1 / 36

Pharmacologically-induced Cytoplasmic NAD(P)+/NAD(P)H Ratio by NQO1 Activator Ameliorates

Pharmacologically-induced Cytoplasmic NAD(P)+/NAD(P)H Ratio by NQO1 Activator Ameliorates the Metabolic Syndrome. Inkyu Lee MD, PhD Department of Internal Medicine, Kyungpook National University, School of Medicine Daegu, Republic of Korea.

inoke
Download Presentation

Pharmacologically-induced Cytoplasmic NAD(P)+/NAD(P)H Ratio by NQO1 Activator Ameliorates

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Pharmacologically-induced Cytoplasmic NAD(P)+/NAD(P)H Ratio by NQO1 Activator Ameliorates the Metabolic Syndrome Inkyu Lee MD, PhD Department of Internal Medicine, Kyungpook National University, School of Medicine Daegu, Republic of Korea

  2. Number of People With Diabetes in the Adult Population Worldwide for 2000 and 2010 World 2000: 151 million 2010: 221 million Increase: 46% 84.5 m 26.5 m 14.2 m 132.3 m 32.9 m 17.5 m ↑57% ↑24% ↑23% 9.4 m 14.1 m 15.6 m 1.0 m ↑50% 22.5 m 1.3 m ↑44% ↑33% Amos et al. Diabet Med. 1997;14:S1-S85; Zimmet et al. Nature. 2001;414:782-787.

  3. Energy Balance Negative Positive Weight Loss Fat Accumulation Fatty Acids Adipose Tissue Liver & Muscle Insulin Resistance “Adipokines” Robust B-cells Weak B-cells Hyperinsulinemia Hyperglycemia Preventing Type 2 Diabetes Three Levels of Opportunity

  4. TZD Treatment • Role of NQO1 and AMPK activator

  5. AMPK (AMPK-activated protein) Calorie restriction, Exercise, Metabolic stress Ca2+ LKB1 CaMKK 5’-AMP AMP Kinase Glucose transport b-acid oxidation HNF4a, SREBP1c HMG-coA reductase p53 TSC2 E2F ACC/FAS Cholesterol synthesis p21 mTOR Fatty acid synthesis CDK Protein synthesis Cell proliferation 혈관재형성 제어 연구실

  6. NAD is a key regulator of cellular energy NAD plays a pivotal role in bioenergetics. When a metabolite is oxidized, NAD+ accepts two electrons plus a hydrogen ion (H+) and NADH results. - NAD+ is reduced to NADH (reduced form) Conversely, NADH can also reduce a metabolite by giving up electrons. - NADH is oxidized to NAD+ ATP Won Gu Jang

  7. Hypothetical Model:Transient Shift of Cytoplasmic Redox State into high NAD+/NADH NAD+/NADH reduction NAD+ NADH Electron transport oxidation cADPR NADH shuttle Complex I Ca++ glycolysis OXPHOS OXPHOS Fatty acid oxidation ATP Cpt1 AMP/ATP AMP kinase Intracellular Lipid adopted from Shong MH slide

  8. NQO1 (NADPH Quinone reductase 1) • NQO1 catalyze metabolic detoxification of quinones • NQO1 protects cells from redox cycling, oxidative stress and neoplasia. • Functions to detoxify quinones by two-electron reduction (hydroquinones) • Functions to stable p53, induces apoptosis Skin tumors that developed in NQO1-null mouse (b) and histotype of tumor (c) Cancer Res. 2005 65;6:2054 혈관재형성 제어 연구실

  9. Oxidative stress NQO1 Quinone metabolism p53 stabilization Endogenous hydroquinones A-tocopherol-quinone Coenzyme Q10 Antioxidant capacity Metabolism of stressor NQO1 (NADPH Quinone reductase 1) II Mutation Research 2004;555:149 혈관재형성 제어 연구실

  10. MB12066 (b-L) • -Known as NAD(P)H:quinone oxidoreductase-1 (NQO1) (E.C. 1.6.99.2), a two-electron oxidoreductase. • Induction of apoptosis in MCF-7:WS8 breast cancer cells by b-L. Cancer Res. 1998. • NAD(P)H:Quinone oxidoreductase activity is the principal determinant of b-L cytotoxicity. J Biol Chem. 2000. • b-L-induced apoptosis is associated with activation of caspase-3 and inactivation of NF-kappaB in human colon cancer HCT-116 cells. Anticancer Drugs. 2003. 혈관재형성 제어 연구실

  11. MB12660 activates AMPK MB660 NQO1 NAD+/NADH NAD+ NADH AMP/ATP AMP ATP Glycolysis AMPK Pyruvate p53 Fatty acid oxidation Acetyl coA p21 Acetyl coA CDK TCA cycle SMC proliferation Sco2 Oxidative phosphorylation ATP ROS Kv1.5 혈관재형성 제어 연구실 Apoptosis

  12. Enhanced cytosolic NADH oxidation by NQO1 stimulates cellular energy metabolism. catalytically-inactive NQO1 (NQO1 C609T) (gray) JW Whang, 2008 Diabetes

  13. βL activates AMPK signaling pathway & fatty acid oxidation in vivo. JW Whang, 2008 Diabetes

  14. βL activates AMPK signaling pathway & fatty acid oxidation in vivo. JW Whang, 2008 Diabetes

  15. βL treatment ameliorates the metabolic sx of DIO mice. JW Whang, 2008 Diabetes

  16. The effects of βL on body weight and food intake of pair-fed DIO mice JW Whang, 2008 Diabetes

  17. SUMMARY & CONCLUSION • Beta-lapachone (βL) stimulated AMP-activated protein kinase (AMPK), subsequently triggers mitochondrial fatty acid oxidation by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase (CPT) in L6 fibroblast and NQO1 MEF. • βL treatment in the rodent models with the metabolic syndrome dramatically ameliorates obesity, glucose intolerance, dyslipidemia, and fatty liver. • Collectively, elevation of NADH oxidation by using NQO1 can be a new therapeutic intervention in treating metabolic diseases.

  18. Activation of NAD(P)H:quinone oxidoreductase 1 (NQO1) prevents arterial restenosis by suppressing vascular smooth muscle cell proliferation

  19. 혈관평활근세포의 증식과 분화(거품세포, 석회화) 동맥경화증의 진행 위험인자 (당뇨병, 고혈압, 고콜레스테롤) • 혈관평활근세포 • 증식, 이주 2) 분화 (거품세포,혈관 석회화) 혈관재형성 제어 연구실

  20. Restenosis의 주범은 혈관평활근세포의 증식 현재 동맥경화증의 유일한 직접치료는 혈관 풍선확장술 STENT Balloon Drug Eluting Stent 260 만원 국내소비 3만 -4만개 /년간 1000억 규모비용/년간 한국 혈관재형성 제어 연구실

  21. (A) (a) Control (b) BI 25 # 20 15 Intima/media ratio * 10 5 ** a d b c 0 (c) BI + bL (100 mg/kg/day) (d) BI + bL (200 mg/kg/day) (B) BI + bL (200 mg/kg/day) Control BI Figure1 SH Kim et al, 2009, Circulation Research

  22. (A) 300 2.5 300 # # # 250 250 2.0 * * 200 200 * * * * * 1.5 Cell number (% of control) * BrdU incorporation (% of control) * Cell number (O.D at 450/655) * * 150 150 * 1.0 100 100 0.5 50 50 0 0 0.0 serum + bL serum G0 PDGF : bL (mM) : serum : bL (mM) : serum : bL (mM) : G1 S G2/M (B) control * serum serum + bL 100 80 # % of total cells # 60 6 6 12 12 0 0 24 24 * 40 0 1 2 3 6 0.5 20 serum + b-Lap control serum 0 + 1 + 1 + 1 + 2 + 2 + 2 - - - - - - + - + - + - + 0.1 + 0.1 + 0.1 + 0.5 + 0.5 + 0.5 (C) (D) bL (2 mM) Time (h) : Time : P-p53 P-pRB p53 pRB cyclin E p21 p27 cyclin D b-actin b-actin Figure 2 SH Kim et al, 2009, Circulation Research

  23. (A) VSMC HeLa DMSO AICAR bL (2 mM) Time (2 h) AICAR bL (mM) : Time (h) : - - + - - + - - + - - + 0 0.5 2 3 6 6 2 1 0 1 0.1 0.5 2 P-ACC (Ser79) P-ACC (Ser79) Total ACC Total ACC P-AMPK (Thr172) P-AMPK (Thr172) AMPKa AMPKa b-actin b-actin (B) AICAR : bL : P-ACC(Ser-79) Total ACC P-AMPK(Thr172) AMPKa P-LKB1 LKB1 NQO1 b-actin Figure 3 (C) 75 DMSO βL Total NAD+/NADH ratio 50 25 0 (min) 20 0 10 30 60 0.3 DMSO βL * AMP/ATP ratio 0.2 * 0.1 * 0.0 60 20 40 50 0 10 30 SH Kim et al, 2009, Circulation Research (min)

  24. CAMKK ? LKB1 βL βL-H2 NQO1 NADH NAD+ [NAD]/[NADH] [AMP]/[ATP] P-AMPK p53 p21 CDK VSMC proliferation SH Kim et al, 2009, Circulation Research

  25. Ad-Null Ad-DN-LKB1 bL : P-ACC - - + + - - + + ACC P-AMPK AMPKa LKB1 NQO1 bL : RASMC + STO609 RASMC + Mock P-ACC(Ser-79) Total ACC P-AMPK(Thr172) AMPKa CaMKKb NQO1 b-actin SH Kim et al, 2009, Circulation Research

  26. Comp C Comp C (A) # # # # # # 250 * * BrdU incorporation (% of control) Cell number (% of control) 200 - + - + 150 100 - - + + 1.0 50 0.8 - - - + - - + + - + + + + - + 0 serum : bL : Comp C : 0.6 - - - + - - + + - + + + + - + serum : bL : Comp C : 0.4 0.2 0.0 (B) (C) bL : bL : P-ACC(Ser79) P-P53 Total ACC P53 P-AMPK(Thr172) P21 AMPKa b-actin b-actin Figure 4 SH Kim et al, 2009, Circulation Research

  27. (E) (D) serum Comp C 250 # bL : 200 # # P-pRb 150 * 18 Cell number (% of control) 16 100 pRb 14 12 50 b-actin 10 8 0 6 4 Ad-DN-AMPK 2 - - + - + + Serum: bL: bL : 0 Ad-Null P-pRb Ad-DN-AMPK - - - - + + - - + + - - + - + + a b c d pRb b-actin (F) (a) CONTROL (b) BI serum # * # Intima/media ratio ** (c) BI + bL + Ad-LacZ (d) BI + bL + Ad-DN-AMPK Figure 4 SH Kim et al, 2009, Circulation Research

  28. (B) (C) VSMC RASMC + Ad-Null RASMC + Ad-si-NQO1 HEK293 + Ad-Null HEK293 + Ad-NQO1 AICAR : bL : - - + - - + - - + - - + P-ACC(Ser-79) + - - + - + + - Total ACC P-AMPK(Thr172) AMPKa P-LKB1 LKB1 NQO1 b-actin (D) ES936 dicoumarol - - + - - + - - + - - + AICAR : bL : - - + - - + - - + - - + P-ACC(Ser 79) Total ACC P-AMPK(Thr172) AMPKa NQO1 b-actin Figure 5 (A) HEK293 bL : P-ACC(Ser-79) Total ACC P-AMPK(Thr172) AMPKa NQO1 b-actin SH Kim et al, 2009, Circulation Research

  29. 1.2 1.0 350 300 0.8 250 250 0.6 200 0.4 200 150 0.2 150 100 0.0 50 100 0 50 - + - + 0 - - - + - - + + - + + + + - + - - + - + - - - - + - + - + + + + - - - + - - + + - + + + + - + control serum serum + bL serum + bL + dicoumarol Figure 6 (A) # # # # # # * # # # * * ## BrdU incorporation (% of control) * Cell number (% of control) Cell number (% of control) Serum: bL: serum : bL : dicoumarol : serum : bL : dicoumarol : Ad-Null Ad-si-NQO1 (B) (C) (D) dicoumarol βL-H2 βL b-Lap : NQO1 P-p53 NAD+ NADH p53 [NAD]/[NADH] P21 [AMP]/[ATP] LKB1 b-actin serum P-AMPK dicoumarol p53 b-Lap : P-pRb p21 pRb CDK b-actin VSMC proliferation SH Kim et al, 2009, Circulation Research

  30. AMPK (AMPK-activated protein) Calorie restriction, Exercise, Metabolic stress Ca2+ LKB1 CaMKK 5’-AMP AMP Kinase Glucose transport b-acid oxidation HNF4a, SREBP1c HMG-coA reductase p53 TSC2 E2F ACC/FAS Cholesterol synthesis p21 mTOR Fatty acid synthesis CDK Protein synthesis Cell proliferation 혈관재형성 제어 연구실

  31. MB12660 prevents atheroscelrosis induced by diabetes in Apo E-deficient mice fed high-Fat diet

  32. 32 STZ Control 350 STZ MB660 25mg/kg 31 STZ MB660 25mg/kg pair fed STZ MB660 50mg/kg STZ MB660 50mg/kg pair fed 300 30 29 250 28 200 27 26 150 25 100 24 8 -4 -3 -2 -1 0 1 2 3 4 5 6 7 0 10 20 30 40 50 60 MB12660 prevents the increase in atheroscelrosis induced by diabetes in Apo E-deficient mice fed high-Fat diet Body weight Glucose level STZ control STZ control MB660 25 mg/ml STZ투여 Plasma glucose (mg/dl) Body weight (g) MB660 25 mg/ml MB660투여 MB660 50 mg/ml MB660 50 mg/ml week day 혈관재형성 제어 연구실

  33. MB12660 prevents the increase in atheroscelrosis induced by diabetes in Apo E-deficient mice fed high-Fat diet (A) MB660 50 mg/kg Control MB660 25 mg/kg Heart aortic valves MB660 25 mg/kg MB660 50 mg/kg Control (B) Aorta 혈관재형성 제어 연구실

  34. Conclusion MB660-H MB660 NQO1 NAD+ NADH AMP ATP AMPK p53 ACC/FAS p21 Fatty acid synthesis CDK SMC proliferation 혈관재형성 제어 연구실

  35. Summary & Conclusion • βL-induced NADH oxidation by NQO1 and by LKB1, at least in part, upregulation of AMPK reduced VSMC proliferation • in vitro and in vivo. • βL-induced NADH oxidation by NQO1 reduced atheroma formation. • This study provides the regulation of NAD+/NADH redox potential may be novel therapeutic target for the prevention of metabolic syndrome

  36. Acknowledgments Sun-Yee Kim Chang-Joo Oh Young-Keun Choi • 충남대학교 의과대학 • 송민호 MD., PhD. • 황정환 • 울산대학교 의과대학 • -이기업 MD., PhD. 전남대학교 호르몬센터 -최흥식 PhD. MD BioAlpha

More Related