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Recent Advances in Vitamin K Metabolism

3 rd International Conference on Nutrition and Food Science (Nutritional Science-2014) Track 1: Nutrition and Basic Science 14:10-14:30, September 23, 2014 Committee Room 1-2 Palacio de Congresos de Valencia, Spain. Kobe Pharmaceutical Univ. Dept. Hygienic Sciences.

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Recent Advances in Vitamin K Metabolism

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  1. 3rd International Conference on Nutrition and Food Science (Nutritional Science-2014) Track 1: Nutrition and Basic Science 14:10-14:30, September 23, 2014 Committee Room 1-2 Palacio de Congresos de Valencia, Spain Kobe Pharmaceutical Univ. Dept. Hygienic Sciences Recent Advances in Vitamin K Metabolism Kobe Pharmaceutical University Toshio Okano

  2. Naturally occurring vitamin K O PK (phylloquinone: vitamin K1） Plant origin （Major dietary source） O O MK-4 (menaquinone-4: vitamin K2) Bacterial origin （Minor dietary source） O MK-7 (menaquinone-7) O O Plasma vitamin K concentrations of healthy Japanese women Tsugawa N, Okano T, et al, Am J Clin Nutr, 2006;83:380-386.

  3. Amounts of dietary intake and plasma concentrations phylloquinone＞＞ MK-4 Tissue concentrations of animals and humans MK-4＞＞ phylloquinone Is phylloquinone converted into MK-4 in the body ? What is the physiological significance of this conversion and what functions dose MK-4 have, and can we develop MK-4 analogues for clinical use ? Suhara Y, Okano T, et al, J Med Chem, 2011;54:4269-4273 Suhara Y, Okano T, et al, J Med Chem, 2011;54:4918-4922 Suhara Y, Okano T, et al, J Med Chem, 2012;55;1553-1558 The aim of our study is to examine the above issues in animals and humans.

  4. Tissue concentrations of PK and MK-4 in mice fed a conventional laboratory chow diet 40 35 PK 30 PKepoxide 25 PK Conc.（pmol/g） 20 15 10 5 0 Fat Eye Skin Liver Bone Plasma Testis Heart Kidney Spleen Uterus Adrenal gland Muscle Ovary Thyroid gland Thymus Lung Stomach Seminal vesicle Cerebrum Duodenum aorta Small intestine Pancreas 2000 1800 ×100 1600 1400 MK-4 1200 MK-4 Conc.（pmol/g） MK-4 epoxide 1000 800 600 400 LC-MS and NMR 200 0 Fat Eye Skin Aorta Bone Ovary Testis Kidney Uterus Spleen Muscle Heart Lung Thymus Liver Plasma Stomach Cerebrum Adrenal gland Thyroid gland Duodenum Seminal vesicle Small intestine Pancreas Okano T. et al., JBC 2008;283:11270-11279

  5. Identification of MK-4 from brain of mice by LC-MS/MS MK-4 fraction Authentic vitamin Ks MK-4 PK MK-4 MK-4 Intensity,cps PK epoxide MK-4 epoxide MK-4 epoxide PK epoxide MK-4 epoxide PK epoxide PK PK PK epoxide PK Time,min Time,min

  6. O 8 1 2 7 2’ 6’ 10’ 14’ 6 3 4 5 1’ O Authentic MK-4 CH3（C-2） 2.17 H-1’ 3.45 H-5,8 8.06 H-2’,6’,10’,14’ 5.03 H-6,7 7.67 MK-4fraction CH3（C-2） 2.17 H-1’ 3.35 H-5,8 8.06 H-2’,6’,10’,14’ 5.08 H-6,7 7.68 3 2 0 4 1 8 7 6 5 d（ppm） Identification of MK-4 from brain of mice by 1H NMR spectrometry

  7. D CD3 D D D O O 3 Experimental design Experiment Oral administration PK-d7 Fast Sacrifice Cerebra 1 w 12 hr 24 hr 0 C57BL/6J mice 7-weeks old n=200 Isolation and purification of MK-4-d7 Analyses by LC-MS/MS and 2H NMR D O PK-d7 -d7 MK-4 CD3 D ？ D O D 3

  8. 194.3 452.5 194.4 452.6 Identification of MK-4-d7 from brain of mice by LC-MS/MS Authentic MK-4-d7 4.5e4 Intensity（cps） 3.0e4 0 MK-4-d7 fraction 3.5e4 Intensity（cps） 1.5e4 0 0 100 200 300 400 500 （m/z） + H D O CD3 D m/z：194 D CH3 D O

  9. D O 8 1 D CD3 2 7 6 D 3 4 5 D O Identification of MK-4-d7 from brain of mice by 2H NMR spectrometry MK-4-d7 Authentic MK-4-d7 2H-5,8 8.10 C2H3 2.14 2H-6,7 7.70 MK-4-d7fraction 2H-5,8 8.08 C2H3 2.14 2H-6,7 7.68 3 2 0 4 1 8 7 6 5 d（ppm）

  10. D O CD3 D D O D 3 Not only PK-d7 but also MK-4-d9 and MK-7-d7 are converted into MK-4-d7 and accumulate in brain of mice D D PK-d7 MK-4-d9 O O MK-7-d7 D O CD3 CD3 CD3 D D D D D D D O D D D O D 3 6 O 3 Fast Sacrifice C57BL/6J ♀mice 8 weeks old MK-4-d7 12 hr 24 hr 0 MK-4 MK-4-d7 120 MK-4 epoxide MK-4-d7 epoxide Endogenous MK-4 100 80 pmol/gof brain 60 40 20 0 MK-7-d7 vehicle PK-d7 MK-4-d9 Single peroral administration (10 g/kg)

  11. MK-4-d9 K1-d9 MK-4-d7 Phylloqunone is converted into MK-4 via integral side-chain removal Unsaturation of side chain Exchange of side chain O O O O 3 3 Phytyl side chain K3 Geranylgeranyl side chain

  12. K vitamins are converted into MK-4 and accumulate in tissues Where does this conversion take place? Following four routes for the conversion of PK or K3 into MK-4, 　　　１．Oral route 　　　２．Enteral route 　　　３．Intravenous route 　　　４．Intra-cerebroventricular route were examined in mice.

  13. D CD3 D D D O -d7 -d8 K1 K3 O -d7 MK-4 O 3 O 3 Experimental design Oral(1), enteral(2), intravenous(3) or intra-cerebroventricular(4) dose K1-d7, K3-d8 Fast Sacrifice Cerebra 1 w 12 hr 24 hr 0 C57BL/6J mice 7 weeks old A single dose of 10 μmol/kg BW for (1), (2), (3) At 0.1 μmol/Kg BW for (4) LC-APCI//MS D CD3 D D D D O CD3 D D D D O

  14. Concentrations of MK-4 in cerebra of mice MK-4-d7 MK-4-d7 epo MK-4 epo MK-4 Endogenous MK-4 Converted MK-4 entero oral 300 200 150 200 Concentration （pmol/g） 100 100 50 0 0 Vehicle K3-d8 PK-d7 Vehicle K3-d8 PK-d7 intravenous 250 250 Intra-cerebroventricular 200 200 150 150 Concentration （pmol/g） 100 100 50 50 0 Vehicle K3-d8 Vehicle K3-d8 PK-d7 PK-d7 0 Okano T, et al, J Biol Chem, 2008; 283:11270-11279

  15. intestine Four Sites Cannulation experiments Inferior vena cava heart serum (IVC) liver Thoracic Lymph duct Lymph(TLD) Bile duct Portal vein Serum (PV) Bile (BD) Po K1-d7 sacrifice Wistar rat 6 (h) 1 2 3 5 0 4

  16. high resolution mass spectrometry (HR-MS) Concentrations of K1-d7, MK-4-d7 and MD-d7 60 K1-d7 50 Authentic MD bile bile bile 100 40 Lymph Lymph Lymph O 80 Serum from IVC Serum from IVC Serum from IVC 173.05982 30 Concentration (pmol/g) Serum from PV Serum from PV Serum from PV [M+H]+ 60 Relative Abundance (%) 20 40 O 10 20 0 0 6 0 1 2 3 4 5 173.00 173.02 173.04 173.06 173.08 173.10 0.05 ( m/z ) MK-4-d7 Authentic MD-d8 100 0.04 D O 181.10525 80 0.03 CD D [M+H]+ Concentration (pmol/g) 3 60 Relative Abundance (%) D D 0.02 40 O D 20 0.01 0 0 181.06 181.08 181.10 181.12 181.14 181.16 0 1 2 3 4 5 6 ( m/z ) MD fraction from lymph Time after administration (h) 1.2 MD-d7 100 80 D O 180.10172 CD D 3 [M+H]+ 60 Relative Abundance (%) 0.8 MD-d7 D Concentration (pmol/mL) 40 O D 20 0.4 0 180.16 180.06 180.08 180.10 180.12 180.14 ( m/z ) 0 0 1 2 3 4 5 6 Time after administration (h)

  17. Time course changes in serum concentrations of K1, MK-4 and MD in humans orally given K1 capsules (40 mg) K1 MD MK-4 600 10 24 (n=5) (n=5) (n=5) 8 18 400 6 MD concentration(pmol/mL) MK-4 concentration(pmol/mL) 12 K1concentration (pmol/mL) 4 200 6 2 0 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Time after administration (h) Time after administration (h) Time after administration (h)

  18. Conversion pathway of MD-d3 to MK-4-d3 demonstrated by HR-MS and 1H NMR analyses (Route 2) GGPP Route 1 MK-4-d2 MD-d3 Route 2 MK-4-d3 GGPP Hirota Y, Okano T, et al, J Biol Chem, 2013; 288:33071-33080

  19. MD(K3) is a catabolic product of oral phylloquinone (K1) in the intestine and a circulating precursor of tissue MK-4 (K2) in mammals K1 (intestine) Removal of side chain Side-chain cleavage enzyme MD (K3) (quinone form)) Lymph and blood stream Reductase (tissues) MD (K3) (hydroquinone form)) Geranylgeranyl pyrophosphate ℗-℗ Prenylation enzyme ℗-℗： MK-4 (K2) Hirota Y, Okano T, et al, J Biol Chem, 2013; 288:33071-33080

  20. O O O Menaquinone-4（MK-4） O O n-1 O 3 R: New paradigm of the metabolic activation of vitamin K in brain and bone Biological actions phylloquinone(PK) GGCX, SXR/PXR, PKA/PKC brain 3 O R O menaquinones(MKn) bone Menadione(MD) Foods active metabolite

  21. O O O Menaquinone-4（MK-4） O O n-1 O 3 R: What is the enzyme(s) involved in MK-4 biosynthesis in mammals ? Biological actions phylloquinone(PK) GGCX, SXR/PXR, PKA/PKC ？ 3 O Enzyme(s) R O menaquinones(MKn) Menadione(MD) foods active metabolite

  22. 1,4 - Dihydroxy - 2 - napthol - CoA O - Succinylbenzoyl - CoA OH OH - O S - CoA O OH P2O73- O OH 1,4 - Dihydroxy - 2 - napthanoate n hydroquinone DMK - n （ ） OH O menG H H n n O OH Biosynthesis of menaquinones in Escherichia. coli - COO Chorismate - O COO OH menF - OH COO O - - O menC menE OOC menD COO menB S - CoA O Isochorismate P2O73- OH yfbB menA n OH Demethlmenaquinone CO2 Menaquinone - n Menaquinone - n hydroquinone MK - n MK - n （ ） （ ）

  23. Alignment of the amino acid sequence of Ubia(E.coli), COQ2(Homo-sapiens), Men A(E.coli) and UBIAD1(Homo-sapiens)

  24. Search for the enzyme(s) responsible for the MK-4 synthesis O O- E.coli OH H n OH MenA human homologs MenA Prenylation domain 311 a.a Chromosome 1 11.25 M UbiA prenyltransferase domain containing 1 （UBIAD1） 338 a.a Prenylation domain Chromosome 4 84.40 M CoenzymeQ2 homolog, prenyltransferase （COQ2） H O 384 a.a Prenylation domain n MK-4 O H 3 n O O Menaquinone-n biosynthesis in E.coli OH MenA MenG OH 1,4-Dihydroxy -2-naphthoate （DHNA） Demethylmenaquinone-n hydroquinone（DMK-n） Menaquinone-n （MK-n） O MD UBIAD1 or COQ2 ? GGPP + P P 3 O

  25. Concentration (pmol/mg protein) Conversion of K3-d8 or MK-4-d12 to MK-4-d7 in siControl-, siUBIAD1- or siCOQ2-transfected MG-63 cells MD-d8 MD-d8 MD-d8 MD-d8

  26. Conversion of PK-d7 and MK-4-d12 into MK-4-d7 in Sf9 cells transfected with siControl or pcDNA3.3-UBIAD1(UBIAD1 expression vector) 90 ** MK4-d7 80 MK4-d7 epoxide 70 60 50 ** Concentration (pmol/mg protein) 40 30 20 10 ** 0 EtOH MD-d8 PK-d7 MK-4-d12 EtOH MD-d8 PK-d7 MK-4-d12 Sf9-UBIAD1 expression vector Sf9-Control Values are means and s.e.m. (n=6). Three asterisks, P<0.001 versus control-infected Sf9 cells with the same compound treatment.

  27. MK-4 biosynthetic activity of UBIAD1 in microsomes prepared from UBIAD1 baculovirus-infected Sf9 cells with geranylgeranyl pyrophosphate (GGPP) and MD 40 50 35 Sf9-control Substrate: K3-d8 (1 M) 30 Sf9-control Sf9-UBIAD1 40 Sf9-UBIAD1 25 UBIAD1 activity (pmol/mg protein/min-1) 30 20 UBIAD1 activity (pmol/mg protein/min-1) 15 20 10 5 10 0 0.01 0.1 1 (M) 0 0 0.01 0.1 1 (M) Substrate: MD-d8 GGPP

  28. Subcellular localization of UBIAD1 in MG-63 cells Merge UBIAD1-GFP Transmitted ER-Tracker Red 50 m Merge BODIPY-TR ceramide Transmitted UBIAD1-GFP 50 m MG-63 cells stably transfected with a UBIAD1–GFP expression vector were stained with ER-tracker Red or BODIPY-TR ceramide (red) and were detected by GFP fluorescence (green). Merged images of GFP fluorescence and by ER-marker or Golgi-marker fluorescence are shown at the right. The control construct (mock-GFP) showed a diffuse fluorescence throughout the cytoplasm. Nakagawa K. et al.,Nature2010; 468:117-121.

  29. UBIAD1 mRNA expression, MK-4 biosynthetic activity, concentrations of MK-4 and its epoxide in tissues of female mice MK-4-d7 biosynthetic activity Amounts of MK-4 and its epoxide UBIAD1 mRNA 2000 1.8 700 1800 1.6 600 1600 MK-4-d7 MK-4 1.4 MK-4-d7 epoxide 500 1400 MK-4 epoxide 1.2 1200 400 1.0 K3-d8からの生成量 (pmol/g組織) 1000 濃度 (pmol/g組織) 相対比 (UBIAD1/-actin) 0.8 300 800 0.6 600 200 0.4 400 100 0.2 200 0 0.0 0 Cerebellum Cerebellum Pancreas Pancreas Cerebellum Cerebrum Uterus Cerebrum Uterus Pancreas Cerebrum Uterus Kidney Kidney Ovary Ovary Kidney Ovary Heart Heart Bone Bone Liver Liver Heart Bone Liver

  30. UBIAD1 is a novel biosynthetic enzyme for MK-4 that may have both side-chain cleavage and prenylation activities GGPP UBIAD1 Prenylation R = PK MK-4 R= Side-chain cleavage MK-4 R= H K3 Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme Nature 2010; 468:117-121. Kimie Nakagawa, Yoshihisa Hirota, Natsumi Sawada, Naohito Yuge, Masato Watanabe, Yuri Uchino, Naoko Okuda, Yuka Shimomura, Yoshitomo Suhara & Toshio Okano Department of Hygienic Sciences, Kobe Pharmaceutical University,

  31. Statin Bisphosphonate MK-4 biosynthesis in tissues is decreased by the treatments with statins and bisphosphonates Mevalonate pathway Acetyl-CoA Vitamin K homologues HMG-CoA HMG-CoA reductase D O D O Mevalonic acid D D CD3 CD3 D D D K3-d8 K1-d7 3 Geranyl pyrophosphate (GPP) D D O O FPP synthase Farnesyl pyrophosphate(FPP) Geranylgeranyl pyrophosphate(GGPP) O D UBIAD1 D CD3 P P D O D 3 3 MK-4-d7 O O O O : P P P P - - O O O O O - - - O O O O

  32. Possible interaction of MK-4 biosynthesis, vitamin K cycle and vitamin K action Mevalonate pathway MK-4 （quinone form） geranylgeranyl pyrophosphate prenylation Vitamin K cycle MK-4 （hydroquinone from） phylloquinone MK-4 cleavage MK-4- (epoxide form) phytol MK-4 synthetic Enz.(UBIAD1) CO2, O2 GGCX VKDP （active form） VKDP （inactive form） SXR/PXR signaling PKA/PKC signaling Biological functions

  33. mTOR exon1 Ubiad1 exon1 Ubiad1 exon2 ATG A Wild-type allele 3.5 kb 1.1 kb 6.7 kb Targeting vector neoR DTA : loxP SacII SalI ClaII NotI KpnI : FRT Generation of Ubiad1knockout mice NheI SpeI 1.7 kb Ubiad1-neo floxed allele neoR Cre recombinase Targeted allele PCR primer F R M.W. +/- +/+ -/- B Posi (A) Schematic presentation of ubiad1 genome, targeting vector and disrupted Ubiad1 genome. (B) PCR genotyping of Ubiad1+/+, Ubiad1+/− and Ubiad1−/− embryos. PCR genotyping of tail DNA of Ubiad1+/+, Ubiad1+/− and Ubiad1−/− embryos. Lane 1, positive controls for Ubiad1+/− allele. Lane 2, PCR bands of Ubiad1+/− embryos. Lane 3, PCR bands of Ubiad1+/+ embryos. Lane 4, PCR bands of Ubiad1−/− embryos. 500 bp 100 bp (bp) 1500 1000 500 300 Nakagawa K, Okano T, et al, PLOS ONE 2014; 9: 1-12, e104078

  34. Morphological examination of Ubiad1+/+, Ubiad1+/− and Ubiad1−/− embryos and weanling mice (postnatal day 1) from pregnant Ubiad1+/− mice orally administered CoQ10 Ubiad1+/+ Ubiad1+/- Ubiad1-/- Ubiad1+/+ Ubiad1+/- Ubiad1-/- A D Day 1 E15.5 B HE staining E * * C N.D. N.D. +/- -/- +/- -/- +/+ +/+ UBIAD1 staining Ubiad1+/+ Ubiad1+/- Ubiad1-/- F UBIAD1 Ubiad1-deficient mouse embryos failed to survive beyond embryonic day 7.5 exhibiting small-sized body and gasturation arrest !!! GAPDH

  35. Generation of a neural cell specific Ubiad1-/- mouse × Cre Nestin-cre Flox/+ Cre/+ Cre Cre +/+ Flox/+ Cre/+ -/+ Cre/+ Flox+/+ +/+ +/+ Cre/+ × Flox/+ Flox/+ +/+ Flox/+ Flox/+ Flox/Flox Impairment of brain function flox/- flox/- cre/+ flox/+ flox/+ cre/+

  36. Co-Workers (Kobe Pharmaceutical University) Kimie Nakagawa, Yoshihisa Hirota,Natsumi Sawada, Yuri Uchino, Masato Watanabe, Syusaku Kimura, Naoto Yuge, Naoko Okuda, Yuka Shimomura, Makiko Yamane (Shibaura Institute of Technology) Yoshitomo Suhara Thank you for your attention! Kobe Pharmaceutical Univ. Dept. Hygienic Sciences

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