Nicotinamide overload may play a role in the development of type 2 diabetes

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1 Online Sumissions: wjg.wjgnet.com Worl J Gstroenterol Decemer 7; 5(5): 57-5 wjg@wjgnet.com Worl Journl of Gstroenterology ISSN 7-7 oi:.7/wjg.5.57 The WJG Press n Bishieng. All rights reserve. ORIGINAL ARTICLE Nicotinmie overlo my ply role in the evelopment of type ietes Shi-Sheng Zhou, D Li, Wu-Ping Sun, Ming Guo, Yong-Zhi Lun, Yi-Ming Zhou, Fu-Cheng Xio, Li-Xin Jing, Shen-Xi Sun, Li-Bin Zhng, Ning Luo, Fu-Ning Bin, Wei Zou, Li-Bin Dong, Zhi-Gng Zho, Sheng-Fn Li, Xio-Jie Gong, Zeng-Guo Yu, Chng-Bin Sun, Cong-Long Zheng, Dong-Ju Jing, Zheng-Ning Li Shi-Sheng Zhou, D Li, Wu-Ping Sun, Yong-Zhi Lun, Fu- Cheng Xio, Li-Xin Jing, Shen-Xi Sun, Ning Luo, Fu-Ning Bin, Sheng-Fn Li, Xio-Jie Gong, Zeng-Guo Yu, Chng- Bin Sun, Cong-Long Zheng, Institute of Bsic Meicl Sciences, Meicl College, Dlin University, Dlin, Lioning Province, Chin Ming Guo, Zheng-Ning Li, College of Environmentl n Chemicl Engineering, Dlin University, Dlin, Lioning Province, Chin Yi-Ming Zhou, Okzki Institute for Integrtive Bioscience, Ntionl Institutes of Nturl Sciences, Okzki -77, Jpn Li-Bin Zhng, Deprtment of Physiology, Xinxing Meicl College, Xinxing 5, Henn Province, Chin Wei Zou, Deprtment of Life Sciences, Lioning Norml University, Dlin, Lioning Province, Chin Li-Bin Dong, Deprtment of Neurology, PLA No. Hospitl, Jiozuo 5, Henn Province, Chin Zhi-Gng Zho, Dong-Ju Jing, Deprtment of Meicine, PLA No. Hospitl, Dlin, Lioning Province, Chin Author contriutions: Zhou SS ws responsile for the stuy concept, esign, irection n supervision, rfte the mnuscript, n otine funing; Li D, Sun WP, Lun YZ, Zhou YM, Xio FC, Jing LX, Sun SX, Zhng LB, Luo N, Bin FN, Zou W, Gong XJ, Yu ZG, Sun CB n Zheng CL contriute to the progrm initition, t cquisition n nlysis/iscussion, s well s writing n eiting the mnuscript; Guo M irecte the HPLC seprtion n nlysis; Dong LB, Zho ZG, Li SF n Jing DJ cquire the clinicl cses n ptient consent, n ssiste in t collection; Li ZN synthesize the compouns. Supporte y Ntionl Nturl Science Fountion of Chin, No. 575; the Fountion of Dlin Technology Bureu, No. ESF; n the Fountion of Key Lortory of Euction Deprtment of Lioning Province, No. S5 Corresponence to: Shi-Sheng Zhou, Professor, PhD, MD, Institute of Bsic Meicl Sciences, Meicl College, Dlin University, Dlin, Lioning Province, Chin. zhouss@ymil.com Telephone: Fx: Receive: Septemer, Revise: Octoer, Accepte: Novemer, Pulishe online: Decemer 7, Astrct AIM: To investigte whether nicotinmie overlo plys role in type ietes. METHODS: Nicotinmie metolic ptterns of ietic n non-ietic sujects were compre using HPLC. Cumultive effects of nicotinmie n N -methylnicotinmie on glucose metolism, plsm HO levels n tissue nicotinmie enine inucleotie (NAD) contents of ult Sprgue-Dwley rts were oserve. The role of humn swet glns n rt skin in nicotinmie metolism ws investigte using sun n urn injury, respectively. RESULTS: Dietic sujects h significntly higher plsm N -methylnicotinmie levels 5 h fter -mg nicotinmie lo thn the non-ietic sujects (. ±. μmol/l vs. ±. μmol/l, P <.). Cumultive oses of nicotinmie ( g/kg) significntly increse rt plsm N -methylnicotinmie concentrtions ssocite with severe insulin resistnce, which ws mimicke y N -methylnicotinmie. Moreover, cumultive exposure to N - methylnicotinmie ( g/kg) mrkely reuce rt muscle n liver NAD contents n erythrocyte NAD/ NADH rtio, n increse plsm HO levels. Decrese in NAD/NADH rtio n increse in HO genertion were lso oserve in humn erythrocytes fter exposure to N -methylnicotinmie in vitro. Sweting eliminte excessive nicotinmie (5.-fol increse in swet nicotinmie concentrtion h fter -mg nicotinmie lo). Skin mge or lehye oxise inhiition with tmoxifen or olnzpine, oth eing notorious for impiring glucose tolernce, elye N - methylnicotinmie clernce. CONCLUSION: These finings suggest tht nicotinmie overlo, which inuce n increse in plsm N - methylnicotinmie, ssocite with oxitive stress n insulin resistnce, plys role in type ietes. The WJG Press n Bishieng. All rights reserve. Key wors: Type ietes; Nicotinmie; N -methylnicotinmie; Insulin resistnce; Oxitive stress; Liver; Swet glns Peer reviewer: Lu Ci, PhD, Associte Professor, Deprtment of Peitrics, University of Louisville School of Meicine, 57 South Preston Street, Suite F, Louisville, KY, Unite Sttes

2 Zhou SS et l. Nicotinmie overlo n ietes 575 Zhou SS, Li D, Sun WP, Guo M, Lun YZ, Zhou YM, Xio FC, Jing LX, Sun SX, Zhng LB, Luo N, Bin FN, Zou W, Dong LB, Zho ZG, Li SF, Gong XJ, Yu ZG, Sun CB, Zheng CL, Jing DJ, Li ZN. Nicotinmie overlo my ply role in the evelopment of type ietes. Worl J Gstroenterol ; 5(5): 57-5 Aville from: URL: com/7-7/5/57.sp DOI: wjg.5.57 sujects, n to investigte the reltionship etween nicotinmie overlo n insulin resistnce. We foun tht ietic sujects exhiite slow N -methylnicotinmie clernce, n tht N -methylnicotinmie coul trigger oxitive stress n insulin resistnce, which suggeste tht the ssocition of nicotinmie overlo with reltively slow N -methylnicotinmie etoxifiction n excretion might e t lest prtilly responsile for the evelopment of type ietes. INTRODUCTION Type ietes, chrcterize y insulin resistnce n oxitive stress [,], hs reche epiemic proportions not only mong ults, ut lso mong olescents in the pst few eces, which hs le to the hypothesis tht type ietes is result of gene-environment (iet) interctions, ecuse the humn genome hs not chnge mrkely in such short time [-5]. However, wht the environmentl/ietry risk fctors re n how they function remin uncler. Nicotinmie, the mie of nicotinic ci, is the precursor for the coenzymes β-nicotinmie enine inucleotie (NAD) n nicotinmie enine inucleotie phosphte (NADP), which function in mny enzymectlyze oxition n reuction rections. Therefore, nicotinmie homeostsis is vitlly importnt for the oy []. In humns, excess nicotinmie is methylte, oxiize or hyroxylte to N -methylnicotinmie, nicotinmie-noxie or -hyroxynicotinmie, respectively, n then N - methylnicotinmie is further oxiize to the pyriones N - methyl--pyrione-5-croxmie (Py) n N -methyl-- pyrione--croxmie y lehye oxise (AOX; EC...). The mjor nicotinmie metolites in humn urine re Py n N -methylnicotinmie [7]. Although the nicotinmie metolism pthwy is well unerstoo, however, the influence of the ctolic efficiency of excess nicotinmie on the oy is not fully unerstoo. Since pellgr ws foun to e relte to vitmin B (nicin) eficiency in s, more ttention hs een pi to the prevention of nicin eficiency. As result, nicotinmie is use extensively s foo itive without hving unergone full forml sfety evlution [], n the chronic effects of nicotinmie overuse re fr from unerstoo []. Nicotinic ci n nicotinmie frequently re reporte to impir glucose tolernce n inuce insulin resistnce [-]. Recent evience suggests tht N - methylnicotinmie is involve in Prkinson s isese []. More importntly, the rupt increse in prevlence of type ietes in the Unite Sttes in the ltter hlf of the th century [] occurre roughly in prllel with the increse in per cpit nicin content []. A similr sitution occurre in Chin, in which foo enrichment with nicin egn in s, followe y shrp increse in the incience of ietes from % in to 5.5% in [5]. However, whether type ietes pthogenesis involves nicotinmie overlo is unetermine. The present stuy ime to compre nicotinmie metolic ptterns etween ietic n non-ietic MATERIALS AND METHODS Chemicls Nicotinmie ws purchse from Sigm (St. Louis, MO, USA). Nicotinmie tlets (5 mg/tlet) were purchse from Lisheng Phrm (Tinjin, Chin). N -methylnicotinmie ws purchse from Tke Chemicl Inustries (Osk, Jpn). Tmoxifen ws from Kunshn Snyou Phrmceuticl Ajuvnt Fctory (Kunshn, Chin). Olnzpine ws otine from Beijing Norhuns Chemicl Technology (Beijing, Chin). N - ethylnicotinmie n -Py were synthesize ccoring to the methos escrie respectively y Hirym et l [] n Holmn et l [7]. Nicotinmie lo test in humns This stuy ws pprove y the relevnt ethics committee, n ll the Chinese prticipnts gve informe consent. Fourteen type ietic ptients from eight fmilies with positive history (eight men n six women, men ge, 55. ±.; rnge, -75 yers) n ge- n sexmtche helthy volunteers without fmily history (men ge, 5. ±.; rnge, -75 yers) prticipte in this stuy. Type ietes ws efine s fsting glucose level 7. mmol/l or current receipt of hypoglycemic meiction. All sujects refrine from rugs, lcohol n cffeinte proucts for t lest h efore the stuy. After n overnight fst, urine ws collecte n quntitte h efore, n, n 5 h fter loing with mg nicotinmie. Venous loo ws collecte into soium citrte tues efore n 5 h fter nicotinmie loing, n seprte y centrifugtion (5 g, min). Aliquots of ech plsm n urine smple were plce irectly in liqui nitrogen n then trnsferre to - n -, respectively. Cumultive nicotinmie n N -methylnicotinmie experiments All niml experiments were conucte in ccornce with institutionl guielines. Mle Sprgue-Dwley rts (- g) were fe stnr rt chow. In nicotinmie experiment, rts were ivie rnomly into three groups (n = ech) n fste for h efore the experiment. In the two nicotinmie-trete groups, nicotinmie ( or mg/kg) ws ministere (intrperitonelly, ip) n repete every h for five oses. Glucose tolernce test ws performe y injection of glucose ( g/kg, ip) h fter the finl nicotinmie injection. Bloo glucose ws mesure h fter glucose injection. Bloo ws collecte

3 57 ISSN 7-7 CN -/R Worl J Gstroenterol Decemer 7, Volume 5 Numer 5 y eye lee into EDTA tues uner urethne nesthesi (.5 g/kg, ip) h fter glucose ministrtion. Plsm ws seprte y centrifugtion (5 g, min). After plsm collection, the uffy cot n top fifth reticulocyte-rich lyer of erythrocytes were iscre. Aliquots of plsm n erythrocytes, n hrveste smples of liver n gstrocnemius muscle were plunge irectly into liqui nitrogen n susequently store t - until ssy. The sme protocol ws use in the N -methylnicotinmie experiment, except tht the two trete groups repetely receive or mg/kg N -methylnicotinmie per injection n totl osge of.5 or g, respectively (ech group, n = ). AOX inhiition Rts in inhiitor-trete groups receive sucutneous tmoxifen (5 mg/kg) or olnzpine ( mg/kg) twice ily for, n rts in ech control group receive vehicle only (ech group, n = ). After n overnight fst, ll the rts receive nicotinmie ( mg/kg, ip). Plsm smples were collecte 5 h fter nicotinmie ministrtion s escrie ove. Chronic AOX inhiition Rts were ivie initilly into two groups: tmoxifentrete ( mg/kg per y, sucutneously, n = ) n vehicle-trete (control, n = ). Eight control n eight tmoxifen-trete rts were scrifice t the en of 7 wk. Liver smples were hrveste n store t - for western lotting. The reminer of tmoxifen-trete rts ws then ivie into two groups trete with tmoxifen ( mg/kg per y) with or without N -methylnicotinmie ( mg/kg per y, sucutneously), respectively. Two weeks fter the tretment, glucose tolernce test ws performe y injection of glucose ( g/kg, ip) fter n overnight fst. Bloo glucose ws mesure efore (fsting) n h fter glucose injection. Smples of plsm, liver n gstrocnemius muscle were hrveste uner urethne nesthesi (.5 g/kg, ip) h fter glucose injection. Therml injury Rts in the urn group (n = ) were given % totl oy surfce re, full-thickness scl urn uner ether nesthesi y immersion of the ck in 5 wter for 5 s, s previously escrie []. Shm rts (n = 7) were sujecte to n ienticl proceure, except tht they were immerse in 5 wter. All rts receive glucose ( g/kg, ip) h fter urning, with n overnight fst. Bloo glucose ws mesure efore (fsting) n h fter glucose osing. Smples of plsm, liver n gstrocnemius muscle were hrveste h fter glucose injection. Swet collection Five helthy young mle volunteers ge - yers prticipte in this stuy. After n overnight fst, whole oy swet ws collecte efore n, n h fter single orl ose of mg nicotinmie, y hving the volunteers sty in plstic g uring sun tretment (, 5 min), with stringent precutions to minimize evportive loss. Aliquots of swet were plce in liqui nitrogen, n trnsferre to - until nlysis. Assys of glucose, insulin n glycogen Bloo glucose ws mesure using glucometer (OneTouch Ultr; LifeScn Inc.). Plsm insulin ws mesure y rioimmunossy using commercil kits (Beijing North Institute of Biologicl Technology, Chin). Muscle n liver glycogen contents were etermine with Glycogen Assy Kits (Nnjing Jincheng Bioengineering Institute, Nnjing, Chin). HO ssy Bloo from helthy ult mle volunteers ws collecte in EDTA-contining tues n centrifuge (5 g, min). The plsm, uffy cot, n top fifth reticulocyte-rich lyer of erythrocytes were iscre, n the remining cells were wshe three times in isotonic sline, n centrifuge (5 g, min). After the superntnt ws iscre, the pcke erythrocytes were trnsferre to Erle s Blnce Slt Solution presturte with 5% O n 5% CO to mke n erythrocyte suspension ( μl pcke erythrocytes/ml). Two hunre microliters of erythrocyte suspension ws e to ech well of -well plte in the sence or presence of N -methylnicotinmie or Py t concentrtions of nmol/l to μmol/l, n incute for h t 7. HO concentrtions in the superntnt of cell cultures n in rt plsm were mesure using n HO Assy Kit (Beyotime Biotechnology, Jingsu, Chin). NAD/NADH ssy Humn erythrocyte suspension ( μl pcke erythrocytes/ml Erle s Blnce Slt Solution) ws incute in.5-ml Eppenorf tues with.5-mm hole in the cover, for h t 7 in the presence or sence of N -methylnicotinmie ( μmol/l). The tues were centrifuge (5 g, 5 min, ) n the superntnt ws iscre. Four hunre microliters of BioVision NAD/ NADH Extrction Buffer (Mountin View, CA, USA) ws e to ech tue for 5 min to lyse the cells. The lystes were ultrfiltere using BioVision -kd cut-off filters ( g, min, ). Assys were performe using the NAD + /NADH Quntifiction Kits ccoring to the mnufcturer s instructions (BioVision). For rt tissue NAD/NADH ssy, mg frozen liver, mg frozen muscle or μl pcke erythrocytes were homogenize in μl BioVision NAD/NADH Extrction Buffer. The homogente ws ultrfiltere using BioVision -kd cut-off filters ( g, min, ). The ssy ws conucte following the mnufcturer s instructions. Western lotting Western lotting nlysis of AOX ws performe ccoring to stnr protocols. Briefly, μg rt liver proteins were seprte on 5%-% SDS polycrylmie gels n trnsferre to polyvinyl ifluorie memrnes (Millipore, Befor, MA, USA). The memrnes were locke in PBS tht contine.% Tween- n 5% non-ft ry milk for

4 Zhou SS et l. Nicotinmie overlo n ietes 577 min t room temperture, n incute with ntioy to AOX (:5; BD Trnsuction Lortories, Lexington, KY, USA) overnight t. Then, the memrnes were wshe y PBS-Tween followe y h incution t room temperture with horserish peroxise (HRP)-conjugte seconry ntioy (:5; Snt Cruz Biotechnology, Snt Cruz, CA, USA) n etecte using the enhnce chemiluminescence (ECL) (Amershm Life Science). Determintion of N -methylnicotinmie, nicotinmie n Py N -methylnicotinmie, nicotinmie n Py were nlyze using n HPLC system tht consiste of n LC-A pump (Shimzu, Kyoto, Jpn), Rheoyne 775i smple injector with -μl smple loop (Rheoyne LLC, Rohnert Prk, CA, USA), Hypersil ODS C column (Thermo, Bellefonte, PA, USA), Wters 7 fluorescence etector (Milfor, MA, USA) for N -methylnicotinmie mesurements, n UV etector (Thermo Seprtions Proucts, Fremont, CA, USA) for Py etection. N -methylnicotinmie concentrtion ws nlyze y etecting its fluorescent,-nphthyriine erivtives ccoring to the metho of Musfel et l [], using nm excittion n nm emission wvelengths, with the moile phse ( mmol/l soium heptnesulfonte, 5 mmol/l triethylmine n % cetonitrile in wter, ph. with 5% HPO) t flow rte of ml/min. Nicotinmie ws quntitte y the sme proceure fter quntittive conversion of nicotinmie to N -methylnicotinmie using ioomethne, ccoring to the metho of Clrk []. For nlyzing urinry Py, 5 μl 5% HPO n.5 ml wter were e to ech urine smple (.5 ml). After rief vortex mixing, the smples were centrifuge ( g, min). The superntnt ws filtere through.5-μm filter for HPLC nlysis. Stnr solutions were prepre for clirtion, which contine,.,, n μg/ml of pure Py in norml urine. The moile phse (5% methnol, mmol/l KHPO, mmol/l triethylmine, ph. juste with 5% HPO) ws elivere t ml/min. UV etection ws performe t nm. Sttisticl nlysis The t re presente s mens ± SD. Sttisticl ifferences in the t were evlute y pire or unpire Stuent s t test, or ANOVA s pproprite, n were consiere significnt t P <.5. RESULTS Slow N -methylnicotinmie clernce is prominent feture of type ietes The 5-h totl urinry Py excretion fter mg nicotinmie lo in the ietic group ws significntly less thn tht in the non-ietic group (. ±.5 mg vs. ±. mg, P <.5, Figure A n C), ut urinry N -methylnicotinmie excretion increse in the ietic group (Figure B n D). The plsm N - methylnicotinmie level 5 h fter nicotinmie lo ws significntly higher in the ietic thn non-ietic group (Figure ). It shoul e note tht there were no sttisticl ifferences in the sl urinry excretions of Py n N -methylnicotinmie n the sl levels of plsm N -methylnicotinmie etween the two groups (Figures n ). These results suggest tht slow plsm N -methylnicotinmie clernce, which cn e revele y nicotinmie lo test, my e potentil iomrker of type ietes. High plsm N -methylnicotinmie inuces insulin resistnce We exmine the effect of nicotinmie overlo on rt glucose metolism. Rts trete with cumultive oses of nicotinmie ( g/kg) exhiite significntly higher levels of loo glucose n plsm insulin, ut significntly lower muscle glycogen content thn control rts fter glucose lo (Figure A). Another notle chnge fter nicotinmie ministrtion ws tht there ws mrke increse in plsm N -methylnicotinmie (Figure A), so we exmine the effects of N -methylnicotinmie. Cumultive oses of N -methylnicotinmie ( g/kg) h comprle effects to nicotinmie (Figure B), which suggeste tht the effects of nicotinmie overlo might hve een meite y N -methylnicotinmie. N -methylnicotinmie triggers oxitive stress Type ietes is ssocite with oxitive stress [,]. We therefore exmine whether nicotinmie overlo n high N -methylnicotinmie levels were implicte in oxitive stress. The results showe tht cumultive effects of nicotinmie ( g/kg) or N -methylnicotinmie ( g/kg) le to significnt increse in rt plsm levels of HO (Figure A n B), mjor rective oxygen species (ROS) n common inictor of oxitive stress []. Such n enhncing effect ws lso oserve in humn erythrocytes in vitro t physiologicl concentrtions of N - methylnicotinmie (Figure C), wheres Py, the en prouct of nicotinmie, i not hve n enhncing effect t the oserve concentrtions ( nmol/l to μmol/l) (t not shown). These results inicte tht high plsm N -methylnicotinmie my inuce systemic oxitive stress. Incresing evience hs inicte tht type ietes hs normlities in the NAD + /NADH reox couple []. We therefore investigte whether N -methylnicotinmie ffecte tissue NAD levels. Cumultive exposure to N - methylnicotinmie significntly reuce rt muscle n liver NAD (NAD + + NADH) contents (Figure D n E). Notly, the erythrocytes of rts trete with cumultive oses of N -methylnicotinmie ( g/kg) exhiite significnt increse in NADH n ecrese in NAD/ NADH rtio (Figure F). A similr effect ws oserve in humn erythrocytes in vitro (Figure G). These results suggest tht N -methylnicotinmie-inuce oxitive stress my originte from imlnce in the NAD + /NADH reox couple. AOX inhiition reuces N -methylnicotinmie clernce AOX is responsile for conversion of N -methylni-

5 57 ISSN 7-7 CN -/R Worl J Gstroenterol Decemer 7, Volume 5 Numer 5 A Before After nicotinmie lo -7 m 7- m - m - m C Nonietic Dietic Urine Py (mg) Urine collection time (m) B Before After nicotinmie lo -7 m 7- m - m - m D Urine NMN (mg) 5 5 Nonietic Dietic P < Urine collection time (m) Figure Urinry excretion ptterns of Py n N -methylnicotinmie in ietic n non-ietic sujects. A n B: Representtive HPLC chromtogrms of urinry excretions of Py (inicte y rrow) n N -methylnicotinmie (NMN) of non-ietic (A n B) n ietic (A n B) suject, efore n fter mg nicotinmie loing t 7: m Urine smples tken t given time were normlize to equl volumes efore HPLC nlysis; C n D: Summries of the results from the mesurements shown in A n B, respectively. Br grphs inicte men ± SD. A c B N -methylnicotinmie (mmol/l)..... Nonietic Dietic P <. Figure Plsm N -methylnicotinmie levels of ietic n non-ietic sujects fter nicotinmie loing. A: Representtive HPLC chromtogrms of plsm N -methylnicotinmie levels from non-ietic (, ) n ietic (c, ) suject efore (, c) n 5 h fter (, ) mg nicotinmie loing. : N -methylnicotinmie; : Internl stnr: N -methylnicotinmie; B: Summry of the results from the mesurements shown in A. Br grph inictes men ± SD... Before After cotinmie-to-py []. We exmine chnges in N - methylnicotinmie clernce fter rt AOX inhiition y the puttive inhiitors tmoxifen n olnzpine [], oth of which re known to impir glucose tolernce [,5]. The rts trete with tmoxifen ( mg/kg per y) or olnzpine ( mg/kg per y) for exhiite significntly higher plsm N -methylnicotinmie levels thn control rts 5 h fter mg/kg nicotinmie

6 Zhou SS et l. Nicotinmie overlo n ietes 57 A Bloo glucose (mmol/l) Glycogen (mg/g muscle) Insulin (pmol/l) 7 5 NMN (mmol/l) 5 Control.5 Control.5 Control.5 Control.5 NM (g) NM (g) NM (g) NM (g) B Bloo glucose (mmol/l) Glycogen (mg/g muscle) Insulin (pmol/l) NMN (mmol/l) 5 Control.5 Control.5 Control.5 Control.5 NMN (g) NMN (g) NMN (g) NMN (g) Figure Effects of nicotinmie n N -methylnicotinmie on glucose metolism of rts. A: Chnges in loo glucose, muscle glycogen, plsm insulin n plsm N -methylnicotinmie in rts trete with or without cumultive nicotinmie (.5 or g/kg) fter glucose loing; B: Comprle effects of cumultive N - methylnicotinmie (.5 or g/kg). NM: Nicotinmie; NMN: N -methylnicotinmie. P <.5 vs control, P <. vs control, P <. vs control. Br grphs show men ± SD. loing (Figure 5A n B). Moreover, chronic tmoxifen tretment for 7 wk significntly reuce rt liver AOX protein expression (P <.5, Figure 5C). To further explore the role of nicotinmie overlo in insulin resistnce, we use tmoxifen plus N -methylnicotinmie to mimic the conitions of reltively low AOX ctivity n excess nicotinmie intke. Rts trete with tmoxifen plus N -methylnicotinmie exhiite significntly higher loo glucose n much lower liver glycogen content thn those trete with tmoxifen lone fter glucose loing (Figure 5D). Skin involvement in N -methylnicotinmie-meite insulin resistnce If N -methylnicotinmie is inee involve in insulin resistnce, then ny fctors relte to N -methylnicotinmie etoxifiction n excretion my ffect insulin sensitivity. Of the fctors, skin my e of prticulr significnce ecuse it tkes prt in N -methylnicotinmie etoxifiction n excretion, respectively, through skin AOX [,7] n swet glns []. We thus explore the role of skin y nlyzing humn swet excretion of nicotinmie n N -methylnicotinmie fter nicotinmie loing, or y ssessing chnges in plsm N -methylnicotinmie clernce of rts with severe skin mge. Importntly, we foun tht nicotinmie concentrtions in the swet h fter mg nicotinmie loing ws 5.-fol higher thn tht in fsting swet, wheres N -methylnicotinmie concentrtions were not significntly ltere uner such conitions (Figure A n B). This inictes tht excess nicotinmie, without neeing ny conversion, is expelle through swet. Moreover, this stuy foun tht rts with severe skin mge h significnt elevtion in plsm N - methylnicotinmie ssocite with high loo glucose n plsm insulin levels, ut lower muscle glycogen content fter glucose loing (Figure C). DISCUSSION The min finings of this stuy were tht: () nicotinmie overlo elevtes plsm levels of N -methylnicotinmie ssocite with oxitive stress n insulin resistnce; () the skin plys n importnt role in expelling excess nicotinmie n etoxifying N -methylnicotinmie; n () ietic sujects hve slow plsm N -methylnicotinmie clernce. These finings my contriute to explin the mechnism of oxitive stress n insulin resistnce in vriety of clinicl conitions.

7 5 ISSN 7-7 CN -/R Worl J Gstroenterol Decemer 7, Volume 5 Numer 5 A HO (mmol/l) 5 B HO (mmol/l) 5 C HO (mmol/l) Control.5 Control.5 Control NM (g) NMN (g) NMN (log mol/l) D NAD (pmol/mg muscle) F NAD (nmol/ml RBC) NADH (nmol/ml RBC) NAD/NADH rtio E NAD (pmol/mg liver) 5 G NAD (nmol/ml RBC) NADH (nmol/ml RBC) NAD/NADH rtio Figure Effects of N -methylnicotinmie on HO genertion n NAD levels. A, B: Cumultive effects of nicotinmie (NM,.5 or g/kg) n N - methylnicotinmie (NMN,.5 or g/kg) on rt plsm HO levels; C: HO concentrtions in the superntnt of culture humn erythrocytes with or without h exposure to ifferent concentrtions of NMN. For ech concentrtion, n = ; D, E: NAD (NAD + n NADH) contents in muscle (D) n liver (E) of rts trete with sline (control) or cumultive ose of g/kg NMN; F: NAD n NADH contents n NAD/NADH rtio in the erythrocytes (RBCs) of rts with or without cumultive exposure to NMN; G: NAD n NADH contents, n NAD/NADH rtio in humn RBCs with (n = ) or without (control, n = ) h exposure to μmol/l NMN in vitro. P <.5 vs control, P <. vs control. Br grphs inicte men ± SD. N -methylnicotinmie is potent trigger of oxitive stress n insulin resistnce Insulin resistnce n oxitive stress re the mjor hllmrks of type ietes [,], ut the mechnisms unerlying the evelopment of systemic oxitive stress n insulin resistnce re uncler. Both nicotinic ci n nicotinmie hve een reporte to inuce insulin resistnce, which my le to elevtion of plsm insulin ue to β-cell compenstion [-]. Coincientlly, our t showe tht nicotinmie overlo inuce cute insulin resistnce in rts ssocite with high plsm levels of N -methylnicotinmie; the methyltion prouct of nicotinmie eing more toxic thn nicotinmie (> -fol) []. Importntly, ietic sujects exhiite significntly higher plsm N -methylnicotinmie levels thn non-ietic sujects fter nicotinmie loing, which suggests its involvement in the toxic effects of nicotinmie overlo. Inee, this stuy emonstrte tht N -methylnicotinmie mimicke the effect of nicotinmie overlo, which suggeste N - methylnicotinmie meition of the toxic effect. Incresing evience suggests tht insulin resistnce is ue to n unfvorle internl environment ecuse muscles resistnt to insulin, when culture in vitro, regin sensitivity to insulin [-]. Further evience revels tht systemic oxitive stress my e responsile for triggering insulin resistnce [,]. Consistent with previous reserch, this stuy foun tht N -methylnicotinmie not only elevte plsm HO levels in vivo, ut lso irectly stimulte HO genertion of humn erythrocytes in vitro t physiologicl concentrtions, which inictes tht N -methylnicotinmie is potent trigger of ietic oxitive stress. Oxitive stress my inuce NAD epletion, mrker of cell injury [,5]. Inee, this stuy foun tht N - methylnicotinmie-inuce high plsm HO level ws ssocite with significnt reuction in NAD content in

8 Zhou SS et l. Nicotinmie overlo n ietes 5 A 5 B C NMN (mmol/l) NMN (mmol/l) Control Tm AOX -ctin Control Tm Control Oln D Bloo glucose (mmol/l) Tm Tm + NMN Glycogen (mg/g liver) 5 Glycogen (mg/g muscle) 5 Insulin (pmol/l) FBG GTT ( h) Tm Tm + NMN Tm Tm + NMN Tm Tm + NMN Figure 5 Effects of lehye oxise (AOX) inhiitors on plsm N -methylnicotinmie levels n glucose metolism in rts. A n B: Plsm N - methylnicotinmie (NMN) levels 5 h fter nicotinmie lo ( mg/kg, ip) in rts trete with or without AOX inhiitors tmoxifen (Tm, A) or olnzpine (Oln, B) (ech group, n = ). P <.5 vs control, P <. vs control; C: Liver AOX expression in rts with or without 7 wk tmoxifen tretment. The lot is representtive of four inepenent experiments; D: Responses to glucose tolernce test in rts fter wk tretment with tmoxifen with or without NMN ( mg/kg per y) tretment in the lst wk. FBG: Fsting loo glucose; GTT ( h): Bloo glucose mesure h fter glucose tolernce test. P <. vs control. Br grph inictes men ± SD. A Before After nicotinmie lo h h h B Concentrtions (mmol/l) NM NMN. t /h C NMN (mmol/l) c Insulin (pmol/l) Glycogen (mg/g muscle) 7 5 c Glycogen (mg/g liver) 5 Glucose (mmol/l) 5 Shm Burn. Shm Burn Shm Burn Shm Burn Shm Burn FBG GTT Figure Role of skin in nicotinmie metolism n insulin resistnce. A: Representtive HPLC chromtogrms showing chnges of swet nicotinmie (NM) n N -methylnicotinmie (NMN) concentrtions in suject efore n, n h fter mg nicotinmie loing. n in A re NM n internl stnr N - ethylnicotinmie, respectively; n in A re NMN n internl stnr N -ethylnicotinmie, respectively; B: Summry of the mesurements shown in A. P <. vs control; C: Comprison of plsm NMN n insulin levels, muscle n liver glycogen contents, n loo glucose etween shm-urn (n = 7) n urn (n = ) rts fter glucose lo. FBG: Fsting loo glucose; GTT: Bloo glucose h fter glucose injection. Br grphs show men ± SD. c P <.5, P <. vs control.

9 5 ISSN 7-7 CN -/R Worl J Gstroenterol Decemer 7, Volume 5 Numer 5 Foo nicotinmie Foo nicotinmie Excess intke Plsm nicotinmie NAD NADP Plsm nicotinmie NAD NADP Plsm N -methylnicotinmie Plsm N -methylnicotinmie NAD/NADH rtio ROS Skin AOX AOX Skin AOX AOX Oxitive stress Swet gln Py Skin loss Col seson Seentry Swet gln Defect or inhiition Py Insulin resistnce Hyperglycemi Swet Urine Swet Urine Figure 7 Propose moel of the role of nicotinmie overlo in the evelopment of type ietes. Normlly, if nicotinmie intke is slightly more thn the oy nees, excess nicotinmie will e etoxifie rpily n eliminte minly vi the N -methylnicotinmie to Py pthwy, which involves liver n skin functions (left). Frequent excess nicotinmie intke, low N -methylnicotinmie etoxifiction, or swet gln inctivity inuces sustntil rise in plsm N - methylnicotinmie concentrtions n resience time fter ech mel, n consequently inuces oxitive stress n insulin resistnce (right). The chnge trens re inicte y re rrows or line thickness. the muscle n liver of rts. Then cme the vitl question: where i the excess systemic ROS originte? NADHepenent ROS genertion is n importnt source of intrcellulr ROS []. Accumulting evience hs inicte tht ietes shows ecrese cytosolic NAD + /NADH rtio in vriety of tissues []. Importntly, this stuy emonstrtes tht N -methylnicotinmie ecreses NAD/NADH rtio in rt erythrocytes in vivo n humn erythrocytes in vitro. Thus, it is likely tht ietic oxitive stress is initite y high plsm N -methylnicotinmie-inuce imlnce in the NAD + /NADH reox couple. Mny cellulr processes re governe y the enzymes using NAD + /NADH s cofctor [,,], therefore, it is not ifficult to unerstn why set of metolic normlities hppen in type ietes. Role of AOX in insulin resistnce Mmmlin AOX is molyo-flvo enzyme involve in the etoxifiction of vrious enogenous n exogenous N-heterocyclic compouns [,]. N -methylnicotinmie is one of the sustrtes of AOX y which N -methylnicotinmie is oxiize to pyriones [7], n thus, etoxifie. AOX is expresse preominntly in the liver. Therefore, severe liver isese might e expecte to reuce plsm N -methylnicotinmie clernce n susequent insulin resistnce. In fct, it is well known tht liver cirrhosis is ssocite with high incience of ietes [,7]. Pumpo et l [] hve foun tht cirrhotic ptients hve high serum N - methylnicotinmie levels, oth in sl vlues n fter nicotinmie loing. Moreover, non-lcoholic stetoheptitis, the most prevlent liver isese in the western worl, typiclly is ssocite with the metolic synrome tht is chrcterize y insulin resistnce, ietes n hypertension []. Tmoxifen, well-known inucer of nonlcoholic stetoheptitis [], is foun to inhiit strongly AOX ctivity [] n its expression (Figure 5C). Collectively, it seems tht the ecrese in N -methylnicotinmie etoxifiction my e involve in heptogenic insulin resistnce. AOX is lso expresse in the skin [], which suggests skin involvement in N -methylnicotinmie etoxifiction. Moreover, s foun in this stuy, humn swet glns cn excrete excess nicotinmie. Therefore, ecrese skin function my e implicte in N -methylnicotinmieinuce insulin resistnce. In fct, severe urns my inuce long-lsting insulin resistnce, well-ocumente ut poorly unerstoo phenomenon [,]. The present stuy emonstrte tht severe urns significntly elye N -methylnicotinmie clernce, which suggests tht long-lsting post-urn insulin resistnce my involve ecrese in nicotinmie etoxifiction n excretion. Reltionship etween lifestyle risk fctors n nicotinmie overlo Environmentl n lifestyle risk fctors my trigger type ietes [,]. From our stuy, it emerges tht the risk fctors my involve nicotinmie overlo. Firstly, excess nicotinmie my le to high plsm N -methylnicotinmie, with susequent oxitive stress n insulin resistnce. In response to insulin resistnce, the pncretic β cells hve to increse insulin secretion (hyperinsulinemi) compenstorily, which my eventully le to β-cell filure n loo sugr levels eing out of control. The result tht tmoxifen-inuce AOX inhiition plus N -methylnicotinmie impire rt glucose tolernce (Figure 5D) implies tht excess nicotinmie intke my e more hrmful to those with low N -methylnicotinmie clernce. Seconly, ietry risk fctors my e relte to nicotinmie contents in foos. For exmple, met, which is rich in nicotinmie, increses the risk of type ietes [,]. Moreover, foo fortifiction with nicin my ply role in nicotinmie overlo. If compring the epiemic of type

10 Zhou SS et l. Nicotinmie overlo n ietes 5 ietes in the Unite Sttes [] with the history of foo fortifiction [], one cn clerly see tht the tren for rpi increse in the incience of ietes hs occurre in prllel with the tren in mntory nicin-fortifiction-inuce increse in the per cpit nicin consumption in the ltter hlf of the th century. These fcts my explin why the western ietry pttern, chrcterize y high intke of met n nicin-fortifie foos, confers such high risk of type ietes. Thirly, seentry lifestyle risk fctor my involve swet gln inctivity. Swet gln ctivity fluctutes ccoring to mient temperture; the most significnt feture of the gln. Therefore, swet gln inctivity is expecte to slow nicotinmie ctolism n therey increse the nger of eveloping insulin resistnce. In fct, the col seson is known to worsen glucose metolism [,], wheres exercising sufficiently to swet my reuce ietic incience []. Therefore, it is likely tht seentry lifestyle risk fctors my e t lest prtilly ue to swet gln inctivity y ir-conitione working/living environments. It shoul e note tht lrge oses of nicotinic ci n nicotinmie my inuce liver mge [5,], therefore, long-term investigtion my e necessry to etermine the reltionship etween chronic nicotinmie overlo n non-lcoholic stetoheptitis. Historiclly, the epiemic of pellgr hs een restricte minly to those who hve performe hevy inustril lor with poor nutrient supply []. Hence, the present stuy gives rise to n importnt socil n pulic helth issue s to whether foos nee to e fortifie with nicin (nicotinmie or nicotinic ci), when the people in evelope countries hve lrey een living in n ge of over-nutrition n swet gln inctivity. In summry, it ppers tht gene-environment (iet) interctions my e reflection, to some extent, of the outcome of comintion of nicotinmie overlo n reltively low N -methylnicotinmie etoxifiction n excretion. As summrize in Figure 7, the pthogenesis of type ietes my e t lest prtilly ue to longterm excess nicotinmie intke, n/or slowness in N - methylnicotinmie etoxifiction, n/or ecrese in excess nicotinmie n N -methylnicotinmie excretion. This my le to high plsm N -methylnicotinmie levels, n susequently oxitive stress n insulin resistnce. Therefore, reucing nicotinmie intke n fcilitting excretion of nicotinmie metolites my e useful preventive n therpeutic intervention in type ietes. COMMENTS Bckgroun Type ietes generlly is ccepte to e result of gene-environment interction, lthough the unerlying mechnism is not cler. Of the environmentl fctors, iet ppers to ply mjor role. In fct, the shrp increses in the incience of ietes in the Unite Sttes in the ltter hlf of the th century n in Chin in the pst two eces of the th century followe foo fortifiction with nicin (i.e. nicotinmie n nicotinic ci) eginning in the erly s in the Unite Sttes n in the erly s in Chin. Moreover, nicin is reporte frequently to impir glucose metolism n cuse liver injury. Thus, there is the possiility tht the high prevlence of type ietes in these countries in the pst fewer eces my involve nicin toxicity. Reserch frontiers Type ietes is ssocite with increse systemic oxitive stress, fctor responsile for the evelopment of insulin resistnce. How the systemic oxitive stress occurs is mjor issue in type ietes. Innovtions n rekthroughs The present stuy emonstrte tht the pthogenesis of type ietes my involve norml nicotinmie metolism. Fctors tht inuce nicotinmie overlo n/or ecrese in the etoxifiction n excretion of nicotinmie metolites my le to systemic oxitive stress n insulin resistnce. The fctors my inclue the frequent use of foos rich in nicotinmie n/or fortifie with nicin, congenitl enzymtic efects, liver iseses, n swet gln loss or inctivity. The present stuy suggests tht gene-environment interctions my reflect the outcome of increse nicotinmie intke n ecrese in its etoxifiction n excretion. Applictions Reucing nicotinmie intke n fcilitting the excretion of excess nicotinmie my e useful preventive n therpeutic intervention in type ietes. Peer review This is n interesting stuy with humn n experimentl t, which investigte cliniclly relevnt issue, n gve n insight into the pthogenic mechnisms involve. The experiments were performe well n re presente well in the pper. REFERENCES Meigs JB, Lrson MG, Fox CS, Keney JF Jr, Vsn RS, Benjmin EJ. Assocition of oxitive stress, insulin resistnce, n ietes risk phenotypes: the Frminghm Offspring Stuy. Dietes Cre 7; : 5-55 Prvicini TM, Touyz RM. NADPH oxises, rective oxygen species, n hypertension: clinicl implictions n therpeutic possiilities. Dietes Cre ; Suppl : S7-S Anressi MG. Metolic synrome, ietes n therosclerosis: influence of gene-environment interction. 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