Deletion of Hepatic FoxO1/3/4 Genes in Mice Significantly Impacts on Glucose Metabolism through Downregulation of Gluconeogenesis and Upregulation of Glycolysis

Dong XC (2013) Deletion of Hepatic FoxO1/3/4 Genes in Mice Significantly Impacts on Glucose Metabolism through Downregulation of Gluconeogenesis and Upregulation of Glycolysis. PLoS ONE 8(8): e74340. doi:10.1371/journal.pone.0074340 Deletion of Hepatic FoxO1/3/4 Genes in Mice Significantly Impacts on Glucose Metabolism through Downregulation of Gluconeogenesis and Upregulation of Glycolysis Xiwen Xiong 0 Rongya Tao 0 Ronald A. DePinho 0 X. Charlie Dong 0 Mengwei Zang, Boston University School of Medicine, United States of America 0 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 2 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center , Houston, Texas , United States of America Forkhead transcription factors FoxO1/3/4 have pleiotrophic functions including anti-oxidative stress and metabolism. With regard to glucose metabolism, most studies have been focused on FoxO1. To further investigate their hepatic functions, we generated liver-specific FoxO1/3/4 knockout mice (LTKO) and examined their collective impacts on glucose homeostasis under physiological and pathological conditions. As compared to wild-type mice, LTKO mice had lower blood glucose levels under both fasting and non-fasting conditions and they manifested better glucose and pyruvate tolerance on regular chow diet. After challenged by a high-fat diet, wild-type mice developed type 2 diabetes, but LTKO mice remained euglycemic and insulin-sensitive. To understand the underlying mechanisms, we examined the roles of SIRT6 (Sirtuin 6) and Gck (glucokinase) in the FoxO-mediated glucose metabolism. Interestingly, ectopic expression of SIRT6 in the liver only reduced gluconeogenesis in wild-type but not LTKO mice whereas knockdown of Gck caused glucose intolerance in both wild-type and LTKO mice. The data suggest that both decreased gluconeogenesis and increased glycolysis may contribute to the overall glucose phenotype in the LTKO mice. Collectively, FoxO1/3/4 transcription factors play important roles in hepatic glucose homeostasis. - Funding: This work was supported by the National Institute of Diabetes And Digestive And Kidney Diseases grant R00DK077505 (to XCD). The funders had no role in study design, data collection and analysis, decision to publish, or presentation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Mammals have four genes encoding the O subfamily of the Forkhead transcription factors: FoxO1/3/4/6 [1,2]. Among them, FoxO1 has been extensively studied. It has been shown that FoxO1 regulates hepatic gluconeogenesis through upregulation of several key genes including phosphoenoylpyruvate carboxykinase (Pck1) and glucose 6phosphatase (catalytic subunit, G6pc) [312]. Under insulin resistance conditions, FoxO1 becomes less phosphorylated at the inhibitory serine/threonine residues and therefore more active to promote expression of these gluconeogenic genes, which may contribute to hyperglycemia in diabetes [13,14]. This notion is generally supported by the data from overexpression and knockout/knockdown of FoxO1. Overexpression of the constitutively active FoxO1 mutant increases blood glucose levels and leads to impaired glucose and insulin tolerance [11,12]. In contrast, knockout or knockdown of hepatic FoxO1 lowers blood glucose levels and improves systemic insulin sensitivity in genetic or diet-induced diabetic mouse models [3,4,6,15]. Recently, two mouse genetic studies have reported inconsistent data on the roles of FoxO1 and FoxO3 in glucose metabolism [16,17]. Haeusler and colleagues have shown that a double deletion of hepatic FoxO1 and FoxO3 genes in mice has similar effects on blood glucose and glucose tolerance as compared to knockout of the FoxO1 gene alone [17]. However, Zhang and coworkers have found that FoxO1 and FoxO3 have significant additive effects FoxO1/3/4 knockout mice also manifest lower serum insulin levels and better glucose tolerance as compared to control mice although animal ages are not specified in the report [17]. Additionally, FoxO6 is predominantly expressed in the brain and also has a significant role in hepatic gluconeogenesis [18,19]. However, molecular mechanisms with regard to the collective roles of FoxOs in hepatic glucose metabolism are still elusive. In this metabolism and the underlying mechanisms. Materials and Methods Animals, blood chemistry, and metabolic analysis FoxO1/3/4 floxed mice were generated and genotyped as previously described [20]. To generate liver-specific FoxO1/3/4 triple knockout mice, the floxed mice were crossed with a line of Albumin-Cre mice (Jackson Lab). Animals were maintained on a mixed genetic background (C57/BL6/129/FVB). Mice were fed either regular chow diet or a high-fat diet (HFD, 60% calories from fat, Harlan Teklad). Adenovirus injections were performed via tail vein as previously described [21]. Blood glucose levels were measured using a glucose meter (Contour from Bayer) under ad libitum (non-fasted) or overnight 16-hour fasting conditions. Plasma insulin was measured using a commercial assay kit (ALPCO). Glucose, pyruvate and insulin tolerance tests were performed as previously described [4], with 2 g glucose or pyruvate per kg body weight and 0.75-1 U insulin (humulin R, Lilly) per kg body weight, respectively. Body composition was analyzed by dual-energy X-ray absorptiometry (DEXA). As males and females had similar phenotype, only male data were presented here. Ethics statement All procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Use and Care Committee of Indiana University School of Medicine (study 10322). Adenovirus preparation SIRT6 and GFP overexpression adenoviruses were prepared in an AdEasy system (Agilent) following the manufacturers manual. The cloning PCR primers for the human SIRT6 coding sequence are: SIRT6-forward, 5'ACTTCCGATATCGCCACCATGTCGGTGAATTACGCGGC-3', and SIRT6-reverse, 5'AAGGAACTCGAGGCTGGGGACCGCCTTG-3'. Gck and GFP shRNA adenoviruses were made in a BLOCK-iT system (Invitrogen). The target mRNA sequences are described in the following: mGck, 5'-GCTGGTAGAGGAGAATCTTCT-3', and GFP, 5'-GCATCAAGGTGAACTTCAAGA-3'. Protein analysis Liver tissue was homogenized in the lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM Sodium Pyrophosphate, 100 mM Sodium Fluoride, and freshly added 100 M Sodium Vanadate, 1 mM PMSF, 10 g/ml Aprotinin, and 10 g/ml Leupeptin). Proteins were resolved on an SDS-PAGE gel and were transferred to nitrocellulose membrane. The membrane was incubated with the following specific antibodies: SIRT6 (Sigma), Gck and Actinin (Santa Cruz Biotechnology). Protein signals were detected by incubating with HRP-conjugated secon (...truncated)