Metabolism Clinical and Experimental 98 (2019) 62–75 Contents lists available at ScienceDirect Metabolism Clinical and Experimental journal homepage: www.metabolismjournal.com PPP1R3C mediates metformin-inhibited hepatic gluconeogenesis Xueying Ji a,1, Shushu Wang a,1,HongjuTanga,b,1,YuqingZhanga, Feiye Zhou a,LinlinZhanga, Qin Zhu a, Kecheng Zhu a,QianqianLiua,YunLiua, Xiao Wang a,⁎, Libin Zhou a,⁎ a Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine andMetabolicDiseases,RuijinHospital,Shang- hai Jiaotong University School of Medicine, Shanghai 200025, PR China b The Department of Geriatrics, The People's of Wenshan Prefecture, Wenshan 663099, PR China article info abstract Article history: Background: Metformin has been widely used to alleviate hyperglycemia in patients with type 2 diabetes mainly Received 28 February 2019 via suppressing hepatic gluconeogenesis. However, the underlying mechanism remains incompletely clear. Here, Accepted 5 June 2019 we aimed to explore the role of PPP1R3C in metformin-mediated inhibition of hepatic gluconeogenesis. Methods: The differentially expressed genes in primary mouse hepatocytes incubated with 8-Br-cAMP and met- Keywords: formin were analyzed by microarrays. Hepatic glucose production and gluconeogenic gene expressions were de- PPP1R3C tected after adenovirus-mediated overexpression or silence of PPP1R3C in vitro and in vivo. The phosphorylation Metformin level and location of transducer of regulated CREB activity 2 (TORC2) were determined by Western blot and im- Gluconeogenesis fl AMPK muno uorescence. TORC2 Results: Metformin and adenovirus-mediated activation of AMPK suppressed 8-Br-cAMP–stimulated Ppp1r3c Liver mRNA expression in primary mouse hepatocytes. Overexpression of PPP1R3C in primary mouse hepatocytes or the livers of wild-type mice promoted hepatic glucose production and gluconeogenic gene expressions. On the contrary, adenovirus-mediated knockdown of PPP1R3C in primary mouse hepatocytes decreased hepatic gluconeogenesis, with the suppression of cAMP-stimulated gluconeogenic gene expressions and TORC2 dephos- phorylation. Notably, Ppp1r3c expression was increased in the liver of db/db mice. After PPP1R3C silence in the livers of wild-type and db/db mice, blood glucose levels and hepatic glucose production were markedly lowered, with decreased expressions of key gluconeogenic enzymes and transcript factors as well as liver glycogen con- tent. Conclusion: Metformin-activated AMPK decreases hepatic PPP1R3C expression, leading to the suppression of he- patic gluconeogenesis through blocking cAMP-stimulated TORC2 dephosphorylation. Hepatic specific silence of PPP1R3C provides a promising therapeutic strategy for type 2 diabetes. © 2019 Elsevier Inc. All rights reserved. 1. Introduction diabetes for several decades [2]. The major effect of metformin is to re- duce hepatic glucose output via inhibiting gluconeogenesis [3,4], but the Inappropriate hepatic glucose production is the major cause of hy- mechanism of its hypoglycemic action remains imperfectly understood. perglycemia in type 2 diabetic patients [1]. Metformin has been widely At the cellular level, metformin is believed to disrupt hepatic gluco- accepted as the first-line therapy of choice for patients with type 2 neogenesis process by antagonizing the glucagon signaling pathways via decreasing production of cyclic adenosine monophosphate (cAMP) or activating AMP-activated protein kinase (AMPK) [5–8]. AMPK, a Abbreviations: cAMP, Cyclic adenosine monophosphate; CREB, cAMP response well-known Serine/Threonine (Ser/Thr) kinase, functions as an intracel- element binding protein; PKA, Protein kinase A; TORC2, Transducer of regulated CREB ac- lular energy sensor activated by an increase in the AMP/ATP ratio [9,10]. tivity 2; AMPK, AMP-activated protein kinase; PEPCK, Phosphoenolpyruvate Once activated by metformin, AMPK inhibits the expressions of two carboxykinase; G6Pase, Glucose-6-phosphatase; PPP1R3C, Protein phosphatase 1 regula- rate-limiting gluconeogenic enzyme genes, phosphoenolpyruvate tory subunit G; 8-Br-cAMP, 8-Bromoadenosine 3′,5′-cyclic monophosphate; PTT, Pyruvate tolerance test; Fbpase, Fructose 1,6-bisphosphatase; SIK2, Salt inducible kinase 2. carboxykinase (PEPCK, encoded by Pck1) and glucose-6-phosphatase ⁎ Corresponding authors at: Shanghai Institute of Endocrine and Metabolic Diseases, (G6Pase, encoded by G6pc), leading to a decrease in gluconeogenesis Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine [11,12]. Although metformin is widely used as an AMPK agonist in bio- and Metabolic Diseases, Ruijin Hospital, Shanghai Jiaotong University School of chemical studies, whether AMPK is actually the key effector of metfor- Medicine, 197 Ruijin 2nd Road, Shanghai 200025, PR China. min on glucose metabolism is uncertain. It was shown that metformin E-mail addresses: [email protected] (X. Wang), [email protected] (L. Zhou). blocked glucose production in mouse primary hepatocytes lacking all 1 These authors contributed equally to this study. AMPK catalytic subunits [13]. https://doi.org/10.1016/j.metabol.2019.06.002 0026-0495/© 2019 Elsevier Inc. All rights reserved. X. Ji et al. / Metabolism Clinical and Experimental 98 (2019) 62–75 63 Pancreatic glucagon promotes hepatic gluconeogenesis by stimulat- production buffer (DMEM without glucose, serum, and phenol red), ing the protein kinase A (PKA)-mediated phosphorylation of cAMP re- supplemented with gluconeogenic substrates (10 mM sodium lactate sponse element binding protein (CREB) at Ser 133 and and 1 mM pyruvate). After 8 h 8-Bromoadenosine 3′,5′-cyclic dephosphorylation of transducer of regulated CREB activity 2 (TORC2) monophosphate (8-Br-cAMP) (Sigma-Aldrich) or metformin treatment, at Ser 171 [14,15]. The subsequent recruitment of CREB-binding protein glucose concentrations in the media were determined by a commer- (CBP) leads to the formation of a CREB-CBP-TORC2 complex [16], which cially available glucose assay kit (Applygen). binds to an cAMP response element site (CRE) to increase transcription of peroxisome proliferator activated receptor γ coactivator-1α (PGC- 2.4. Microarray analysis 1α), Pck1, and G6pc [17]. Increasing evidence suggests that TORC2 is the crucial factor in the regulation of glucose homeostasis and hepatic Total RNA was extracted from isolated primary mouse hepatocytes gluconeogenesis [18]. Metformin has been reported to stimulate the incubated with 100 μM 8-Br-cAMP in the presence or absence of phosphorylation and nuclear exclusion of TORC2 [17]. However, the un- 2 mM metformin for 8 h using TRIzol (Thermo Fisher Scientific). Sample derlying molecular mechanism has not been fully elucidated. labeling and array hybridization were performed according to the The reversible phosphorylation of proteins is accomplished by the Agilent One-Color Microarray-Based Gene Expression Analysis protocol coordinated activation of Ser/Thr protein kinases and repression of (Agilent Technology) with minor modifications. The arrays were counteracting phosphatases. Protein phosphatase 1 (PP1) consists of a scanned by the Agilent Scanner G2505C. Agilent Feature Extraction soft- catalytic subunit (PP1c) and a regulatory subunit (PP1r) [19]. There ware (version 11.0.1.1) was used to analyze the acquired array images. are seven genes (PPP1R3A to PPP1R3G) encoding PP1 regulatory sub- Quantile normalization and subsequent data processing were per- unit. As a glycogen-targeting regulatory subunit of PP1, PPP1R3C targets formed using the GeneSpring GX v12.0 software package (Agilent Tech- PP1 to glycogen, where it inhibits glycogen phosphorylase activity and nologies). Differentially expressed mRNAs between two groups were activates glycogen synthase activity [20]. It has been demonstrated identified through Volcano Plot filtering (Fold Change ≥ 1.5, P value b that overexpression of PPP1R3C in hepatocytes dramatically increases 0.01). Gene Set Enrichment Analysis (GSEA) is supported by the Broad glycogen synthesis and storage [21,22] and might be involved in Institute website (http://www.broadinstitute.org/gsea/index.jsp)[24] whole body energy metabolism [23]. However, little is known about and performed using the Java GSEA implementation. the physiologic function of PPP1R3C upon hepatic gluconeogenesis. In the present study, PPP1R3C was identified as a potential target gene of 2.5. Generation of recombinant adenoviruses metformin in suppressing cAMP-stimulated hepatic gluconeogenesis by microarray assay. Overexpression of PPP1R3C increased hepatic glu- For overexpression, adenoviruses expressing mouse Ppp1r3c (Ad- coneogenesis in vitro and in vivo while knockdown of PPP1R3C showed Ppp1r3c), Creb (Ad-Creb) or green fluorescent protein (Ad-GFP) were an opposite result. We further explored the molecular mechanism un- constructed with a full-length Ppp1r3c, Creb or GFP cDNA coding se- derlying PPP1R3C-regulated gluconeogenesis. quence. To knockdown Ppp1r3c expression in hepatocytes, adenovi- ruses carrying short hairpin (sh) RNA targeting Ppp1r3c (shPpp1r3c- 2. Materials and methods 34: 5′-AAGTGAAGAATGTGAGCTT-3′;shPpp1r3c-35:5′-CCTCCTATCGA TGAGTTAG-3′; shPpp1r3c-36:5′-CATTTCTTATCACGCTAAT-3′)ora 2.1. Animal experiments scrambled sequence (shCtrl) were generated. Adenoviruses encoding full length mouse dominant-negative AMPK protein [AMPK-DN Male C57BL/6 mice aged 6–8 weeks were purchased from Shanghai (D157A)] and a constitutively