Epigallocatechin Gallate Inhibits Hepatic Glucose Production in

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Bioactive Constituents, Metabolites, and Functions

Epigallocatechin gallate inhibits hepatic glucose production in primary hepatocytes via downregulating PKA signaling pathway and transcriptional factor FoxO1 Xiaopeng Li, Yunmei Chen, Zheng Shen, Quan Pan, Wanbao Yang, Hui Yan, Weiqi Ai, Liao Wang, and Shaodong Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00395 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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Journal of Agricultural and Food Chemistry

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Epigallocatechin gallate inhibits hepatic glucose production in

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primary hepatocytes via downregulating PKA signaling pathway and

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transcriptional factor FoxO1

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Xiaopeng Li

†,‡ ,

Yunmei Chen †＃ , James Zheng Shen † , Quan Pan † , Wanbao Yang † ,

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Hui Yan†, Huimin Liu†, Weiqi Ai†, Wang Liao†, Shaodong Guo*,†

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†Department of Nutrition and Food Science, College of Agriculture and Life Science,

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Texas A&M University, College Station, TX 77843, USA

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‡ College of Food Science and Technology, Huazhong Agricultural University,

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Wuhan, 430070, China

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＃ School

of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003,

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China

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*Corresponding

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Shaodong Guo

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Department of Nutrition and Food Science

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College of Agriculture and Life Sciences, Texas A&M University

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373 Olsen Blvd., Cater-Mattil Hall Rm. 123A, TAMU2253,

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College Station, TX 77843, USA. Tel: 979-845-0850

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Keywords

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(-)-Epigallocatechin gallate / FoxO1 / Hepatic glucose production (HGP) / Glucagon /

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AMPK

author:

Fax: 979-862-6842

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ACS Paragon Plus Environment


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ABSTRACT: Forkhead/winged helix transcription factor O-class member 1 (FoxO1)

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is a key mediator of insulin and glucagon signaling in control of glucose homeostasis.

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Although epigallocatechin gallate (EGCG) has attracted interests owing to its

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potential to combat hyperglycemic diabetes, molecular mechanisms underlying its

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antihyperglycemic effect, in particular the effect on FoxO1 is poorly understand. This

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study aims to assess the impact of EGCG on the glucagon signaling pathway in

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regulating glucose metabolism. Primary hepatocytes from wild-type (WT),

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liver-specific FoxO1 knock out (FKO) and FoxO1-S273D knock-in (KI) mice were

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isolated, cultured, and treated with EGCG or/and glucagon. Our data showed the

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treatment of 10 μM EGCG for 6 h decreased hepatic glucose production by 20% and

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23% in WT and FKO primary hepatocytes, respectively. EGCG repressed both

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gluconeogenesis and glycogenolysis in primary hepatocytes, coupled with activating

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AMPK. In addition, EGCG decreased mitochondrial oxygen consumption. We further

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investigated the effects of EGCG on glucagon-stimulated cAMP/PKA signaling

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pathway. EGCG reduced p-PKA-T197/T-PKA and p-CREB-S133/T-CREB level by

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39% and 20%, blocked p-FoxO1-S273 and suppressed nuclear FoxO1 translocation,

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suggesting that FoxO1 and CREB were possible downstream targets. A novel

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mechanism of EGCG in restraining hepatic glucose production (HGP) is through

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antagonizing glucagon signaling and suppressing FoxO1 via Ser273. EGCG may

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serve as a promising compound for regulating glucose homeostasis.

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■INTRODUCTION

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Diabetes is characterized by the deregulation of glucose homeostasis partly owing to

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increased hepatic glucose production (HGP). And now diabetes is becoming a global

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health issue. An estimated 30.2 million people in the United States in 2017 according

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to the International Diabetes Federation (IDF) and more than 86 million Americans

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are considered pre-diabetic.1 In China, the number of people with diabetes reached

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114.4 million in 2017. The healthcare expenditure of people with diabetes is expected

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to continue to grow. The highest yearly cost per person with diabetes is the US with

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11,638 USD (IDF, 2017, http://diabetesatlas.org). Moreover, diabetes dramatically

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elevated comorbidities of other chronic health issues, including cardiovascular

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disease, kidney disease, and diabetic eye disease.2

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Two important transcriptional factors regulate gluconeogenesis: FoxO1 and element

binding

protein

(CREB).1,

3

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cAMP-responsible

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gluconeogenic genes expression, PEPCK, and G-6-Pase, as well as contributes to the

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regulation of cell metabolism.4 As a transcriptional factor in response to insulin,

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FoxO1 is inhibited by phosphorylating at S256 in human, equivalent to S253 of

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FoxO1 in mice, through insulin/Akt signaling pathway.5 The phosphorylation of

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FoxO1 at S256 resulted in promoting transport from nuclear to cytoplasmic and

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reducing gluconeogenesis. In contrast, our lab has found that glucagon could improve

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FoxO1 nuclear translocation and stability through phosphorylation of FoxO1-S273 by

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PKA activation, suggesting that FoxO1-S273 also participated in glucagon/PKA

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signaling pathway.6-7 CREB binding protein (CBP) complex is another transcriptional


FoxO1

promotes


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factor regulated by cAMP/PKA through the activation at site Ser133 phosphorylation.

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Such activation further leads to the recruitment of CREB-binding protein (CBP) to

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activate the CREB-CBP complex in response to glucagon signaling. CREB-CBP

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complex can directly bind to cAMP response element site (CRE) to induce PGC1α

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transcription and its downstream target genes such as the rate-limiting

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gluconeogenetic enzymes genes, PEPCK and G-6-Pase.8-9 Additionally, insulin can

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directly abrogate the cAMP signaling pathway by phosphorylation CBP at Ser 436,

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interfering the binding of CBP to CREB.10 Metformin has shown suppressed the

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gluconeogenesis through phosphorylation of CBP at Ser 436 by AMPK activation.11

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EGCG, a major polyphenol from green tea, has been investigated with the

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prevention of insulin resistance and diabetes.12-16 The plasma concentration of EGCG

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after tea catechin intake could reach to 10 μM in animal study (10 mg/kg intravenous

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administration).17 After a high dose of tea catechins in human oral administration, the

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EGCG concentration in plasma could reach around 10 μM.18 Previous studies

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demonstrated EGCG suppressed HGP through activating AMPK in primary

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hepatocytes.19-20 AMPK activation reduced glucagon-stimulated cAMP signaling via

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activation of cyclic nucleotide phosphodiesterase 4B (PDE4B).21 AMPK activation

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also suppresses hepatic gluconeogenesis by destroying CREB-CBP complex through

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CBP-S436 phosphorylation and interfering FoxO1 by class Ⅱ a histone deacetylases

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(HDACs) phosphorylation.11,

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gluconeogenesis. Metformin also inhibited hepatic gluconeogenesis in mice lacking

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hepatic AMPK through changing the energy state.23 Meanwhile, EGCG has also been

22

However, AMPK is not necessary for regulating


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reported to inhibit ATP synthase and downregulate mitochondrial complex I-V

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activities, resulting in accumulating of ADP or AMP.24 AMP could directly activate

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AMPK and bind to the P-site at the adenylate cyclase and suppress its activity,

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causing a decrease accumulation of cAMP once stimulated by glucagon.25-26 The

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inhibition of mitochondrial function may directly or indirectly interfere cAMP/PKA

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signaling pathway. Thus, mitochondria seem to be at the crossroad in regulating

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AMPK and cAMP/PKA signaling.

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Under fasting condition, glucagon is secreted and activates cAMP/PKA signaling,

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resulting in elevated gene expression of glycogenolytic and gluconeogenetic enzymes.

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7

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elusive. Based on these backgrounds, our objective was to determine the underlying

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purpose of EGCG on cAMP/PKA/FoxO1 signaling pathway in regulating glucose

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metabolism.

However, the role of EGCG in glucose homeostasis related to cAMP/PKA remains

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■MATERIALS AND METHODS

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Chemicals and antibodies

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EGCG, glucagon, and the CBP-CREB interaction inhibitor were obtained from

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Millpore Sigma (MA, USA). Metformin was got from Enzo, and PKA inhibitor H89

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was obtained from Tocris (MN, USA).

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(MA, USA). p-AMPK-T172, AMPKα, p-CREB-S133, CREB, p-Akt-S473, Akt,

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p-PKA-T197, PKA, T-FoxO1, Histone, and GAPDH were bought from Cell

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Signaling Technology (MA, USA). p-FoxO1-S273 was generated in our lab as

Compound C was purchased from Abcam



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previously reported 7. NE-PER Nuclear and cytoplasmic extraction reagents and

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pierce lactate dehydrogenase (LDH) cytotoxicity assay kit were supplied by Thermo

113

Scientific (MA, USA).

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Isolation of primary hepatocytes

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Mice deficient for FoxO1 gene in the liver (FKO mice) and FoxO1-S273D KI mice

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were generated as previously described.7, 27 All mice used for isolation of hepatocytes

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were kept in a controlled environment (22 ℃ and 65 ± 3% humidity) and fed with

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normal chow diet. Animal protocols were approved by institutional animal care and

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use committee (IACUC) at Texas A&M University (2015-0316). Under anesthesia

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state with isoflurane, livers were infused with Hanks' balanced solution containing 10

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mM Hepes, 0.5 mM EGTA, 5.5 mM glucose and 1% penicillin-streptomycin

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(perfusion solutionⅠ) at 5.5 ml/min for 5 min, followed by Hanks' balanced solution

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containing 0.055% collagenase (Worthington, typeⅡ), 10 mM Hepes, 1.5 mM CaCl2,

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5.5 mM glucose and 1% penicillin-streptomycin ( perfusion solutionⅡ) at 5.5 ml/min

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for 5 min. Livers were collected and mashed slowly, and then hepatocytes were

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purified with Percoll (Sigma-Aldrich, St. Louis, MO) and centrifugation. Hepatocytes

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were plated onto collagen-coated 6 well plates at 3 × 105 cells/well in DMEM with

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2% FBS for HGP study. The primary hepatocytes were kept at 37 ℃ with 5% CO2 in

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the air.

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Gluconeogenesis, glycogenolysis, and HGP study

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For HGP studies, primary hepatocytes were cultured in DMEM with 2% FBS. After

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3-4 hours attachment, cells were washed with warmed PBS for 3 times and the


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medium was changed to HGP buffer (119 mM NaCl, 5.0 mM KCl, 2.6 mM KH2PO4,

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2.6 mM MgSO4, 2.0 mM CaCl2, 24.6 mM NaHCO3, 10 mM HEPES, 0.5% BSA, 10

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mM sodium L-lactate, and 5 mM pyruvate, pH=7.4). Subsequently, cells were treated

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with EGCG, glucagon or both. It should be noted that the CREB-CBP interaction

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inhibitor (40 μM) and compound C (5 μM) were used to pre-treat hepatocytes for 30

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min before adding EGCG or glucagon in this study.

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To test the effects of EGCG on glycogenolysis and gluconeogenesis, HGP buffer

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with or without substrates (pyruvates and sodium L-lactate) were used in this study.

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The glucose production hydrolyzed from glycogenolysis was determined in the

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absence

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gluconeogenesis is defined as the increase between the glucose production in HGP

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buffer and HGP buffer without substrates. Cell culture medium was collected at 1, 3,

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and 6 h after treatment for glucose content determination by using Amplex Red

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Glucose Assay kit (Invitrogen, Carlsbad, CA). Glucose production was normalized by

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protein concentration in cell lysates determined by BCA assay (Invitrogen, Carlsbad,

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CA).

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LDH release assay

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The primary hepatocytes were seeded as 4,000 cells/well in 100 μL of medium in a

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96-well tissue culture plate, followed by the incubation at 37 ℃ and 5% CO2

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overnight. Add 10 μL of PBS to one group of cells (spontaneous LDH activity

153

controls), and nothing to another group of cells (maximum LDH activity controls).

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Add 10 μL of PBS containing different concentration of EGCG (1-200 μL) to the

of

pyruvate/lactate

(substrates).

The


glucose

production

from


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cells. Incubate the plate in an incubator for 3 h, and add 10 μL of lysis buffer (10×)

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and mix by gentle tapping. After 45 min, transfer 50 μL of each medium to a 96-well

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plate and transfer 50 μL of the reaction mixture to each sample well. Incubate the

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plate at room temperature for 30 min protected from light and add 50 μL of stop

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solution. The absorbance was measured at 490 nm and 680 nm, and subtract the 680

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nm absorbance value (background) from the 490 nm absorbance before calculation

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of % Cytotoxicity.

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% Cytotoxicity =

EGCG - treated LDH activity － Spontaneous LDH activity Maximum LDH activity － Spontaneous LDH activity

× 100

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AMPK activation

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For AMPK activation test, primary hepatocytes were cultured in completed medium

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(DMEM with 10% FBS). After 3 hours of attachment, cells were washed with

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warmed PBS for 3 times and the medium was changed to DMEM (fasting medium).

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Then the cells were cultured with/without glucagon and co-treated with or without

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EGCG for 1 h and were collected for Western blot analysis.

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Mitochondria activity measurement

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Mitochondrial function was measured by monitoring oxygen consumption rate (OCR)

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using XF Cell Mito Stress Test Kit (Agilent technology). Isolated primary hepatocytes

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were seeded in an XF96 cell culture microplate. The primary hepatocytes were

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cultured in completed medium (DMEM with 10% FBS). After 3 hours attachment,

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cells were washed with warmed PBS for 3 times and the medium was changed to

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DMEM (fasting medium). The primary hepatocytes were treated with EGCG or

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metformin. Seahorse assay was run in XF96 Extracellular Flux Analyzer (Agilent


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technology). During the OCR measurement, cells were exposed sequentially to

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Oligomycin (0.5 μmol/L), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone

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(FCCP, 1 μmol/L), and Rotenone (0.5 μmol/L). The OCR measurements were

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recorded after every injection. To normalize the results, the protein of each well was

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quantified using BCA protein assay kit (Thermo Scientific, MA, USA).

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Nuclear and cytoplasmic protein extraction

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Nuclear and cytoplasmic proteins from primary hepatocytes were extracted by

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NE-PER Nuclear and cytoplasmic extraction kit according to manufactory's

185

instruction. Primary hepatocytes were seeded in completed medium and fasted for 2 h

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with DMEM, and then treated with EGCG for 1 h. The cells were washed with PBS

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twice and harvested with trypsin-EDTA. Then centrifuge at 500 × g for 5 min and

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wash the cells with PBS. Add 300 μL ice-cold CERⅠ, vortex and incubate the tubes

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on ice for 10 min. Ice-cold CERⅡwas then added, mixed and centrifuged for 5 min at

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16,000 × g. The supernatant section (cytoplasmic extract) was immediately

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transferred to a clean pre-chilled tube. The insoluble fraction was suspended in 150

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μL of ice-cold NER and continued vortex for 15 seconds every 10 min (a total of 40

193

min), and then centrifuged at 16,000 × g for 10 min. The supernatant was quickly

194

transferred to a clean tube and store at -80 ℃ until use.

195

Western blot analysis

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Protein was extracted from primary hepatocytes in RIPA lysis buffer (Sigma-Aldrich,

197

St. Louis, MO). The nuclear and cytoplasmic protein were extracted using NE-PER

198

nuclear and cytoplasmic extraction reagents according to instructions provided by the



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manufacturer (Thermo Scientific, MA, US). And the protein concentrations were

200

determined by BCA protein assay kit (Thermo Scientific, MA, USA). Subsequently,

201

protein extracts were diluted in sample buffer and heated for 10 min in boiling water

202

bath. The protein samples were separated by SDS-polyacrylamide gel electrophoresis

203

(SDS-PAGE), and then electro-transferred to a PVDF membrane (Millipore, Bedford,

204

MA, USA). The membranes were then blocked with 5% BSA in TBST for 2 h at

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room temperature in a shaker and incubated overnight at 4 ℃

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antibodies in TBST (p-AMPK, 1:1000; AMPK, 1:1000; p-PKA-T197, 1:1000;

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T-PKA, 1:1000; p-Akt-S473, 1:1000; T-Akt, 1:1000; p-CREB-S133, 1:1000;

208

T-CREB, 1:1000; p-FoxO1-S273, 1:800; GAPDH, 1:1000), followed by the

209

incubation of horseradish peroxidase (HRP)-conjugated secondary antibodies

210

(1:5000) for 2 h at room temperature in a shaker. Immunoreactive bands were

211

visualized by the enhanced chemiluminescent reagents (ECL). The intensity of bands

212

was measured using ChemiDoc imaging system (Bio-Rad LaboratoriesInc., Hercules,

213

CA).

214

Quantitative Real-Time PCR

215

Primary hepatocyte RNA was extracted with Trizol reagent (Invitrogen, Carlsbad,

216

CA),

217

(Bio-RadLaboratoriesInc., Hercules, CA). Gene expression was measured using the

218

SYBR Green Supermix according to manufacturer's protocol in Real-time PCR

219

system (Bio-RadLaboratoriesInc., Hercules, CA). The mRNA levels were normalized

220

to 18S using double-delta Ct method and presented as relative fold changes. Real-time

and

cDNA

was synthesized

using

iScript


cDNA

with primary

Synthesis

kit

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PCR primers were listed in Supplementary Table 1.

222

Statistical analysis

223

All results are calculated as the mean ± SD. Statistical significance between two

224

groups was calculated using unpaired, two-tailed t-tests. Data form more than two

225

groups was compared using a one-way analysis of variance (ANOVA). Values of

226

p

Epigallocatechin Gallate Inhibits Hepatic Glucose Production in

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