biomedicines Article GADD45β Regulates Hepatic Gluconeogenesis via Modulating the Protein Stability of FoxO1 Hyunmi Kim 1,2 , Da Som Lee 1,2 , Tae Hyeon An 1,2 , Tae-Jun Park 1, Eun-Woo Lee 1 , Baek Soo Han 1,2, Won Kon Kim 1,2, Chul-Ho Lee 3 , Sang Chul Lee 1,2, Kyoung-Jin Oh 1,2,* and Kwang-Hee Bae 1,2,* 1 Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea; [email protected] (H.K.); [email protected] (D.S.L.); [email protected] (T.H.A.); [email protected] (T.-J.P.); [email protected] (E.-W.L.); [email protected] (B.S.H.); [email protected] (W.K.K.); [email protected] (S.C.L.) 2 Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34141, Korea 3 Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea; [email protected] * Correspondence: [email protected] (K.-J.O.); [email protected] (K.-H.B.) Abstract: Increased hepatic gluconeogenesis is one of the main contributors to the development of type 2 diabetes. Recently, it has been reported that growth arrest and DNA damage-inducible 45 beta (GADD45β) is induced under both fasting and high-fat diet (HFD) conditions that stimulate hepatic gluconeogenesis. Here, this study aimed to establish the molecular mechanisms under- lying the novel role of GADD45β in hepatic gluconeogenesis. Both whole-body knockout (KO) mice and adenovirus-mediated knockdown (KD) mice of GADD45β exhibited decreased hepatic gluconeogenic gene expression concomitant with reduced blood glucose levels under fasting and HFD conditions, but showed a more pronounced effect in GADD45β KD mice. Further, in primary hepatocytes, GADD45β KD reduced glucose output, whereas GADD45β overexpression increased it. Mechanistically, GADD45β did not affect Akt-mediated forkhead box protein O1 (FoxO1) phospho- Citation: Kim, H.; Lee, D.S.; An, T.H.; Park, T.-J.; Lee, E.-W.; Han, B.S.; Kim, rylation and forskolin-induced cAMP response element-binding protein (CREB) phosphorylation. W.K.; Lee, C.-H.; Lee, S.C.; Oh, K.-J.; Rather it increased FoxO1 transcriptional activity via enhanced protein stability of FoxO1. Further, et al. GADD45β Regulates Hepatic GADD45β colocalized and physically interacted with FoxO1. Additionally, GADD45β deficiency po- Gluconeogenesis via Modulating the tentiated insulin-mediated suppression of hepatic gluconeogenic genes, and it were impeded by the Protein Stability of FoxO1. restoration of GADD45β expression. Our finding demonstrates GADD45β as a novel and essential Biomedicines 2021, 9, 50. https:// regulator of hepatic gluconeogenesis. It will provide a deeper understanding of the FoxO1-mediated doi.org/10.3390/biomedicines9010050 gluconeogenesis. Received: 8 December 2020 Keywords: GADD45β; gluconeogenesis; FoxO1; protein stability; cAMP signaling Accepted: 7 January 2021 Published: 8 January 2021 Publisher’s Note: MDPI stays neu- 1. Introduction tral with regard to jurisdictional clai- ms in published maps and institutio- Gluconeogenesis, the de novo glucose synthesis, is important in maintaining blood nal affiliations. glucose levels to meet the whole-body energy requirements during the state of energy exhaustion [1]. However, excessive gluconeogenesis significantly contributes to type 2 diabetes, which increases the risk of many complications, such as cardiovascular disease, kidney disease, and cancer [2]. Copyright: © 2021 by the authors. Li- Master transcription factors for regulating hepatic gluconeogenesis include cAMP censee MDPI, Basel, Switzerland. response element-binding protein (CREB) and forkhead box protein O1 (FoxO1) [3]. They This article is an open access article are mainly regulated by insulin and glucagon, the main counter regulatory hormones distributed under the terms and con- involved in balancing blood glucose levels [4]. Glucagon activates protein kinase A (PKA) ditions of the Creative Commons At- by raising the cAMP levels and subsequently phosphorylates CREB, which induces the tribution (CC BY) license (https:// expression of gluconeogenic genes including glucose-6-phosphatase catalytic subunit creativecommons.org/licenses/by/ (G6PC) and phosphoenolpyruvate carboxykinase-1 (PCK1) [5]. On the other hand, FoxO1 is 4.0/). Biomedicines 2021, 9, 50. https://doi.org/10.3390/biomedicines9010050 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 50 2 of 13 negatively regulated by insulin [6]. Insulin inhibits FoxO1 activity by promoting the nuclear exclusion and cytoplasmic retention of FoxO1 via the AKT-mediated phosphorylation of FoxO1 at ser256 [7,8]. Phosphorylated FoxO1 is sequestered in the cytoplasm by binding to the 14-3-3 proteins [9–11], eventually becoming a target of the ubiquitin-mediated degradation [12]. FoxO1 can also be phosphorylated and acetylated by glucagon for regulating hepatic gluconeogenesis [5,13,14]. Additionally, glucagon can regulate FoxO1 protein stability and nuclear localization [14]. Growth arrest and DNA damage-inducible 45 beta (GADD45β) is a scaffold protein involved in DNA damage, apoptosis, and oxidative stress [15–18]. As a starvation response gene, GADD45β expression is increased 2 to 5 times by fasting in mouse liver [19]. Recently, it has been reported that fasting-induced hepatic GADD45β expression regulates hepatic lipid metabolism by inhibiting hepatic fatty acid uptake [20]. On the other hand, hepatic GADD45β is suppressed by signal transducer and activator of transcription 3 (STAT3), which inhibits gluconeogenesis, and is a direct transcriptional target of STAT3 [21]. The STAT3-mediated suppression of hepatic gluconeogenesis is associated with FoxO1 activ- ity [22–24]. Therefore, we hypothesized GADD45β might be related with the regulation of hepatic gluconeogenesis. Here, we demonstrated the role of GADD45β in hepatic gluconeogenesis by using the adenovirus-mediated GADD45β knockdown (KD) and GADD45β overexpression system as well as GADD45β knockout (KO) mice. Collectively, our findings described that GADD45β is an essential regulator of cAMP-induced hepatic gluconeogenesis in a FoxO1- dependent manner. This study will deepen understanding of the molecular mechanism of cAMP/PKA-induced and FoxO1-mediated gluconeogenesis. 2. Experimental Section 2.1. Animal Experiments Eight-week-old male C57BL/6 mice were purchased from ORIENT BIO. Mice lacking the whole-body expression of GADD45β were obtained from Chul-Ho Lee’s Lab in the Korea Research Institute of Bioscience and Biotechnology (KRIBB). All mice were housed and maintained in a 12 h light/12 h dark cycle under temperature- and humidity-controlled conditions with free access to food and water. Mice were fed either a standard chow diet or an HFD (60 kcal % fat diet: D12492 of Research Diets) for 12 weeks. For all animal experiments involving adenoviruses, 10-week-old male C57BL/6 mice were tail vein- injected with Ad-shGADD45β or Ad-US (control) at 0.25–0.5 × 109 pfu per mice. To induce fasting conditions, mice were fasted for 6 h, 16 h, or 24 h with free access to water. All animal procedures were approved by the Institutional Animal Care and Use Committee of the KRIBB (KRIBB-AEC-20155, 15 June 2020) and were performed according to the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.2. Plasmids and Recombinant Adenoviruses The full-length sequences for mouse GADD45β, FoxO1, and FoxO1-ADA were re- verse transcribed using liver RNA derived from the C57BL/6 mice. The sequences were amplified via PCR and inserted into the pcDNA3-Flag or pcDNA3-HA expression vector. The 6X-insulin response elements (IRE) and G6PC promoter sequences were amplified by PCR using genomic DNA from the C57BL/6 mice and inserted into the pGL4-luciferase re- porter vector. Adenoviruses expressing GFP control, GADD45β, unscrambled nonspecific RNAi control (US), and shGADD45β were used as previously described [25]. For animal experiments, the adenoviruses were purified on a CsCl gradient, dialyzed against PBS buffer containing 10% glycerol, and stored at −80 ◦C. 2.3. Culture of Primary Hepatocytes Primary hepatocytes were isolated from 8-week-old male C57BL/6 mice by collage- nase perfusion method [25]. 1 × 106 cells were plated in 6-well plates with medium 199 Biomedicines 2021, 9, 50 3 of 13 (Sigma-Aldrich, St Louis, MO, USA) supplemented by 10% FBS, 1% antibiotics, and 10 nM dexamethasone. After attachment, cells were infected with adenoviral vectors for 48 h or 72 h. Cells were maintained in medium 199 without 10% FBS for 16 to 18 h and then treated with 10 uM forskolin or 100 nM insulin. 2.4. Quantitative PCR Total RNA from primary hepatocytes or mouse liver was extracted using the easy-spin Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea); 2 µg of total RNA was reverse transcribed into cDNA with the M-MLV Reverse Transcriptase (Promega, Madison, WA, USA). The cDNA was analyzed by quantitative PCR using the SYBR green PCR kit in a C1000 Touch™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). All data were normalized to the expression of ribosomal L32 in the corresponding sample. 2.5. Glucose Production in Primary Hepatocytes Glucose production was assayed, as previously described [25]. Primary hepatocytes were infected with adenoviral vectors and incubated in serum-free media for 16 h. The cells were then stimulated with 10 uM forskolin and 1 nM dexamethasone in the Krebs-Ringer Buffer (KRB) containing gluconeogenic substrates, 20 mM lactate, and 2 mM pyruvate, for 8 h. Glucose concentrations were measured using a Glucose Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). 2.6. Seahorse Analysis Mitochondrial functions, manifested as the oxygen consumption rate and extracellular acidification rate were determined with an XF24 extracellular flux analyzer (Seahorse Bio- science, North Billerica, MA, USA). The glycolysis in Ad-US or Ad-shGADD45β-infected primary hepatocytes was assessed by analyzing their respective extracellular acidification rate (ECAR). Hepatocytes were seeded on collagen-coated XF24 cell culture microplate 72 h before the experiment.