PowerPoint

Cyanidin-3-O-glucoside ameliorates lipid and glucose accumulation in C57BL/6J mice via activation of PPAR- and AMPKFood Biomedical Science Lab.Yaoyao JiaSep 23th, 20143rdInternational Conference and Exhibition onNutrition & Food SciencesSeptember 23-25, 2014 Valencia, Spain

1characteristics

Background AntioxidantAnti-inflammatory effects Cardiovascular Diseases Cancer Obesity DiabetesGrapesBerriesCherriesApplesPlumsRed cabbageRed onion

Naturalorganic pigment

functions

objectivesTo investigate the effects and molecular mechanisms of cyanidin-3-O-glucoside (C3G)?Molecular target

2Background * RXR: retinoid X receptor PPRE: peroxisome proliferator hormone response elements

PPRENucleus

ligandsPeroxisome proliferator-activated receptors (PPARs):Nuclear receptorsContaining 3 isoforms: PPAR, PPAR, PPAR/

PPAR:A major regulator of lipid metabolism in the liverFatty acid uptake (fatty acid transport)Fatty acid utilization (fatty binding and activation)Fatty acid catabolism (peroxisomal and mitochondrial fatty acid -oxidation)KetogenesisTriglyceride turnoverLigands:Synthetic ligands include the fibrate drugs (hyperlipidemia) Endogenous ligands include fatty acids and various fatty acid-derived compoundsAMP-activated protein kinase (AMPK):An enzyme plays a role in cellular energy homeostasis Consists of three proteins (subunits): , , and Three subunits together make a functional enzyme

AMPK: Stimulate:Fatty acid oxidation Ketogenesis Inhibit:LipogenesisTriglyceride synthesisGluconeogenesis

AMPKPThr172Lipid metabolism

Glucose metabolismAMP:ATP ratio Exercise (muscle stimulation)

Berries containing C3G regulate lipid and glucose metabolisms3Experimental design Molecular targets of C3GBIAcore Surface plasmon resonance (SPR)Time resolution-fluorescence resonance energy transfer (TR-FRET) coactivator assayAMPK activity assayPhysiological relevance & molecular mechanisms of C3GBody & organ weight measurementPlasma lipid, glucose, insulin & hormone measurementLiver & adipose tissue histology & analysisLiver lipid concentration measurementOral glucose tolerance test (OGTT)Insulin tolerance test (ITT)Autophagy pathway analysisqPCR & immunobloting

Animal/cell experimental design

HFD: High fat diet (45%)FF: 100 mg/kg body weight of fenofibrate (FF)C3G: 100 mg/kg body weight of cyanidin-3-O-glucoside (C3G) SacrificeSample (plasma & organs) collection

HepG2 cellsLipid-loading for 24 hTreatment with C3G for 24 hLipid contentsFatty acit oxidation/synthesisAutophagy analysisGluconeogenesis

ABDCKD values and EC50 values of C3G and positive controlsC3GPositive controlsPPARPPARPPARPPARPPARPPARGW7647TTGW0742SPR456 nM1.36 M4.96 M13.2 nM377 nM102 nMTR-FRET1126 nM10.8 M31.05 M26.9 nM82.3 nM10.25 nMC3G induces PPAR coactivator activity via direct binding to PPARKD, the equilibrium dissociation constant ('binding constant');EC50, Half maximal effective concentration

Surface Plasmon Resonance (BIAcore SPR)

Time resolution-fluorescence resonance energy transfer (TR-FRET) coactivator assayC3GC3GC3G

AB

C3G induces AMPK1 activity via direct interaction with AMPK1 EC50 (nM)A1/B1/G1A2/B1/G1A-769662262457C3G5991010AMPK

A-769662C3G directly activates PPAR and AMPK6

C3G reduces lipid accumulation in mouse livers & hepatocytes

ABDCAST, Aspartate Aminotransferase;ALT, Alanine Aminotransferase

B

AD

C3G induces hepatic fatty acid oxidation and ketogenesis while decreases fatty acid synthesis via regulation of PPAR & AMPK1

CEACC, acetyl-CoA carboxylaseCPT1, carnitine palmitoyltransferase 1; LPL, lipoprotein lipase;HMGCS2, 3-hydroxy-3-methylglutaryl-CoA synthase 2C3G reduces lipid accumulation via increases fatty acid oxidation, ketogenesis, whereas inhibits fatty acid synthesis

8

C3G induces phosphorylation of AMPK thus blocks the mTOR-S6K1 axismTOR, mammalian target of rapamycin; S6K1, P70-S6 Kinase 1

p-mTORT2446mTORp-S6K1T389S6K1p-AMPKT172AMPK

HFDFFC3G

-actin

C 1 10 50LC3ILC3II

-actin

GW7647 C3G (M)

HFD FF C3G

-tubulin

LC3ILC3IIABC

DC3G induces hepatic autophagy pathwaySTF, STF-62247

C3G reduces lipid accumulation via activates hepatic autophagy pathway

ABCD

C3G reduces plasma glucose & insulin concentrations and improves insulin sensitivityHOMA-IR, Homeostatic Model Assessment - Insulin Resistance;AUC, Area under the curve

ABC3G reduces gluconeogenesisCE-TOF & QqQMSSelected component analysis

p-AMPKAMPKp-CRTC2CRTC2p-HDAC5HDAC5HFDFFC3G

-actinC3G reduces gluconeogenesis via increases plasma adiponectin concentration & inhibits FOXO and CREB activity

AB

FOXO1, Forkhead box protein O1; CREB, cAMP response element-binding protein; HDAC5, Histone deacetylase 5; CRTC2, CREB regulated transcription coactivator 2; PEPCK, Phosphoenolpyruvate carboxykinas; G6Pase, Glucose 6-phosphatase

C

PEPCKG6PaseCRTC2HDAC5

XC3G reduces glucose accumulation via inhibits hepatic gluconeogenesis

Organ weight of mice HFDFFC3GEpididymal Fat (g)2.450.16a2.430.26a2.410.19aVisceral Fat (g)1.670.11a0.710.09bc0.980.19cPerirental Fat (g)1.520.10a1.020.09bc1.190.15acTotal White Adipose Tissue (WAT, g)5.630.20a4.160.42bc4.580.52acBrown Adipose Tissue (BAT, g)0.290.03ab0.220.03a0.360.04bWAT/BAT20.812.07a19.901.52a12.900.87bSkeletal Muscle (g)0.680.04a0.550.08a0.760.06aWAT/Skeletal Muscle8.430.49a7.980.58ab5.850.80bLiver (g)1.590.13a1.460.05a1.370.17aLiver/Body weight0.0360.002a0.0400.001a0.0340.003aC3G reduces body weight, visceral fat weight & adipocyte sizeAC

B

ABC3G increases energy expenditure via induces thermogenesis gene expressions in brown adipose tissue (BAT) PGC-1, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; UCP1, uncoupling protein 1C3G reduces body weight via increases energy expenditure and thermogenesis in brown adipose tissueConclusion

C3G

C3GFatty acid oxidationEnergy expenditureAutophagy Gluconeo-genesisFatty acid synthesisInsulin sensitivity Body weight, visceral fat weight & adipocyte size Lipid accumulation in liver Glucose & insulin concentrations in plasmaImproves insulin sensitivity

AtherosclerosisAcknowledgementFood Biomedical Science Lab.

Supervisor Prof. Sung-Joon Lee

FBS lab. MembersJi Hae LeeChunyan Wu Bobae kimJi Ah KimSoyoung KimBoram Mok

Rural Development Administration of Korea

Ewha Womens University

Prof. Young-Suk Kim

Minyoung So

Thank you for your attention!