Long-term dietary nitrite and nitrate deficiency causes the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice

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1 Dietologi (217) 6: DOI 1.17/s ARTICLE Long-term dietry nitrite nd nitrte defiieny uses the metoli syndrome, endothelil dysfuntion nd rdiovsulr deth in mie Mik Kin-Tnd 1,2 & Myuko Sknshi 1 & Akihide Tnimoto 3 & Tdshi Knme 4 & Toshihiro Mtsuzki 1 & Ktsuhiko Noguhi 1 & Tro Uhid 1 & Junko Nksone 1 & Chisyo Kozuk & Msyoshi Ishid 1,6 & Hruki Kuot 1 & Yuji Tir 1 & Yuihi Totsuk 1 & Shin-ihiro Kin 2 & Hjime Sunkw 2 & Junihi Omur 7 & Kimio Stoh 7 & Hiroki Shimokw 7 & Nouyuki Yngihr 8 & Shiro Med 4 & Yusuke Ohy 9 & Msyuki Mtsushit 1 & Hiroki Msuzki & Akir Arski 2 & Msto Tsutsui 1 Reeived: 16 Septemer 216 /Aepted: 27 Ferury 217 /Pulished online: 28 Mrh 217 # Springer-Verlg Berlin Heidelerg 217 Astrt Aims/hypothesis Nitri oxide (NO) is synthesised not only from L-rginine y NO synthses (NOSs), ut lso from its inert metolites, nitrite nd nitrte. Green lefy vegetles re undnt in nitrte, ut whether or not defiieny in dietry nitrite/nitrte spontneously uses disese remins to e lrified. In this study, we tested our hypothesis tht long-term dietry nitrite/nitrte defiieny would indue the metoli syndrome in mie. Methods To this end, we prepred low-nitrite/nitrte diet () onsisting of n mino id-sed low-nitrite/nitrte how, in whih the ontents of L-rginine, ft, rohydrtes, protein nd energy were identil with regulr how, nd potle ultrpure wter. Nitrite nd nitrte were undetetle in oth the how nd the wter. Results Three months of the did not ffet food or wter intke in wild-type C7BL/6J mie ompred with regulr diet (). However, in omprison with the, 3 months of the signifintly eliited viserl diposity, dyslipidemi nd gluose intolerne. Eighteen months of the signifintly provoked inresed ody weight, hypertension, insulin resistne nd impired endothelium-dependent relxtions to etylholine, while 22 months of the signifintly led to deth minly due to rdiovsulr disese, inluding ute Mik Kin-Tnd nd Myuko Sknshi ontriuted eqully to this work. Eletroni supplementry mteril The online version of this rtile (doi:1.17/s ) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. Msto Tsutsui tsutsui@med.u-ryukyu..jp Deprtment of Phrmology, Grdute Shool of Mediine, University of the Ryukyus, 27 Uehr, Nishihr, Okinw 93-21, Jpn Deprtment of Orl nd Mxillofil Funtionl Rehilittion, University of the Ryukyus, Okinw, Jpn Deprtment of Pthology, Kgoshim University Grdute Shool of Medil nd Dentl Sienes, Kgoshim, Jpn Deprtment of Advned Genomi nd Lortory Mediine, Grdute Shool of Mediine, University of the Ryukyus, Okinw, Jpn Seond Deprtment of Internl Mediine, Grdute Shool of Mediine, University of the Ryukyus, Okinw, Jpn Regenertive Mediine Reserh Center, Grdute Shool of Mediine, University of the Ryukyus, Okinw, Jpn Deprtment of Crdiovsulr Mediine, Tohoku University Grdute Shool of Mediine, Sendi, Jpn Deprtment of Phrmology, Shool of Mediine, University of Ouptionl nd Environmentl Helth, Kitkyushu, Jpn Third Deprtment of Internl Mediine, Grdute Shool of Mediine, University of the Ryukyus, Okinw, Jpn Deprtment of Physiology, Grdute Shool of Mediine, University of the Ryukyus, Okinw, Jpn

2 Dietologi (217) 6: myordil infrtion. These normlities were reversed y simultneous tretment with sodium nitrte, nd were signifintly ssoited with endothelil NOS downregultion, diponetin insuffiieny nd dysiosis of the gut miroiot. Conlusions/interprettion These results provide the first evidene tht long-term dietry nitrite/nitrte defiieny gives rise to the metoli syndrome, endothelil dysfuntion nd rdiovsulr deth in mie, inditing novel pthogeneti role of the exogenous NO prodution system in the metoli syndrome nd its vsulr omplitions. Keywords Aute myordil infrtion. Crdiovsulr deth. Diet. Endothelil dysfuntion. Metoli syndrome. Mie. Nitrte. Nitri oxide. Nitrite Arevitions AMPK Adenosine monophosphte-tivted protein kinse CT Computed tomogrphy enos Endothelil nitri oxide synthse EWAT Epididyml white dipose tissue Glyerldehyde-3-phosphte dehydrogense inos Induile nitri oxide synthse Low-nitrite/nitrte diet nnos Neuronl nitri oxide synthse NOS Nitri oxide synthse PPAR-γ Peroxisome prolifertor-tivted reeptor-γ Regulr diet WAT White dipose tissue WT Wild-type Introdution The metoli syndrome is defined s onstelltion of interrelted rdiovsulr risk ftors of metoli origin, inluding viserl oesity, dyslipidemi, hypertension, gluose intolerne nd insulin resistne [1]. The metoli syndrome is highly prevlent in industril ountries worldwide, nd it hs een reported tht, in ordne with the most reent hrmonised definition of the metoli syndrome, 23% of the dult popultion ( 2 yers of ge) in the USA suffered from the metoli syndrome in [2, 3]. Greter glol industrilistion is ssoited with rising rtes of oesity, whih re expeted to drmtilly inrese the prevlene of the metoli syndrome worldwide, espeilly s the popultion ges [1]. The metoli syndrome is ssoited with inresed risks of myordil infrtion, stroke, rdiovsulr disese mortlity nd ll-use mortlity [4]. It lso onfers higher risks of peripherl vsulr disese, type 2 dietes, renl disese, hepti disese nd ner [ 9]. Severl ftors, inluding exessive food energy intke, lk of physil tivity, geneti suseptiility nd geing, hve een thought to e involved in the pthogenesis of the metoli syndrome. However, the preise mehnisms in its development remin to e fully eluidted [1]. Nitri oxide (NO) exerts multiple iologil tions, nd is one of the most ruil signlling moleules in mmmlin physiology nd pthology [1 1]. It is endogenously synthesised from preursor L-rginine y fmily of NO synthses (neuronl [nnos], induile [inos] nd endothelil [enos]) with stoihiometri prodution of L-itrulline. NO hs very short hlf-life of severl seonds nd is rpidly oxidised to nitrite (NO 2 ) nd susequently to nitrte (NO 3 ). Although nitrite nd nitrte were in the pst regrded s mere inert metolites of NO, reent studies hve reveled tht nitrte is reduedtonitritendthentono,sotheyservesnodonors [16 18]. Green lefy vegetles, suh s spinh nd lettue, nd eetroot, re undnt in nitrte, nd vegetles re the dominnt soure of dietry nitrte in humns, ontriuting to 6 8% of dietry nitrte intke [19, 2]. Potle tp wter lso ontins nitrte nd smll quntity of nitrite, nd 1 2% of dietry nitrte intke is derived from tp wter [19, 2]. It hs een reported tht rdi nd hepti ishemi reperfusion injury in mie [21 23], rdi llogrft rejetion in rts [23] nd pltelet ggregtion in mie [24] re exerted y lownitrite/nitrte diet () ( ommerilly ville low-nitrite/ nitrte how plus potle ultrpure wter, or the low-nitrite/ nitrte how lone) ompred with regulr diet (). These results suggest tht dietry nitrite/nitrte defiieny modultes disese onditions. Whether or not this defiieny spontneously uses disese, however, remins to e lrified. In this study, we tested our hypothesis tht long-term dietry nitrite/nitrte defiieny would give rise to the metoli syndrome in mie. Methods Mie This study ws pproved y the Animl Cre nd Use Committee, University of the Ryukyus, Jpn, nd ws rried out ording to the Institutionl Poliy on the Cre nd Use of the Lortory Animls. The experiments were performed in 6-week-old mle wild-type (WT) C7BL/6J mie (Kyudo, Tosu, Jpn). All the mie were mintined in temperture- nd humidity-ontrolled rooms illuminted from 8: h to 2: h. Food nd wter intke ws mesured y pling the nimls in metoli ges for 24 h (see eletroni supplementry mteril [ESM] Methods for further detils). Diet We prepred purified mino id-sed low-nitrite/nitrte how in whih the ontents of L-rginine, ft, rohydrtes, protein nd energy were identil to regulr how (Purin 1; LDiet, St Louis, MO, USA), nd potle ultrpure Milli-Q wter in whih nitrite nd nitrte levels were

3 114 Dietologi (217) 6: undetetle (Merk Millipore, Drmstdt, Germny) (Tles 1 nd 2). We rndomly ssigned the mie to diet groups, nd either the low-nitrite/nitrte how plus ultrpure wter () or the regulr how plus tp wter () ws fed d liitum to the mie from 6 weeks of ge for 22 months (see ESM Methods). Nitrite nd nitrte levels The nitrite nd nitrte ontents of the hows were nlysed using the dizotistion method nd the dmium redution-dizotistion method, respetively (Jpn Food Reserh Lortories, Tokyo, Jpn). The nitrite nd nitrte levels in the plsm nd drinking wter were ssessed using the HPLC-Griess system (ENO-2; Eiom, Kyoto, Jpn) (see ESM Methods). Blood pressure Systoli lood pressure ws mesured y the til-uff method under onsious onditions in linded mnner (Model MK-2; Muromhi Kiki, Tokyo, Jpn) (see ESM Methods). Gluose tolerne test Gluose 1 g/kg ody weight ws intrperitonelly injeted into the mie under generl nesthesi with sodium pentoritl ( mg/kg, i.p.; Sigm-Aldrih, St Louis, MO, USA) fter 18 h of fsting. Whole lood smples were olleted from the til, nd lood gluose levels were evluted using portle lood gluose nlyser (Gluord MyDi; Arkry, Kyoto, Jpn) (see ESM Methods). Insulin tolerne test nd plsm insulin levels The mie reeived.3 U/kg ody weight of insulin (solule humn insulin, Humulin R; Eli Lilly, Indinpolis, IN, USA) injeted into the intrperitonel vity under generl nesthesi with sodium pentoritl ( mg/kg, i.p.). The fsting plsm insulin levels were ssessed using ommerilly ville ELISA kit (AKRIN-31; Shiygi, Gunm, Jpn) (see ESM Methods). Viserl ft weight After euthnsi, epididyml white dipose tissue (EWAT) ws removed nd weighed (see ESM Methods). Tle 1 Composition of regulr nd low-nitrite/nitrte how Constituent Regulr how Low-nitrite/nitrte how Protein (% kj) Ft (% kj) Crohydrte (% kj) L-rginine (g/kg) Nitrite (μmol/kg) <6. <6. Nitrte (μmol/kg) 48.3 <4.8 Vlues re expressed s the men vlues of two smples Tle 2 Nitrte nd nitrite levels in tp wter nd ultrpure wter Constituent Tp wter Ultrpure wter Nitrite (μmol/l) 3.3 <.1 Nitrte (μmol/l) 6.7 <.1 Vlues re expressed s the men vlues of two smples Adipoyte hypertrophy nd inflmmtion Epididyml nd peri-renl white dipose tissue (WAT) ws stined with n H&E solution. The irumferentil length of eh dipoyte ws mesured using light mirosope equipped with CCD mer nd morphometri nlysis softwre (DS-Ri1CCD mer nd NIS-Elements D 3.2 softwre; Nikon, Tokyo, Jpn). To evlute inflmmtion in the dipose tissues, ggregtes of inflmmtory ells (inflmmtory foi onsisting of more thn ten inflmmtory ells) were ounted in the mximl ut surfe of the EWAT nd peri-renl WAT setions on light mirosope t 4 mgnifition (see ESM Methods). Plsm lipid profile Plsm lipid profile ws ssessed using Dri-Chem utonlyser (FDC4; Fuji Film, Tokyo, Jpn). Plsm LDL-holesterol levels were determined y HPLC (Skylight Bioteh, Akit, Jpn) [2] (seeesm Methods). Western lot nlysis Western lot nlysis ws performed s previously reported [26] to detet nnos, inos nd enos, phosphorylted enos t serine 1177 nd t threonine 49 (BD Trnsdution Lortories, Frnklin Lkes, NJ, USA), diponetin, peroxisome prolifertor-tivted reeptor-γ (PPAR-γ), denosine monophosphte-tivted protein kinse (AMPK), sirtuin 1 (Cell Signling Tehnology, Dnvers, MA, USA), p-ampk (Snt Cruz Biotehnology, Dlls, TX, USA) nd glyerldehyde-3-phosphte dehydrogense () (Sigm-Aldrih) (see ESM Methods). The vlidtion of ntiodies ws performed y the supplier. Serum ytokine nd hemokine levels Serum ytokine/ hemokine levels were mesured using Bio-Plex system (23-Plex, M6-9PD; Bio-Rd, CA, USA) [27]. 16S riosoml RNA gene sequening The fel DNA smples were run through next-genertion sequener, s previously reported [28]. The numer of eh teril strin ontined in the fel ontents ws estimted s the genome equivlent y quntittive rel-time PCR of 16S riosoml RNA genes, followed y pyrosequening of the 16S mplions [28] (see ESM Methods). Orgn hmer experiment Thori orti rings were mounted in mirotissue orgn th hmers (MTBO-1; Lo Support, Osk, Jpn) filled with Kres-Henseleit

4 Dietologi (217) 6: solution, nd the isometri ontrtile fore of the rings ws mesured (see ESM Methods). Miro-omputed tomogrphy imging Mie were nesthetised with 2% isoflurne (Wko Pure Chemil Industries, Osk, Jpn), nd omputed tomogrphy (CT) imges were quired y three-dimensionl miro-ct (R_mCT2; Rigku Corportion, Tokyo, Jpn) (see ESM Methods). Sttistil nlyses Most of our results re expressed s men ± SEM. Sttistil nlyses were performed using Student s t test, or ANOVA followed y Bonferroni s post ho test. The results of sequening reds of gut teri re expressed s the medin nd interqurtile rnge, nd the sttistil nlysis ws rried out using Wiloxon rnk-sum test [29]. Kpln Meier survivl urves were ompred using the logrnk test. A vlue of p <. ws onsidered to e sttistilly signifint (see ESM Methods). Results Three month mrkedly redued plsm nitrite/ nitrte levels The 3 month mrkedly redued plsm nitrite/nitrte levels ompred with the (Fig. 1). To exmine the underlying mehnism, we evluted NOS levels in isolted ort nd viserl ft. enos protein levels in the ort were omprle in the two diets (Fig. 1), wheres, intriguingly, levels in EWAT were mrkedly lower with the thn the (Fig. 1), ounting for the mrkedly redued plsm nitrite/nitrte levels indued y the. No nnos or inos ws found in EWAT with either diet (ESM Fig. 1). Three month resulted in metoli syndrome-like onditions There were no signifint differenes in food intke (Fig. 1d), wter intke (ESM Fig. 1), or ody weight (ESM Fig. 1) etween the 3 month nd. However, the 3 month signifintly inresed EWAT weight (Fig. 1e), epididyml white dipoyte size (Fig. 1f) nd plsm levels of totl holesterol (Fig. 2), LDL-holesterol (Fig. 2) nd smll Fig. 1 Effets of 4. months of the on plsm nitrite/ nitrte, enos levels, food intke nd viserl ft in WT mie. () Plsm nitrite/nitrte levels (n =8 12). (, ) enosprotein levels in the ort (; n =6)nd EWAT indued y 3 months of the (; n =1).(d, e) Food intke (n =1 12) nd reltionship of EWAT to ody weight (n =1 12). (f) Epididyml white dipoyte size indued y 3 months of the (n = 1). Sle rs, μm. White rs, ; lk rs,. p <.,p <.1, p <.1 Plsm nitrite/nitrte (μmol/l) enos/ (fold) months 3 months 4. months enos 14 kd 1.. enos/ (fold) d Food intke (g/dy) enos kd months 3 months 4. months e.2.1 EWAT/ody weight (wt/wt).3 months 3 months 4. months f Epididyml white dipoyte size (μm 2 )

5 1142 Dietologi (217) 6: Fig. 2 Effets of 4. months of the on plsm lipid, lood gluose nd insulin response. () Plsm totl holesterol levels (n =1 12). White rs, ; lk rs,. () Plsm LDL-holesterol (LDL-) levels indued y 3 months of the (n =12).( e) Blood gluose levels fter i.p. injetion of 1 g/kg gluose fter (), 3 (d) nd 4. (e) months of the (n =11 12). (f h) Perentge hnge in lood gluose levels fter i.p. injetion of.3 U/kg insulin fter (f), 3 (g) nd 4. (h) months of the (n =8 12). White rs nd white irles, ; lk rs nd lk squres,. p <., p <.1, p <.1 Plsm totl holesterol (mmol/l) Blood gluose (mmol/l) months 3 months 4. months d Blood gluose (mmol/l) Plsm LDL- (mmol/l) e Blood gluose (mmol/l) f 1 g 1 h 1 Chnge in lood gluose (%) 1 Chnge in lood gluose (%) 1 Chnge in lood gluose (%) dense LDL-holesterol (ESM Fig. 1d), nd non-signifintly elevted plsm triylglyerol levels ompred with the (p =.4) (Tle 3). Furthermore, the 3 month signifintly ugmented lood gluose levels fter i.p. gluose injetion nd ggrvted lood gluose-lowering responses to i.p. insulin injetion (Fig. 2d, g). Arteril lood pressure levels were similr with oth diets (ESM Fig. 2). We next studied the time ourse of the metoli effets t, 3 nd 4. months fter the strt of the. The effets on EWAT weight (Fig. 1e), plsm totl holesterol levels (Fig. 2), lood gluose levels fter gluose injetion (Fig. 2 e) nd lood gluose-lowering responses to insulin (Fig. 2f h, ESM Fig. 1e g) were time-dependent nd ppered to reh plteu t 3 months. There were no signifint differenes in fsting plsm insulin levels etween the mie fed the or the t ny of the time points (ESM Fig. 2). Tle 3 Plsm triylglyerol levels t, 3 nd 4. months fter the strt of or Diet Plsm triylglyerol levels (mmol/l) months 3 months 4. months (n =6 12) 1.23 ±.7.94 ±. 1.6 ±.7 (n = 6 12) 1.2 ± ± ±.19 Sttistil nlysis ws performed y two-wy ftoril ANOVA followed y Bonferroni s post ho test for multiple omprisons, nd no signifint differenes were seen

6 Dietologi (217) 6: Adiponetin levels in EWAT were mrkedly lower for the 3 month ompred with the (Fig. 3). The EWAT levels of PPAR-γ, totl AMPK nd p-ampk, ut not sirtuin 1, were lso mrkedly redued fter 1 week of the in omprison to the (Fig. 3 e). Simultneous orl tretment with 2 mmol/l sodium nitrte for 3 months signifintly reversed the redued plsm nitrite/ nitrte levels indued y the (Fig. 4). It lso nonsignifintly improved the -indued gin in EWAT weight (p =.6) (Fig. 4), nd signifintly meliorted the epididyml white dipoyte hypertrophy, hyper-smll dense LDL-holesterolemi, impired gluose tolerne, redued lood gluose-lowering responses to insulin, enos downregultion nd diponetin insuffiieny indued y the (Fig. 4 h, ESMFig.2). Dysiosis of gut miroiot ws noted in the -fed mie Although there ws no signifint differene etween the two diets in the numer of totl sequening reds (ESM Fig. 3), there were signifintly fewer opertionl txonomi units, whih represent the kind of gut teri, with the thn the, suggesting less diversity of gut miroiot (Fig. ). Signifintly different gut teri in eh rnk re shown in Fig., nd ESM Fig. 3 j. In the phylum rnk, there were more Atinoteri with the thn the (ESM Fig. 3). In the lss rnk, there were more Atinoteri nd fewer Betproteoteri with the (ESM Fig. 3, d). In the order rnk, Bifidoteriles ws more numerous with the, nd Burkholderiles nd Billles were less numerous (ESM Fig. 3e, f). In the fmily (ESM Fig. 3g, h), the genus (Fig., ) nd the speies (ESM Fig. 3i, j) rnks, more thn three gut teri were more prevlent, nd more thn two gut teri were less prevlent with the. On the other hnd, Bteroides frgilis ws to ertin degree present with the, ut sent with the (medin nd interqurtile rnge 312 [ ] for the vs for the ; n = ). There were no signifint differenes etween the two diets in Firmiutes, Bteroidetes or Firmiutes/Bteroidetes rtio, whih might hnge in disese sttes of the metoli syndrome [3 33], or Akkermnsi muiniphil, whih might improve the metoli syndrome [34, 3] (dt not shown). Bteroides uniformis CECT 7771, whih might improve the metoli syndrome [36], ws not deteted in either diet. An 18 month resulted in more severe metoli syndrome nd endothelil dysfuntion Although the metoli effets of the ppered to reh plteu t 3 months, we explored the effets of n extremely long period of (18 months) for onfirmtion. There were no signifint differenes in food or wter intke etween the 18 month nd (ESM Fig. 4, ), ut, notly, 18 months of the resulted in more severe metoli syndrome, eliiting signifint gin in ody weight, whih ws not seen until 4. months fter the strt of the (Fig. 6). Miro-CT Adiponetin 27 kd PPAR-γ 7 kd Adiponetin/ (fold) 1.. PPAR-γ/ (fold) nitrte Totl AMPK/ (fold) Totl AMPK kd + nitrte d p-ampk/ampk (fold) p-ampk AMPK Fig. 3 Effets of 3 months of the nd nitrte supplementtion on diponetin, nd effets of 1 week of the nd nitrte supplementtion on PPAR-γ, totl AMPK, p-ampk nd sirtuin 1 levels in EWAT. () kd 62 kd e Sirtuin 1/ (fold) Sirtuin 1 12 kd + nitrte + nitrte 1.. Adiponetin levels (n = ).( e) PPAR-γ, totl AMPK, p-ampk nd sirtuin 1 levels (n =7).p <.,p <.1, p <.1

7 1144 Dietologi (217) 6: Fig. 4 Effets of 3 months of nitrte supplementtion on metoli syndrome-like onditions indued y the. () Plsm nitrite/nitrte levels (n =6 12). () Reltionship of EWAT to ody weight (n =7 8). () Epididyml white dipoyte size (n = 1). Sle r, μm. (d) Plsm smll dense LDL-holesterol (LDL-) levels (n =1 12). (e) Blood gluose levels fter i.p. injetion of 1 g/kg gluose (n =8).(f) Perentge hnge in lood gluose levels fter i.p. injetion of.3 U/kg insulin (n = 9 1). Blk squres, ; white tringles, + nitrte. (g, h)enos(n = 6) nd diponetin (n =3)levelsin EWAT. p <.,p <.1, p <.1 Plsm nitrite/nitrte (μmol/l) Epididyml white dipoyte size (μm 2 ) nitrte + nitrte EWAT/ody weight (wt/wt) d Plsm smll dense LDL- (mmol/l) nitrte + nitrte e Blood gluose (mmol/l) 1 1 f Chnge in lood gluose (%) g enos 14 kd h Adiponetin 27 kd enos/ (fold) nitrte Adiponetin/ (fold) nitrte imging indited tht ody ft ws mrkedly inresed, y 3.2 times, in the 18 month -fed mie (Fig. 6, ), nd this ws speifilly due to n inrese in viserl ft (Fig. 6d)nd not suutneous ft (ESM Fig. 4). The 18 month elevted levels of plsm smll dense LDL-holesterol nd lood gluose following gluose injetion, nd signifintly lunted lood gluose-lowering responses to insulin (Fig. 6e g,esm Fig. 4d). In ddition, it signifintly inresed levels of fsting lood gluose, fsting plsm insulin nd rteril lood pressure (Fig. 7 ), findings tht were not oserved until 4. months fter the strt of the. enos protein levels in the EWAT were lso signifintly deresed with the 18 month (ESM Fig. 4e). As the 18 month indued severe metoli syndrome, we next exmined vsulr retivity. In isolted orts, ontrtions in response to phenylephrine, n drenergi α 1 -reeptor gonist, nd endothelium-independent relxtions to diethylmine NONOte, n NO donor, were omprle etween the two diets (ESM Fig. 4f, g), wheres endotheliumdependent relxtion in response to etylholine, physiologil enos tivtor, ws signifintly impired with the (Fig. 7d). enos protein levels were signifintly diminished in

8 Dietologi (217) 6: Fig. Effets of 3 months of the on gut miroiot. () Opertionl txonomi units (n =eh).(, ) Signifintly higher () or lower () numers of gut teri in the genus rnk (p <.,n =eh).white oxes, ; grey oxes,. Insets re the mgnified views of sequening reds of the rightmost three gut teri in prts nd. p <. Opertionl txonomi units Sequening reds Sequening reds Sequening reds Sequening reds Bteroides Euterium Prteroides Bifidoterium Anerotrunus Adlerreutzi Brnesiell Prsutterell Butyrivirio Roinsoniell Prprevotell Desulfovirio Desulfitoterium Desulfosporosinus Butyriious the orts (Fig. 7e), nd there ws non-signifint tendeny with the towrds lower levels of phosphoryltion of enos t serine 1177 (p =.12), whih is n index of enos tivtion, without ffeting the phosphoryltion levels of enos t threonine 49, whih is n index of enos intivtion (ESM Fig. 4h, i). Simultneous tretment with sodium nitrte for 18 months inhiited these metoli normlities, the endothelil dysfuntion nd the orti nd EWAT enos downregultion indued y the (Figs 6, 7 e, ESM Fig. 4 e). There were no signifint inreses in the serum levels of 23 inflmmtory mrkers mesured using the Bio-Plex system etween the month nd (Tle 4). There were lso no signifint differenes in the numer of inflmmtory foi in the EWAT nd peri-renl WAT etween the 3 month nd (dt not shown). However, the 18 month non-signifintly inresed the numer of inflmmtory foi in the EWAT (p =.8) nd signifintly ugmented those in the peri-renl WAT ompred with the (Fig. 7f, g). A 22 month led to rdiovsulr deth We experiened sudden deth in some of the -fed mie. During the 22 months of follow-up, none (/24) of the -fed mie, ut 31.8% (7/22) of the -fed mie, died. The survivl rte ws signifintly worse in the -fed thn in the -fed mie, nd o-tretment with sodium nitrte improved the redued survivl (Fig. 8). We performed post mortem histopthologil nlysis to identify the use of deth in the seven ded -fed mie. We were not le to investigte the use of deth in one mouse euse of strong post mortem putreftion. We judged tht one mouse hd died of n ute nterior wll myordil infrtion (Fig. 8), nd tht one mouse died of mlignnt lymphom of the lung, liver (Fig. 8g), kidney nd spleen. In the other four mie, we noted oronry perivsulr firosis, pulmonry ongestion nd ute renl tuulr nerosis (Fig. 8d f), findings tht re oserved in rdi sudden deth. Myordil firosis, whih might hve resulted from myordil infrtion, ws seen in one mouse (Fig. 8), mlignnt lymphom of the liver nd spleen ws oserved in one mouse, nd no other pthologil findings tht ould explin the use of deth were seen in ny of the mie. Disussion In previous studies, the effets of n on rdi nd liver ishemi reperfusion injury [21 23], rdi llogrft rejetion [23] nd pltelet ggregtion [24] were investigtedusing ommerilly ville low-nitrite/nitrte how, ut the ontents of L-rginine, ft, rohydrtes, protein nd energy were

9 1146 Dietologi (217) 6: nitrte Body weight (g) Horizontl Coronl + nitrte d Body ft/ody mss (vol./vol. %) nitrte Viserl ft/ody mss (vol./vol. %) nitrte e f g Plsm smll dense LDL- (mmol/l) nitrte Blood gluose (mmol/l) Chnge in lood gluose (%) Fig. 6 Effets of 18 months of the nd nitrte supplementtion on ody weight, ody ft, plsm smll dense LDL-holesterol, lood gluose nd insulin sensitivity. () Body weight (n =12 16). () Miro-CT imges. Yellow, viserl ft; ornge, suutneous ft. () Reltionship of ody ft to ody mss (n =11 12). (d) Reltionship of viserl ft to ody mss (n = 11 12). (e) Levels of plsm smll dense LDL-holesterol (LDL-; n =7 9). (f) Blood gluose levels fter i.p. injetion of 1 g/kg gluose (n =12 14). (g) Perentge hnge in lood gluose levels fter i.p. injetion of.3 U/kg insulin (n = 1). White irles, ; lk squres, ; white tringles, + nitrte. p <., p <.1 for vs ; p <., p <.1 for vs + nitrte onsiderly different etween the ommerilly ville low-nitrite/nitrte how nd regulr how. In this study, we employed low-nitrite/nitrte how in whih the ontents of those ingredients were identil with the regulr how. We previously red mie in whih ll three NOS isoforms were ompletely disrupted (triply n/i/enoss / mie) [37], nd indited tht oth their plsm nitrite/ nitrte onentrtions nd urinry nitrite/nitrte exretion were extremely low, t less thn 1% of the norml levels of WT mie [38]. These results suggested tht in vivo NO synthesis is predominntly regulted y endogenous NOSs, nd tht the ontriution of the exogenous NO prodution system to tht regultion might e minor. However, ontrry to these suggestions, nother study showed tht 1 week, ompred with n, mrkedly deresed plsm nitrite/nitrte levels in WT mie showing norml NOS tivities [21]; however, the possile underlying mehnisms remin to e lrified. In this study, we otined similr finding in tht 4. months of the mrkedly redued plsm nitrite/ nitrte levels in WT mie. We then exmined the ustive mehnisms, nd found tht enos levels in the viserl ft, ut not in the ort, were mrkedly suppressed in the 3 month -fed mie, ounting for the mrkedly depressed plsm

10 Dietologi (217) 6: Fsting lood gluose (mmol/l) nitrte Fsting plsm insulin (pmol/l) nitrte d Systoli BP (mmhg) 1 1 Relxtion (%) e enos/ (fold) enos nitrte + nitrte 14 kd f Numer of inflmmtory foi per 4 field Fig. 7 Effets of 18 months of the nd nitrte supplementtion on fsting lood gluose, lood pressure, vsulr retivity, enos levels nd inflmmtion. () Fsting lood gluose levels (n = 12 14). () Fsting plsm insulin levels (n = 6 7). () Systoli BP(n = 12 1). (d) Endothelium-dependent relxtion in response to etylholine ACh ( log 1 mol/l) g Numer of inflmmtory foi per 4 field nitrte + nitrte (ACh) (n = 6). White irles, ; lk squres, ; white tringles, + nitrte. (e) enos protein levels in the ort (n =6).(f, g) Numer of inflmmtory foi in EWAT (f) nd peri-renl WAT (g) (n = 12 14). p <., p <.1 for vs ; p <., p <.1, p <.1 for vs + nitrte nitrite/nitrte levels indued y the. We lso found tht diponetin levels in the viserl ft were remrkly low in the 3 month -fed mie. Adiponetin hs een reported to upregulte enos [39], so the diponetin insuffiieny my hve medited the viserl dipose enos downregultion indued y the. It hs lso een reported tht enos enhnes diponetin levels in dipoytes [4]. Thus, there my e viious yle of diponetin insuffiieny nd enos downregultion, nd this viious yle my ontriute to the mrkedly diminished plsm nitrite/nitrte levels indued y the. The levels of PPAR-γ, totl AMPK nd p-ampk were signifintly lower in the EWAT in the 1 week -fed mie ompred with the -fed mie. As it hs een reported tht PPAR-γ nd AMPK inrese levels of diponetin levels in dipoytes [41, 42], it is oneivle tht redued PPAR-γ nd AMPK in the viserl ft were involved in the diponetin insuffiieny indued y the. The 3 month -fed mie exhiited viserl oesity with dipoyte hypertrophy, hyper-ldl-holesterolemi nd hyper-smll dense LDL-holesterolemi nd gluose intolerne, nd the 18 month -fed mie mnifested ody weight gin, hypertension, insulin resistne nd endothelil dysfuntion. These hnges eventully resulted in deth due to rdiovsulr disese, inluding ute myordil infrtion. These normlities were reversed y onurrent nitrte supplementtion, inditing tht the oserved effets were indeed used y dietry nitrite/nitrte defiieny. It is thus evident tht long-term dietry nitrite/nitrte defiieny n use the metoli syndrome, endothelil dysfuntion nd rdiovsulr deth in mie. The 3 nd 4. month signifintly redued lood gluose-lowering responses to insulin, ut did not signifintly ffet fsting plsm insulin levels. Thus, efore 18 months, the effets of on gluose tolerne ould e due to the result of those on pnreti et ell funtion.

11 1148 Dietologi (217) 6: Tle 4 Serum levels of 23 inflmmtory mrkers in mie fed the nd for months Mrker Serum levels (pg/ml) p vlue IL-1α 31.3 ± ± 3.. IL-1β ± ± IL ± ± IL ± ± IL ± ± IL- 89. ± ± IL ± ± IL ± ± IL ± ± IL-12p ± ± IL-12p ± ± IL ± ± IL ± ± TNF-α ± ± IFN-γ ± ± MCP ± ± MIP-1α 74.4 ± ± MIP-1β ± ± G-CSF ± ± GM-CSF 46.9 ± ± KC ± ± Eotxin ± ± RANTES 34.2 ± ± n =8 1 mie for eh group G-CSF, grnuloyte-olony stimulting ftor; GM-CSF, grnuloyte/ mrophge-olony stimulting ftor; KC, kertinoyte-derived hemokine; MCP, monoyte hemotti protein; MIP, mrophge inflmmtory protein; RANTES, regulted on tivtion norml ell expressed nd sereted A lustering of rdiovsulr risk ftors (i.e. the metoli syndrome) ould hve ontriuted to the development of endothelil dysfuntion nd rdiovsulr deth in the fed mie. On the other hnd, hyper-ldl-holesterolemi nd hyper-smll dense LDL-holesterolemi, oth of whih re independent rdiovsulr risk ftors [43], were lso noted in the -fed mie. The smll dense LDL-holesterol prtile n esily penetrte the vsulr wll euse of its smll prtile size, nd is relted more strongly to the risk of rdiovsulr disese. It is thus likely tht those ftors my lso hve een independently involved in the ourrene of endothelil dysfuntion nd rdiovsulr deth in the -fed mie. The 18 month -fed mie displyed n impirment of endothelium-dependent relxtion in response to etylholine, physiologil enos tivtor, long with orti enos downregultion, suggesting the presene of oronry vsospsm. Coronry vsospsm-eliited myordil ishemi n led to ftl rdi rrhythmi nd/or rdiogeni shok. On the other hnd, the ded -fed mie showed ute myordil infrtion, myordil firosis tht might hve resulted from myordil infrtion nd oronry perivsulr firosis. They lso exhiited pulmonry ongestion nd ute renl tuulr nerosis, oth of whih re seen in sudden rdi deth. Tking these findings together, we thought tht 83.3% (/6) of the ded -fed mie showed hnges onsistent with rdiovsulr deth. The dt otined t 3 nd 4. months re the most importnt nd signifint, s the hnges in gluose metolism, lipid iology, ody ft distriution nd gut miroiome ourred independent of food intke nd ody weight. On the other hnd, the moridity nd mortlity experiments t 18 nd 22 months, respetively, re omplited y greter weight gin in the group, mking it diffiult to seprte the effets of oesity from those of the nitrite/nitrte defiieny. We onduted the gluose nd insulin tolerne tests under nesthesi. Wheres it hs een reported tht high-ft dietfed C7BL/6J mie showed gluose intolerne ompred with their -fed ounterprts whether they were tested under onsious or nesthetised onditions [44], it hs lso een indited tht the use of nesthesi ould influene gluose tolerne in C7BL/6J mie [4]. Therefore, the use of nesthesi ould e limittion of this study. An inresed numer of inflmmtory foi, deresed enos levels nd lower diponetin levels were noted in the viserl ft of the -fed mie, nd improvements in the -indued metoli syndrome y nitrte supplementtion were linked to meliortions of these hnges. As it hs een reported tht inflmmtion, enos downregultion nd diponetin insuffiieny ontriute to the ourrene nd progression of the metoli syndrome [46 48], it is possile tht those ftors were involved in the development of the metoli syndrome indued y the. The following lines of evidene suggest usl role of dysiosis of the gut miroiot in the development of the metoli syndrome. First, it hs een reported tht the omposition of gut miroiot differs lrgely etween len individuls nd ptients with the metoli syndrome [33]. Seond, it hs een indited tht trnsplnttion of gut miroiot from oese humns with metoli normlities into germ-free mie results in the development of oesity nd metoli normlities in the mie [49]. Third, it hs een shown tht the trnsfer of gut miroiot from len helthy humn prtiipnts into individuls with the metoli syndrome improves insulin resistne in the ltter group [33]. In our study, there were signifintly fewer opertionl txonomi units with the thn with the, suggesting less diversity of gut miroiot in the -fed mie. There lso were signifintly different onstituents of gut miroiot in vriety of the hierrhy rnks with the nd the. In

12 Dietologi (217) 6: Fig. 8 Effets of 22 months of the nd nitrte supplementtion on survivl rte nd post mortem findings in -fed mie. () Survivl rte (n =1 24). Grey line, ; lk line, ; dotted line, + nitrte. p <.1.() Aute myordil infrtion. () Myordil firosis. (d) Coronry perivsulr firosis. (e) Pulmonry ongestion. (f) Aute renl tuulr nerosis. (g) Mlignnt lymphom. Sle rs, 1 μm Survivl rte (%) Administrtion period (months) Control hert Aute myordil infrtion Control hert Myordil firosis d e Control oronry rtery Coronry perivsulr firosis Control lung Pulmonry ongestion f g Control renl tuules Aute renl tuulr nerosis Mlignnt lymphom in lung Mlignnt lymphom in liver greement with these results, Vrieze et l. indited tht less diversity nd fewer distint onstituents of gut miroiot were reognised in individuls with the metoli syndrome, nd tht the improvement of insulin resistne fter trnsfer of the gut miroiot ws ompnied y n meliortion of the redued diversity nd distint onstituents of gut miroiot [33]. It is thus possile tht dysioti gut miroiot were involved in the pthogenesis of the metoli syndrome indued y the. We ompletely mthed the energy ontents in the two diets, nd food onsumption ws omprle etween the nd t, 3, 4. nd 18 months fter the strt of the diet, suggesting similr energy intke in the nd. Nevertheless, the -fed mie developed the metoli syndrome. Therefore, we my hve sueeded for the first time in identifying speifi dietry ingredients tht use the metoli syndrome even in the sene of exessive intke of energy. In summry, we were le to demonstrte tht long-term dietry nitrite/nitrte defiieny gve rise to the metoli syndrome, endothelil dysfuntion nd eventully rdiovsulr deth in mie, inditing novel pthogeneti role of the exogenous NO prodution system in the metoli syndrome nd its vsulr omplitions.

13 11 Dietologi (217) 6: Funding This work ws supported in prt y Grnt-in-Aids for Reserh Ativity Strt-up (1H619) nd Sientifi Reserh (C) (16K919) from the Jpn Soiety for the Promotion of Siene, Speil Aount Budgets for Edution nd Reserh grnted y the Jpn Ministry of Edution, nd Grnts from the Okinw Medil Siene Reserh Foundtion, the Ryukyu University Support Foundtion, the Promotion Projet of Medil Clustering of Okinw Prefeture, the Okinw Prefeture for Promotion of Advned Mediine nd the University of the Ryukyus, Jpn. Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Dt vilility The dtsets generted during nd/or nlysed during the urrent study re ville from the orresponding uthor on resonle request. Contriution sttement MK-T. nd MS designed the study, quired, nlysed nd interpreted dt, nd drfted the rtile. AT quired, nlysed nd interpreted dt, nd drfted the rtile. TK, TM, KN, TU, JN, CK, MI, HK, YT, Yto, S-iK, JO nd KS quired, nlysed, nd interpreted dt. HSu nd HM designed the study, nlysed nd interpreted dt. HSh, NY, SM, YO, MM nd AA nlysed nd interpreted dt. MT designed the study, nlysed nd interpreted dt, drfted the rtile. All uthors ritilly revised the rtile for importnt intelletul ontent nd gve finl pprovl of the version to e pulished. MT is the gurntor of this work. Referenes 1. Ekel R (21) The Metoli Syndrome. In: Ksper DL, Fui AS, Huser SL, Longo DL, Jmeson JL, Losldo J (eds) Hrrison's Priniples of Internl Mediine. 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