The Protective Effect of Antioxidants Consumption on Diabetes and Vascular Complications

51 Pages·2016·3.37 MB·English

diseases
Review
The Protective Effect of Antioxidants Consumption
on Diabetes and Vascular Complications
StéphanieDalandSéverineSigrist*
DIATHECEA7294UMRCentreEuropéend’EtudeduDiabète(CeeD),UniversitédeStrasbourg(UdS),
boulevardRenéLeriche,Strasbourg67200,France;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:MaurizioBattino
Received:30March2016;Accepted:23June2016;Published:11July2016
Abstract: Obesity and diabetes is generally accompanied by a chronic state of oxidative stress,
disequilibriumintheredoxbalance,implicatedinthedevelopmentandprogressionofcomplications
such as micro- and macro-angiopathies. Disorders in the inner layer of blood vessels, the
endothelium, play an early and critical role in the development of these complications. Blunted
endothelium-dependentrelaxationand/orcontractionsarequietlyassociatedtooxidativestress.
Thus,preservingendothelialfunctionandoxidativestressseemstobeanoptimizationstrategyin
thepreventionofvascularcomplicationsassociatedwithdiabetes. Dietisamajorlifestylefactor
thatcangreatlyinfluencetheincidenceandtheprogressionoftype2diabetesandcardiovascular
complications. Thenotionthatfoodsnotonlyprovidebasicnutritionbutcanalsopreventdiseases
andensuregoodhealthandlongevityisnowattainedgreaterprominence. Somedietaryandlifestyle
modificationsassociatedtoantioxidativesupplycouldbeaneffectiveprophylacticmeanstofight
against oxidative stress in diabesity and complications. A significant benefit of phytochemicals
(polyphenolsinwine,grape,teas),vitamins(ascorbate,tocopherol),minerals(selenium,magnesium),
andfruitsandvegetablesinfoodsisthoughttobecapableofscavengingfreeradicals,loweringthe
incidenceofchronicdiseases. Inthisreview,wediscusstheroleofoxidativestressindiabetesand
complications,highlighttheendothelialdysfunction,andexaminetheimpactofantioxidantfoods,
plants,fruits,andvegetables,currentlyusedmedicationwithantioxidantproperties,inrelationto
thedevelopmentandprogressionofdiabetesandcardiovascularcomplications.
Keywords: diabetes;complications;oxidativestress;antioxidants;plants;prevention
1. Introduction
Today,WHOandIDF(InternationalDiabetesFederation)drawsattentiontothesimilarityoftrends
inobesityanddiabetesintheWorld. Theterm“diabesity”iscommonlyusedtodaytodescribethis
epidemicorpandemicwithexponentialdramaticgrowthobservedinallcountries[1]. Ourchangeof
lifestyletoasedentaryattitudeandmassiveindustrializationwithaccessfromanearlyagetofood
andbeveragesrichinenergy, fat, sugar, oracombinationthereofispartlythecauseofmillionsof
obeseanddiabeticpeople[2]. Despitetechnicalandtechnologicalprogressaccompanyingtherapeutic
arsenalavailableandpublichealthplans, wefailtodaytostoptheprogressionofdiabetesandits
complications. Infact,diabetesisasilentandsneakydisease. Therefore,itisassociatedwithmany
complications. Cardiovasculardiseasesarethemajorcauseofdeathanddisabilityamongdiabetic
people[3],particularlyforwomanwhohavelostcardiovascularprotectionaffordedbytheclassically
femalesex. Diabeticvascularcomplicationsareanimportantpathologicalissueindiabetesthatleads
tothefurtherfunctionaldeteriorationofseveralorgansandcausedmicro-andmacro-angiopathy[4].
Endothelialdysfunction,thelossofabalancebetweenvasodilatorsandvasoconstrictorsfactorsinthe
bloodvessels,haslargelybeenassociatedinseveralregionsofthevasculatureinT2D[5].
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Diseases2016,4,24 2of51
A common point of all these cardio-metabolic disorders is the appearance of oxidative stress.
Oxidativestressisduetoanimbalancebetweenantioxidants(enzymes,vitamins,proteins,etc.) and
pro-oxidants(UVradiations,alcohol,smoking,etc.)[6]. Oxidativestressalongwithchroniclow-grade
inflammation may initiate changes in cardiovascular structure and function such as endothelial
dysfunction, cardiac hypertrophy, cardiac fibrosis, and ventricular contractile dysfunction [7].
Manystudieshaveshownthatdiabeticpatientsundergochronicoxidativestress,particularlydueto
hyperglycemia[8,9]. Thus,astrategyfocusonbothoxidativestressandendothelialfunctioncould
helptopreventordelaytheonsetofvascular-relatedtype2diabetescomplications.
Muchevidenceshowsthatconsumptionofnaturalsourcesubstancesconferschemopreventive
and cytoprotectant activities. In fact, epidemiological studies suggest that consumption of fruits,
vegetables[10–14],andplants[15]maybeassociatedwithareducedriskofdiabetesoraprotective
effect [16]. Some observations have also revealed an inverse relationship between the risk of
cardiovascularmortalityormorbidityandtheconsumptionofpolyphenol-richproducts(redwine,
cocoa and tea) [17–20]. Their consumption brings several exogenous antioxidants and vitamins,
increasingtheantioxidantstatusoftheorganism,inadditiontotheirdirecteffectonbloodvesselsand
inparticularontheendothelium[21].
Many plants are also used for their benefits in traditional medicines. Some of them are at
theoriginofthedevelopmentofdrugs[16]suchasbiguanide,metformin,antidiabeticdrugs,and
Galegaofficinalis. Indevelopedcountries,traditional,complementary,andalternativemedicinesare
becomingincreasinglypopularandarecommonlyusedtotreatorpreventchronicdiseasesandare
improvingqualityoflife[15].
Therefore,wewillseethroughthisreviewthatmanycompoundssurroundinguscanbeareal
asset in the prevention of “diabesity” but also a valuable aid in addition to current treatments to
preventtheoccurrenceofsuchcomplications. Wewillalsodiscusstheappealfortheuseofsingle
moleculestothedetrimentoftotalextracts,therebypromotingmolecularsynergy.
2. DiabesityandCardiovascularComplications
2.1. TheEvolutionofObesityandDiabetes
Developedsocietiesfacetwocrucialhealthproblems: overweightandobesity. Obesityisthe
most common metabolic disease, and the number of individuals who are overweight or obese is
fast increasing worldwide [22]. Overweight and obesity are defined as abnormal or excessive fat
accumulationthatmayimpairhealth. Bodymassindex(BMI)isasimpleindexofweight-for-height
thatiscommonlyusedtoclassifyoverweightandobesityinadults. Itisdefinedasaperson'sweight
inkilogramsdividedbythesquareofhisheightinmeters(kg/m2). BMIiswidelyusedasameasure
ofweightstatusanddiseaseriskandiswidelyusedforroutinecharacterizationofweightstatusin
epidemiology,clinicalnutrition,andresearch. Moreover,fatmassandfat-freemassasassessedby
validatedtechniques(densitometry,dualimpedanceanalysis,etc.) arealsocurrentlyused. Thus,the
termobesityinourreviewisusedinabroadsensethatincludesBMI,fatmass,%bodyfat,etc.
Obesityhasmorethandoubledsince1980. Theprevalenceofoverweightandobeseyouthhas
increaseddramaticallyoverthepastthreedecades[23]. In2014,morethan1.9billionadults,18years
andolder,wereoverweight. Oftheseover600millionwereobese. 39%ofadultsaged18yearsand
overwereoverweightin2014,and13%wereobese. Mostoftheworld’spopulationlivesincountries
whereoverweightandobesitykillsmorepeoplethandoesunderweight.
Overweightandobesityhavereachedepidemicproportionsgloballyalongwithanadoptionofa
Westernizedlifestylecharacterizedbyacombinationofexcessivefoodintakeandinadequatephysical
activity. Raised BMI is a major risk factor for noncommunicable diseases such as cardiovascular
diseases(mainlyheartdiseaseandstroke),whichweretheleadingcauseofdeath.
The dramatic rise in the prevalence of obesity and changes in lifestyle-related factors such as
areductioninphysicalactivityhavebeenaccompaniedbyalarmingincreasesintheincidenceand
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prevalenceoftype2diabetes[24]. Diabetesisachronicdiseasethatoccurseitherwhenthepancreas
does not produce enough insulin or when the body cannot effectively use the insulin it produces.
Insulin is a hormone that regulates blood sugar [25]. Hyperglycemia, or raised blood sugar, is a
commoneffectofuncontrolleddiabetesand,overtime,leadstoseriousdamagetomanyofthebody's
systems,especiallythenervesandbloodvessels. In2014,9%ofadults18yearsandolderhaddiabetes.
In2012,diabeteswasthedirectcauseof1.5milliondeaths. Morethan80%ofdiabetesdeathsoccurin
low-andmiddle-incomecountries.
Epidemiologicalstudieshaveconfirmedastrongpositiveassociationbetweenexcessadiposity
andriskofdevelopingtype2diabetes. BasedonthedatafromtheBehavioralRiskFactorsSurveillance
SystemconductedbytheUnitedStates,Mokdadetal.[26]estimatedthat,foreverykilogramincrement
inself-reportedbodyweight,theriskofdiabetesincreasesbyabout9%. Theterm“diabesity”hasbeen
coinedtoillustratethecloserelationshipbetweenobesityanddiabetes[27,28].
2.2. Lifestyle
Lifestylehabitshavedeterioratedovertimewithincreasesinobesity,centralobesity,anddiabetes
andstagnatingratesofpersistentsmoking. Anincreaseinobesityanddiabeteshasparalleledthe
growth of urbanization and globalization in the region. For example, in China, the prevalence
ratesofdiabetesinlargeprovincialcapitalcitiesrangefromahighof8%(intheEasternregion)to
4.6%(inthelowestintheWesternregion)[29]. Behavioralriskfactorsincludetobaccouse,alcohol
consumption,unhealthydiet,andphysicalinactivity. Physicalinactivityisthe4thmortalityriskfactor
formortality[30]withanincreaseof20–30%ofdeathcomparedwithpeoplewhopractice30minof
exerciseaday[31].
Finally,advancesinagricultureandfoodsystems,consequentincreasesinfoodavailability,anda
shiftindietaryconsumptionpatternswitheconomicdevelopmentandurbanizationofdeveloping
societiespromotesoverweightandobesity. This“new”dietfavorsconsumptionoffats, saturated
fatslargelyfromanimalsourcesandsugars. Theessenceofthesechangesiscapturedbytheterm
“nutrition transition” which accompanies the demographic and epidemiologic transition in these
countrieswitheconomicdevelopment[32].
Epidemiologicalstudiesindicatethatweightloss,evenmoderate,canimproveinsulinsensitivity,
improveinsulinaction,anddecreasetheriskofdevelopingtype2diabetes. Improvementsininsulin
actionafteranaverageof10%weightreductionwerelostwithweightregainbutlargelypreserved
withweightmaintenance[33].
Physicalactivityisassociatedwithasignificantreductionintheriskoftype2diabetes,whereasa
sedentarylifestyleisassociatedwithanincreasedrisk[34,35]. Thereisa20%increasedriskofdiabetes
foreach2-hdailyincrementinwatchingtelevision[36].However,somestudieshavedemonstratedthe
feasibilityandefficiencyoflifestyleinterventionprogramsinthepreventionofdiabetesinindividuals
withimpairedglucosetolerance[37,38]. Thelifestyleinterventionprogrampermitsareductionin
weightwithmoderateexerciseandacontrolledfoodintake(reductionoffat,increaseinfiber,and
frequentconsumptionoffruits,vegetables,etc.)
2.3. DiabeticComplications: LinkwithOxidativeStressandInflammation
Chronichyperglycemia,disturbancesofcarbohydrate,andlipidandproteinmetabolismlead
to metabolic derangements in diabetes and various complications including both macro- and
microvascular dysfunctions [22]. Over time, diabetes can damage the heart, blood vessels, eyes,
kidneys, and nerves. The incidence of cardiovascular diseases in people with diabetes, one of the
majorcomplications,isthreetofourtimesthatinnon-diabeticindividuals. Inamultinationalstudy,
50%ofpeoplewithdiabetesdieofcardiovasculardisease(primarilyheartdiseaseandstroke)[39],
with a twofold increase in risk of heart failure in male patients, and a fivefold increase in female
patients[40]. Likewise,diabetesincreasedincidenceofcoronaryarterydiseaseandatherosclerotic
lesionsatayoungerage,oftenassociatedwithmultivesseldiseaseandinvolvementofdistalcoronary
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segments. Hypertension is also commonly found in both type 1 and type 2 diabetes [41]. Finally,
diabetescancausedistinctpathologicalterationsinthemyocardium,independentofitseffectonblood
pressureandcoronaryatherosclerosis,termed“diabeticcardiomyopathy”(DMC)[42]. Wewillfocus
oncardiovascularcomplicationsbelowinthisreview.
Combinedwithreducedbloodflow,neuropathy(nervedamage)inthefeetincreasesthechance
offootulcers,infection,andtheeventualneedforlimbamputationandaffectsalmost30%to50%
ofpatientswithdiabetes. Onepercentofglobalblindnessisattributedtodiabeticretinopathy[43]
duetoalong-termaccumulateddamagetothesmallbloodvesselsintheretina,andtheoverallrisk
ofdyingamongpeoplewithdiabetesisatleastdoubletheriskoftheirpeerswithoutdiabetes[44].
Anotherimportantmicrovascularcomplicationsisdiabeticnephropathy,ofwhichthereisaninefold
higherriskinpatientswithdiabetes,leadingtoend-stagerenaldiseaserequiringchronicdialysisand
transplantation[23].
Oxidative stress has been suggested to be a common pathway for the pathogenesis of
complicationsindiabetes[24,25]. Forexample,(1)theproductionofhydrogenperoxidebymesangial
cellsandlipidperoxidation,activationofproteinkinaseC(PKC),mitogen-activatedprotein(MAP)
kinases,andcytokineproductionleadtorenalinjury[26];(2)theredox-sensitivenucleartranscriptional
factor, NFκB, accumulation of advanced-glycation end-products (AGEs) localized in sub-retinal
membranes, and microvessels are activated earlier in the course of diabetic retinopathy [23,27] in
addition to polyol accumulation and glycation associated to cataract [28]; (3) AGEs inhibit axonal
regeneration[29],anincreaseinDNAdamageandthestimulationofthePKCpathway,andNFκB
andTGF-βincreasedepositionoftheextracellularmatrix[30], andallmechanismshaveinvolved
inneuropathy. Moreover,HbA1c,abiomarkeroftheoverallglycemicexposure,isthemostknown
diabeticparameterlinktooxidativestress.Infact,itisduetotheglycationofhemoglobin.Theincrease
inHb1AcvariabilitypredictstheriskofmicrovascularcomplicationsinT1D[31–33]andtheriskof
nephropathyandcardiovasculardiseasesinT2D[34–36].
In addition to oxidative stress, inflammation stands out as a determinant process in the
developmentofdiabeticcomplications[37]. Itisdifficult,infact,tounderstandtheimpactofthese
factorswithouteachother,sincenumerousinterplaysexistbetweeninflammationandoxidativestress
and vice versa [38,39]. Hyperglycemia increases the levels of pro-inflammatory proteins [37], and
infiltratedmacrophagessecreteinflammatorycytokines(correlatewithahigherbodymassindex:
IL-6,IL-8,MCP-1[43]),therebyleadingtoalocalandsystemicinflammation. Increasedproductionof
TNF-αhasalsobeenwidelyassociatedwithobesity-relatedinsulinresistanceandabnormalvascular
reactivity,thevasculaturebeinganimportanttargetofTNF-α[44]andcloselylinkedtodiabeticmicro-
andmacro-complications[40,45].
3. OxidativeStressandCardiovascularComplications
Theconceptthatoxygen,whichisessentialtolife,couldbecausingcelldamageandinvolved
inmanydiseases,wasdiscoveredinrecentyears. Today,manyepidemiologicalandclinicalstudies
stronglysuggesttheinvolvementofreactiveoxygenspecies(ROS)inthegenesisandevolutionof
chronicdiseases,includingdiabetesanditscomplications[7](Figure1).Chronichyperglycemiacaused
amajoroxidativestress[22],andYubero-Serranoetal.[46]recentlyproposedSODactivityasthemost
relevantoxidativestressbiomarkerinpatientssufferingfrommetabolicsyndrome. Itcouldbeusedas
apredictivetooltodeterminethedegreeoftheunderlyingoxidativestressinthispathology.
Diseases2016,4,24 5of51
Diseases 2016, 4, 24 5 of 48

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3.1. Oxidative Stress: A Question of Balance
3.1. OxidativeStress: AQuestionofBalance
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presence of CO2, it form nitrate anion (NO3−) and nitrogen dioxide (NO2) (Figure 2).
Diseases 2016, 4, 24 6 of 48
In diabetes, the alteration of the first sites in the mitochondrial membrane lead to the activation
of the complex II [59] and contribute to the formation of excessive O2.− by a leakage of electrons [60].
NADPH oxidases (Nox’s), a family of enzymes with the sole function of producing ROS, are
implicated in the pathophysiology of many cardiovascular diseases [61–64] and are the major source
of glucose-induced ROS production in the vasculature [65,66], kidney [65], liver [66,67], and β cells
[68], confirming this enzyme as a mediator of diabetic complications. Recently, Brandes et al. [69]
described molecular mechanisms of Nox activation and supported their implications in diabetes,
hyperglycemia, and hyperinsulinemia through complex pathways involving NADPH oxidases.
Xanthine oxidase is also implicated in diabetes and vascular complications [70], whereas treatment
Diseases2016,4,24 6of51
of T2D patients with Allopurinol, a XO inhibitor, reduces the level of oxidized lipids in plasma and
improves blood flow [70]. Glucose itself, as well as its metabolites, is known to react with hydrogen
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3 2

Figure 2. Oxidative defense and complications. AGEs: advanced glycated end-products;
Figure 2. Oxidative defense and complications. AGEs: advanced glycated end-products; COX:
COX: cyclooxygenases; H O : hydrogen peroxide; LOX: lipoxygenases; NO: nitric
cyclooxygenases; H2O2: hydrogen2 pe2roxide; LOX: lipoxygenases; NO: nitric oxide; NOS: NO synthase;
oxide; NOS: NO synthase; NADPH oxydase: nicotinamide adenine dinucleotide oxidase;
NADPH oxydase: nicotinamide adenine dinucleotide oxidase; MDA: malondialdehyde (lipid
MDA:malondialdehyde (lipid peroxidation); SOD: superoxyde dismutases; GPx: glutathione
peroxidation); SOD: superoxyde dismutases; GPx: glutathione peroxydase; GSH gluthathione;
peroxydase;GSHgluthathione;O .´:superoxideanion;ONOO.´:peroxynitrite;OH.hydroxylradical;
O2.−:superoxide anion; ONOO.−: p2eroxynitrite; OH. hydroxyl radical; 8-OHdG: 8-hydroxy-2’-
8-OHdG:8-hydroxy-2’-deoxyguanosine(DNAdamages).
deoxyguanosine (DNA damages).
3.1.3. AInntidoixaibdeatnest,s tDheefaelntesreast ionofthefirstsitesinthemitochondrialmembraneleadtotheactivationof
thecomplexII[59]andcontributetotheformationofexcessiveO .´ byaleakageofelectrons[60].
2
The body has a number of very effective antioxidant defense systems to lower the concentration
NADPH oxidases (Nox’s), a family of enzymes with the sole function of producing ROS, are
of free radicals in the body. The term antioxidant refers to “any substance that, when present at low
implicatedinthepathophysiologyofmanycardiovasculardiseases[61–64]andarethemajorsourceof
concentration compared with that of an oxidizable substrate, significantly delays or inhibits oxidation
glucose-inducedROSproductioninthevasculature[65,66],kidney[65],liver[66,67],andβcells[68],
of the substrate” [71]. Nature of the antioxidant systems differs depending on the cell types, tissues,
confirmingthisenzymeasamediatorofdiabeticcomplications. Recently,Brandesetal.[69]described
and localization in the intracellular or extracellular medium [72]. There are different types of
molecularmechanismsofNoxactivationandsupportedtheirimplicationsindiabetes,hyperglycemia,
molecules, natural or synthetic, with enzymatic or scavenging activities (Figure 3).
and hyperinsulinemia through complex pathways involving NADPH oxidases. Xanthine oxidase
is also implicated in diabetes and vascular complications [70], whereas treatment of T2D patients
withAllopurinol,aXOinhibitor,reducesthelevelofoxidizedlipidsinplasmaandimprovesblood
flow[70]. Glucoseitself,aswellasitsmetabolites,isknowntoreactwithhydrogenperoxideinthe
presenceofironandcopperionstoformhydroxylradicalduringauto-oxidation,describedindiabetes
andcomplicationsbyWolffandDeanin1987(Figure2).
3.1.3. AntioxidantsDefenses
Thebodyhasanumberofveryeffectiveantioxidantdefensesystemstolowertheconcentration
offreeradicalsinthebody. Thetermantioxidantrefersto“anysubstancethat,whenpresentatlow
concentrationcomparedwiththatofanoxidizablesubstrate,significantlydelaysorinhibitsoxidation
ofthesubstrate”[71]. Natureoftheantioxidantsystemsdiffersdependingonthecelltypes,tissues,
andlocalizationintheintracellularorextracellularmedium[72].Therearedifferenttypesofmolecules,
naturalorsynthetic,withenzymaticorscavengingactivities(Figure3).
DDiisseeaasseess 22001166,, 44,, 2244 77 ooff 4581

Figure 3. Oxidative defense strategies. CAT: catalase; GPx: glutathione peroxydase; SOD:
Figure 3. Oxidative defense strategies. CAT: catalase; GPx: glutathione peroxydase; SOD: superoxyde
superoxydedismutases.
dismutases.
TThhee ffiirrsstt lilninee ofo dfedfeenfesnesse asgaaignasitn fsrteefr reaedricaadlisc galrsougpros uthpesseth eenszeyemnaztyicm saytsitcemsyss t(eSmODs,( SCOADT,, GCPAxT),
(GFPigxu)r(eFsi g2 uarneds 32) aanndd a3r)ea aniddeadr ebya imdeicdrobnyumtriiecnrotsn (uctorpiepnetrs, z(cionpc,p seerl,enziinucm,)s e[7le3n] iausm co)f[a7c3to]rass. Tcohfearcet oarres.
tThhreeree iasroefotrhmrese fiosro ftohrem sSOfoDr tdheesScrOibDedd eisnc rmibaemdminalms a[m74m]: atlhse[ 7m4]a:ntghaenmesaen-SgOanDe s(eM-SnOSDOD(M) ninS OthDe)
minittohcehmonidtorciah,o cnodprpiae,r c(oCpup)e, ror(C zuin),c o(rZnzi)n icn (Zthne) ciyntothpelacsymto apnlads mthea nmdittohcehomnidtoricah, oannddr ibao,tahn dCub/oZtnh
eCxutr/aZcenlleuxltarra cSeOllDul a(rCSuO/ZDn (CSuO/DZ) ninS OvDes)sienlsv. eCssAelTs .iCs AeTssiesnetisaslelyn tiparlleysepnrte sienn tpienropxeirsooxmiseosm aensda nind
einryethryrothcyrotecsy te[7s5[]7. 5G].PxG Pisx pisrepsreenste nint intheth eexetxratrcaeclleullluarl arflufliudi d(b(lboloodo)d )anandd inin tthhee ccyyttooppllaassmm aanndd
mmeemmbbrraanneess ooff cceellllss [[7766]] aanndd ffoorrmmss aa ccoouuppllee wwiitthh gglluuttaatthhiioonnee rreedduuccttaassee ((GGRR)) pprroovviiddiinngg gglluuttaatthhiioonnee
((GGSSHH)) bbiiooaavvaaiillaabbiilliittyy [[77]]..
TThhee sseeccoonndd lliinnee ooff ddeeffeennsseess iinnvvoollvveess nnoonn--eennzzyymmaattiicc aannttiiooxxiiddaannttss,, ssuucchh aass nnaattuurraallllyy nnuuttrriieennttss
pprroovviiddeedd bbyy ffoooodd,, wwiitthh aa ssccaavveennggiinngg eeffffeecctt ((ccaappttuurree ooff ffrreeee eelleeccttrroonn aanndd ffoorrmmaattiioonn ooff mmoorree ssttaabbllee
eennttiittiieess)),, aa ssttimimuulalatotorryy eefffefectc toonn enednodgoegneonuosu sanatniotixoixdiadnatn etneznyzmyems,e os,r obrotbho t[h77[]7. 7M].aiMn aminolmecoulleecsu alrees
GarSeHG, SvHit,amviitna mEi n(tEhe( tmheosmt oascttiavcet ivfoermfo:r mα:-toαc-otpochoeprohle),r ovl)i,tavmitianm Cin (CL-a(Lsc-oasrbcoicr baiccida)c,i dv)i,tavmitainm iAn
(Aca(rcoatreontoeindosi)d, sb),utb uatlsaol sopoplyoulynusnatsuartuatreadte dfatftaytt yacaicdisd soro reexxooggeennoouus sflflaavvoonnooididss ((qquueerrcceettiinn, ,rruuttiinn,,
rreessvveerraattrrooll,, eettcc..)),, wwhhiicchh ccaann ssttrreennggtthheenn tthhee aannttiiooxxiiddaanntt ddeeffeennsseess ooff tthhee bbooddyy [[7733]].. FFoorr eexxaammppllee,,
iinnccrreeaassiinngg ccoonncceennttrraattiioonn ooff GGSSHH wwiitthh tthheessee pprroodduuccttss ccaann pprrootteecctt aaggaaiinnsstt ccaanncceerr [[7788]] aanndd ddiiaabbeettiicc
ccoommpplliiccaattiioonnss [[7799]].. VViittaammiinn EE ttrraappss oorrggaanniicc ffrreeee rraaddiiccaallss ffrroomm tthhee ooxxiiddaattiioonn ooff lliippiiddss aanndd hheellppss rreedduuccee
lliippiidd ppeerrooxxiiddaattiioonn..
ΒB--cceellllss aarree ppaarrttiiccuullaarrllyy sseennssiittiivvee ttoo RROOSS bbeeccaauussee tthheeyy aarree llooww iinn ffrreeee rraaddiiccaall qquueenncchhiinngg
((aannttiiooxxiiddaanntt)) eennzzyymmeess ssuucchh aass CCAATT,, SSOODD,, aanndd GGPPxx [[8800––8822]] aanndd hhaavvee aa lloowweerr lleevveell ooff GGSSHH [[8822,8,833]]. .
HHoowweevveerr,, tthhee bbaallaannccee bbeettwweeeenn ffrreeee rraaddiiccaallss aanndd aannttiiooxxiiddaanntt ddeeffeennssee ssyysstteemmss iiss ccrruucciiaall ttoo mmaaiinnttaaiinniinngg
hhoommeeoossttaassiiss;; iiff iittss eeqquuiilliibbrriiuumm iiss bbrrookkeenn iinn ffaavvoorr ooff tthhee pprroo--ooxxiiddaanntt eennttiittiieess,, ppaatthhoollooggiiccaall ooxxiiddaattiivvee
ssttrreessss aappppeeaarrss [[8844]] ((FFiigguurree 44))..
Diseases2016,4,24 8of51
Diseases 2016, 4, 24 8 of 48

Figure4.Oxidativestress:Aquestionofbalance.
Figure 4. Oxidative stress: A question of balance.
3.2. FreeRadicals: GoodandBadBoys?
3.2. Free Radicals: Good and Bad Boys?
ROS and RNS are well recognized for playing a dual role as both deleterious and beneficial
ROS and RNS are well recognized for playing a dual role as both deleterious and beneficial
species,sincetheycanbeeitherharmfulorbeneficialtolivingsystems[85],butitisawell-known
species, since they can be either harmful or beneficial to living systems [85], but it is a well-known
featurethatcellsarecapableofgeneratingendogenouslyandconstitutivelyROS[6].
feature that cells are capable of generating endogenously and constitutively ROS [6].
3.2.1. PhysiologicalRoles: TheGoodBoySide
3.2.1. Physiological Roles: The Good Boy Side
Oxygenhomeostasisatthetissuelevelisvitalfordevelopment,growth,andsurvival,andcells
Oxygen homeostasis at the tissue level is vital for development, growth, and survival, and cells
hence have evolved a number of mechanisms to sense and respond to low oxygen levels. Under
hence have evolved a number of mechanisms to sense and respond to low oxygen levels. Under
physiologicalconditions,beneficialeffectsoffreeradicalsoccuratlowormoderateconcentrations
physiological conditions, beneficial effects of free radicals occur at low or moderate concentrations
andinvolvephysiologicalrolesintheregulationofcellularsignalsimplicatedinproliferationand
and involve physiological roles in the regulation of cellular signals implicated in proliferation and
cell adhesion, apoptosis, inflammatory responses, and the regulation of transcription factors [6].
cell adhesion, apoptosis, inflammatory responses, and the regulation of transcription factors [6]. ROS,
ROS, in low concentration, are generating when cells are stimulated by cytokines, growth factors,
in low concentration, are generating when cells are stimulated by cytokines, growth factors, and
and hormones [86], and ROS can thus play a role as a secondary messengers [87,88] like the
hormones [86], and ROS can thus play a role as a secondary messengers [87,88] like the mitogen-
mitogen-activatedproteinkinase(MAPK)pathways[89],probablythemostsignificanteffectofmetals
activated protein kinase (MAPK) pathways [89], probably the most significant effect of metals and
andROS.Thisinvolvestheactivationofnucleartranscriptionfactorsandcontroloftheexpression
ROS. This involves the activation of nuclear transcription factors and control of the expression of
ofprotectivegenesthatrepairdamagedDNA,powertheimmunesystem,arresttheproliferationof
protective genes that repair damaged DNA, power the immune system, arrest the proliferation of
damagedcells,andinduceapoptosis[89]. Celladhesionplaysanimportantroleinembryogenesis,
damaged cells, and induce apoptosis [89]. Cell adhesion plays an important role in embryogenesis,
cell growth, differentiation, wound repair, and others, depending on redox regulation [90] and
cell growth, differentiation, wound repair, and others, depending on redox regulation [90] and the
theinvolvement ofNADPHoxidase [91]. Inan inflammatoryenvironment, activated neutrophils
involvement of NADPH oxidase [91]. In an inflammatory environment, activated neutrophils and
and macrophages produce a large quantity of ROS via NADPH oxidase and myeloperoxidase.
macrophages produce a large quantity of ROS via NADPH oxidase and myeloperoxidase. This
This“oxidativeburst”playsakeyroleinthedefenseagainstenvironmentalpathogens[92]. Lowand
“oxidative burst” plays a key role in the defense against environmental pathogens [92]. Low and
moderatelevelsofROSalsoplayimportantrolesinregulatingautophagyandapoptosis,therefore
moderate levels of ROS also play important roles in regulating autophagy and apoptosis, therefore
controlling cell death and survival [93,94], and ROS generated during ischemic preconditioning
controlling cell death and survival [93,94], and ROS generated during ischemic preconditioning
(alternation of short periods of ischemia and reperfusion) confer cardiac protection by reducing
(alternation of short periods of ischemia and reperfusion) confer cardiac protection by reducing
necrosis and the severity of arrhythmias, improving functional recovery when challenged with a
necrosis and the severity of arrhythmias, improving functional recovery when challenged with a
longerperiodofischemia[95]. Thismechanismisverycomplexandinvolvestriggers, mediators,
longer period of ischemia [95]. This mechanism is very complex and involves triggers, mediators,
andmultiplesecondmessengers’pathways[96–98],butitisaninnatephysiologicadaptiveprocess
and multiple second messengers’ pathways [96–98], but it is an innate physiologic adaptive process
against potentially lethal ischemic injury. NO stimulates soluble guanylyl cyclase, leading to the
against potentially lethal ischemic injury. NO stimulates soluble guanylyl cyclase, leading to the
relaxation of vascular smooth muscle [99] and the essential role of NO in endothelium-induced
relaxation of vascular smooth muscle [99] and the essential role of NO in endothelium-induced
relaxation was discovered by Furchgott and Zawadzki in 1980 [100]. Nowadays, various studies
relaxation was discovered by Furchgott and Zawadzki in 1980 [100]. Nowadays, various studies
reportapivotalroleofNOonvascularhomeostasis(anti-thrombotic,anti-aggregate,anti-migration,
report a pivotal role of NO on vascular homeostasis (anti-thrombotic, anti-aggregate, anti-migration,
andrelaxation)[101–103]. ROSplayacrucialroleintheactivationofmechanotransductionsignaling
and relaxation) [101–103]. ROS play a crucial role in the activation of mechanotransduction signaling
pathwaysandincardiaccontractionandrelaxation[104]. Inaddition,incardiovascularhealth,insulin
pathways and in cardiac contraction and relaxation [104]. In addition, in cardiovascular health,
sensitivityplaysavitalrole,andROSinterveneintheinsulinsignalingpathway. H O inducestypical
insulin sensitivity plays a vital role, and ROS intervene in the insulin signali2ng2 pathway. H2O2
metabolicactionsofinsulin,linkingROStoinsulin[105],increasesglucoseuptakeinadipocytesand
induces typical metabolic actions of insulin, linking ROS to insulin [105], increases glucose uptake in
adipocytes and muscles [106], is involved in the modulation of vascular endothelial function [107],
Diseases2016,4,24 9of51
muscles[106],isinvolvedinthemodulationofvascularendothelialfunction[107],andstimulates
GLUT4 translocation and lipids synthesis in adipocytes [108]. However, ROS levels are the major
determinantsofimpairedversusenhancedinsulinsensitivity[109]throughaROS-inducedincreasein
PI3K/Aktsignaling[110].
3.2.2. PathologicalRoles: TheBadBoySide
Certainlynecessaryinmanyphysiologicalpathways,theirexcessiveproductioncausesdirect
damage to biological molecules (DNA oxidation, proteins, lipids, and carbohydrates) as well as
secondary damage due to cytotoxic and mutagenic character of metabolites released in particular
during the lipid oxidation (Figures 2 and 5). The body may also react against these abnormal
compoundsbyproducingantibodies,whichunfortunatelymaybeautoantibodiescreatingathird
Diseases 2016, 4, 24 10 of 48
waveofattack.

FFiigguurree 55.. RReeaaccttiivvee ooxxyyggeenn ssppeecciieess ((RROOSS)) aanndd ccoommpplliiccaattiioonnss.. IImmppaacctt ooff RROOSS oonn lliippiiddss,, DDNNAA,, pprrootteeiinnss,,
gglluuccoossee,, aanndd vveesssseellss..
3.3. Oxidative Stress, Diabetes, and Vascular Complications
While DNA is the memory of all the biochemical live composition, it is very sensitive to free
radicIanlc“raetatsaecdk .o”xAidtatthiveev setrryeslse ahsat,s fibveeenm parionpcolasesdse tsoo bfeo xoindea toifv ethdea mmaajgoer mcaeudseiast eodf hbyypOerHgl‚yccaenmbiae-
ginednuercaetde dtr.iAggmerosn goft hdeimabeatriec ocxoimdipzleidcabtiaosness,, aimbapsliiccastietes ss,einvterraa-lc amteencharayniasdmdsu [c1ts2,5s]t, raanndd bisr eaa kbsip,aonladr
DprNoAce-spsr. oTtehien fbirrsitd igse tsh[e1 1g1e]n.eIrnaatidodni toifo nROtoSR, aOnSd, RthNeS sesucocnhda sisp ae rdoexcyrneaitsreit eins apnladsmniatr aicnotixoixdiedhanavtse saulscoh
baes evnitiammpilnic aEt,e vditianmDinN AC,d laipmoaicg eac[1id1,2 ]a.nTdh eglmutoasthtieoxnteen [s1i2v6e]l.y Bsotuthd ihedavDe NbAeenle soibosnerisvethde ifno rdmiaabteiotinc
opfa8ti-eOnHts -[G12,7a,n1d28t]h wesiethc hmaincgroe-s aanred tmheacfirrosvtasstceuplsaor fdciaarbceitnico gceonmepsilsic[a8t5io];nist [i3s,n12o9c],o liinnckidinegn cmeetthaabtotlhice-
cgaernceinraotgeedn iRcOagS etnot sthaere dpeovweleorpfumlefnret eorfa ddiiacbaletgiec nceormatpolrisca(UtioVnasn [d24i]o.n Tizhiinsg roraled ioaft ihonyp,semrgolykcee,malicao hhoals,
absebeens teosstafibbliesrhse,dca brcyi nlaorggeen-siccamlee ptarloss,ppeoclyticvyec slitcuhdyiedsr foocra brboothn sT,1eDtc .a)n(Fdi gTu2rDe, 5t)h.e DCCT/EDIC (Diabetes
ContrTohl eancdar Cboomnprleiacacttiivonesc Tomriapl)o u[1n3d0s],( tRhCe CUsK),PsDucSh (aUsKm Parloosnpdeciatilvdee Dhyiadbeet(eMs SDtAud)ya)n [d1341-h],y adnrdo xtyhne oSnteennoal-
(22 -sHtuNdEy )[,1a3r2e].f oDrimabeedtiecn cdaordgieonvoausscluyladru croinmgplliipcaidtiopnesr oaxpipdeaatiro tno abned mgulyltciofaxcitdoartiiaoln ino forciagribno [h1y3d3,r1a3t4es],.
Tbhuet,y inre apcatrtwiciuthlarth, egltyiscsoul-eoxainddatcivelel usltarrespsr ohtaesi nbseeton fsourgmgeAstGedE sto(a dbev atnhcee udngiflyyicnagti olinnke nbde-tpwroeednu ctthse)
various molecular disorders in diabetes mellitus [59,135]. In fact, it is well established that
hyperglycemia and acute glucose fluctuations have many side effects: modifying the redox balance,
increasing circulating FFA, increasing NADPH oxidase activity and TNFα [126], and decreasing
NADPH levels and glutathione, all of which generate by-products, activate oxidative, and
inflammatory signaling. Hyperglycemia induces (1) an increase in glucose and other sugar fluxes
through the polyol pathway, (2) an increase in advanced-glycation end-products (AGEs) formation
through the hexosamine pathway, (3) expression of their receptor (RAGE) [136], and (4) the
stimulation of protein kinase C (PKC) pathway. These mechanisms lead to increase production of
glycative, glycoxidative, and carbonyl free radicals [22,137,138], which altered enzymatic and non-
enzymatic antioxidant defenses. For example, oxidative stress increases mitochondrial DNA
damages and causes axons cell death, leading to neuropathies [139]. Accumulation of sorbitol, due to
an enzymatic conversion of excessive glucose, disrupts osmotic balance [140], a higher fructose
production induces AGEs formation [141], and all participate in peripheral insulin resistance
development [142,143] and β-cells injury [144]. Elevated AGEs may be a significant risk factor for
T1D [145] and induce the progression of pre-diabetes to diabetes [146] and some complications such
Diseases2016,4,24 10of51
and ALEs (advanced lipid peroxidation end-products), inducing protein dysfunctions (loss of
activity,increasedsensitivitytoproteases)[113,114]anddamageincellularresponses—inparticular,
ininflammatoryresponsesandapoptosis[114,115]. Lipids,mainlypolyunsaturatedfattyacids,arethe
maintargetoftheattackbyOH‚andformconjugateddieneradical[116]. Thesemodificationsconcern
circulatinglipoproteinsormembranephospholipids. Thesederivativesareoftenhydrophobicand
willthereforeforminandaroundabnormalclustersofendothelialcells. TheseRCCs,MDA,4-HNE,or
oxidized-LDLwerefoundinlargequantitiesduringmechanismsofcarcinogenesisinvariousstagesof
cardiovasculardiseases[117]suchasatherosclerosis[118,119],metabolicsyndrome[7],diabetesand
complications[120],obesity,andinsulinresistance[121],andinchronicinflammatorydiseasessuch
aslupus[122],asthma,chronicinflammationofthelungs,andrespiratoryallergies[123,124],andin
degenerativediseases[120](Figure5).
3.3. OxidativeStress,Diabetes,andVascularComplications
Increased oxidative stress has been proposed to be one of the major causes of
hyperglycemia-inducedtriggersofdiabeticcomplications,implicatesseveralmechanisms[125],and
is a bipolar process. The first is the generation of ROS, and the second is a decrease in plasma
antioxidantssuchasvitaminE,vitaminC,lipoicacid,andglutathione[126]. Bothhavebeenobserved
indiabeticpatients[127,128]withmicro-andmacrovasculardiabeticcomplications[3,129],linking
metabolic-generatedROStothedevelopmentofdiabeticcomplications[24].Thisroleofhyperglycemia
has been established by large-scale prospective studies for both T1D and T2D, the DCCT/EDIC
(DiabetesControlandComplicationsTrial)[130],theUKPDS(UKProspectiveDiabetesStudy)[131],and
the Steno-2 study [132]. Diabetic cardiovascular complications appear to be multifactorial in
origin [133,134], but, in particular, glycol-oxidative stress has been suggested to be the unifying
linkbetweenthevariousmoleculardisordersindiabetesmellitus[59,135]. Infact,itiswellestablished
that hyperglycemia and acute glucose fluctuations have many side effects: modifying the redox
balance, increasing circulating FFA, increasing NADPH oxidase activity and TNFα [126], and
decreasing NADPH levels and glutathione, all of which generate by-products, activate oxidative,
andinflammatorysignaling. Hyperglycemiainduces(1)anincreaseinglucoseandothersugarfluxes
throughthepolyolpathway,(2)anincreaseinadvanced-glycationend-products(AGEs)formation
throughthehexosaminepathway,(3)expressionoftheirreceptor(RAGE)[136],and(4)thestimulation
of protein kinase C (PKC) pathway. These mechanisms lead to increase production of glycative,
glycoxidative,andcarbonylfreeradicals[22,137,138],whichalteredenzymaticandnon-enzymatic
antioxidant defenses. For example, oxidative stress increases mitochondrial DNA damages and
causesaxonscelldeath,leadingtoneuropathies[139]. Accumulationofsorbitol,duetoanenzymatic
conversionofexcessiveglucose,disruptsosmoticbalance[140],ahigherfructoseproductioninduces
AGEsformation[141],andallparticipateinperipheralinsulinresistancedevelopment[142,143]and
β-cells injury [144]. Elevated AGEs may be a significant risk factor for T1D [145] and induce the
progressionofpre-diabetestodiabetes[146]andsomecomplicationssuchasdiabeticretinopathy[147].
Asshownbefore,oxidativestressiscloselylinktoinflammation.Indeed,circulatingTNF-αmayimpair
vascularfunctionbyalteringthebalancebetweenendothelial-derivedvasodilatorandvasoconstrictor
substancesbecauseitdownregulatedtheexpressionofeNOSandupregulatedET-1productionin
endothelialcells[148]. Moreover,itmayalsodirectlyactivateNADPHoxidaseandthenincreasethe
productionofROSinthevasculature[149].
Oxidativestresscanbemeasuredinvivoinmultipletypesincludingcells,solidtissues,urine,
blood,andsaliva. Severalinvestigationscorrelatedoxidativestressobservedinserumandinsaliva,
and,today,salivacanbeconsiderateasanoxidativestressdiagnosticfluid[150,151]. Somehuman
studies highlight reactive compounds in saliva in some pathologies, such as T1D [152–155] and
T2D[153,156],withthedetectionofbiomarkerssuchas8-oxodG[152],MDA,andTBARS,proteins
carbonyl [152], and total antioxidant capacity [153–156]. Recently, Wang et al. published a critical
reviewofsalivarybiomarkersofoxidativestress[157],highlightingtheproblemofstandardizationin