Research Article Vitamin B6 Prevents Endothelial Dysfunction...

Research ArticleVitamin B6 Prevents Endothelial DysfunctionInsulin Resistance and Hepatic Lipid Accumulation inApoeminusminus Mice Fed with High-Fat Diet

Zhan Liu1 Peng Li2 Zhi-Hong Zhao1 Yu Zhang1 Zhi-Min Ma3 and Shuang-Xi Wang24

1Department of Clinical Nutrition and Gastroenterology The First Affiliated Hospital (Peoplersquos Hospital of Hunan Province)Hunan Normal University Changsha 430070 China2College of Pharmacy Xinxiang Medical University Xinxiang 453003 China3Division of Endocrinology The Second Affiliated Hospital Soochow University Suzhou 215000 China4The Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education andChinese Ministry of Health Qilu Hospital School of Medicine Shandong University Jinan 250012 China

Correspondence should be addressed to Shuang-Xi Wang shuangxiwangsdueducn

Received 16 July 2015 Revised 8 October 2015 Accepted 21 October 2015

Academic Editor Hiroshi Okamoto

Copyright copy 2016 Zhan Liu et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Backgrounds VitB6 deficiency has been associated with a number of adverse health effects However the effects of VitB6 inmetabolic syndrome are poorly understoodMethodsVitB6 (50mgkgday) was given to119860119901119900119890minusminus mice with hkdigh-fat diet (HFD)for 8 weeks Endothelial dysfunction insulin resistance and hepatic lipid contents were determined ResultsVitB6 administrationremarkably increased acetylcholine-induced endothelium-dependent relaxation and decreased random blood glucose level in119860119901119900119890minusminus mice fed with HFD In addition VitB6 improved the tolerance of glucose and insulin normalized the histopathology

of liver and reduced hepatic lipid accumulation but did not affect the liver functions Clinical and biochemical analysis indicatedthat the levels of VitB6 were decreased in patients with fatty liverConclusionsVitamin B6 prevents endothelial dysfunction insulinresistance andhepatic lipid accumulation in119860119901119900119890minusminusmice fedwithHFD Supplementation ofVitB6 should be considered to preventmetabolic syndrome

1 Introduction

Vitamin B6 (VitB6) includes pyridoxal pyridoxine and pyri-doxamine which function as essential cofactors for enzymesinvolved in variousmetabolic activities which include aminoacid fat and glucose metabolism [1] The phosphate esterderivative pyridoxal 51015840-phosphate (PLP) is the biologicallyactive form of this vitamin and reflects long-term bodystorage [2] Studies have shown that low plasma PLP concen-trations are associated with increased risk of cardiovasculardisease (CVD) [3 4]

Nutrient overload is associated with high incidence ofchronic metabolic diseases including obesity insulin resis-tance and type 2 diabetes [5] Prolonged exposure to high

concentrations of saturated fatty acids leads to oxidativestress and endoplasmic reticulum stress which may impairinsulin signaling [6] Moreover supplementation of a high-fat diet (HFD) with branched-chain amino acids causedinsulin resistance as a part of metabolic syndrome [7]Metabolic syndrome is associated with a risk of CVD andis a common early abnormality in the development of type2 diabetes In patients with nonalcoholic fatty liver disease(NAFLD) metabolic abnormalities have been reported in33 to 100 of cases [8] Patients presenting with NAFLDneed to be examined for the presence of the components ofthe metabolic syndrome and their complications [9] We alsopreviously reported that apoptosis of liver cells contributes toliver dysfunction [10]

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2016 Article ID 1748065 8 pageshttpdxdoiorg10115520161748065

2 Journal of Diabetes Research

The identification of the link between VitB6 and meta-bolic syndrome including insulin and NAFLD might helpto define novel nutritional and pharmacological approachesfor the treatment of diabetes obesity and insulin resistanceHere we reported that administration of VitB6 preventsendothelial dysfunction insulin resistance and hepatic lipidaccumulation in 119860119901119900119890minusminus mice fed with high-fat diet Clini-cally deficiency of VitB6 should be considered as a high riskfactor of NAFLD

2 Materials and Methods

21 Materials Human recombinant insulin was purchasedfrom Sigma-Aldrich (St Louis MO) Antibodies againstAkt (pAkt) GLUT4 glycogen synthase kinase-3120573 (GSK3)forkhead box protein O (FOXO) and GAPDH were pur-chased from Santa Cruz Biotechnology (Dallas TX)The sec-ondary antibodies were obtained from Jackson ImmunoRe-search Laboratories (West Grove PA) VitB6 acetylcholine(ACh) sodiumnitroprusside (SNP) and phenylephrine werefrom Sigma-Aldrich Company All drug concentrations areexpressed as final working concentrations in the buffer

22 Animals and Experimental Protocols Male 119860119901119900119890minusminusmice were purchased from Hua-Fu-Kang Animal Company(Beijing China) All animals were housed in temperature-controlled cageswith a 12-hour light-dark cycle and given freeaccess to water and normal chow This study was carried outin strict accordance with the recommendations in the Guidefor the Care and Use of Laboratory Animals of the NationalInstitutes of Health

Model of hyperlipidemia was induced by feeding micewithHFD containing 021 cholesterol and 21 fat (ResearchDiets Inc D12079B)This diet was administered at 6 weeks ofage and continued for 8 consecutive weeks At 6 weeks of ageVitB6 (50mgkgday) was also added to the drinking waterfor 8 weeks The animal protocol was reviewed and approvedby the Animal Care and Use Committee of Hunan NormalUniversity

23 Determinations of Serum Lipid Profiles and Liver Func-tions Blood was sampled from mice for determinationof total bilirubin (TB) aspartate aminotransferase (AST)alanine aminotransferase (ALT) alkaline phosphatase (ALP)and albumin (ALB) Serum levels of TB AST ALT AP andALBwere determined by commercial kits (Nanjing JianchengBiology Company Nanjing China)

24 Organ Chamber In vivo or ex vivo organ chamber studywas performed as described previously [11] Mice were sacri-ficed under anesthesia by intravenous injection with pento-barbital sodium (30mgkg)The descending aorta isolated byremoving the adhering perivascular tissue carefully was cutinto rings (2-3mm in length) Aortic rings were suspendedandmounted to organ chamber by using two stainless hooksThe rings were placed in organ baths filled with Krebs bufferof the following compositions (in mM) NaCl 1183 KCl47 MgSO

4

06 KH2

PO4

12 CaCl2

25 NaHCO3

250

EDTA 0026 pH 74 at 37∘C and they were gassed with95 O

2

plus 5 CO2

under tension of 10 g for 90-minuteequilibration period During this period the Krebs solutionwas changed every 15min After the equilibration aorticrings were challenged with 60mM KCl After washing andanother 30-minute equilibration period contractile responsewas elicited by phenylephrine (1 120583M) At the plateau ofcontraction accumulative ACh (001 003 01 03 1 and3 120583M) or SNP (001 003 01 03 1 3 and 10120583M) was addedto induce the relaxation

25 Glucose Tolerance Test (GTT) and Insulin Tolerance Test(ITT) As described previously [12] glucose (20 gkg) wasgiven tomice (ip) after an overnight fast Blood glucose (BG)levels were then measured at indicated times with a portableglucosemeter (LifeScanMilpitas CA) after tail snipping ForITT mice were injected with insulin (055 IUkg ip) after 6-hour fast BG levels were measured at indicated times witha portable glucose meter (LifeScan Milpitas CA) after tailsnipping

26 HE or Oil Red O Staining Histological specimens weretaken at the end of the study period for all mouse groupsas described previously [13] For each mouse liver segmentswere fixed in 4 buffered formaldehyde and embeddedin paraffin for histological analysis Sections (5120583m) werestained with either hematoxylin or eosin Degree of severityof liver fibrosis was derived from blind analysis of eachof the animals in each group To determine hepatic lipidaccumulation frozen liver sections were stained with 05Oil Red O for 10min washed and counterstained withMayerrsquos hematoxylin for 45 sec Data for Oil Red O stainingwere presented as the mean percentage of stained area to atotal hepatic region in 10 fields from each liver section Quan-titative analysis was performedusing analySIS-FIVEprogram(Olympus Soft Imaging System Munster Germany)

27 Western Blotting The protocol for western blot wasdescribed as previously with some modifications [14] Livertissues were homogenized and the protein content in super-natant was assayed by BCA protein assay reagent (PierceUSA) 20 120583g proteins were loaded to SDS-PAGE and thentransferred to membrane Membrane was incubated with1 1000 dilution of primary antibody followed by 1 2000dilution of horseradish peroxides-conjugated secondary anti-body Protein bands were visualized by ECL (GE HealthcareUSA) The intensity (area times density) of the individual bandson western blots was measured by densitometry (model GS-700 Imaging Densitometer Bio-Rad) The background wassubtracted from the calculated area The average of densityfor the bands in control group is considered as 100

28 Measurement of Cholesterol and Triglyceride Contentsin Liver Lipids in mouse liver were extracted as describedby Folch et al [15 16] Cholesterol and triglyceride levelsin extracted lipids were measured enzymatically using thereagents from Cayman Chemical (Ann Arbor MI) accordingto the manufacturerrsquos instruction

Journal of Diabetes Research 3

ControlVitB6

lowast

lowast

lowast

lowast

100

80

60

40

20

0Re

laxa

tion

()

minus8 minus6 minus5 minus4minus7

ACh (log M)

(a)

ControlVitB6

120

100

80

60

40

20

0

Rela

xatio

n (

)


SNP (log M)

(b)

Figure 1 Administration of VitB6 prevents endothelial dysfunction in 119860119901119900119890minusminus mice fed with high-fat diet Male 119860119901119900119890minusminus mice at the age of6 weeks received high-fat diet and VitB6 (50mgkgday) administration in drinking water for 8 weeks At the end of experiments mice weresacrificed under anaesthesiaThe descending aortas were isolated and cut into rings (a) ACh-induced endothelium-dependent relaxation and(b) SNP-induced endothelium-independent relaxation were determined by organ chamber as described in Section 2 All data were expressedas mean plusmn SEM119873 is 10ndash15 in each group lowast119875 lt 005 versus control

29 Statistical Analysis The results were expressed asmean plusmnSEM One-way ANOVA followed by 119905-test was used for twogroupsrsquo comparison 119875 lt 005 was considered significant

3 Results

31 VitB6 Prevents Endothelial Dysfunction in Apoeminusminus MiceFed with HFD Endothelial dysfunction has been identifiedas an early hallmark of CVD such as atherosclerosis andhypertension [17ndash20] We firstly determined whether VitB6prevents endothelial dysfunction in mice with metabolicsyndromes The hyperlipidemia model was induced by feed-ing 119860119901119900119890minusminus mice with HFD [21] As indicated in Table 1HFD in 119860119901119900119890minusminus mice dramatically increased serum levels oftriglyceride cholesterol and LDL indicating that the modelis successfully established Importantly the random level ofblood sugar was also increased in 119860119901119900119890minusminus mice fed withHFD However treatment of these mice with VitB6 did notalter the levels of triglyceride cholesterol and LDL exceptfor random level of blood glucose

The endothelial function was determined by using AChAs shown in Figure 1(a) ACh-induced vasorelaxation wassignificantly improved byVitB6The SNP-induced vasorelax-ation was not affected by VitB6 (Figure 1(b)) demonstratingthat the protective effects of VitB6 on vascular function arelimited to endothelium

32 VitB6 Enhances Insulin Sensitivity in Apoeminusminus Mice Fedwith HFD Insulin resistance is a high risk factor of endothe-lial dysfunction in CVD [22] We next examined whether

Table 1 Serum sugar and lipid levels in Apoeminusminus mice

WT Apoeminusminus

ND ND HFD HFD + VitB6Glucose (mM) 65 plusmn 13 82 plusmn 13 135 plusmn 08 102 plusmn 09lowast

Cholesterol (mM) 37 plusmn 05 105 plusmn 21 284 plusmn 53 264 plusmn 49Triglyceride (mM) 07 plusmn 02 08 plusmn 02 16 plusmn 03 15 plusmn 05HDL-C (mgL) 128 plusmn 15 252 plusmn 30 267 plusmn 38 257 plusmn 39LDL-C (mgL) 109 plusmn 14 249 plusmn 23 417 plusmn 53 435 plusmn 67After 8-week administration of VitB6 inApoeminusminus mice fed with high-fat dietserum sugar levels and lipid levels were determined WT wild-type NDnormal diet HFD high-fat diet HDL high density lipoprotein LDL lowdensity lipoprotein All data were expressed as mean plusmn SEM 119873 is 10ndash15 ineach group lowast119875 lt 005 versus Apoeminusminus mice fed with HFD

VitB6 improves insulin sensitivity in HFD-fed119860119901119900119890minusminus miceAs shown in Figure 2(a) injection of D-glucose dramaticallyincreased the levels of blood glucose (BG) in HFD-fed119860119901119900119890minusminus mice The peak level of BG was about 600mgdL

after 30 minutes The level of BG was back to the basal levelafter 90 minutes However administration of VitB6 delayedand lowered the peak levels of BG (470mgdL) After 90minutes of glucose injection the level of BG was also backto the basal level These data indicate that VitB6 increases thetolerance of glucose

The protective effect of VitB6 on glucose metabolism wasfurther confirmed by measuring the sensitivity of insulin(Figure 2(b)) By injecting exogenous insulin into HFD-fed119860119901119900119890minusminus mice the levels of BG were reduced to 40 of basal

level at the 60th minute and then went back to 80 of basal


ControlVitB6

lowast

lowast

lowast

0

100

200

300

400

500

600

700Bl

ood

gluc

ose (

mg

dL)

20 40 60 80 100 1200Time (min)

(a)

ControlVitB6

lowast

lowast lowast

lowast

0

20

40

60

80

100

120

Basa

l glu

cose

()

20 40 60 80 100 1200Time (min)

(b)

pAkt

GAPDH

GLUT4

GSK3

Control VitB6

FOXO

(c)

Figure 2 VitB6 improves insulin resistance in 119860119901119900119890minusminus mice fed with high-fat diet Male 119860119901119900119890minusminus mice at the age of 6 weeks receivedhigh-fat diet and VitB6 (50mgkgday) administration in drinking water At the 8th weekend after VitB6 treatment (a) GTT and (b) ITTwere evaluated as described in Section 2 All data were expressed as mean plusmn SEM 119873 is 10ndash15 in each group lowast119875 lt 005 versus control (c)Homogenates of liver tissues were subjected to perform western blotting analysis to assay the levels of pAKt GLUT4 GSK3 and FOXOThepicture is a representative blot from 10ndash15 mice

level at the 120thminute However VitB6 further reduced thelevel of BG at the 90thminute to 25 After the 120thminutethe level of BG was 55 of the basal level Collectively thissuggests that VitB6 enhances insulin sensitivity in mice

33 Increased Hepatic Levels of pAkt GSK3 and GLUT4Proteins and Decreased FOXO Protein Expression in VitB6-Treated Apoeminusminus Mice The beneficial effects of VitB6 oninsulin resistance were further examined by assaying thehepatic levels of pAkt GSK3 GLUT4 and FOXO which areproteins related to glucose metabolism [23] As depicted inFigure 2(c) compared to HFD-fed 119860119901119900119890minusminus mice the levels

of pAkt GSK3 GLUT4 and BG were increased and the levelof FOXO was reduced in HFD-fed 119860119901119900119890minusminus mice with VitB6further supporting the notion that VitB6 improves insulinresistance in mice

34 VitB6 Treatment Prevents Hepatic Lipid Accumulationin Mice NAFLD is characterized by insulin resistance [24]Thus we detected the liver function in these mice In Table 2the markers of liver function such as ALB ALP ALT ASTand TB were comparable in HFD-fed 119860119901119900119890minusminus mice with orwithout VitB6 treatment Histological analysis of HE stainingin liver sections from mice at the end of the experiment


Table 2 The indexes for liver function in mice

Control VitB6AST (IUL) 1322 plusmn 279 1574 plusmn 318ALT (IUL) 1867 plusmn 213 1957 plusmn 286TB (mgdL) 013 plusmn 007 015 plusmn 009ALP (IUL) 2555 plusmn 328 2798 plusmn 358ALB (gL) 172 plusmn 14 206 plusmn 168After 8-week administration of VitB6 inApoeminusminus mice fed with high-fat dietserum levels of AST ALT TB ALP and ALB were determined All data wereexpressed as mean plusmn SEM119873 is 10ndash15 in each group

Table 3The levels of serumVitB6 homocysteine folate andVitB12in patients with fatty liver

Control (57) Patients (49)VitB6 (PLP nM) 558 plusmn 107 239 plusmn 81lowast

VitB12 (pgmL) 6867 plusmn 213 5187 plusmn 286lowast

folic acid (ngmL) 83 plusmn 07 77 plusmn 09Homocysteine (nM) 158 plusmn 28 228 plusmn 54lowast

Serum levels of VitB6 VitB12 folic acid and homocysteine were determinedin patients with fatty liver and control subjects All data were expressed asmean plusmn SEM lowast119875 lt 005 versus control

(Figure 3(a)) revealed that HFD caused marked neurosis andfibrosis which was reversed by VitB6 treatment suggestingthat VitB6 is effective to protect the liver

The typical feature of NAFLD is the elevated hepatic lipidaccumulation [25 26] We next investigated whether VitB6prevents hepatic lipid accumulation in hyperlipidemia miceby Oil Red O staining (Figure 3(a)) Compared to controlHFD-fed 119860119901119900119890minusminus mice the contents of liver triglycerides(Figure 3(b)) and cholesterol (Figure 3(c)) were decreaseddemonstrating that VitB6 prevents hepatic lipid accumu-lation in mice and is potentially considered to serve asprevention of NAFLD

35 Plasmatic Lower Levels of VitB6 in Patients with FattyLiver Finally in order to establish the clinical associationbetween VitB6 deficiency andNAFLD we performed clinicaland biochemical analysis As described in Table 3 fifty-sevenhealthy humans and forty-nine patients had a clinical andbiochemical analysis completed in the study Compared tothe healthy human subjects the levels of folic acid weresimilar in patients with fatty liver The levels of VitB12 werelightly increased However the levels of homocysteine inNAFLD patients were significantly increased consistent withother reports [27 28] Most importantly we found that thelevels of VitB6 were lower in NAFLD than control healthyhumansThese results indicate that deficiency of VitB6mightbe a risk factor of NAFLD clinically

4 Discussion

In the present study we provide the first evidence thatadministration of VitB6 prevents endothelial dysfunctioninsulin resistance and hepatic lipid accumulation in119860119901119900119890minusminusmice fedwithHFD in vivo Clinically the serum level ofVitB6

is low in patients with NAFLD Our data not only indicatethat VitB6 protects endothelial function and improves insulinresistance but also imply that lowVitB6 statusmight be a riskfactor of NAFLD as a component of metabolic syndrome

The major discovery in the present study is that VitB6produces several beneficial effects to prevent metabolic syn-drome such as insulin resistance and NAFLD TraditionallyVitB6 in the form of PLP is the coenzyme of 5 enzymes inthese metabolic pathways cystathionine-120573-synthase (CBS)cystathionine-120574-lyase (CGL) cytoplasmic andmitochondrialserine hydroxymethyltransferase (cSHMT and mSHMT)and glycine decarboxylase (GDC) in the mitochondria [29]In this way VitB6 regulates the transsulfuration pathwaywhich contributes to homocysteine regulation and providescysteine synthesis and consists of sequential reactions cat-alyzed by CBS and CGL CBS catalyzes the condensationof homocysteine and serine to form cystathionine in areaction that is subject to positive allosteric regulation byS-adenosylmethionine (SAM) whereas CGL catalyzes thecleavage of cystathionine to yield 120572-ketobutyrate ammoniaand cysteine Because both CBS and CGL require PLP as acoenzyme inadequate VitB6 status might lead to impairedregulation of cellular homocysteine concentration Highlevels of homocysteine impair endothelial function and causemetabolic syndrome including insulin resistance and lipidaccumulation in liver HHCY might play a role in thepathogenesis of vascular disorders and is considered as anindependent risk factor for atherosclerosis [30] From ourobservations supplementation of VitB6 should be a helpfultherapy to improve endothelial dysfunction and metabolicsyndrome Of course the mechanism of VitB6 in preventionof metabolic syndrome needs further investigations

We also identified VitB6 deficiency as a new risk factorof NAFLD Obesity metabolic syndrome and type 2 diabetesmellitus are strictly related and are key pathogenetic factors ofNAFLD the most frequent liver disease worldwide NAFLDis a clinicopathological syndrome including a wide spectrumof liver damage instances ranging from hepatic steatosis tononalcoholic steatohepatitis (NASH) to cirrhosis [31] Epi-demiologic studies showed that low VitB6 nutritional statusis associated with increased risk of CVD venous thrombosisstroke and possibly colon cancer [32] Although a connectionbetween VitB6 status and lipid metabolism has appearedperiodically for more than 80 years there is no evidence tosupport the role of PLP in NAFLD To our knowledge thisis the first study to investigate whether marginal VitB6 defi-ciency affects hepatic lipid accumulation in human adultsWeobserved a significant decrease of plasma PLP concentrationin patients with NAFLD A potential mechanism responsiblefor the observations of lower plasma VitB6 level linking toNAFLD is impairment of PUFA interconversion because ithas been reported that marginal VitB6 deficiency decreasesplasma (n-3) and (n-6) PUFA concentrations in healthy menand women [33] Further investigation should focus on thedirect target of VitB6 on regulation of lipid metabolism inliver

A limitation of this study is that 119860119901119900119890minusminus mouse is suit-able for studying atherosclerosis resulting from hypercholes-terolemia Additionally this mouse has several intriguing


VitB

6C

ontro

l

HE Oil Red O

(a)

Control VitB6

lowast

0

08

16

24

Live

r trig

lyce

ride c

onte

nt (m

gg)

(b)Control VitB6

lowast

0

3

6

9

Live

r cho

leste

rol c

onte

nt (m

gg)

(c)

Figure 3 VitB6 reduces hepatic lipid accumulation in119860119901119900119890minusminus mice fed with high-fat diet Male119860119901119900119890minusminus mice at the age of 6 weeks receivedhigh-fat diet and VitB6 (50mgkgday) administration in drinking water At the end of experiments mice were sacrificed under anaesthesia(a)Histological analysis of liver tissue byHEorOil RedO staining (b and c) Liver lipidswere extracted andhepatic triglyceride and cholesterollevels were assayed using a commercial kit The quantitative data were expressed as mean plusmn SEM119873 is 10ndash15 in each group lowast119875 lt 005 versusControl

characteristics First 119860119901119900119890minusminus mice show obesity-resistantphenotype resulting in remarkable insulin sensitivity Sec-ond this mouse has hepatic steatosis due to impairmentof VLDL secretion from liver Third this mouse basicallypossesses endothelial dysfunction damaged from excess beta

lipoprotein It would be better to investigate the metaboliceffects of vitamin B6 on wild-type mice with diet-inducedmetabolic disorders such as C57B16 strain

In summary the results of this study have shown that lowVitB6 status has substantial effects on metabolism including


glucose and fatty acid The results of this study also demon-strate that the deficiency of VitB6 might be a risk factor ofNAFLD

Conflict of Interests

The authors confirm that there is no conflict of interests

Authorsrsquo Contribution

Zhan Liu and Peng Li designed and performed all exper-iments analyzed the data and wrote the paper Zhi-HongZhao Yu Zhang and Zhi-Min Ma collected the clinical sam-ples Zhan Liu and Shuang-Xi Wang conceived the projectand wrote the paper Zhan Liu and Peng Li contributedequally to this work

Acknowledgments

Thisworkwas supported byNational 973 Basic Research Pro-gram of China (2013CB530700) andNational Natural ScienceFoundation of China (81570723 81470591 and 81370411)Thisproject was also sponsored by Program for New CenturyExcellent Talents inUniversity (NCET-13-0351) the ScientificResearch Foundation for the Returned Overseas ChineseScholars State Education Ministry and Program of ClinicalInvestigation (Nanshan Group) Qilu Hospital ShandongUniversity (2014QLKY15) Shuang-Xi Wang is a recipient ofQilu Professional Scholar of Shandong University

References

[1] D B Coursin ldquoPresent status of vitamin B6 metabolismrdquo TheAmerican Journal of Clinical Nutrition vol 9 pp 304ndash314 1961

[2] X Jouven M-A Charles M Desnos and P DucimetiereldquoCirculating nonesterified fatty acid level as a predictive riskfactor for sudden death in the populationrdquo Circulation vol 104no 7 pp 756ndash761 2001

[3] V Lotto S-W Choi and S Friso ldquoVitamin B6 a challenginglink between nutrition and inflammation in CVDrdquo The BritishJournal of Nutrition vol 106 no 2 pp 183ndash195 2011

[4] E B Rimm W C Willett F B Hu et al ldquoFolate and vitaminB6 from diet and supplements in relation to risk of coronaryheart disease among womenrdquo Journal of the American MedicalAssociation vol 279 no 5 pp 359ndash364 1998

[5] L Lionetti M P Mollica A Lombardi G Cavaliere GGifuni and A Barletta ldquoFrom chronic overnutrition to insulinresistance the role of fat-storing capacity and inflammationrdquoNutrition Metabolism and Cardiovascular Diseases vol 19 no2 pp 146ndash152 2009

[6] M Cnop ldquoFatty acids and glucolipotoxicity in the pathogenesisof Type 2 diabetesrdquoBiochemical Society Transactions vol 36 no3 pp 348ndash352 2008

[7] C B Newgard J An J R Bain et al ldquoA branched-chainamino acid-relatedmetabolic signature that differentiates obeseand lean humans and contributes to insulin resistancerdquo CellMetabolism vol 9 no 4 pp 311ndash326 2009

[8] N Alkhouri C Carter-Kent and A E Feldstein ldquoApoptosisin nonalcoholic fatty liver disease diagnostic and therapeuticimplicationsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 5 no 2 pp 201ndash212 2011

[9] M Li C M Reynolds S A Segovia C Gray and M HVickers ldquoDevelopmental programming of nonalcoholic fattyliver disease the effect of early life nutrition on susceptibilityand disease severity in later liferdquo BioMed Research Internationalvol 2015 Article ID 437107 12 pages 2015

[10] Z Liu S Wang H Zhou Y Yang and M Zhang ldquoNa+H+exchanger mediates TNF-120572-induced hepatocyte apoptosis viathe calpain-dependent degradation of Bcl-xLrdquo Journal of Gas-troenterology and Hepatology vol 24 no 5 pp 879ndash885 2009

[11] X-H Yang P Li Y-L Yin et al ldquoRosiglitazone via PPAR120574-dependent suppression of oxidative stress attenuates endothelialdysfunction in rats fed homocysteine thiolactonerdquo Journal ofCellular and Molecular Medicine vol 19 pp 826ndash835 2015

[12] T-Y Xu L-L Guo PWang et al ldquoChronic exposure to nicotineenhances insulin sensitivity through 1205727 nicotinic acetylcholinereceptor-STAT3 pathwayrdquo PLoS ONE vol 7 no 12 Article IDe51217 2012

[13] S Wang C Zhang M Zhang et al ldquoActivation of AMP-activated protein kinase alpha2 by nicotine instigates forma-tion of abdominal aortic aneurysms in mice in vivordquo NatureMedicine vol 18 no 6 pp 902ndash910 2012

[14] S Wang B Liang B Viollet and M-H Zou ldquoInhibition of theAMP-activated protein kinase-1205722 accentuates agonist-inducedvascular smoothmuscle contraction and high blood pressure inmicerdquo Hypertension vol 57 no 5 pp 1010ndash1017 2011

[15] H Li Q Min C Ouyang et al ldquoAMPK activation pre-vents excess nutrient-induced hepatic lipid accumulation byinhibitingmTORC1 signaling and endoplasmic reticulum stressresponserdquo Biochimica et Biophysica ActamdashMolecular Basis ofDisease vol 1842 no 9 pp 1844ndash1854 2014

[16] J Folch M Lees and G H Sloane Stanley ldquoA simple methodfor the isolation and purification of total lipides from animaltissuesrdquo The Journal of Biological Chemistry vol 226 no 1 pp497ndash509 1957

[17] S Wang Q Peng J Zhang and L Liu ldquoNa+H+ exchanger isrequired for hyperglycaemia-induced endothelial dysfunctionvia calcium-dependent calpainrdquo Cardiovascular Research vol80 no 2 pp 255ndash262 2008

[18] S Wang M Zhang B Liang et al ldquoAMPK1205722 Deletion causesaberrant expression and activation of NAD(P)H Oxidase andconsequent endothelial dysfunction in vivo role of 26S protea-somesrdquo Circulation Research vol 106 no 6 pp 1117ndash1128 2010

[19] S Wang J Xu P Song et al ldquoAcute inhibition of guano-sine triphosphate cyclohydrolase 1 uncouples endothelial nitricoxide synthase and elevates blood pressurerdquo Hypertension vol52 no 3 pp 484ndash490 2008

[20] S Wang J Xu P Song B Viollet and M-H Zou ldquoIn vivoactivation of AMP-activated protein kinase attenuates diabetes-enhanced degradation of GTP cyclohydrolase Irdquo Diabetes vol58 no 8 pp 1893ndash1901 2009

[21] A K Leamy R A Egnatchik and J D Young ldquoMolecularmechanisms and the role of saturated fatty acids in the pro-gression of non-alcoholic fatty liver diseaserdquo Progress in LipidResearch vol 52 no 1 pp 165ndash174 2013

[22] M Iantorno U Campia N Di Daniele et al ldquoObesity inflam-mation and endothelial dysfunctionrdquo Journal of BiologicalRegulators and Homeostatic Agents vol 28 no 2 pp 169ndash1762014

[23] M Yuan E Pino L Wu M Kacergis and A A Soukas ldquoIden-tification of Akt-independent regulation of hepatic lipogenesisby mammalian target of rapamycin (mTOR) complex 2rdquo The


Journal of Biological Chemistry vol 287 no 35 pp 29579ndash29588 2012

[24] T Khoury A Ben Yarsquoacov Y Shabat L Zolotarovya R Snirand Y Ilan ldquoAltered distribution of regulatory lymphocytes byoral administration of soy-extracts exerts a hepatoprotectiveeffect alleviating immune mediated liver injury non-alcoholicsteatohepatitis and insulin resistancerdquo World Journal of Gas-troenterology vol 21 no 24 pp 7443ndash7456 2015

[25] H Malhi and G J Gores ldquoMolecular mechanisms of lipotoxic-ity in nonalcoholic fatty liver diseaserdquo Seminars in Liver Diseasevol 28 no 4 pp 360ndash369 2008

[26] J Wang C Zhang Z Zhang et al ldquoBL153 partially preventshigh-fat diet induced liver damage probably via inhibitionof lipid accumulation inflammation and oxidative stressrdquoOxidative Medicine and Cellular Longevity vol 2014 Article ID674690 10 pages 2014

[27] S C R de Carvalho M T C Muniz M D V Siqueira etal ldquoPlasmatic higher levels of homocysteine in non-alcoholicfatty liver disease (NAFLD)rdquo Nutrition Journal vol 12 article37 2013

[28] E Bravo S Palleschi P Aspichueta et al ldquoHigh fat diet-induced non alcoholic fatty liver disease in rats is associatedwith hyperhomocysteinemia caused by down regulation of thetranssulphuration pathwayrdquo Lipids in Health and Disease vol10 article 60 2011

[29] H F Nijhout J F Gregory C Fitzpatrick et al ldquoAmathematicalmodel gives insights into the effects of vitaminB-6 deficiency on1-carbon and glutathione metabolismrdquo Journal of Nutrition vol139 no 4 pp 784ndash791 2009

[30] K S McCully ldquoVascular pathology of homocysteinemia impli-cations for the pathogenesis of arteriosclerosisrdquo The AmericanJournal of Pathology vol 56 no 1 pp 111ndash128 1969

[31] H Azzam and S Malnick ldquoNon-alcoholic fatty liver diseasemdashthe heart of the matterrdquoWorld Journal of Hepatology vol 7 pp1369ndash1376 2015

[32] C P Lima S R Davis A DMackey J B Scheer J Williamsonand J F Gregory III ldquoVitamin B-6 deficiency suppressesthe hepatic transsulfuration pathway but increases glutathioneconcentration in rats fed AIN-76A or AIN-93G dietsrdquo TheJournal of Nutrition vol 136 no 8 pp 2141ndash2147 2006

[33] M Zhao Y Lamers M A Ralat et al ldquoMarginal vitamin B-6 deciency decreases plasma (n-3) and (n-6) PUFA concentra-tions in healthy men and womenrdquoThe Journal of Nutrition vol142 no 10 pp 1791ndash1797 2012

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Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The identification of the link between VitB6 and meta-bolic syndrome including insulin and NAFLD might helpto define novel nutritional and pharmacological approachesfor the treatment of diabetes obesity and insulin resistanceHere we reported that administration of VitB6 preventsendothelial dysfunction insulin resistance and hepatic lipidaccumulation in 119860119901119900119890minusminus mice fed with high-fat diet Clini-cally deficiency of VitB6 should be considered as a high riskfactor of NAFLD

2 Materials and Methods

21 Materials Human recombinant insulin was purchasedfrom Sigma-Aldrich (St Louis MO) Antibodies againstAkt (pAkt) GLUT4 glycogen synthase kinase-3120573 (GSK3)forkhead box protein O (FOXO) and GAPDH were pur-chased from Santa Cruz Biotechnology (Dallas TX)The sec-ondary antibodies were obtained from Jackson ImmunoRe-search Laboratories (West Grove PA) VitB6 acetylcholine(ACh) sodiumnitroprusside (SNP) and phenylephrine werefrom Sigma-Aldrich Company All drug concentrations areexpressed as final working concentrations in the buffer

22 Animals and Experimental Protocols Male 119860119901119900119890minusminusmice were purchased from Hua-Fu-Kang Animal Company(Beijing China) All animals were housed in temperature-controlled cageswith a 12-hour light-dark cycle and given freeaccess to water and normal chow This study was carried outin strict accordance with the recommendations in the Guidefor the Care and Use of Laboratory Animals of the NationalInstitutes of Health

Model of hyperlipidemia was induced by feeding micewithHFD containing 021 cholesterol and 21 fat (ResearchDiets Inc D12079B)This diet was administered at 6 weeks ofage and continued for 8 consecutive weeks At 6 weeks of ageVitB6 (50mgkgday) was also added to the drinking waterfor 8 weeks The animal protocol was reviewed and approvedby the Animal Care and Use Committee of Hunan NormalUniversity

23 Determinations of Serum Lipid Profiles and Liver Func-tions Blood was sampled from mice for determinationof total bilirubin (TB) aspartate aminotransferase (AST)alanine aminotransferase (ALT) alkaline phosphatase (ALP)and albumin (ALB) Serum levels of TB AST ALT AP andALBwere determined by commercial kits (Nanjing JianchengBiology Company Nanjing China)

24 Organ Chamber In vivo or ex vivo organ chamber studywas performed as described previously [11] Mice were sacri-ficed under anesthesia by intravenous injection with pento-barbital sodium (30mgkg)The descending aorta isolated byremoving the adhering perivascular tissue carefully was cutinto rings (2-3mm in length) Aortic rings were suspendedandmounted to organ chamber by using two stainless hooksThe rings were placed in organ baths filled with Krebs bufferof the following compositions (in mM) NaCl 1183 KCl47 MgSO

4

06 KH2

PO4

12 CaCl2

25 NaHCO3

250

EDTA 0026 pH 74 at 37∘C and they were gassed with95 O

2

plus 5 CO2

under tension of 10 g for 90-minuteequilibration period During this period the Krebs solutionwas changed every 15min After the equilibration aorticrings were challenged with 60mM KCl After washing andanother 30-minute equilibration period contractile responsewas elicited by phenylephrine (1 120583M) At the plateau ofcontraction accumulative ACh (001 003 01 03 1 and3 120583M) or SNP (001 003 01 03 1 3 and 10120583M) was addedto induce the relaxation

25 Glucose Tolerance Test (GTT) and Insulin Tolerance Test(ITT) As described previously [12] glucose (20 gkg) wasgiven tomice (ip) after an overnight fast Blood glucose (BG)levels were then measured at indicated times with a portableglucosemeter (LifeScanMilpitas CA) after tail snipping ForITT mice were injected with insulin (055 IUkg ip) after 6-hour fast BG levels were measured at indicated times witha portable glucose meter (LifeScan Milpitas CA) after tailsnipping

26 HE or Oil Red O Staining Histological specimens weretaken at the end of the study period for all mouse groupsas described previously [13] For each mouse liver segmentswere fixed in 4 buffered formaldehyde and embeddedin paraffin for histological analysis Sections (5120583m) werestained with either hematoxylin or eosin Degree of severityof liver fibrosis was derived from blind analysis of eachof the animals in each group To determine hepatic lipidaccumulation frozen liver sections were stained with 05Oil Red O for 10min washed and counterstained withMayerrsquos hematoxylin for 45 sec Data for Oil Red O stainingwere presented as the mean percentage of stained area to atotal hepatic region in 10 fields from each liver section Quan-titative analysis was performedusing analySIS-FIVEprogram(Olympus Soft Imaging System Munster Germany)

27 Western Blotting The protocol for western blot wasdescribed as previously with some modifications [14] Livertissues were homogenized and the protein content in super-natant was assayed by BCA protein assay reagent (PierceUSA) 20 120583g proteins were loaded to SDS-PAGE and thentransferred to membrane Membrane was incubated with1 1000 dilution of primary antibody followed by 1 2000dilution of horseradish peroxides-conjugated secondary anti-body Protein bands were visualized by ECL (GE HealthcareUSA) The intensity (area times density) of the individual bandson western blots was measured by densitometry (model GS-700 Imaging Densitometer Bio-Rad) The background wassubtracted from the calculated area The average of densityfor the bands in control group is considered as 100

28 Measurement of Cholesterol and Triglyceride Contentsin Liver Lipids in mouse liver were extracted as describedby Folch et al [15 16] Cholesterol and triglyceride levelsin extracted lipids were measured enzymatically using thereagents from Cayman Chemical (Ann Arbor MI) accordingto the manufacturerrsquos instruction


ControlVitB6

lowast

lowast

lowast

lowast

100

80

60

40

20

0Re

laxa

tion

()


ACh (log M)

(a)

ControlVitB6

120

100

80

60

40

20

0

Rela

xatio

n (

)


SNP (log M)

(b)



3 Results





WT Apoeminusminus








ControlVitB6

lowast

lowast

lowast

0

100

200

300

400

500

600

700Bl

ood

gluc

ose (

mg

dL)

20 40 60 80 100 1200Time (min)

(a)

ControlVitB6

lowast

lowast lowast

lowast

0

20

40

60

80

100

120

Basa

l glu

cose

()

20 40 60 80 100 1200Time (min)

(b)

pAkt

GAPDH

GLUT4

GSK3

Control VitB6

FOXO

(c)

















4 Discussion







VitB

6C

ontro

l

HE Oil Red O

(a)

Control VitB6

lowast

0

08

16

24

Live

r trig

lyce

ride c

onte

nt (m

gg)

(b)Control VitB6

lowast

0

3

6

9

Live

r cho

leste

rol c

onte

nt (m

gg)

(c)











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlVitB6

lowast

lowast

lowast

lowast

100

80

60

40

20

0Re

laxa

tion

()


ACh (log M)

(a)

ControlVitB6

120

100

80

60

40

20

0

Rela

xatio

n (

)


SNP (log M)

(b)



3 Results





WT Apoeminusminus








ControlVitB6

lowast

lowast

lowast

0

100

200

300

400

500

600

700Bl

ood

gluc

ose (

mg

dL)

20 40 60 80 100 1200Time (min)

(a)

ControlVitB6

lowast

lowast lowast

lowast

0

20

40

60

80

100

120

Basa

l glu

cose

()

20 40 60 80 100 1200Time (min)

(b)

pAkt

GAPDH

GLUT4

GSK3

Control VitB6

FOXO

(c)

















4 Discussion







VitB

6C

ontro

l

HE Oil Red O

(a)

Control VitB6

lowast

0

08

16

24

Live

r trig

lyce

ride c

onte

nt (m

gg)

(b)Control VitB6

lowast

0

3

6

9

Live

r cho

leste

rol c

onte

nt (m

gg)

(c)











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlVitB6

lowast

lowast

lowast

0

100

200

300

400

500

600

700Bl

ood

gluc

ose (

mg

dL)

20 40 60 80 100 1200Time (min)

(a)

ControlVitB6

lowast

lowast lowast

lowast

0

20

40

60

80

100

120

Basa

l glu

cose

()

20 40 60 80 100 1200Time (min)

(b)

pAkt

GAPDH

GLUT4

GSK3

Control VitB6

FOXO

(c)

















4 Discussion







VitB

6C

ontro

l

HE Oil Red O

(a)

Control VitB6

lowast

0

08

16

24

Live

r trig

lyce

ride c

onte

nt (m

gg)

(b)Control VitB6

lowast

0

3

6

9

Live

r cho

leste

rol c

onte

nt (m

gg)

(c)











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























4 Discussion







VitB

6C

ontro

l

HE Oil Red O

(a)

Control VitB6

lowast

0

08

16

24

Live

r trig

lyce

ride c

onte

nt (m

gg)

(b)Control VitB6

lowast

0

3

6

9

Live

r cho

leste

rol c

onte

nt (m

gg)

(c)











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VitB

6C

ontro

l

HE Oil Red O

(a)

Control VitB6

lowast

0

08

16

24

Live

r trig

lyce

ride c

onte

nt (m

gg)

(b)Control VitB6

lowast

0

3

6

9

Live

r cho

leste

rol c

onte

nt (m

gg)

(c)











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Vitamin B6 Prevents Endothelial Dysfunction...

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Transcript of Research Article Vitamin B6 Prevents Endothelial Dysfunction...