Cyanidin-3-O-glucoside ameliorates lipid and glucose accumulation in C57BL/6J mice via activation of PPAR-α and AMPK

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Harry Booth
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1 3 rd Interntionl Conference nd Exhiition on Nutrition & Food Sciences Septemer 23-25, 214 Vlenci, Spin Cynidin-3-O-glucoside meliortes lipid nd glucose ccumultion in C57BL/6J mice vi ctivtion of PPAR-α nd AMPK Food Biomedicl Science L. Yoyo Ji Sep 23 th, 214

effects Crdiovsculr Diseses Cncer Oesity Dietes To

2 Bckground Nturl orgnic pigment Grpes Berries Cherries Apples Plums Red cge Red onion Antioxidnt Anti-inflmmtory effects Crdiovsculr Diseses Cncer Oesity Dietes To investigte the effects nd moleculr mechnisms of cynidin- 3-O-glucoside ()

3 Nucler Contining A Ftty Ftty Ftty Triglyceride Synthetic Endogenous Bckground Nucleus AMP:ATP rtio Exercise (muscle stimultion) Berries contining regulte lipid nd glucose metolisms lignds PPRE Thr172 P γ α β AMPK Lipid metolism Glucose metolism Peroxisome prolifertor-ctivted receptors (PPARs): receptors 3 isoforms: PPARα, PPARγ, PPARδ/β PPARα: mjor regultor of lipid metolism in the liver cid uptke (ftty cid trnsport) cid utiliztion (ftty inding nd ctivtion) cid ctolism (peroxisoml nd mitochondril ftty cid β-oxidtion) Ketogenesis turnover Lignds: lignds include the firte drugs (hyperlipidemi) lignds include ftty cids nd vrious ftty cid-derived compounds AMP-ctivted protein kinse (AMPK): An enzyme plys role in cellulr energy homeostsis Consists of three proteins (suunits): α, β, nd γ Three suunits together mke functionl enzyme AMPK: Stimulte: Ftty cid oxidtion Ketogenesis Inhiit: Lipogenesis Triglyceride synthesis Gluconeogenesis RXR: retinoid X receptor PPRE: peroxisome prolifertor hormone response elements

Experimentl design Moleculr trgets of BIAcore Surfce plsmon resonnce (SPR) Time resolution-fluorescence resonnce energy trnsfer (TR-FRET) coctivtor ssy AMPK ctivity ssy Animl/cell experimentl design

4 Experimentl design Moleculr trgets of BIAcore Surfce plsmon resonnce (SPR) Time resolution-fluorescence resonnce energy trnsfer (TR-FRET) coctivtor ssy AMPK ctivity ssy Animl/cell experimentl design HepG2 cells Lipid-loding for 24 h Tretment with for 24 h Scrifice Smple (plsm & orgns) collection Lipid contents Ftty cit oxidtion/synthesis Autophgy nlysis Gluconeogenesis : High ft diet (45%) : 1 mg/kg ody weight of fenofirte () : 1 mg/kg ody weight of cynidin-3-o-glucoside () Physiologicl relevnce & moleculr mechnisms of Body & orgn weight mesurement Plsm lipid, glucose, insulin & hormone mesurement Liver & dipose tissue histology & nlysis Liver lipid concentrtion mesurement Orl glucose tolernce test (OGTT) Insulin tolernce test (ITT) Autophgy pthwy nlysis qpcr & immunoloting

5 A 15 induces PPARα coctivtor ctivity vi direct inding to PPARα PPARα/ 8 PPARα/GW7647 Time resolution-fluorescence resonnce Surfce energy trnsfer Plsmon (TR-FRET) Resonnce coctivtor (BIAcore ssy SPR) Response Unit (RU) B Concentrtion (µm): Time (s) PPARγ/ Response Unit (RU) Concentrtion (µm): Time (s) PPARγ/TT Response Unit (RU ) C Response Unit (RU) D Concentrtion (µm): Time (s) PPARδ/β/ Concentrtion (µm): Time (s) PPARα GW7647 Response Unit (RU) Response Unit (RU) Concentrtion (µm): Time (s) PPARδ/β/GW742 Concentrtion (µm): Time (s) PPARγ Troglitzone (TT) K D vlues nd EC 5 vlues of nd positive controls PPARδ GW742 Positive controls PPARα PPARγ PPARδ PPARα PPARγ PPARδ GW7647 TT GW742 SPR 456 nm 1.36 µm 4.96 µm 13.2 nm 377 nm 12 nm TR-FRET 1126 nm 1.8 µm 3 µm 26.9 nm 82.3 nm 1.25 nm K D, the equilirium dissocition constnt ('inding constnt'); EC 5, Hlf mximl effective concentrtion TR-FRET Rtio Concentrtion (nm) EC 5 : = 1126 nm GW7647 = 26.9 nm TR-FRET Rtio Concentrtion (nm) EC 5 : = 185 nm TT = 82.3 nm TR-FRET Rtio 1..5 EC 5 : = 3146 nm GW742 = 1.25 nm Concentrtion (nm)

6 induces AMPKα1 ctivity vi direct interction with AMPKα1 A Luminescence (RLU) AMPK (α1/β1/γ1) ctivity Concentrtion (nm) A Luminescence (RLU) AMPK (α1/β1/γ1) ctivity (nm) + A (1 nm) + A (1 nm) B Luminescence (RLU) AMPK (α2/β1/γ1) ctivity Concentrtion (nm) A AMPK α γ β A EC 5 (nm) A1/B1/G1 A2/B1/G1 A directly ctivtes PPARα nd AMPK

7 reduces lipid ccumultion in mouse livers & heptocytes A Fold chnge 1..5 Intrcellulr Triglyceride B Triglyceride (mg/dl) Plsm triglyceride. Lipid (µm) GW7647 (µm) C 15 Heptic Triglyceride /dl) Triglyceride (mg/ 1 5 D 4 Plsm AST 2 Plsm ALT AST (U/L) ALT (U/L) c c AST, Asprtte Aminotrnsferse; ALT, Alnine Aminotrnsferse

8 induces heptic ftty cid oxidtion nd ketogenesis while decreses ftty cid synthesis vi regultion of PPARα & AMPKα1 A Fold chnge Ftty cid oxidtion Liver β-oxidtion (µmol/g/min) Ftty cid oxidtion c Lipid (µm) GW7647 (µm)o B Ftty cid synthesis Ftty cid synthesis C 4 Mlonyl CoA Fold chnge 1..5 fold chnge 1..5 ng/ml µm. (µm) (µm) O GW7647 (µm)o D. (µm) GW7647 (µm)o mrna expression in liver β-hydroxyutyrte reduces lipid ftty cid oxidtion,. inhiits ftty cid synthesis PPARα CPT1 LPL HMGCS2 E Fold chnge lipid ccumultion vi increses ketogenesis, wheres ACC, cetyl-coa croxylse CPT1, crnitine plmitoyltrnsferse 1; LPL, lipoprotein lipse; HMGCS2, 3-hydroxy-3-methylglutryl-CoA synthse 2

chnge 4 3 2 1 p-ampk/ampk p-mtor/mtor p-s6k1/s6k1 Fold chnge 5 4

9 induces phosphoryltion of AMPK thus locks the mtor-s6k1 xis p-ampk T172 AMPK p-mtor T2446 mtor p-s6k1 T389 S6K1 β-ctin Fold chnge p-ampk/ampk p-mtor/mtor p-s6k1/s6k1 Fold chnge Fold chnge mtor, mmmlin trget of rpmycin; S6K1, P7-S6 Kinse 1

10 induces heptic utophgy pthwy A B C LC3I LC3II β-ctin GW7647 (µm) C LC3I LC3II α-tuulin Fold chnge LC3 II Fold chnge LC3 II Fluorescence (R Reltive Units) Autophgy c d. (µm) GW7647 (µm)o Tmoxifen (µm) (µm) D STF, STF No. of Autolysosomes/Cell Numer of Autolysosomes 5 reduces STF62247 (µm) lipid (µm) reduces lipid ccumultion vi ctivtes heptic utophgy pthwy

11 reduces plsm glucose & insulin concentrtions nd improves insulin sensitivity A Glucose (mg/dl) Plsm glucose c c ng/ml Plsm insulin B Plsm glucose level (mg/dl) OGTT AUC 1..5 AUC of OGTT Time(min). C Plsm glucose level (mg/dl) ITT Time(min) AUC AUC of ITT D Insulin-sensitive Index c c HOMA-IR c c HOMA-IR, Homeosttic Model Assessment - Insulin Resistnce; AUC, Are under the curve

12 A reduces gluconeogenesis B 25 2 Gluconeogenesis camp Metformin Compound C CE-TOF & QqQMS:Selected component nlysis

13 reduces gluconeogenesis vi increses plsm diponectin concentrtion & inhiits FOXO nd CREB ctivity A p-ampk AMPK HDAC5 p-crtc2 CRTC2 p-hdac5 HDAC5 β-ctin CRTC2 Fold chnge PEPCK X 1 G6Pse p-ampk/ampk Fold chnge p-crtc2/crtc2 Fold chnge p-hdac5/hdac5 B Luciferse ctivity (%) FOXO ctivity CRE-Luc ctivity mrna expression in liver. A (µm) Foskolin (µm) (µm) reduces 5 PEPCK G6Pse glucose Fold chnge 1..5 c c µg/ml Adiponectin FOXO1, Forkhed ox protein O1; CREB, camp response element-inding protein; HDAC5, Histone decetylse 5; CRTC2, CREB regulted trnscription coctivtor 2; PEPCK, Phosphoenolpyruvte croxykins; G6Pse, Glucose 6-phosphtse C reduces glucose ccumultion vi inhiits heptic gluconeogenesis

14 reduces ody weight, viscerl ft weight & dipocyte size A 5 Body weight B Body weight (g) 4 3 C Adipocyte size (µm 2 ) Weeks Adipocytes Orgn weight of mice Epididyml Ft (g) 2.45 ± ± ±.19 Viscerl Ft (g) 1.67 ± ±.9 c.98 ±.19 c Perirentl Ft (g) 2 ± ±.9 c 1.19 ±.15 c Totl White Adipose Tissue (WAT, g) 5.63 ± ±.42 c 4.58 ±.52 c Brown Adipose Tissue (BAT, g).29 ±.3.22 ±.3.36 ±.4 WAT/BAT 2.81 ± ± ±.87 Skeletl Muscle (g).68 ±.4.55 ±.8.76 ±.6 WAT/Skeletl Muscle 8.43 ± ± ±.8 Liver (g) 9 ± ± ±.17 Liver/Body weight.36 ±.2.4 ±.1.34 ±.3

15 increses energy expenditure vi induces thermogenesis gene expressions in rown dipose tissue (BAT) A VO2 (ml/min/kg) VO2 (ml/min/kg) Totl Light Drk CON CON RQ(Respirtory Quotient) RQ(Respirtory Quotient) CON Totl Light Drk CON Energy Expenditure (kcl/dy/kg) Energy Expenditure (kcl/dy/kg) CON Totl Light Drk CON B 4 mrna expression in BAT 3 2 reduces 1 energy rown PPARα PGC-1α dipose UCP1 Fold chnge reduces ody weight vi increses energy expenditure nd thermogenesis in rown dipose tissue PGC-1α, Peroxisome prolifertor-ctivted receptor gmm coctivtor 1-lph; UCP1, uncoupling protein 1

16 Conclusion Ftty cid oxidtion Ftty cid synthesis Insulin sensitivity Energy expenditure Autophgy Gluconeogenesis Body weight, viscerl ft weight & dipocyte size Lipid ccumultion in liver Glucose & insulin concentrtions in plsm Improves insulin sensitivity Atherosclerosis

17 Acknowledgement Food Biomedicl Science L. Ewh Women s University Supervisor Prof. Sung-Joon Lee FBS l. Memers Ji He Lee Chunyn Wu Boe kim Ji Ah Kim Soyoung Kim Borm Mok Prof. Young-Suk Kim Minyoung So Rurl Development Administrtion of Kore

18 Thnk you for your ttention!

* * * * * liver kidney ileum. Supplementary Fig.S1

* * * * * liver kidney ileum. Supplementary Fig.S1 Supplementry Fig.S1 liver kidney ileum Fig.S1. Orlly delivered Fexrmine is intestinlly-restricted Mice received vehicle or Fexrmine (100mg/kg) vi per os (PO) or intrperitonel (IP) injection for 5 dys (n=3/group).