prostaglandin (PG) F2a is a bioactive lipid metabolite of hepatic gluconeogenesis via the CaMKIIg/p38/FOXO1 arachidonic acid produced through the sequential reaction signaling pathway, indicating that the FP receptor might of cyclooxygenases and PGF synthase. PGF2a exerts a wild be a promising therapeutic target for type 2 diabetes. range of pathological and physiological functions, including 1Department of Pharmacology, School of Basic Medical Sciences, 2011 Collab- 5Department of Microbiology and Immunology, University of Michigan Medical orative Innovation Center of Tianjin for Medical Epigenetics, Key Laboratory of School, Ann Arbor, MI Immune Microenvironment and Disease (Ministry of Education), Tianjin Medical Corresponding author: Ying Yu, [email protected] or [email protected], or ’ University, Tianjin, People s Republic of China Yujun Shen, [email protected]. 2Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Received 14 December 2017 and accepted 1 May 2018. Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, People’sRepublic This article contains Supplementary Data online at http://diabetes of China .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1521/-/DC1. 3State Key Laboratory for Medical Genomics, School of Life Science and © 2018 by the American Diabetes Association. Readers may use this article as Biotechnology, Shanghai Jiao Tong University, Shanghai, People’s Republic long as the work is properly cited, the use is educational and not for profit, and the of China work is not altered. More information is available at http://www.diabetesjournals 4Department of Pediatric Cardiology, Xinhua Hospital affiliated to Shanghai Jiao .org/content/license. Tong University School of Medicine, Shanghai, People’s Republic of China See accompanying article, p. 1742. diabetes.diabetesjournals.org Wang and Associates 1749 reproduction (8), blood pressure (9), and bone remodeling Thermal Cycler (Bio-Rad) using SYBR Green PCR mix (10), by binding to its receptor (F-prostanoid [FP]). Exper- (TaKaRa). Values were normalized to actin mRNA or 18S imental and clinical studies showed that PGF2a is involved rRNA. See Supplementary Tables 1 and 2 for primer in different inflammatory conditions, including rheumatic sequences. diseases, asthma, atherosclerosis, ischemia-reperfusion, Immunofluorescence septic shock, and obesity (11). Interestingly, increased PGF 2a The frozen sections from liver or chamber slides with metabolite production is found in urine from patients with hepatocytes were washed with PBS and fixed in cold both type 1 diabetes (12) and type 2 diabetes (13), and plasma acetone. To block nonspecific binding of antibodies, sam- PGF2a levels can be used as a marker to predict glycemic ples were incubated with PBS containing 3% BSA and control and oxidation status (14). Moreover, hepatic PGF 2a 0.1% Triton X-100 (for permeabilization) for 30 min. The production is also significantly increased in fasted, high- samples were then incubated with mCherry fluorescent fat diet (HFD)–fed, or diabetic animals (7). FP receptor is protein (1:200; Abbkine, Redlands, CA) or FOXO1 (1:500; expressed in hepatocytes (15); however, whether an acti- Cell Signaling Technology, Danvers, MA) overnight at 4°C. vated PGF /FP axis is implicated in aberrant glucose 2a Slides were then washed with PBS three times and in- metabolism in diabetes remains largely unclear. cubated with secondary antibodies conjugated with Alexa In this study, we demonstrated that the activation of Fluor 594 or Alexa Fluor 633 (Invitrogen) for 1 h at room FP receptor increased fasting-induced hepatic gluconeo- temperature. ProLong Gold Antifade Reagent with DAPI genesis in mice by upregulating gluconeogenic genes (PCK1 (Invitrogen) was applied to mount and counterstain the and G6Pase) in a FOXO1-dependent manner. FP activation slides. Images were obtained using confocal microscopy promoted FOXO1 nuclear translocation by stimulating 2+ (IX51; Olympus, Center Valley, PA). Gq-mediated Ca /calmodulin-activated protein kinase IIg (CaMKIIg) signaling, with p38 required for FP/CaMKIIg- Western Blot mediated FOXO1 nuclear translocation. Moreover, the silenc- Total protein and cytosolic and nuclear protein fractions ing of hepatic FP receptor–ameliorated glucose metabolism were isolated from tissues or cultured cells using a protein in obese mice. Therefore, FP activation facilitated hepatic extraction kit (Beyotime, Shanghai, People’s Republic gluconeogenesis through the CaMKIIg/p38/FOXO1 signaling of China), followed by separation by SDS-PAGE, transfer pathway under both fasting and diabetic conditions. to nitrocellulose membranes, and probing with different primary antibodies against PCK1, p38, and lamin B (Pro- RESEARCH DESIGN AND METHODS teintech, Wuhan, People’s Republic of China); G6Pase and g a Mouse Models CaMKII (Santa Cruz Biotechnology, Dallas, TX); -tubulin, phospho-Thr287 CaMKIIg, phospho-p38, FOXO1, and Human FP (hFP) hepatocyte transgenic (HCTG) and hemagglutinin (HA)-Tag (Cell Signaling Technology); hFP nontransgenic (NTG) littermates were obtained from b the mating of Tg-hFP-STOPFlox (16) and AlbCre mice. (Epitomics, Burlingame, CA); and -actin (Sigma-Aldrich). F/F The membranes were then conjugated with a horseradish FP exon2-floxed mice (FP ) were generated through peroxidase–labeled secondary antibody in blocking buffer CRISPR/Cas9 technology by Shanghai Biomodel Organism for 2 h at room temperature. Blots were developed using Science & Technology Development Co., Ltd. (Shanghai, Cre enhanced chemiluminescence reagents (Pierce, Rockford, People’s Republic of China) and crossed with Alb mice to F/F cre IL), followed by densitometric quantification using ImageJ obtain hepatic FP-deficient mice (FP Alb ). All mice used (National Institutes of Health, Bethesda, MD). See Supple- were from a C57BL/6 background. For inhibitor treatment, mentary Table 3 for antibodies. 8-week-old HCTG and NTG mice were intraperitoneally injected with KN-93 (3 mg/kg) (MedChem Express, Mon- Live Imaging mouth Junction, NJ) three times weekly for 1 week (17) or Male mice (8 weeks old) were injected with G6P-Luc with SB202190 (2.5 mmol/L/kg) (Sigma-Aldrich, St. Louis, adenoviruses (1 3 108 plaque-forming units) by tail vein. MO) daily for 1 week (18). All animals were maintained and On day 5 after adenovirus delivery, mice were subjected to used in accordance with the guidelines of the Institutional fasting for 8 h before imaging, followed by intraperitoneal Animal Care and Use Committee of the Institution for Nu- injection with 100 mg/kg sterile