Branched-Chain and Aromatic AminoAcidsArePredictorsof InsulinResistancein Young AdultsPETER W€URTZ, PHD

1,2

PASI SOININEN, PHD2,3

ANTTI J. KANGAS, MSC2

TAPANI RÖNNEMAA, MD, PHD4

TERHO LEHTIMÄKI, MD, PHD5

MIKA KÄHÖNEN, MD, PHD6

JORMA S. VIIKARI, MD, PHD4

OLLI T. RAITAKARI, MD, PHD7,8

MIKA ALA-KORPELA, PHD2,3

OBJECTIVEdBranched-chain and aromatic amino acids are associatedwith the risk for futuretype 2 diabetes; however, the underlying mechanisms remain elusive. We tested whether aminoacids predict insulin resistance index in healthy young adults.

RESEARCHDESIGNANDMETHODSdCirculating isoleucine, leucine, valine, phenyl-alanine, tyrosine, and six additional amino acids were quantified in 1,680 individuals from thepopulation-based Cardiovascular Risk in Young Finns Study (baseline age 32 6 5 years; 54%women). Insulin resistance was estimated by homeostasis model assessment (HOMA) at baselineand 6-year follow-up. Amino acid associations with HOMA of insulin resistance (HOMA-IR) andglucose were assessed using regression models adjusted for established risk factors. We furtherexamined whether amino acid profiling could augment risk assessment of insulin resistance(defined as 6-year HOMA-IR .90th percentile) in early adulthood.

RESULTSdIsoleucine, leucine, valine, phenylalanine, and tyrosine were associated withHOMA-IR at baseline and for men at 6-year follow-up, while for women only leucine, valine,and phenylalanine predicted 6-year HOMA-IR (P, 0.05). None of the other amino acids wereprospectively associated with HOMA-IR. The sum of branched-chain and aromatic amino acidconcentrations was associated with 6-year insulin resistance for men (odds ratio 2.09 [95% CI1.38–3.17]; P = 0.0005); however, including the amino acid score in prediction models did notimprove risk discrimination.

CONCLUSIONSdBranched-chain and aromatic amino acids are markers of the develop-ment of insulin resistance in young, normoglycemic adults, with most pronounced associationsfor men. These findings suggest that the association of branched-chain and aromatic amino acidswith the risk for future diabetes is at least partly mediated through insulin resistance.

Diabetes Care 36:648–655, 2013

Insulin resistance is a core defect in thepathogenesis of type 2 diabetes, andthe condition is associated with in-

creased risk for diabetes already in earlyadulthood (1–4). In addition, impairedinsulin sensitivity in young adults fre-quently precedes a dyslipidemic profileand contributes to development of

cardiovascular disease (5,6). Studies onthe etiology of insulin resistance have pre-dominantly focused on lipid-inducedmechanisms (1) and the interplay withobesity (2). Nonetheless, several studieshave indicated a prominent amino acidsignature of insulin resistance, even innormoglycemic individuals (7–12).

Furthermore, the circulating concentra-tions of branched-chain amino acids (iso-leucine, leucine, and valine) and aromaticamino acids (phenylalanine and tyrosine)were recently shown to be associated withthe risk of future hyperglycemia and overtdiabetes (13–15). However, it remains in-completely understood how these aminoacids mediate the risk for development ofdiabetes. In order to illuminate the role offasting state amino acid levels in patho-genesis of insulin resistance and type 2diabetes, we investigated whether aminoacids are predictors of insulin resistanceindex as well as fasting glucose levelscross-sectionally and after 6-year follow-up in young, apparently healthy individ-uals from a population-based cohort.

RESEARCH DESIGN ANDMETHODSdThe Cardiovascular Riskin Young Finns Study was designed toinvestigate the contribution of early liferisk factors to the risk of cardiovasculardiseases (16). The first survey in 1980 in-cluded 3,596 children and adolescentsaged 3–18 years representatively selectedfrom rural and urban parts of Finland.Participants attending follow-up surveysin both 2001 (baseline) and 2007 wereeligible for inclusion in the present longi-tudinal study (n = 1,809). This study pop-ulation was representative of the originalcohort (17). BMI and blood pressure weremeasured, and current smoking statusand physical activity were obtained fromself-reported questionnaires. Physical ac-tivity index was computed based on thefrequency and intensity of physical activ-ity as previously described (18). Bloodsamples were drawn after a 12-h fast. Tri-glycerides and HDL cholesterol werequantified by nuclear magnetic resonance(NMR) spectroscopy (19), and apolipo-protein (apo)A1 and -B andhigh-sensitivityC-reactive protein were measured by stan-dard assays (16). Fasting plasma insulinwas analyzed by microparticle enzyme im-munoassay kit (Abbott Laboratories) andglucose enzymatically (Olympus AU400).Insulin resistance index was estimated byhomeostasis model assessment (HOMA) ofinsulin resistance (HOMA-IR) using the

c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c

From the 1Institute forMolecularMedicine Finland, University of Helsinki, Helsinki, Finland; 2ComputationalMedicine, Institute of Clinical Medicine, University of Oulu, Oulu, Finland; the 3NMR MetabolomicsLaboratory, School of Pharmacy, University of Eastern Finland, Kuopio, Finland; the 4Department ofMedicine, University of Turku and Turku University Hospital, Turku, Finland; the 5Department of ClinicalChemistry, University of Tampere and Tampere University Hospital, Tampere, Finland; the 6Department ofClinical Physiology, University of Tampere and Tampere University Hospital, Tampere, Finland; the7Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Turku, Finland;and the 8Department of Clinical Physiology, Turku University Hospital, Turku, Finland.

Corresponding author: Peter W€urtz, peter.wurtz@helsinki.fi.Received 9 May 2012 and accepted 9 August 2012.DOI: 10.2337/dc12-0895© 2013 by the American Diabetes Association. Readers may use this article as long as the work is properly

cited, the use is educational and not for profit, and thework is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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HOMA2 calculator (www.dtu.ox.ac.uk/homacalculator/), which applies a struc-turalmodel used to solve the nonlinear em-pirical equations describing glucoseregulation (20). Individuals missing insu-lin, glucose, or amino acid quantification(n = 17); pregnant women (n = 80); andstudy participants on lipid-lowering medi-cations (n = 7) or treatment for diabetes atbaseline (n = 18) or follow-up (n = 7) wereexcluded from analyses, leaving 1,680 in-dividuals for the current study. Of these, 2individuals had type 2 diabetes at baseline,as did an additional 14 individuals at 6-yearfollow-up based on fasting glucose .7.0mmol/L. All participants gave written in-formed consent, and the study was ap-proved by the local ethics committees andconducted in accordance with the Declara-tion of Helsinki.

Amino acid quantificationAmino acid concentrations were quanti-fied by high-throughput serum NMRspectroscopy (19). The serum sampleswere stored at 2808C and thawed over-night in a refrigerator prior to samplepreparation. A proton NMR spectrumwas acquired where spectral signalsfrom the macromolecules and lipoproteinlipids were suppressed to enhance detec-tion of the amino acid signals. Nine aminoacids (isoleucine, leucine, valine, phenyl-alanine, and tyrosine as well as alanine,glutamine, glycine, and histidine) werequantified in millimoles per liter byNMR. Details of the spectroscopy andamino acid quantification have previouslybeen described (19,21,22). In addition,arginine (n = 1,678) and tryptophan (n =742) concentrations were determined byisocratic andreverse-phasehigh-performanceliquid chromatography, respectively, aspreviously described (23,24).

Statistical analysesClinical characteristics for men andwomen were compared using two-tailedt test and Kolmogorov-Smirnov test fornormally distributed and skewed vari-ables, respectively. Amino acid concen-trations quantified from fasting serumsamples collected in 2001 were used toassess associations with HOMA-IR andfasting glucose at baseline as well as at6-year follow-up. Analyses were stratifiedby sex, as amino acids are known to ex-hibit sex-specific associations with insulinresistance (9,12). Amino acids, triglycer-ides, and HOMA-IR were log transformedprior to linear regression analyses. Foreach amino acid, a linear regression

model was fitted with HOMA-IR or fast-ing glucose as outcome and the aminoacid as explanatory variable with BMI,systolic blood pressure, HDL cholesterol,triglycerides, smoking status, and physi-cal activity index as covariates. Prospec-tive associations with 6-year HOMA-IRand 6-year fasting glucose as outcomeswere further adjusted for baselineHOMA-IR and glucose, respectively. Toassess the combined effect of thebranched-chain and aromatic aminoacids, an amino acid score was calculatedas the sum of isoleucine, leucine, valine,phenylalanine, and tyrosine concentra-tions in millimoles per liter. Regressioncoefficients are reported in standardizedunits of 1-SD difference in HOMA-IRor fasting glucose (outcome) per 1-SDdifference in amino acid concentration(predictor).

For prediction of the highest extent of6-year insulin resistance evidenced inyoung adults, a dichotomous score wasdefined as 6-year HOMA-IR .90th per-centile using sex-specific values. This def-inition of insulin resistance corresponds

to fasting insulin$17.7 IU/L for men and$15.9 IU/L for women in this study pop-ulation. Odds ratios (ORs) of amino acidswith 6-year insulin resistance were as-sessed using logistic regression models.The incremental value of amino acid pro-filing for prediction of insulin resistancewas further examined. A reference modelof established risk factors was derived bybackward stepwise selection of the fol-lowing variables: baseline HOMA-IR,age, BMI, triglycerides, HDL cholesterol,apoA1 and apoB, C-reactive protein, sys-tolic blood pressure, smoking status, andphysical activity index (P , 0.05 for re-tention). The best-fitting model was com-pared with a model with the amino acidscore included. The ability to discriminaterisk for 6-year insulin resistance in thetwo models was compared using area un-der the receiver operating characteristiccurve (AUC), net reclassification im-provement (NRI), and integrated dis-crimination improvement (IDI) (25).For NRI, participants were assigned tofour categories (,5, 5–10, 10–20, and.20%) that reflected their 6-year risk for

Table 1dBaseline characteristics of the study population

Clinical characteristics Men Women P

n 769 911Age (years) 31.8 (5.0) 32.1 (4.9) 0.32BMI (kg/m2) 25.6 (3.9) 24.4 (4.5) 4 3 1028

Systolic blood pressure (mmHg) 121 (12) 113 (13) 1 3 10239

Triglycerides (mmol/L) 1.2 (0.9–1.8) 1.0 (0.8–1.3) 7 3 10218

HDL cholesterol (mmol/L) 1.2 (0.3) 1.4 (0.3) 2 3 10256

Fasting glucose (mmol/L) 5.1 (4.9–5.4) 4.90 (4.6–5.1) 3 3 10226

Fasting insulin (IU/L) 6 (4–9) 6 (5–9) 0.89HOMA-IR 0.8 (0.5–1.2) 0.8 (0.6–1.2) 0.776-year fasting insulin (IU/L) 7.2 (4.5–10.8) 6.8 (4.1–10.5) 0.186-year HOMA-IR 1.0 (0.6–1.4) 0.9 (0.5–1.4) 0.08Branched-chain amino acidsIsoleucine (mmol/L) 65 (56–76) 50 (43–58) 3 3 10275

Leucine (mmol/L) 97 (85–110) 77 (68–88) 1 3 10281

Valine (mmol/L) 230 (210–260) 190 (170–220) 4 3 10269

Aromatic amino acidsPhenylalanine (mmol/L) 76 (69–85) 75 (68–83) 5 3 1024

Tyrosine (mmol/L) 59 (52–67) 52 (45–61) 4 3 10226

Gluconeogenic amino acidsAlanine (mmol/L) 410 (370–460) 410 (370–460) 0.75Glutamine (mmol/L) 550 (510–600) 500 (450–550) 5 3 10236

Glycine (mmol/L) 290 (270–330) 300 (270–360) 5 3 10212

Other amino acidsHistidine (mmol/L) 72 (66–79) 70 (63–78) 2 3 1026

Arginine (mmol/L) 110 (85–130) 110 (89–130) 0.09Tryptophan (mmol/L) 92 (83–100) 84 (76–93) 2 3 10210

Data are means (SD) and median (25th–75th percentile) for skewed variables. P values for comparison ofmen and women are t tests for normally distributed variables and Kolmogorov-Smirnov tests for skewedvariables.

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insulin resistance. Log-likelihood ratio x2

was used to estimate improvement inglobal model fit. Prediction models wereevaluated using 10-fold cross-validationto avoid an individual’s risk assessment be-ing influenced by outcome status (22).Analyses were performed with MATLAB7.10 (MathWorks, Natick, MA), and statis-tical significance was inferred as two-tailedP , 0.05.

RESULTSdA total of 1,680 individualsfree of treatment for diabetes (of whom911 were women [54%]) were includedin this study. Clinical characteristics areprovided in Table 1. The median baselineinsulin level of 6 IU/L reflects the youngage and generally healthymetabolic status

of the study participants. Associations ofthe amino acid score, defined as the sumof branched-chain and aromatic aminoacid concentrations, and the 11 assayedamino acids with insulin resistance indexat baseline and 6-year follow-up areshown in Fig. 1. Cross-sectionally,branched-chain and aromatic aminoacids as well as alanine were associatedwith HOMA-IR after adjustment for con-ventional metabolic risk factors. Forwomen, glutamine, glycine, and histidinewere also associated with baseline insulinresistance index. The associations ofbranched-chain and aromatic aminoacids were most pronounced for men;the magnitude of association for thebranched-chain and aromatic amino

acid score was twice as strong for men(b = 0.24) than for women (b = 0.12),where the b-regression coefficient indi-cates difference in SDs of HOMA-IR per1-SD difference in amino acid concentra-tion. This corresponds to a difference of1.2 IU/L fasting insulin for men and 0.65IU/L for women per 1-SD difference in theamino acid score when adjusting for es-tablished risk factors.

Prospective associations of the aminoacids with 6-year HOMA-IR are illus-trated in Fig. 1. The longitudinal analyseswere adjusted for baseline HOMA-IR inaddition to conventional metabolic riskfactors. The amino acid score as well asthe five branched-chain and aromaticamino acids were predictors of 6-year

Figure 1dCross-sectional and prospective associations of amino acids with HOMA-IR in the Cardiovascular Risk in Young Finns Study. Linearregression models were adjusted for baseline age, BMI, systolic blood pressure, HDL cholesterol, triglycerides, smoking status, and physical activityindex. Prospective associations (filled circles) for insulin resistance index at 6-year follow-up were further adjusted for baseline HOMA-IR.Association magnitudes are in standardized units of 1 SD HOMA-IR per 1 SD amino acid concentration. Error bars indicate 95% CIs. Amino acidscore: sum of isoleucine, leucine, valine, phenylalanine, and tyrosine concentrations. *P , 0.05; +P , 0.005.

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HOMA-IR for men, whereas only leucine,valine, and phenylalanine displayed sig-nificant prospective associations forwomen (P , 0.05). The magnitudes ofassociation in longitudinal analyses wereapproximately half of the effect sizesfound cross-sectionally when accountingfor baseline insulin resistance index. Im-portantly, none of the other six assayedamino acids were predictors of HOMA-IR at 6-year follow-up (Fig. 1).

Cross-sectional and prospective asso-ciations of amino acids with fasting glu-cose are shown in Fig. 2. Several aminoacids were associated with baselineglycemia for both men and women, in-cluding the branched-chain and aromaticamino acid score and alanine; yet, the as-sociation magnitudes were less prominentthan those observed for HOMA-IR. In pro-spective analyses, however, none of thebranched-chain and aromatic amino

acids were predictors of 6-year fastingglucose in these young adults (P . 0.05for all) in contrast to the results observedfor insulin resistance index. The only sig-nificant association with 6-year glucoselevels was the inverse association of glu-tamine observed for women (P = 0.005).

Essentially similar results were foundthroughout when limiting analyses tonormoglycemic subjects (fasting glucose,5.6 mmol/L; n = 1,508) or individuals

Figure 2dAssociations of amino acids with baseline and 6-year fasting glucose. Linear regression models were adjusted for baseline age, BMI,systolic blood pressure, HDL cholesterol, triglycerides, smoking status, and physical activity index. Prospective associations (filled squares) werefurther adjusted for baseline glucose. Association magnitudes are in standardized units of 1 SD glucose per 1 SD difference in amino acid con-centration. Error bars indicate 95% CIs. *P , 0.05; +P , 0.005.

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without the metabolic syndrome (n =1,486) as defined by the Harmonized def-inition (26) (data not shown). All resultswere virtually identical when HOMA-IRwas replaced by fasting plasma insulinas outcome.

Prediction of high extent of insulinresistanceA high extent of insulin resistance in thisyoung study population was defined as6-year HOMA-IR.90th percentile. Withuse of this definition, 168 individualswere classified with insulin resistance atfollow-up. ORs of each amino acid for6-year insulin resistance are shown inFig. 3. For men, the ORs were in linewith the results from the continuous ana-lyses, whereas for women no significant

associations with a high degree of 6-yearinsulin resistance were observed (Fig. 3).The multivariate OR for 6-year insulin re-sistance of the amino acid score for men(OR 2.09 [95% CI 1.38–3.17]; P =0.0005) was greater than the OR of BMI(1.72 [1.23–2.39]; P = 0.001) and com-parable with that of baseline HOMA-IR(2.07 [1.45–2.96]; P = 0.00007). Isoleu-cine was the most pronounced predictorof high extent of insulin resistance at6-year follow-up, as in the case of thecontinuous associations.

Predictionmodels for a high degree of6-year insulin resistance in young adultswith and without a score of branched-chain and aromatic amino acids are com-pared in Table 2. The best-fitted referencemodelwas composed of baselineHOMA-IR

and BMI, and for men themodel addition-ally included apoB and physical activityindex. The reference model AUCs wererelatively high for both men and women,indicating that it is difficult to improve riskassessment. Indeed, inclusion of theamino acid score did not result in a signif-icant change in risk discrimination (AUC)or improved reclassification (NRI andIDI), although in the case of men themodel fit was improved (x2 = 10.4, P =0.002). Because there is no consensus onwhat signifies clinically significant insulinresistance in young adults, we furtherexamined the predictive performance ofthe models using 6-year HOMA-IR$85thand 95th sex-specific percentiles to denoteinsulin resistance, with similar results ob-tained (data not shown).

CONCLUSIONSdThis study demon-strates that circulating branched-chainand aromatic amino acid levels predictinsulin resistance index 6 years later innormoglycemic individuals even whenaccounting for baseline insulin resistanceand conventional risk factors. A set of fivebranched-chain and aromatic aminoacids have recently been linked with therisk for hyperglycemia and diabetes inolder populations (13–15); our resultssuggest that the amino acids mediate therisk for future diabetes because they aremarkers for the development of insulinresistance. Despite the prospective associ-ations with impaired insulin sensitivity,our findings also indicate limited addedvalue of amino acid profiling for the pre-diction of insulin resistance in youngadults.

Accumulating evidence suggests thatbranched-chain and aromatic aminoacids are associated with the extent ofinsulin resistance and hyperglycemia at asingle time point (8–15,27,28). Alreadyseveral decades ago, a study found thatobese individuals had elevated concentra-tions of branched-chain and aromaticamino acids and that these amino acidscorrelated with insulin levels (7). Weand others have recently demonstratedpronounced cross-sectional associationsof amino acids with HOMA-IR with sex-and obesity-related effects (8–12). Here,our prospective analyses indicate thatbranched-chain and aromatic aminoacids predict future impairment in insulinsensitivity in young adults. Circulatingamino acid concentrations have been re-ported to be lower in overweight adoles-cents with type 2 diabetes in comparisonwith normal-weight peers (29). Our

Figure 3dORs and 95% CIs for insulin resistance at 6-year follow-up (HOMA-IR $90th per-centile) per 1 SD difference in baseline amino acid concentration. Logistic regression models wereadjusted for baseline HOMA-IR, age, BMI, systolic blood pressure, HDL cholesterol, triglycerides,smoking status, and physical activity index. *P , 0.05; +P , 0.005.

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population-based results could, however,suggest that elevated branched-chain andaromatic amino acid levels precede thedevelopment of insulin resistance alreadyin early adulthood. In a smaller studywithsimilar follow-up time, but consisting ofolder Finnish individuals, we have shownthat branched-chain and aromatic aminoacids predict future glycemia in the gen-eral population (13). In contrast, no asso-ciations were observed with fastingglucose at 6-year follow-up in theseyoung adults. Taken together, these re-sults indicate that altered branched-chainand aromatic amino acid metabolism isassociated with impairment of insulinsensitivity prior to effects observed forimpaired glucose levels.

Intravenous administration and oralingestion of multiple amino acids areknown to modulate insulin secretionacutely (30,31); however, such effectshave not been reported for fasting aminoacid levels in prospective settings. For in-stance, arginine is an effective stimulus forinsulin secretion (30); yet, no associationwas observed between arginine and insu-lin resistance index. Indeed, none of theother assayed amino acids predicted6-year insulin resistance (Fig. 1). Al-though insulin secretion may potentiallyconfound the reported associations withHOMA-IR, we have previously shownthat circulating amino acid concentra-tions in the fasting state are associated pri-marily with insulin sensitivity rather thaninsulin secretion (13,14). The majority ofthe study population (90%) was withinnormoglycemic range, and exclusion ofsubjects with impaired fasting glucosedid not alter our findings. b-cell dysfunc-tion is therefore unlikely to underpin the

associations established in this study (32).Hence, our findings support the notionthat the risk for future diabetes inducedby the amino acids is at least in part me-diated through insulin resistance.

Whether branched-chain and aro-matic amino acids are involved in thedevelopment of insulin resistance in afunctional manner remains to be estab-lished. Despite the prospective associa-tions with HOMA-IR, these findings donot imply that the amino acids are mech-anistically involved in the pathogenesis.Several physiological and animal studieshave suggested direct promotion of in-sulin resistance by branched-chain aminoacids (8,27,28); however, genetic evi-dence has thus far not provided supportfor a causal role of branched-chain aminoacids in the pathogenesis (12). It is plau-sible that the prospective associationswith insulin resistance occur as a resultof indirect effects rather than beingcaused by altered amino acid levels. Sincefasting insulin and glucose are regulatedby compensatory mechanisms, elevationsin branched-chain and aromatic aminoacid concentrations could potentially pre-cede observations of high insulin re-sistance index and thereby serve aspredictors of impaired insulin sensitivity(13). The observational nature of thisstudy, however, precludes assessment ofcause and effect, and further functionalstudies are required to elucidate themechanisms underlying the links be-tween amino acids and the risk of insulinresistance and overt diabetes.

In addition to branched-chain andaromatic amino acids, alanine and gluta-mine (inversely) have also recently beenassociated with future hyperglycemia and

the risk for diabetes (11,13,14). While wefound significant cross-sectional associa-tions of these amino acids with insulinresistance index, the prospective associa-tions were nonsignificant after adjust-ment for baseline HOMA-IR (Fig. 1).Nonetheless, glutamine was inversely as-sociated with 6-year glycemia in women(Fig. 2). Insulin resistance has also beensuggested to underpin the associationwith future diabetes in the case of alanineand glutamine (14). Our results couldsuggest that altered concentrations ofthese gluconeogenic amino acids arelinked to impairment of insulin sensitivityin a later stage of the disease progressionthan perturbed branched-chain and aro-matic amino acid metabolism.

Associations of amino acids withHOMA-IR were markedly different formen and women (Figs. 1 and 3). Thesefindings corroborate prior reports on sexinteractions of certain amino acids withinsulin resistance (9,12). While the abso-lute concentrations of branched-chainand aromatic amino acid were signifi-cantly higher in men, a similar range ofinsulin resistance was observed for bothsexes (Table 1). We have previously dem-onstrated that association of branched-chain amino acids with insulin resistanceis significant for young women only ifthey are obese (12); yet, although similarcross-sectional findings were observedhere, no such obesity interaction wasfound in prospective analyses (data notshown). Differences in gestational program-ming have been suggested to underpinthe sex interactions for branched-chainamino acids with insulin resistance (9);however, the molecular underpinningsare still to be determined, and our findings

Table 2dPrediction of 6-year insulin resistance with and without branched-chained and aromatic amino acid score

AUC (95% CI) PAUC† NRI (%) PNRI† IDI (%) PIDI† x2 Px2†

MenReference model: baselineHOMA-IR, BMI, apoB,and physical activity index 0.868 (0.826–0.911)

Reference model plus aminoacid score 0.876 (0.836–0.915) 0.34 5.8 0.29 1.7 0.08 10.4 0.002

WomenReference model: baselineHOMA-IR and BMI 0.858 (0.815–0.900)

Reference model plus aminoacid score 0.856 (0.813–0.899) 0.08 21.2 0.36 20.10 0.25 21.6 0.40

Amino acid score: sum of isoleucine, leucine, valine, phenylalanine, and tyrosine concentrations in millimoles per liter. Insulin resistance was defined as HOMA-IR$90th percentile at 6-year follow-up. x2: Log-likelihood ratio compared with reference model. †Comparison between reference model and model including aminoacid score.

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highlight attention to sex differences in therole of amino acids in the pathogenesis ofinsulin resistance.

Early identification of individuals athigh risk for diabetes is of importance forprevention (33). The sum of branched-chain and aromatic amino acid concen-trations was recently shown to predictincidence of diabetes independent ofbaseline metabolic risk factors (15). Fur-ther, adding the amino acid score toestablished risk factors improved risk iden-tification in a study sample of matchedhigh-risk individuals; however, risk dis-crimination was not augmented in a gen-eral population setting (15). In line withthese findings, our results indicate thatbranched-chain and aromatic amino acidsare predictors of a high extent of insulinresistance for men (Fig. 2). However, de-spite an improvement in model fit by in-clusion of the amino acid score in theprediction model, our results suggest thatthe incremental value for predictionmay belimited in young adults (Table 2). Never-theless, the clinical utility of amino acidprofiling for the prediction of incident di-abetes in older populations remains to bedetermined (34).

A limitation in this study is the use ofHOMA-IR as a surrogate of insulin re-sistance. However, it has been shown thatfasting measures are adequate surrogatesof insulin resistance assessed by clamptest in the Finnish population (35). Theabsence of insulin secretion assessmentlimits correction of the prediction modelsto establish independent prospective as-sociations with insulin resistance; yet, wehave previously shown that fasting aminoacid concentrations are primarily associ-ated with insulin sensitivity rather thaninsulin secretion (13,14). We acknowl-edge that there is no established definitionof what constitutes clinically significantinsulin resistance, in particular for theyoung age-group studied; however, simi-lar results were obtained using alternativedefinitions to signify the highest extent ofinsulin resistance. Due to the apparentlyhealthy status of the young study popula-tion, statistical power was inadequate toassess the 6-year risk for type 2 diabetes.Strengths of the study include the youngpopulation-based cohort free of medica-tion and prospective data to address asso-ciations of amino acids in an early stage ofthe pathogenesis of insulin resistance.

In summary, we have shown thatcirculating branched-chain and aromaticamino acids from fasting serum are predic-tors of insulin resistance index, but not

glycemia, at 6-year follow-up in youngadults, with the most pronounced associ-ations observed for men. Adding a scoreof branched-chain and aromatic aminoacids to established risk factors for riskassessment of early insulin resistance didnot improve discrimination or classifi-cation. These results suggest that alteredbranched-chain and aromatic amino acidmetabolism precedes the development ofinsulin resistance already in early adult-hood prior to the occurrence of impairedfasting glucose, and this could at least partlyexplain how these amino acids are linkedwith the risk of future type 2 diabetes.

AcknowledgmentsdThisworkwas supportedby the Academy of Finland (grants 250422,137870, 126925, 121584, and 124282) andthe Research Programme of the Academy ofFinland Responding to Public Health Chal-lenges (grants 129269, 129429, and 129378),the Instrumentarium Science Foundation, thePaulo Foundation, the Juho Vainio Founda-tion, the Paavo Nurmi Foundation, the Jennyand Antti Wihuri Foundation, the Social In-surance Institution of Finland, Kuopio Tam-pere and Turku University Hospital MedicalFunds, the Finnish Foundation for Cardio-vascular Research, and the Strategic ResearchFunding from the University of Oulu.No potential conflicts of interest relevant to

this article were reported.P.W. researched data, wrote the manu-

script, and reviewed, commented on, and ac-cepted themanuscript. P.S., A.J.K., andM.A.-K.designed the amino acid profiling platform,analyzed NMR experiments, and reviewed,commented on, and accepted the manuscript.T.R., T.L., M.K., J.S.V., and O.T.R. providedclinical data and interpretations and re-viewed, commented on, and accepted themanuscript. P.W. is the guarantor of thiswork and, as such, had full access to all thedata in the study and takes responsibility forthe integrity of the data and the accuracy ofthe data analysis.

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