Risk of type 2 diabetes in patients with non-alcoholic fatty liver disease: Causal association or epiphenomenon?

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Availableonlineat

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Diabetes&Metabolism42(2016)142–156

Review

Risk

of

type

2 diabetes

in

patients

with

non-alcoholic

fatty

liver

disease:

Causal

association

or

epiphenomenon?

G. Targher

a,∗

,

G. Marchesini

b

,

C.D.

Byrne

c,d

a_Section_of_{Endocrinology,}_Diabetes_and_Metabolism,_Department_of_Medicine,_University_and_Azienda_Ospedaliera_{Universitaria}_Integrata_of_Verona,

PiazzaleStefani,1,37126Verona,Italy

b_Unit_of_Metabolic_Diseases_and_Clinical_Dietetics,_“Alma_Mater_Studiorum”_University,_Bologna,_Italy c_Nutrition_and_Metabolism,_Faculty_of_Medicine,_University_of_Southampton,_Southampton,_UK

d_Southampton_National_Institute_for_Health_Research_Biomedical_Research_Centre,_University_Hospital_Southampton,_Southampton,_UK

Received29March2016;accepted11April2016 Availableonline30April2016

Abstract

Non-alcoholicfattyliverdisease(NAFLD)hasbecometheleadingcauseofchronicliverdiseasesworldwide,causingconsiderableliver-related mortalityandmorbidity.Overthelast10years,ithasalsobecomeincreasinglyevidentthatNAFLDisamultisystemdisease,affectingmany extra-hepaticorgansystemsandinteractingwiththeregulationofmultiplemetabolicpathways.NAFLDispotentiallyinvolvedintheaetiologyand pathogenesisoftype2diabetesviaitsdirectcontributiontohepatic/peripheralinsulinresistanceandthesystemicreleaseofmultiplehepatokines thatmayadverselyaffectglucosemetabolismandinsulinaction.Inthisupdatedreview,wediscusstherapidlyexpandingbodyofclinicaland epidemiologicalevidencethatsupportsastronglinkbetweenNAFLDandtheriskofdevelopingtype2diabetes.Wealsobrieflyexaminethe conventionalandthemoreinnovativepharmacologicalapproachesforthetreatmentofNAFLDthatmayinfluencetheriskofdevelopingtype2 diabetes.

Keywords: Diabetesrisk;Epidemiology;NAFLD;Non-alcoholicfattyliverdisease;Type2diabetes

1. Introduction

Non-alcoholic fattyliverdisease (NAFLD) encompassesa

spectrum of liver pathology including simple steatosis,

non-alcoholic steatohepatitis (NASH) with varying amounts of

fibrosisandcirrhosis[1].NAFLDhasemergedasapublichealth

problem of epidemicproportions inmany parts of the world

(affectingupto30%oftheadultpopulationintheUnitedStates

andEurope) [1,2].TheprevalenceofNAFLDismuchhigher

inpatientswithtype2diabetes(rangingfromapproximately50

to75%).Notably,patientswithtype2diabetesandNAFLDare

alsomorelikelytodevelopthemoresevereformsofNAFLD,

∗_{Corresponding}_author.

E-mailaddress:giovanni.targher@univr.it(G.Targher).

suchasNASH,advancedfibrosis,cirrhosis,andinsomecases

hepatocellularcarcinoma[1,2].

Over the past decade, it has become increasingly clear

that NAFLD is not onlyassociated withan increased risk of

liver-relatedmorbidityormortality,butalsoitisamultisystem diseasethataffectsavarietyofextra-hepaticorgansystems,and

interacts with the regulation of multiple metabolic pathways

[3]. Strong evidence indicates that cardiovascular disease is

the leadingcause ofmortality inpatients withNAFLD [3,4].

As detailed below, there is now also convincing evidence

suggestingthatNAFLDmayoftenprecedethedevelopmentof

type2diabetes.Thissupportstheassertionthattheconventional

paradigmofNAFLDrepresentingthesimple“hepatic

manifes-tation”ofthemetabolicsyndromeisoutdated,andthatNAFLD

might be regarded as an early predictor and determinant for

the development of diabetes and other clinical traits of the

metabolic syndrome [5]. This finding may have potentially

http://dx.doi.org/10.1016/j.diabet.2016.04.002

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relevantclinicalimplicationsforthediagnosis,preventionand treatmentoftype2diabetes.

Specifically,inthisnarrativereviewwediscusstherapidly

expanding body of clinical evidence that supports a strong

association between NAFLD and the risk of new-onset type

2diabetes,andtheputativebiologicalmechanismsunderlying

thisassociation.Wealsobrieflydiscusssomeofthetreatment optionsforNAFLDthatmayinfluencetheriskofdiabetes.

Tothisend,althoughthisisnotaformalsystematicreview,

weextensivelysearchedPubMeddatabasetoidentifyrelevant

articlespublisheduptoJanuary2016,usingthekeywords

“non-alcoholic fatty liver disease” or “fatty liver” combined with

“incidenttype2diabetes”or“diabetesrisk”.

2. EpidemiologicalevidencelinkingNAFLDtotherisk ofnew-onsettype2diabetes

In thisreview,we havenotdiscussed the largenumberof

prospective,population-basedstudiesthatusedserumlevelsof liverenzymes(orothersurrogatemarkersofNAFLDsuchasthe fattyliverindex)todiagnoseNAFLD.Thesestudieshave

consis-tentlyshownthatmildlyelevatedserumliverenzymes(mainly

serumgamma-glutamyltransferase)areindependent,long-term

predictorsofnew-onsettype2diabetesinvariousethnic

popu-lations(Asian,AmericanandEuropeanpeople)[6–8].

Datafromobservationalcohort(prospectiveorretrospective)

or case-control studies that used non-invasive imaging

tech-niques(predominantlyultrasonographythathashighsensitivity andspecificity fordetectingmoderateandseveresteatosis)to

diagnoseNAFLD are summarizedinTable1.Thereare now

about20observationalstudiesthat haveassessedthe

associa-tionbetweenNAFLDandtheriskofnew-onsettype2diabetes

[9–25].

Themaincharacteristicsofthestudypopulations,thelength offollow-upperiod,thediagnosticcriteriafordiabetes,the rel-ativerisksofdevelopingdiabetesandthecovariatesincludedin multivariateregressionmodelshavebeenspecifiedinthetable.

Amongstthesepublishedstudies,nearlyall(exceptforthe

studybyOkamotoetal.[9])haveshownthatNAFLDwas

asso-ciatedwithasubstantiallyincreasedrisk of new-onsettype2

diabetes.The risk of diabetes amongthese published studies

variedmarkedlyfroma33%increase[14]toa5.5foldincrease inrisk[11].Thiswideinterstudyvariationinriskmightreflect differencesinNAFLDseverity,sincethestudybyParketal.[15]

showedthatthe5-yearincidencerateoftype2diabetesincreased

progressively according to the ultrasonographic severity of

NAFLDat baseline(normal: 7.0%,mild:9.8%,

moderate-to-severe:17.8%)inacohortof25,232SouthKoreannon-diabetic

middle-agedmen.Evenafteradjustingformultiple

confound-ingvariables,thehazardratios(HRs)fordiabetesdevelopment

werehigherinthemild-NAFLDgroup(adjusted-HR1.09;95%

confidence interval [CI] 0.81–1.48) and in the

moderate-to-severe-NAFLDgroup(adjusted-HR1.73; 95%CI 1.00–3.01)

comparedwiththeno-NAFLDgroup,respectively.Inaddition,

thiswideinterstudyvariabilityinNAFLD-associatedriskoftype 2diabetesmightalsoreflectdifferencesinthedemographic char-acteristicsofthestudypopulations,inthelengthoffollow-up

(rangingfromameanperiodof3to12.8years)andinthevarying degreeofconfounderadjustmentinindividualstudies(Table1).

Using data from an occupational cohort study of over

12,000SouthKoreanindividualswithmeasurementsofinsulin

resistance (by homeostasis model assessment [HOMA-IR]),

overweight/obesityandNAFLD(asdetectedby

ultrasonogra-phy)atbaseline,Sungetal.[16]havecomparedtheeffectof thesethreeriskfactors(singlyorincombination)ontheriskof new-onsetdiabetesat5-yearfollow-up.Thesedatashowedthat eachofthesethreeriskfactorswasassociatedwithan

approx-imate doubling of the diabetesrisk evenafter adjustmentfor

other established risk factors (for example, with an adjusted

oddsratio[OR]of2.42,95%CI1.74–3.36,forNAFLD).When

allthreerisk factorsoccurredtogetherinthe sameindividual (andthisoccurredinapproximately50%ofpatientswith inci-dentdiabetesatfollow-up),therewasanapproximately14-fold increaseindiabetesriskafteradjustingforpotentialconfounding variables[16].

Notably, andmostinterestingly, in thesame observational

cohortofKoreanindividuals,theauthorshavefurtherassessed theriskofnew-onsettype2diabetesat5yearsoffollow-upin thefollowingthreesubgroups:

• insubjectsinwhomtherewasresolutionoffattyliverover 5years,i.e.,fattyliverthathadbeenpresentatbaselinewas

notpresentatfollow-upexamination;

• insubjects inwhom therewasadevelopmentof newfatty

liverbetweenbaselineandthefollow-upexaminations;

• insubjectsinwhomtherewasanincreaseinseverityoffatty liverstatus,frommildfattylivernotedatbaselineto moderate-severefattyliver,identifiedatfollow-upexamination[20].

Interestingly,thesedatashowedthatchangingfattyliver sta-tusovera5-yearperiodwasassociatedwithmarkedlydifferent risksofincidenttype2diabetes.Inparticular,asalsoshownin

Fig.1,therewasasignificantdiabetesriskreduction inthose

subjectsinwhomfattyliveronultrasonographyresolvedover

time. Inparticular, inthosesubjects,risk of incidentdiabetes

decreased tothebackgroundrisk of someone whohadnever

hadfattyliver.Conversely,thesubjectsinwhomtheseverityof fattyliverworsenedover5years(frommildtomoderate-severe) showedamarkedincreaseinriskofincidentdiabetescompared withtheriskinpeoplewithresolutionoffattyliver,supporting thenotionthatmoresevereformsofNAFLDareassociatedwith higherriskofincidentdiabetes[20].Similarly,Yamazakietal.

[21]recentlyassessedtherelationshipbetweenimprovementof

NAFLDandriskofincidenttype2diabetesinaretrospective

cohortof 4604non-diabeticJapaneseindividualsfollowed-up

foraperiodof10years.TheyfoundthatNAFLDatbaselinewas associatedwithanapproximately2.5-foldincreasedriskof

inci-dentdiabetes,andthatNAFLDimprovementwassignificantly

associatedwitha70%riskreductionofdevelopingdiabetesover time,evenafteradjustingforage,sex,familyhistoryofdiabetes,

BMI,impairedfastingglycaemia,dyslipidemia,hypertension,

and physicalexercise[21]. However,these two latterstudies arenotinterventiontrialsthatfocusedontreatingNAFLD;so cautionisneededininterpretingtheseresults.

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Table1

Principalobservational(retrospective/prospectiveorcase-control)studiesoftheassociationbetweenNAFLDandtheincidenceoftype2diabetes(orderedby publicationyear).

Authors,year (ref.)

Studydesign,sample size,population, follow-up,and NAFLDdiagnostic tool Diagnosisofincident diabetes

No.incidentcasesof diabetes;%in non-NAFLDvs. NAFLDcases Adjustments considered Mainresults Okamotoetal., 2003[9] Retrospectivecohort study n=840non-diabetic Japanesesubjects; 10years;liver ultrasonography Fastingglucose ≥7.8mmol/lor HbA1c≥6.5%or drugtreatment n=82incidentcases 7.6%vs.22.5%

Age,sex,BMI,family historyofdiabetes, fastingglucose, HbA1c,alcohol intake,frequencyof check-upsand changesofBMI duringfollow-up

NAFLDwasassociated withincidentdiabetes inunivariateanalysis (OR2.62,95%CI 1.6–4.3).This associationdisappeared afteradjustingfor confounders(aOR1.83, 95%CI0.9–3.5) Fanetal.,2007 [10] Nestedcase-control study n=358Chinese NAFLDpatientsand 788no-NAFLD, matchedforageand sex;6years;liver ultrasonography Fastingglucose ≥7.8mmol/l,clinical historyordrug treatment n=102incidentcases 5.2%vs.20.3%

Age,sex,BMI NAFLDwas

independently associatedwithincident diabetes(aOR4.63, 95%CI3.0–7.1, P<0.001) Shibataetal., 2007[11] Retrospectivecohort studywithanested case-controlanalysis

n=3189male Japaneseworkerswith normalglucose tolerance,noviral hepatitis,noalcohol; 4years;liver ultrasonography Fastingglucose ≥7.0mmol/lor2-h glucose≥11.1mmol/l on75gOGTT n=109incidentcases 1.8%vs.8.1%

AgeandBMI(inthe wholecohort analysis);age,BMI, smoking,blood pressure,physical activity,durationof follow-upand prevalenceof metabolicsyndrome (inthenested case-controlanalysis)

NAFLDwas

independently associatedwithincident diabetesbothinthe wholecohort(aHR 5.50,95%CI3.6–8.5) andinthenested case-controlanalysis (aHR4.60,95%CI 3.0–6.9,P<0.001) Kimetal.,2008 [12] Retrospectivecohort study n=5372non-diabetic SouthKorean subjects,noviral hepatitisorexcessive alcohol;5years;liver ultrasonography Fastingglucose ≥7.0mmol/l,clinical historyordrug treatment n=234incidentcases 2.3%vs.8.5%

Age,sex,BMI,family historyofdiabetes, smoking,fasting glucose, HDL-cholesterol, triglycerides,serum ALT NAFLDwas independently associatedwithincident diabetes(aHR1.51, 95%CI1.04–2.2,

P=0.034). Moderate/severe NAFLDhadhigher HRsvs.mildNAFLD. Exclusionofdrinkers didnotattenuatethe association Yamadaetal., 2010[13] Retrospectivecohort study n=12,375 non-diabeticJapanese subjectswithout hepaticdiseasesand viralhepatitis;5years; liverultrasonography Fastingglucose ≥7.0mmol/l,clinical historyordrug treatment n=96incidentcases ofdiabetes;0.5%vs. 2.5% n=728incidentIFG cases;4%vs.10%

Age,sex,BMI,family historyofdiabetes, hypertension,alcohol intake,smokingstatus

NAFLDwas

independently associatedwithincident IFGand/ordiabetesin bothsexes(aOR1.91, 95%CI1.6–2.3inmen andOR2.15,95%CI 1.5–3.0inwomen) Baeetal.,2011 [14] Retrospectivecohort study n=7849non-diabetic SouthKorean subjects;5years;liver ultrasonography Fasting glucose≥7.0mmol/l orHbA1c≥6.5%, clinicalhistoryor drugtreatment n=435incidentcases 3.7%vs.9.9%

Age,sex,BMI, triglycerides,HDL cholesterol,systolic bloodpressure, smokingstatus, physicalactivity, alcoholintake,IFG status

NAFLDwas

independently associatedwithincident diabetes(aHR1.33, 95%CI1.1–1.7,

P<0.05).This associationwasmuch strongerinpre-existing IFG

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Table1(Continued) Authors,year (ref.)

No.incidentcasesof diabetes;%in non-NAFLDvs. NAFLDcases Adjustments considered Mainresults Parketal.,2012 [15] Prospectivecohort study(health check-up) n=25,232 non-diabeticSouth Koreanmen,noviral hepatitis,orexcessive alcohol;5years;liver ultrasonography Fastingglucose ≥7.0mmol/l,HbA1c ≥6.5%,clinical history n=2108incident cases 7%inno-steatosisvs. 9.8%inmildsteatosis vs.17.8%in moderate-severe steatosis Age,waist circumference,HDL cholesterol, triglycerides,systolic bloodpressure, C-reactiveprotein, HOMA-IR,serum creatinine,family historyofdiabetes, physicalexercise, metabolicsyndrome NAFLDwas independently associatedwithincident diabetes;theHRs(95% CI)wereincreasedin mildsteatosis(1.09; 0.8–1.5)andin moderate/severe steatosis(1.73;1.0–3.0) vs.no-steatosis Sungetal.,2012 [16] Retrospectivecohort study n=12,853 non-diabeticSouth Koreansubjects; 5years;liver ultrasonography Fastingglucose ≥7.0mmol/l,clinical historyordrug treatment n=223incidentcases 0.8%vs.4.3%

Age,sex,BMI, educationalstatus, smoking,physical activity,alcohol intake,HOMA-IR, serumtriglycerides, serumALT Theclusteringof increasedHOMA-IR, overweight/obesity,and NAFLDmarkedly increasestheoddsof developingdiabetes, witheffects independentofeach otherandof confoundingfactors. NAFLDwasassociated withincidentdiabetes (aOR2.42,95%CI 1.7–3.4,P<0.001) Kasturiratneetal., 2013[17] Retrospectivecohort study n=2276non-diabetic SriLankan individuals,noviral hepatitisorexcessive alcoholconsumption; 3years;liver ultrasonography Fastingglucose ≥7.0mmol/l,clinical historyordrug treatment n=242incidentcases 10.5%vs.19.7%

Age,sex,family historyofdiabetes, BMI,waist circumference, hypertension, dyslipidemia,IFG status,serumALT

NAFLDwas

independently associatedwithincident diabetes(aHR1.64, 95%CI1.2–2.2). NAFLDwastheonly predictorofincident diabetesinthosewith IFGatbaseline Changetal.,2013 [18] Prospectivecohort study(health check-up) n=38,291 non-diabeticSouth Koreansubjects,no knownliverdiseases, viralhepatitisor excessivealcohol; 5years;liver ultrasonography Fasting glucose≥7.0mmol/l, HbA1c≥6.5%,or drugtreatment n=2025incident cases 3.5%inno-NAFLD vs.7.4%inNAFLD withlowfibrosisscore (NFS)vs.15.3%in NAFLDwith intermediate/high NFS

Age,sex,smoking status,alcoholintake, regularexercise, familyhistoryof diabetes,cholesterol, triglycerides,HDL cholesterol, HOMA-IR,C-reactive protein

TheaHRs(95%CI)for incidentdiabetesin NAFLDwithlowNFS andNAFLDwith intermediateorhigh NFSvs.noNAFLD were2.0(1.8–2.2)and 4.74(3.7–6.1), respectively.This associationremained significantinsubjects withfasting glucose<100mg/dL andwithHbA1c<5.8% Choietal.,2013 [19] Retrospectivecohort study n=7849non-diabetic SouthKorean subjects,noviral hepatitisorexcessive alcohol;4years;liver ultrasonography Fasting glucose≥7.0mmol/l, HbA1c≥6.5%,or drugtreatment n=435incidentcases 3.5%incontrolsvs. 4.6%intheincreased ALTvs.7.3%inthe steatosisvs.11.8%in thecombined abnormalitygroup

Age,sex,BMI, systolicblood pressure, triglycerides,HDL cholesterol,IFG status,physical activity,smoking, alcoholconsumption TheHRs(95%CI)of incidentdiabeteswas stepwiseincreasedin theelevatedALT,the steatosis,andthe combinedabnormality group.Subjectsinthe combinedabnormality grouphada

significantlyincreased aHRof1.64(95%CI 1.3–2.1,P<0.001)

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No.incidentcasesof diabetes;%in non-NAFLDvs. NAFLDcases Adjustments considered Mainresults Sungetal.,2013 [20] Retrospectivecohort study n=13,218 non-diabeticSouth Koreansubjects; 5years;liver ultrasonography Fastingglucose ≥7.0mmol/l,clinical historyordrug treatment n=234incidentcases 0.8%innosteatosis vs.3.4%inmild steatosisvs.12.3%in moderate-severe steatosis

Age,sex,BMI, glucose,insulin, triglycerides,HDL cholesterol,systolic bloodpressure, alcoholuse,smoking, physicalactivity, changeinBMI betweenbaselineand follow-up,serum ALT,AST,GGT

Fattyliverresolvedin 828,developedin1640, andprogressedfrom mildtomoderate/severe in324people. Resolutionwasnot associatedwithriskof incidentdiabetes(aOR 0.95,95%CI0.5–1.9). Developmentofnew fattyliverwas associatedwithincident diabetes(aOR2.49, 95%CI1.5–4.1,

P<0.001).In individualswith worseningfattyliver (frommildto moderate/severe), diabetesriskmarkedly increased(aOR6.13, 95%CI2.6–14.7,

P<0.001vs.subjects withfattyliver resolution) Yamazakietal., 2015[21] Retrospectivecohort study n=3074non-diabetic Japanesesubjects,no viralhepatitisor excessivealcohol; 11.3years;liver ultrasonography Fasting glucose≥7.0mmol/l, HbA1c≥6.5%, clinicalhistoryor drugtreatment n=189incidentcases 3.1vs.16.1%

Age,sex,family historyofdiabetes, BMI,IFGstatus, dyslipidemia, hypertension,physical exercise

NAFLDwas

independently associatedwithincident diabetes(aOR2.37, 95%CI1.6–3.5, P<0.001).NAFLD improvementwas associatedwitha reductionofincident diabetes(aOR0.27, 95%CI0.1–0.6, P<0.001) Fukudaetal., 2015[22] Retrospectivecohort study n=4629non-diabetic Japanesesubjects,no liverdiseaseor excessivealcohol; 12.8years;liver ultrasonography Fasting glucose≥7.0mmol/l, HbA1c≥6.5%,or drugtreatment n=351incidentcases 3.2%in non-overweight, no-NAFLDvs.14.4% innon-overweight NAFLDvs.8.0%in overweight no-NAFLDvs.26.4% inoverweightNAFLD

Age,sex,family historyofdiabetes, alcoholintake, smoking,regular exercise,HbA1c

aHRsforincident diabetesvs.

non-overweightwithout NAFLDgroupwere: 3.59(95%CI 2.14–5.76)inthe non-overweightwith NAFLDgroup,1.99 (95%CI1.5–2.7)inthe overweightwithout NAFLDgroupand6.77 (95%CI5.2–8.9)inthe overweightwith NAFLDgroup Mingetal.,2015 [23] Retrospectivecohort study n=508non-diabetic Chinesesubjects,no chronicliverdiseases orexcessivealcohol; 5years;liver ultrasonography Fasting glucose≥7.0mmol/l, 2-hglucose ≥11.1mmol/lon75g OGTTordrug treatment n=20incidentcases 2.4%vs.10.3%

Age,sex,educational level,smoking, alcoholintake, physicalactivity, familyhistoryof diabetes,BMI,blood pressure,fasting glucose,2-hglucose, triglycerides,HDL cholesterol NAFLDwas independently associatedwithincident diabetes(aHR4.46, 95%CI1.9–10.7,

P<0.001)butnotwith incidentprediabetes (aHR1.64,95%CI 0.97–2.8)

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No.incidentcasesof diabetes;%in non-NAFLDvs. NAFLDcases Adjustments considered Mainresults

Lietal.,2015[24] Retrospectivecohort study n=4736non-diabetic Chinesesubjects,no viralhepatitisor excessivealcohol; 4years;liver ultrasonography Fastingglucose ≥7.0mmol/l,clinical historyordrug treatment n=380incidentcases 4.1vs.17.4%

Age,sex,blood pressure,lipids,ALT, uricacid,creatinine

NAFLDwas

independently associatedwithincident diabetes(aHR3.37, 95%CI2.4–4.3) Shahetal.,2015 [25] Prospectivecohort study n=3153non-diabetic individualsfromthe Multi-EthnicStudyof Atherosclerosis; 9.1years;liver computedtomography Fastingglucose ≥7.0mmol/l,clinical historyordrug treatment

n=216incidentcases Age,sex,race,family historyofdiabetes, BMI,waist circumference, systolicblood pressure, triglycerides,HDL cholesterol,glucose, C-reactiveprotein, exercise,statinuse

NAFLD(definedas1st quartileofhepatic attenuationon computedtomography) wasindependently associatedwithincident diabetes(aHR2.06, 95%CI1.5–2.8,

P<0.001)

ALT:alanineaminotransferase;AST:aspartateaminotransferase;BMI:bodymassindex;CI:confidenceinterval;GGT:gamma-glutamyltransferase;HDL: high-densitylipoprotein;HOMA-IR:homeostasismodelassessment-insulinresistance;IFG:impairedfastingglycaemia;aHR:adjustedhazardratio;aOR:adjustedodds ratio.

Todate,thereareveryfewstudiesexaminingtheassociation

betweenhistology-diagnosedNAFLDandtheriskofincident

diabetes.Inacohortstudyof129Swedenadultswith

biopsy-provenNAFLDandelevatedserumliverenzymes,whowere

followedforapproximately14years,Ekstedtetal.[26]found

thattheprevalenceofpreviouslyknowndiabeteswas8.5%at

baseline.Notably, attheendof the follow-up,approximately

80% of thesepatients developedeither new-onsettype2

dia-betesorimpairedglucosetolerance.Again,patientswithNASH

hadanapproximatelythreefoldhigherrisk ofdeveloping

dia-betesthanthosewithsimplesteatosisoverthefollow-upperiod

[26].

Fig.1.Incidenttype2diabetesat5-yearfollow-upaccordingtochangesinfattyliverstatusonultrasonographybetweenbaselineandfollow-upinanoccupational cohortof13,218SouthKoreannon-diabeticindividuals.Changeinfattyliverstatusovertimewasassociatedwithmarkedlyvariablerisksofincidenttype2diabetes (P<0.001whencomparedwiththe“nosteatosis”category).

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Collectively,althoughthereisnowconvincingevidencethat

NAFLDisassociatedwithanincreasedriskofnew-onsettype

2diabetes[10–25],andthereisemergingevidencethatthisrisk variesaccordingtotheseverityofNAFLD(asdetectedeitherby ultrasonographyorbyuseofnon-invasivefibrosisscoring sys-tems)[9,12–14],furtherevidenceisunquestionablyneededin non-Asianpopulations,asnearlyallofthestudiessummarised inTable1havebeenconductedinvariousAsianpopulations,

andmost of theevidence obtainedto datehasbeen obtained

inSouthKorean individuals.However,arecentanalysis from

theMulti-EthnicStudyofAtherosclerosisstudyinvolvingmore than3000UnitedStatesnon-diabeticindividualshasconfirmed

thatNAFLDasdetectedbycomputedtomographywas

indepen-dentlyassociatedwithatwo-foldincreasedriskofdeveloping type2diabetes[25].In addition,as alsoreportedabove,

sev-eral population-basedcohort studies,involving European and

American middle-agednon-diabetic individuals, have

repeat-edlyshown that moderately elevated serumliver enzymes (a

proxy of NAFLD) are stronglyand independentlyassociated

withanincreasedriskofnew-onsettype2diabetes[6–8].

Anotherimportantcaveattokeepinmindwhenexamining

theresultsfrompublishedstudiesisthattheadjustmentfor estab-lishedriskfactorsofdiabetesandotherpotentialconfounding factorswasoftenincomplete.Forexample,dataonwaist circum-ferencewasoftenlacking,whereasfamilyhistoryofdiabetes, insulinresistanceindexvalues andphysicalactivity werenot

alwaysincludedinmultivariateregressionmodels.Additional

follow-upstudieswithalargerpanelofestablishedand emerg-ingdiabetesriskfactors(e.g., higherserumferritinandlower adiponectinlevelsthathavebeenalsoassociatedwiththe

pres-enceandseverityofNAFLD)areneededtofirmlyestablishan

independentcontributionofNAFLDpersetotheriskof

new-onsettype2diabetes.Finally,itisimportanttounderlinethatin noneofthepublishedstudies,exceptforthestudiesbyShibata etal.[11]andbyMingetal.[23];thediagnosisofdiabeteswas

basedon2-hourpostloadplasmaglucoseconcentrations.

3. PutativebiologicalmechanismsbywhichNAFLD contributestodiabetesdevelopment

Whenimbalance occursbetweenenergy intakeandenergy

expenditure,orwhenthereisanintrinsicproblemwithstoring excessenergyastri-acylglycerolinadiposetissuedepots,lipid accumulatesinotherorgansthroughoutthebody.Iflipid accu-mulatesintissuesororgansnotdesignedtoaccumulatefat,e.g., liveroromentum,theterm‘ectopicfataccumulation’infersthat lipidaccumulationhasoccurredinanonphysiologicalsitewith thepotentialforincreasingriskofcertaincommondiseasessuch

asdiabetes,CVDandNAFLD[27].

NAFLDisanexample ofectopicfataccumulationinliver

andthislipidaccumulationisusuallyassociatedwith: • insulinresistance,abnormalsubstratefluxes[28]; • increasedsecretionofhepatokines[29];

• increasedgluconeogenesis;

• decreased glycogen synthesis and inhibition of insulin

signallingpathways[30,31].

Whenexcesshepaticlipidaccumulates(NAFLD),itnotonly

hasthepotentialforcausinginsulinresistancebutthereisalso thepotentialforchronicliverinflammation,increasing riskof progressive fibrotic liver disease,cirrhosis andhepatocellular carcinoma.Suchapathologicalstatealsohasthepotentialfor

increasing both very low-density lipoprotein (VLDL)

secre-tionandhepaticglucoseoutput.Markedliverfataccumulation,

increasedVLDLs,increasedhepaticglucoseoutputandinsulin

resistanceareallassociatedwithincreasedriskofdiabetes.Fig.2

illustratesthepotentialmechanismsleadingtoalteredlipidand

glucosemetabolismresultingfromandcontributingtoinsulin

resistanceandincreasedhepaticglucoseproductioninNAFLD.

Thefigureschematicallyillustratessomeofthekeymechanisms relevanttothepathwaysthatoperateintheadiposetissue(panel A),theliver(panelB),andtheintestine(panelC).

Itisnowclearthatadiposetissuedysfunction/inflammation

isalsoimportantinNAFLDpathogenesis,andincreasing

evi-dence now suggeststhat the intestineis also involved in the

developing disease process. Alteration of the normal

intesti-nal microbiota(dysbiosis)also playsakey role inregulating

severalintrahepaticmetabolicandinflammatorypathwaysthat

are involvedinthedevelopmentofNAFLDanddiabetes.For

example,dataobtainedinmiceillustratetheimportanceofthe gutmicrobiotaforaffectinghepaticlipidlevels,andserum glu-coseandinsulinconcentrations.Suchdatahasshownthatoral

treatment of lean germ-free mice with the caecal microbiota

fromobesemicecausedanincreaseinhepatictriglyceride

accu-mulation, andinsulinresistancewithincreased seruminsulin

andglucoseconcentrations[32].Severaldifferentmechanisms

have been suggestedthat are involved inthe pathogenesis of

dysbiosis-inducedNAFLDandthatcontributetothe

develop-mentandprogressionofliverdiseaseinNAFLD.Forexample,

increased intestinal absorptionof multiple bacterial products, such as short-chain fattyacids (e.g.,butyrate, propionateand

acetate), lipopolysaccharide and endotoxins, have the

poten-tial to influence both hepatic glucose and lipid metabolism,

promote hepatic inflammation and increase gut permeability,

thusadverselyinfluencingtheriskofinflammationbeyondthe liver[27,33,34].Intestinaldysbiosisperturbsbiliarycholesterol metabolism, andalteredbileacidsintheintestinecanmodify thegutmicrobiota.Bileacidsarederivedinitiallyfromhepatic cholesterolmetabolismandarecapableofactingassignalling moleculeswhicharecapableofactivatingsomereceptors,e.g.

farnesoid X receptor (FXR), and modifying signalling

path-ways that influence both liver lipid metabolism and hepatic

glucosemetabolism.Recently,therehasbeenconsiderable inter-est in this field, with the appreciated that subtle changes in the bile acidstructure canprofoundly influencetheirefficacy

as signalling molecules. For adding an ethyl group at

car-bon position6 inchenodeoxycholicacid, it markedly affects

the ligand-bindingcapabilityofchenodeoxycholicacidtothe

nuclear hormone FXR. Treatment with obeticholic acid has

recently shownpromiseintreatingNASHinthe FarnesoidX

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Adipose A LCFAs

Glycerol 3 P

LPA _PA DAG TAG

Increased FFAs

Increased Glycerol Inflammation

LCFAs from adipose ssue

Dietary Glucose/fructose Glycerol 3 P

LPA PA DAG TAG Increased VLDL

Hepac inﬂammaon & lipid droplets Ceramide Insulin resistance (PKC ε)

Liver B FFAs / glycerol 1 bile acids Hepac inﬂammaon /lipid/ﬁbrosis/cancer Lipopolysaccaride-induced inflammation

Altered FXR acvaon TLR-4 acvaon

Intestine C

Increased gut permeability Intesnal microbiota dysbiosis

Increased 2 bile acids

Altered incren (e.g. GLP-1 producon) and SCFAs (e.g.

butyrate, propionate, acetate) Glucose intolerance Increased hepa_{Increased hepa}c glucose_c output lipogenesis/gluconeogenesis Hepatokines

Increased hepac glucose output Decreased hepac glycogen

synthesis Dietary Glucose/fructose

TAG hydrolysis

Increased hepac gluconeogenesis and hepac glucose output

Fig.2.Potentialmechanismsleadingtoalteredlipidandglucosemetabolismresultingfromandcontributingtoinsulinresistanceandincreasedhepaticglucose productioninNAFLD.PanelA:adiposetissue,panelB:liver,andpanelC:intestine.(A)Adiposetissue.Synthesisoflipidintermediatesfromlong-chainfattyacids (LCFAs),e.g.,lyso-phosphatidicacid,phosphatidicacid(PA),di-acylglycerol(DAG)andtri-acylglycerol(TAG).SynthesisofvariousspeciesofDAGinparticular maypromoteadiposetissueinflammation.Increasedproductionoffreefattyacids(FFAs)andglycerolfromhydrolysisofTAGmaypromotehepaticgluconeogenesis andincreasehepaticglucoseoutput/production.(B)Liver.SynthesisoflipidintermediatesfromLCFAs,e.g.,ceramide,lyso-phosphatidicacid,phosphatidicacid (PA),di-acylglycerol(DAG),tri-acylglycerol(TAG)andverylow-densitylipoprotein(VLDL)secretion.SynthesisofvariousspeciesofDAG,inparticular,may promotehepaticinflammation;andDAGandpossiblyceramideanddi-palmitoyl-phosphatidicacid(Di-PPA)increasehepaticinsulinresistancebydecreasing efficientinsulinsignalling(e.g.viaDAG/PKC␧inhibitionofinsulinreceptorkinaseactivity)todecreaseglycogensynthesis.(C)Intestine.Dysbiosis,perhapscaused byalterationsindietandgeneticfactors,increasessynthesisofsecondarybileacidswithconsequenthepatictoll-likereceptor-4(TLR-4)activation,alteredfarnesoid Xreceptor(FXR)activityandsubsequentincreasedhepaticinflammation,fibrosis,increasedriskofcancerandincreasedhepaticglucoseproduction.InNAFLD andinsulinresistance,dysfunctionofglucagon-likepeptide(GLP-1)secretionandalteredshortchainfattyacid(SCFA)productionmayalsoadverselyinfluence hepaticgluconeogenesisandlipogenesis.

trialwithimprovementinliverhistologyinapproximately50% ofpatientswithnon-cirrhoticNASH[35].

3.1. Hepaticlipidaccumulation,insulinresistance,insulin clearance,adiposetissuelipolysisandhepaticglucose production

Althoughobesityisstronglyassociatedwithhepatic

steato-sis, excess body fat accumulation is not necessarily a

requirementfordevelopingNAFLD.Peoplewithlipodystrophy

havemarkedinsulinresistanceandcommonlydevelophepatic

steatosisandtype2diabetes,stronglysuggestingthatitisnot body fatquantity perse that is important,but it isabnormal

adipose tissue function that is a key contributor to NAFLD

pathogenesis [36]. In insulin sensitive individuals, postpran-dial increases in insulin concentrations are very effective in suppressinglipaseactivityinadiposetissuetodecreaseserum freefattyacid(FFA)concentrationsandtherebydecreasedFFA

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fluxes tothe liver.In contrast, ininsulinresistant individuals

there is decreased inhibition of insulin-mediated suppression

of FFAconcentrations withresultant increasedFFA fluxes to

the liver. Specifically, in the presence of insulin resistance, increased FFA fluxes tothe liver increase the availability of

long-chainfattyacyl-CoAsforhepaticlipidmetabolism.Such

aneffectoccursinsedentaryobeseindividuals,whodonot ben-efit fromthe ameliorating effectof increased muscle activity

andincreasedmitochondrialfattyacidoxidation,whichwould

normallybuffer theliverfrom theburden of highFFAlevels

[33].Withthe increasedfluxes oflong-chainfatty acylCoAs through the liver, evidenceis accumulatingthat hepatic lipid accumulationiscapableofpromotinghepaticinsulinresistance andhepaticinflammationthroughaccumulationofdi-acyl glyc-erols(DAGs)andproteinkinaseC␧(PKC-␧)activity,inhibiting

the insulin signalling pathway and promoting insulin

resis-tance(reviewed indetail in[28]).The conversionfrom TAG

to DAG is mediated by adipose triglyceride lipase (ATGL)

andcomparativeGeneIdentification-58(CGI-58)isan

activa-torofATGL.DAGactivatesPKC␧membranetranslocationto

inhibittheinsulinreceptorkinaseanddecreaseinsulinsignalling

[37].

With decreasedeffective insulinsignallingactivity in sub-jectswithinsulinresistance,thereisalsodecreasedpostprandial glycogensynthesis.Increasedfluxesofhepaticlong-chainfatty

acyl-CoAs also promote hepatic glucose production, thereby

increasingtheriskofglucoseintoleranceanddiabetes.Increased fluxesoflong-chainfattyacyl-CoAseffectsallostericactivation

of pyruvate carboxylase-phosphoenolpyruvate carboxykinase

activity withresultingstimulation ofhepatic gluconeogenesis

andincreased hepatic glucoseoutput.Additionally, increased

adiposetissuetri-acylglycerollipolysisalsoproducesincreased levelsofglycerolfromthehydrolysisofmoretri-acylglycerol molecules.Onceliberatedfromtri-acylglycerol,glycerolisnot re-esterifiedbackintotri-acylglycerolbytheadipocyte,because theadipocytedoesnotpossesstherequiredglycerolkinase activ-ity.Consequently,increasedtri-acylglycerolhydrolysisresults inincreasedglycerolfluxestotheliver,increasingthe

produc-tionofdihydroacetonephosphate,hepaticgluconeogenesisand

theproductionofhepaticglucose[28].

Hepatic lipid synthesis stimulated by increased fluxes of

long-chain acyl Co-As to the liver, increases production

of other lipid intermediates, besides DAGs, such as,

di-palmitoyl phosphatic acid and other lipid products, such as

ceramides [38]. Many of these lipid intermediates promote

hepatic inflammation[39–42] andincreasing risk of

progres-sive liverdisease that occurswithNASH. Hepaticlipids that

are not esterified also induce endoplasmic reticulum stress,

leading to the activation of c-Jun N-terminal kinases and

nuclearfactorkappa(NF-␬B)-light-chain-enhancerofactivated

B cells [43], which are two major regulators of

inflamma-tory pathways that also inhibit phosphorylation of insulin

receptorsubstrate(IRS)-1[44],potentiallyfurtheraggravating hepaticinsulinresistanceandincreasingintrahepaticcytokine production.

In the liver, ceramides utilise long-chain FAs [45] and

ceramidescanaccumulateintothecellsviathreemainroutes:

• thehydrolysisofthemembranephospholipidsphingomyelin,

whichiscoordinatedbytheenzymesphingomyelinase;

• denovosynthesisfromlong-chainFAssuchaspalmitateand

serine;

• a ‘salvage’ pathway that utilises sphingosine and forms

ceramide[46,47].

Althoughinthepast,itwasthoughtthatceramidewassimply astructuralmolecule,thereissomeevidencethatincreasesin

membraneceramidemaycauseinsulinresistance[48],

poten-tiallyprovidingamechanismtolinkhepaticlipidmetabolism

andhepaticinflammation.Recently,ithasbeensuggestedthat

differences in ceramide metabolism may also explain why

NAFLD withinsulinresistance isassociated withfeatures of

the metabolic syndrome; whereas the patatin-like

phospholi-pasedomain-containingprotein3(PNPLA3148MM)genotype,

associatedwithNAFLD,isnotassociatedwithmetabolic

syn-drome(andtherebywithnoincreasedriskoftype2diabetes)

[49].Insuchstudy,theliverlipidomewasanalysedfromtissue

obtainedfromliverbiopsiesin125subjectswhoweredivided

intosimilarlysizedgroupsbasedonmedianHOMA-IR(‘high

vs. lowHOMA-IR’, n=62andn=63)or PNPLA3genotype

(PNPLA3 148MM/MI,n=61vs. PNPLA3148II,n=64).The

results of this small experimental study showed that for the

‘highHOMA-IR’vs.‘lowHOMA-IR’groupcomparison,inthe

moreinsulinresistantgroup(thehighHOMA-IRgroup)theliver

wasmarkedlyenrichedwithsaturatedandmonounsaturated

tri-acylglycerols andfreefattyacids, dihydroceramides(markers

ofdenovoceramidesynthesis)andceramides[49].Clearly,the resultsofthisstudysuggestthatfurtherworkisneededtobetter

understandhepatic processingandremodelling of liverlipids

such as ceramides, andhow ceramides cause hepatic insulin

resistanceinpeoplewithgenotypessuchas PNPLA3148MM

thatisknowntoinfluenceliverdiseaseinNAFLD.

Besides increased insulinresistance in NAFLD, it is also

plausiblethatpartofthereasonforpatientswithtype2diabetes

andNAFLDoftenrequirelargedosesofexogenousinsulinto

helpcontrolplasmaglucoseconcentrations,isthatinsulin

clear-ance may alsobe increased inpatients withNAFLD. Recent

datasuggestthatincreasedinsulinclearancemayoccurin sim-plesteatosisandalsoinNASH[50].Theseinvestigatorsstudied hyperinsulinemiaanditsrelationshiptoliverhistologyin con-trolsubjects,patientswithsimplesteatosisandinpatientswith

NASH.PatientswithsimplesteatosisandNASHhadasimilar

∼30%reduction inhepaticinsulinclearance,whencompared

topatientswithoutNAFLD.Reducedhepaticinsulinclearance

was notassociated withseverityof inflammation,ballooning,

andfibrosis.Incontrasttohepaticinsulinclearance,worse histo-logicalinflammationandballooning(butnothepaticsteatosisor fibrosis)wereassociatedwithaprogressivereductionin whole-bodyinsulinclearance[50].

3.2. Secretedhepatokines,andtheroleofincretinsand glucagoninliverdiseaseandimpairedglucosemetabolism

Recently, it has been proposed that there are molecules

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Table2

PharmacologictreatmentsofNAFLD,withpotentialimpactonglucosemetabolism,testedinrandomizedclinicaltrialswithhistologicoutcomes. Author,year[Ref] Treatment,numberofcases.

Patientswithdiabetes

Treatment duration

Experimentaldrugeffecton liverdiseaseathistology

Effectsonglucosemetabolism anddiabetesparameters Bugianesietal.,

2005[72]

MET2g,55;Vit.E,28;Diet, 27.Only10caseswithT2DM

12months Vit.EandDietdidnot

producehistologicaleffects andwerecombinedas controls.METdecreasedliver fat(P<0.001),fibrosisand necro-inflammation (P=0.012)atfollow-upvs. controls

OnaverageMETdecreased fastingglucoseby10mg/dL andinsulinby4.5mU/mLvs.a limiteddecreaseofonly 5mg/dLand1.5mU/mLin otherarms,respectively

Belfortetal.,2006

[73]

PIO45mg,29;counselling,25. 55withIGT/T2DM

6months PIOsignificantlyimproved

steatosis,inflammation, ballooningandfibrosisvs. counselling(P<0.01forall)

PIOdecreasedfastingglucose by20mg/dL,A1cby0.7%and fastinginsulinby5mU/mLvs. unchangedinPL(P<0.01) Ratziuetal.,2008 [74] FLIRTtrial RSG8mg,32;PL,31.16with T2DM

12months RSGimprovedsteatosis

(P=0.014),without differencesinfibrosisor necro-inflammation RSGdecreasedglucoseby 16mg/dL,A1cby0.2%,insulin by5.5mU/mLvs.unchanged orincreasedinPL Aithaletal.,2008 [75] PIO30mg,37;PL,37.Cases withT2DMwereexcluded

12months PIOdecreased

necro-inflammation(PIOvs. PL,0.005)andMallory-Denk bodies(P=0.004)

HighPIOdecreasedglucoseby 7mg/dL,A1cby0.2%vs. +7mg/dLand+0.1%inPL (P<0.02) Haukelandetal., 2009[76] MET2.5–3g,24;Controls,24. Allcaseshadprediabetesor T2DM

6months Nodifferencesinsteatosis,

necro-inflammationorfibrosis wereobservedbetweenMET andcontrols

METproducedamoderate reductionofglucose (−5mg/dL)andHbA1c (−0.2%) Shieldsetal., 2009[77] MET0.5–1g9;Counselling, 10.T2DMsubjectswere excluded

12months METdidnotchangeNAFLD

activityscoreandits individualcomponents

ImprovedHOMA-IRvaluesin allpatients,withoutdifferences betweengroups Ratziuetal.,2010 [78] FLIRT2trial RSG,53(RSG-RSG,25; PL-RSG,28).Open-label extensionofFLIRT[72]

24months RSGdidnotproduceany

additionalhistological improvementbeyondthe1 yearliverbiopsy

Nochangesinbiochemistry wereobservedduringthe extensionperiod Sanyaletal.,2010

[79]

PIVENStrial

PIO30mg,87;Vit.E,84;PL, 83.AllsubjectshadNASH. Patientswithdiabeteswere excluded

96weeks Bothdrugswerebetterthan

PLonsteatosis(P<0.001) andlobularinflammation (P<0.01),notonfibrosis. HigherratesofNASH resolutionwerereportedon bothexperimentaldrugs

Improvedfastingglucoselevels andHOMA-IRwereobserved onPIOvs.PL

Lavineetal.,2011

[80]

TONICtrial

Paediatricstudy:Vit.E800UI, 57;MET1g,57;PL,58.No subjectshaddiabetesatentry

96weeks NASscoreimprovedinall

groups.HigherratesofNASH resolutionwereobservedon VitEinsubjectswithNASH atentry(58%vs.28%;

P=0.006).Onlyballooning improvedonMET

Noremarkabledifferencesin thetime-coursesoffasting glucoseorHOMA-IRwere reportedbetweengroups

Neuschwander-Tetrial.2014

[35]FLINT trial

OCA25mg,141;PL142.Trial stoppedforsuperiority:OCA, 102;PL,98.52%withT2DM

72weeks Thestudywasdiscontinued

earlyforefficacy:improved histology(steatosis,lobular inflammation,ballooning, fibrosis)in45%OCAvs.21% PL.DefiniteNASHresolution, 22%vs.13%(P=0.08)

Nochangesinfastingglucose betweengroups,butOCAwas associatedwithincreased insulinlevels(P=0.02vs.PL) andHOMA-IR Armstrongetal., 2015[58] LEANprogram LIRA1.8mg,26;PL,26.33% ofcaseshadT2DMandwere onchronicMET(+SULFAin 4%)

48weeks (extensionto 72weeks)

LIRAwasassociatedwith higherratesofimprovementin steatosis,ballooningand fibrosis.NASHresolution occurredin39%ofcaseson LIRAvs.9%inPL;P=0.02)

Weightloss,5.3kgonLIRAvs. 0.6onPL(P=0.003). Significantlyreducedglucose (−18mg/dL),A1c(−0.5%)vs. PL(P=0.005andP=0.03, respectively)

A1c:glycosylatedhaemoglobin;HOMA-IR:homeostasismodelassessment-insulinresistance;IGT:impairedglucosetolerance;LIRA:liraglutide;MET:metformin; OCA:obeticholicacid;PIO:pioglitazone;PL:placebo;RSG:rosiglitazone;T2DM:type2diabetesmellitus;VitE:vitaminE.

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glucosemetabolisminNAFLD.Thesefactors(orhepatokines) include fetuinA [51],adropin[52], angiopoietin-likeprotein

[53],fibroblastgrowthfactor-21[54],andselenoprotein[55].All ofthesehepatokineshavebeensuggestedaspotentialendocrine

mediators of insulin resistance and/or glucose intolerance in

NAFLD. Assuch, it hasbeen proposed that thesemolecules

havethe potentialtoadversely influenceglucosetolerancein

patientswithNAFLD.

Recentinteresthasalsofocussedonfetuin-Basahepatokine thatiscapableofhavinganendocrinefunctionbeyondtheliver toinfluenceadverselyinsulinsensitivityandcauseglucose

tol-erance.Arecentstudy of168 hepatokines,of which32 were

differentiallysecretedinsteatoticversusnon-steatotic

hepato-cytesshowedthatfetuinBwasincreasedinhumanswithliver

steatosisandinpatientswithtype2diabetes[56].These investi-gatorsthenshowedbothininvitroexperimentsandinmicethat fetuinBimpairedinsulinactionexvivoinmyotubesandin hepa-tocytes,andalsocausedglucoseintoleranceinvivoinmice.As proofofconceptthatfetuinBwasresponsibleforglucose intol-eranceinvivo,theythenshowedthatsilencingoffetuinBinthe

obesemiceimprovedglucosetoleranceintheseanimals[56].

Thus, thesedata strongly suggest that steatotic andinflamed

liverisabletosecreteahepatokinethatiscapableofhavingan endocrinefunctionbeyondthelivertoinfluenceadversely glu-cosetolerance.Itisplausiblethattheproteinsecretoryprofile

ofhepatocytesinNAFLDisalteredwithdevelopingsteatosis,

andtheproductionofhepatokinesislinkedtoinflammationand

insulinresistanceinNAFLD.

Theregulation of glucagonsecretioninvolvesthe gut

hor-moneglucagon-likepeptide-1(GLP-1),andintype2diabetes

fastinghyperglucagonaemiamaycontributetoincreasedhepatic glucoseproduction[57].Althoughthepreciseroleofglucagon

inNAFLDremainsuncertain,NAFLDisfrequentlyassociated

withabnormal glucose toleranceandthere has beenrecently

considerableinterestintheroleofincretins,suchasGLP-1,in

NAFLD.LiraglutideisonesuchGLP-1agonistthatisusedas

aoncedailytreatmentfor hyperglycaemiaintype2 diabetes.

AlthoughthereisnotgoodevidenceofGLP-1receptor

expres-sioninthehumanliver,thesafetyandefficacyofliraglutideat adoseof1.8mg/daywasrecentlytestedinasmall-randomised clinicaltrialinpatientswithbiopsy-confirmedNASH[58],as specifiedinthesectionbelow.

4. TreatmentoptionsforNAFLDthatmayinﬂuence riskoftype2diabetes

Thepastdecadehasbeencharacterizedbyanintensivesearch ofeffectivetreatmentsforNAFLD,fosteredbytheevidenceofa significantburdenofdiseaseforallnationalhealthcaresystems. Behaviouralchanges,includingdietaryrestrictionandincreased

physicalactivity, anddrug therapy arethe pillars of NAFLD

treatment.Theassessmentoftheireffectsontheriskofdisease progressionhasbeenlimitedbyseveralfactors:

• the lack of valid surrogate markers, making liver biopsy

mandatorytodemonstratesolidimprovements;

• treatment outcomes undefined at regulatory levels. Only

recentlytheoutcomeof NASHimprovementorregression,

withoutworseningoffibrosis,hadgainedgeneralconsensus

[59];

• limitedtreatmentduration(maximum,2years).Nonetheless,

asignificantbenefitofbothweightlossanddrug treatment onliverhistologyhasbeenreportedinrecentreviews[60,61], butonlyafewdataareavailableontheriskofprogressionto diabetes.

4.1. WeightlossanddecreaseddiabetesriskinNAFLD

Considering that overweight/obesity andinsulinresistance

are a hallmark of NAFLD, there is no surprise that weight

loss mayconsiderably affectdiabetesrisk.In mostcases, the

methodologyusedforpromotingweightlosswasthesameor

was strictly derived from the classical methodology used in

the Diabetes Prevention Program [62],wherea58% reduced

risk oftype2 diabeteswasmeasuredinsubjectswith

predia-betesover a4-yearfollow-upperiod [63].Inthe samestudy, therewasaconsistentdeclineofserumALTlevelsinrelation

to weightloss inthe entire cohort, andweight loss was

sig-nificantly associated with a decreased risk of abnormal liver

enzymesinsubjectswithnormalALTlevelsatbaseline[64].No datawerereportedonprogressiontodiabetesinrelationtoserum

ALTchanges,andcirculatingALTlevelsremain averycrude

markerof NAFLDandNAFLDprogression.In1087subjects

withultrasonography-detectedNAFLD,randomlyassignedto

alifestylemodificationinterventionbasedonphysicalactivity,

diet andbehaviourtherapywerecomparedwithusualcareon

NAFLDremission[65].Weightlosswaslargerinthe

interven-tion groupandwas associatedwithasignificant reduction of

serumliverenzymes,aswellasa30-mg/dLreductioninblood

glucose.Nodatawerereportedonriskofincidentdiabetes. Unfortunately,trialsof dietary orbehavioural treatment of

NAFLD rarely classify their population in terms of glucose

regulation as normal, prediabetes or diabetes, to verify the

effectiveness of thistreatment ondiabetes risk,andthe short

duration of intervention precludes any analysis.Vilar-Gomez

et al.recently reportedthe resultof an intensive, prospective behaviouralinterventionaimedatweightlossina1-year follow-upof293NASHpatients,observedatatertiarymedicalcentrein

Havana,Cuba[66].TheoutcomemeasurewasNASHresolution

withoutworseningoffibrosisinpairedliverbiopsies(available in261participants).Weightloss≥5%wasobservedin30%and theprobabilitytoachieveNASHresolutionwasstrictly

depend-entontheamountofweightloss(58%insubjectswithweight

loss≥5%;90% withweightloss≥10%).Only33% of cases

had type 2 diabetes, with another33% withprediabetes and

33% withnormal glucoseregulation [66].Follow-upglucose

datahavenotbeenreported,butitwillbelargelyimpossibleto demonstrateiftheexpectedreductionofincidentdiabetesisdue toNASHresolutionortoweightlossperse.

Many more data are available on diabetes risk in

rela-tion to bariatricsurgery-induced weightloss. In the Swedish

ObeseSubjectsinterventionstudy,surgeryproduceda

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[67];theeffectwasdirectlyassociatedwiththeamountofweight loss [68], notwithbaseline BMI [69].In the samecohort of

morbidlyobesesubjects undergoingsurgery, weightlosswas

accompaniedbyasystematicreductionofserumliverenzymes

at2-yearand10-yearfollow-up,consideredsurrogatemarkers

ofsteatosis,alsoproportionaltothedegreeofweightloss[70].

Inanotherrecentstudy,follow-upbiopsiesinmorbidlyobese

patients withNASHat baselinehaveshownthat the massive

weightlossinduced bybariatricsurgerywasaccompaniedby

NASHresolutionin85% ofcases, anda10-mg/dLreduction

ofbloodglucose.Notably,thosepatientswhodidnotachieve

NASHresolutionwerecharacterizedbylowerweightlossand

maintainedhighervaluesofinsulinresistance[71].In conclu-sion,alargebodyofevidencesuggeststodatethatweightloss, independentlyofthetherapeuticstrategy,significantlyimproves

NAFLDandNASHandreducestheriskofincidenttype2

dia-betes,buttheindependenteffectofimprovedliverdiseasehas

neverbeendemonstrated.

4.2. DrugtreatmentofNAFLDwithadeﬁnedimpacton glucosemetabolism

Considering that insulin resistance is the hallmark of

NAFLD,drugsactingonglucosemetabolism(Table2)havelong

beenconsideredforNAFLD treatment[35,58,72–80].Insulin

sensitizershavebeentestedinseveralclinicaltrialsofdifferent duration.Theyareverylikelytoreduceboththeriskofincident

diabetesandtoimprovemetaboliccontrolintheNAFLD

popu-lationwithoutandwithdiabetes,respectively.Thistherapeutic effecthasbeendemonstratedwithmetformin,whichisthebasis ofdiabetestreatmentandsignificantlyreducestheprogressionof prediabetestodiabetesinseverallargeinterventiontrials. How-ever,initialevidenceforaneffectofmetforminonNAFLD his-tologywasnotconfirmed[72,76,77,80],andtheeffectsof met-forminonliverdiseaseprogressionhavebeenquestioned[81].

Many more data are available with the

peroxisome-proliferatoractivatorreceptor-␥(PPAR-␥)agonists(glitazones) as insulin sensitizers (Table 2). They definitely reduce

pro-gression to diabetes in persons at risk, via enhanced insulin

sensitivity in skeletal muscles and in the liver as well as in

theadipose tissue,wheretheyalsopromoteadipocyte

differ-entiation. The first evidencewith glitazoneson NAFLD was

producedin2001, asmall pilot trial reported reduced serum

ALTlevels in10patients treatedwithtroglitazone,butminor orno effectson liverhistology[82].Ninecaseswerefree of diabetes,andtroglitazonewashighlyeffectiveinreducing pro-gressionfromprediabetestodiabetesinatreatmentarminside

theDiabetes PreventionProgram [83].Troglitazonewas later

withdrawnfromthemarket,butbothrosiglitazoneand

piogli-tazonetreatmentinthe earlyprediabetesphasehaveshowna

beneficialeffecton diabetesrisk prevention[84],andare the mostextensivestudieddrugsonNASHtreatment[73–75,78,79].

Alsoacarbosetreatment reducesthe riskof diabetes

develop-ment[85],butnodatahavebeenraisedonhistologicoutcomes

ofNAFLDandnoeffectshavebeendemonstratedonhepatic

biochemistry.

Otherdrugshavemorerecentlyenteredthearena.Thedual

PPAR-␣/␦agonistGFT-505(Elafibranor®)showedpotentially

useful resultsin asmall pilot study inobesity, where it also demonstratedaninsulin-sensitizingactivity[86].GFT-505has

recently completed a phase IIb trial withpositive results on

NAFLDhistology,andthepublicationoffinalresultsiseagerly awaited.Bileacidshavebeenreportedtoregulatehepaticlipid

andglucosemetabolismthroughFXR,amemberofthenuclear

hormonereceptorsuperfamily,andTGR5,aGprotein-coupled

bile acid receptor 1. Whereas no solid data are available for

a beneficial effect of ursodeoxycholic acid on histology in

NASH patients, obeticholic acid has recently been reported

to promote NASH remission in aphase II study [35].

How-ever, no advantage on glucosemetabolism was reported and

the use of this compound may be limited by some

impor-tantsideeffects(e.g.,pruritusandincreaseinLDL-cholesterol concentrations).

Themostinterestingdrugsforapotentiallyfavourableeffect onbothglucoseandliverdiseaseistheclassofincretin mimet-ics.Therationalefortheiruse(boththeglucagon-likepeptide-1

receptor agonists [GLP-1Ra] and the dipeptidyl peptidase-4

inhibitors [DPP-4i]) in NAFLD comes from a report of a

reduced expression of GLP-1 receptors in the biopsies from

NASHpatients,incomparisonwithcontrolbiopsies[87].

GLP-1Ra and DPP-4i are also likely to improve NAFLD through

improvedinsulinsensitivity[88].InitialstudiesinNASHwere carriedoutwiththeshort-actingGLP-1Raexenatide;inapilot study it produced apositiveeffect onliver histopathologyin

three out of 8 cases [89]. The long-acting GLP-1Ra

liraglu-tidehasnowcompletedaphaseIIbtrial,showingasignificant

positive effecton NASHremission, independent of the

pres-ence of diabetes [58]. The mechanism(s) might be mediated

by improved insulin sensitivity andreduced hepatic de novo

lipogenesis[90].

Atpresent,severalphaseIIstudiesarebeingplannedto

mea-sure the long-term effects of different GLP-1Ra in NAFLD,

havingthestrictlydefinedhistologicaloutcomeofNASH remis-sionasprimaryoutcome.Futurestudiesshouldalsocomparethe

relativeeffectsofGLP-1Ratreatmentincomparisontoweight

loss programs inobesesubjects,whereas drug treatment will

remainthetreatmentofchoiceinleanNAFLD[91].

5. Conclusions

Inthisupdatedreviewofthepublishedstudies,weprovide

furthersupportforthestrongassociationbetweenNAFLDand

increased risk of new-onsettype 2 diabetes,thoughcausality

remainstobeproveninwell-controlledprospectiveand

inter-vention studies. Future studies are also needed to assess the

potentialaddedvalueofusingthepresenceofNAFLDto

pre-dict therisk ofincident type2diabetesbeyondthat provided

by commonrisk factorsfor diabetes.Finally, inthe presence

ofNAFLD,therapeuticstrategiesshouldtackleboththeriskof advancedliverdiseaseandtheriskoftype2 diabetes,inturn responsibleforliverdiseaseprogression.

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Authorcontributions

GT conceivedand designed the study.GT, GM andCDB

researcheddataandwrotethemanuscript.

Disclosureofinterest

Theauthorsdeclarethattheyhavenocompetinginterest.

Acknowledgments

GTissupportedinpartbygrantsfromtheUniversitySchool

of Medicine of Verona, Verona, Italy. CDB is supported in

partbytheSouthamptonNationalInstituteforHealthResearch

BiomedicalResearchCentre.

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