the study was composed of two finnish population - based cohorts , the pieksmki cohort and the health 2000 study . the pieksmki cohort originally consisted of 1,294 subjects from the town of pieksmki in eastern finland ( 13,14 ) . the eligible population consisted of all individuals born in 1942 , 1947 , 1952 , 1957 , and 1962 , of which 71% ( 923 ) participated in the initial examination in 1997 and 690 of these attended a follow - up survey in 20032004 . the health 2000 study is a finnish cross - sectional health survey carried out in 20002001 ( 15 ) . the overall health 2000 study ( 8,028 persons ) was representative of the finnish population 30 years of age and above . a supplemental study of 1,353 participants 4676 years of age was carried out in order to study diabetes risk factors more thoroughly ; this group was analyzed in the current study . in total , 2,090 participants from the two cohorts had glucose tolerance and comprehensive metabolite data measured . all individuals treated for diabetes ( n = 46 ) or on lipid medication ( n = 162 ) were excluded from analyses . the two studies served as mutual replication of cross - sectional findings . in the longitudinal substudy , 618 individuals ( 59% women ) from the pieksmki cohort had data on oral glucose tolerance test ( ogtt ) at the 6.5-year follow - up ( 19972003 ) and were not treated pharmacologically for diabetes . all participants gave written informed consent , the study protocols were approved by the local ethics committees , and the studies were conducted in accordance with the declaration of helsinki . all participants underwent a 2-h ogtt using a 75-g glucose load with the zero time point in a fasting condition . plasma glucose concentrations were determined using an automated hexokinase method ( intercoefficient of variation [ inter - cv ] 1.9% ) ( peridochrom glucose god - pap ; boehringer ingelheim , mannheim , germany ) for the pieksmki cohort and a dehydrogenase assay for the health 2000 study ( inter - cv 1.7% ) ( merck , darmstadt , germany ) . insulin concentrations at 0 , 30 , and 120 min during the ogtt were determined by radioimmunoassay ( inter - cv 5.7% ) ( phadeseph insulin ria ; pharmacia , uppsala , sweden ) in the health 2000 study . alanine aminotransferase was assayed by a kinetic method using a cobas 6000 analyzer ( hitachi , tokyo , japan ) . circulating metabolites were quantified from fasting serum samples using high - throughput nuclear magnetic resonance ( nmr ) spectroscopy ( 16 ) . this nmr platform has been applied in various epidemiological and genetic studies ( 6,1721 ) . detection of small molecule solutes from native serum , such as various amino acids and glycolysis substrates , was enabled by spectroscopy settings that suppress the broad spectral signals from lipoprotein particles . lipid constituents and the diversity of fatty acid saturation were measured from serum lipid extracts ( 17 ) . baseline and follow - up samples from the pieksmki cohort were measured in a single measurement series ( 13 ) . details of the nmr experimentation and metabolite quantification have been described previously ( 1618 ) . peripheral insulin sensitivity index was estimated based on matsuda isi ( 22 ) and insulin secretion index calculated as total insulin area under the curve per total glucose area under the curve during the ogtt ( 3 ) . metabolite tracking ( the likelihood of maintaining the original fractile over time ) was assessed with spearman correlation coefficients between the baseline and follow - up concentrations . associations of circulating metabolites with glucose levels as outcomes were assessed with linear regression models separately for each metabolite . all models were adjusted for sex , age , bmi , systolic blood pressure , hdl cholesterol ( hdl - c ) , and triglycerides . adjustment for hdl - c and triglycerides was omitted for serum lipid extract measurements to minimize collinearity . cross - sectional analyses were conducted separately for the two cohorts and combined using inverse variance  weighted meta - analysis assuming fixed effect size . for these cross - sectional analyses , an additional criterion was that each metabolite was nominally significant ( p < 0.05 ) in both cohorts with concordant direction of effect . we tested for sex differences but did not find significant metabolite  sex interactions . to ease comparison of effects , -regression coefficients are reported throughout in units of 1-sd increase in glucose levels ( outcome ) per 1-sd increase in metabolite concentration ( predictor ) . metabolites displaying cross - sectional associations with glycemia were analyzed for association with insulin sensitivity and insulin secretion indices for 975 individuals from the health 2000 study . the same metabolites were further analyzed for the subset of 618 individuals with longitudinal data on glucose tolerance at the 6.5-year follow - up . changes in metabolite levels during the follow - up period were tested for association with changes in glucose levels . finally , the metabolites were analyzed to assess whether baseline metabolite levels would be predictive of fasting and postload glucose at the 6.5-year follow - up . these longitudinal analyses were also adjusted for baseline glucose and insulin levels , and statistical significance was inferred for two - tailed p < 0.05 . all participants underwent a 2-h ogtt using a 75-g glucose load with the zero time point in a fasting condition . plasma glucose concentrations were determined using an automated hexokinase method ( intercoefficient of variation [ inter - cv ] 1.9% ) ( peridochrom glucose god - pap ; boehringer ingelheim , mannheim , germany ) for the pieksmki cohort and a dehydrogenase assay for the health 2000 study ( inter - cv 1.7% ) ( merck , darmstadt , germany ) . insulin concentrations at 0 , 30 , and 120 min during the ogtt were determined by radioimmunoassay ( inter - cv 5.7% ) ( phadeseph insulin ria ; pharmacia , uppsala , sweden ) in the health 2000 study . alanine aminotransferase was assayed by a kinetic method using a cobas 6000 analyzer ( hitachi , tokyo , japan ) . circulating metabolites were quantified from fasting serum samples using high - throughput nuclear magnetic resonance ( nmr ) spectroscopy ( 16 ) . this nmr platform has been applied in various epidemiological and genetic studies ( 6,1721 ) . detection of small molecule solutes from native serum , such as various amino acids and glycolysis substrates , was enabled by spectroscopy settings that suppress the broad spectral signals from lipoprotein particles . lipid constituents and the diversity of fatty acid saturation were measured from serum lipid extracts ( 17 ) . baseline and follow - up samples from the pieksmki cohort were measured in a single measurement series ( 13 ) . details of the nmr experimentation and metabolite quantification have been described previously ( 1618 ) . peripheral insulin sensitivity index was estimated based on matsuda isi ( 22 ) and insulin secretion index calculated as total insulin area under the curve per total glucose area under the curve during the ogtt ( 3 ) . metabolite tracking ( the likelihood of maintaining the original fractile over time ) was assessed with spearman correlation coefficients between the baseline and follow - up concentrations . associations of circulating metabolites with glucose levels as outcomes were assessed with linear regression models separately for each metabolite . all models were adjusted for sex , age , bmi , systolic blood pressure , hdl cholesterol ( hdl - c ) , and triglycerides . adjustment for hdl - c and triglycerides was omitted for serum lipid extract measurements to minimize collinearity . cross - sectional analyses were conducted separately for the two cohorts and combined using inverse variance  weighted meta - analysis assuming fixed effect size . for these cross - sectional analyses , an additional criterion was that each metabolite was nominally significant ( p < 0.05 ) in both cohorts with concordant direction of effect . we tested for sex differences but did not find significant metabolite  sex interactions . to ease comparison of effects , -regression coefficients are reported throughout in units of 1-sd increase in glucose levels ( outcome ) per 1-sd increase in metabolite concentration ( predictor ) . metabolites displaying cross - sectional associations with glycemia were analyzed for association with insulin sensitivity and insulin secretion indices for 975 individuals from the health 2000 study . the same metabolites were further analyzed for the subset of 618 individuals with longitudinal data on glucose tolerance at the 6.5-year follow - up . changes in metabolite levels during the follow - up period were tested for association with changes in glucose levels . finally , the metabolites were analyzed to assess whether baseline metabolite levels would be predictive of fasting and postload glucose at the 6.5-year follow - up . these longitudinal analyses were also adjusted for baseline glucose and insulin levels , and statistical significance was inferred for two - tailed p < 0.05 . baseline characteristics of the 1,873 individuals from the two population - based studies are shown in table 1 . the study population represents middle - aged , community - dwelling men and women free of lipid - lowering medication and diabetes treatment . absolute concentrations and interquartile ranges of the fasting metabolite levels in both cohorts and at the follow - up survey are given in supplementary table 1 along with tracking coefficients of the metabolites over the 6.5-year follow - up . most metabolites displayed tracking comparable to that of glucose during the follow - up period , suggesting that the metabolite levels are not solely reflecting immediate dietary intake . baseline characteristics of the study population metabolites associated with fasting and/or 2-h glucose at baseline are listed in table 2 . in total , 19 metabolite measures were cross - sectionally associated with glycemia ( meta - analyzed p < 0.001 ) and nominally significant with the same direction of effect in both cohorts . the significant metabolites comprise a spectrum of gluconeogenesis substrates , branched - chain and aromatic amino acids , 1-acid glycoprotein , as well as fatty acid species and saturation measures . metabolites were positively associated with increased glycemia with the exceptions of glycine , -6 fatty acids / total fatty acids , and double bonds / fatty acid chain . association magnitudes reached up to  = 0.19 for fasting as well as postload glucose , where the unit of -regression coefficient indicates increase in sds of glucose ( 0.9 and 2.1 mmol / l for fasting and 2-h glucose , respectively ) per 1-sd increase in metabolite concentration . for comparison , the association magnitude of bmi with glycemia was  = 0.21 for both fasting and postload glucose , yet the metabolite associations were adjusted for this established risk factor . the majority of metabolites displayed similar magnitude of association for fasting and postload glucose ; however , fatty acid metabolites tended to be more strongly associated with postload glycemia . further , the associations were generally stronger for amino acids and gluconeogenesis precursors than for the fatty acid measures . to assess whether the fasting state metabolite levels reflect postload glycemia independently of fasting glucose , we tested 2-h glucose associations with additional adjustment for fasting glucose , as shown in table 2 . albeit attenuated , most metabolite associations remained significant , in particular for gluconeogenic substrates and fatty acid measures . associations of all analyzed metabolites with fasting and 2-h glucose are illustrated separately for the two cohorts along with cross - sectional associations at the follow - up survey in supplementary fig . the metabolite associations were coherent for both cohorts , and the associations remained essentially unaltered at the follow - up survey , thus indicating consistency of the associations across study setting and time . cross - sectional associations of metabolites with fasting and postload glucose deficiencies of insulin action and insulin secretion may underpin the metabolite associations with fasting and postload glucose ( 6,23 ) . associations of the metabolites with peripheral insulin sensitivity and total insulin secretion indices in the health 2000 study are shown in supplementary fig . most of the metabolites were associated with both insulin sensitivity and total insulin secretion during ogtt ; however , all associations with insulin secretion vanished when conditioning on insulin sensitivity , whereas the insulin sensitivity associations remained significant upon conditioning on insulin secretion . the regulation of fasting metabolite levels may follow changes in glucose tolerance over time ( 24 ) . associations between the changes in metabolites and changes in glucose during the follow - up period are shown in fig . the changes in the majority of the metabolites were associated with changes in fasting glucose during the 6.5-year follow - up period , and changes in fatty acid metabolites also paralleled changes in postload glucose . notably , changes in branched - chain amino acids and phenylalanine levels were not associated with changes in glycemia during follow - up . changes in metabolite concentrations vs. change in fasting glucose ( blue triangle ) and 2-h glucose ( red triangles ) during 6.5-year follow - up adjusted for sex , age , baseline bmi , systolic blood pressure , hdl - c , triglycerides , insulin , and glucose . association magnitudes are in units of 1-sd change in glucose level per 1-sd change in metabolite concentration . the metabolites linked with glycemia in cross - sectional analyses ( table 2 ) could potentially be markers of perturbations in glucose homeostasis associated with glucose tolerance later in life . to address this question , we tested whether the metabolites would be predictive of 6.5-year fasting and 2-h glucose in 618 individuals from the pieksmki cohort who attended follow - up . results of these longitudinal analyses are shown for significant metabolites ( p < 0.05 ) in table 3 . branched - chain amino acids predicted both 6.5-year fasting and postload glycemia (  = 0.14/0.11 for fasting/2-h glucose for the sum of leucine , isoleucine , and valine ) . the most prominent association was observed for leucine (  = 0.18/0.15 , corresponding to 0.11/0.24 mmol higher glucose per 17 mol / l leucine higher than the median ) . in addition , phenylalanine and 1-acid glycoprotein were prospectively associated both with fasting and 2-h glucose ( p < 0.05 ) . in contrast , alanine , lactate , and pyruvate as well as tyrosine were exclusive predictors of 6.5-year postload glucose ( p < 0.05 for all ) . of notice , none of the fatty acid measures exhibited significant associations in prospective settings . interestingly , the prospective associations displayed a pattern opposite to that observed for the changes of metabolites with glycemia ; the changes in those metabolites , which were predictors of either fasting or postload glycemia , were not associated with the changes in the corresponding glucose level during the follow - up period ( fig . the ability of the metabolites to predict 6.5-year glycemia may be compared with the predictive ability of baseline glucose for estimating glycemia at follow - up . the prospective metabolite associations were up to 40% of the magnitude of that for baseline glucose versus 6.5-year glycemia ( table 3 ) . the moderate association magnitudes are partly accounted for by the adjustment for baseline glucose as well as insulin and other established risk factors . thus , the associations were not solely driven by the small number of individuals with overt diabetes . the results were virtually unchanged when excluding individuals on blood pressure  lowering medication and hormone treatment ( data not shown ) . this metabolic profiling study identified 19 circulating metabolite measures associated with fasting or postload glucose in a general population setting of middle - aged men and women . amino acids , gluconeogenesis precursors , and fatty acids displayed metabolic footprints of both elevated fasting glucose and attenuated glucose tolerance . alanine , lactate , pyruvate , and tyrosine were selective predictors of 6.5-year postload glucose , suggesting that these metabolites could be markers of deteriorating glucose tolerance later in life . branched - chain amino acids predicted both fasting and postchallenge glucose levels , confirming their suspected role as markers of future glycemia ( 7 ) . multiple metabolic abnormalities are associated with the progression toward type 2 diabetes ( 25 ) . hence , it is to be expected that circulating levels of numerous metabolites are reflective of the degree of glycemia ( 5 ) . our results exemplify the diversity of metabolites associated with glucose levels in a nondiabetic population independent of conventional metabolic risk factors ( table 2 ) . despite differences in cardiovascular morbidity and mortality between individuals with impaired fasting glucose and impaired glucose tolerance , their cardiometabolic risk factor profiles are similar ( 4,26 ) . correspondingly , we found the cross - sectional metabolite associations with baseline fasting and 2-h glucose to be of comparable magnitude . importantly , the fasting metabolite concentrations were reflecting the degree of postchallenge glucose , thus indicating that glucose tolerance may partly be observed in the fasting metabolite profile . however , the small effect sizes also suggest that ogtt response can not be accurately inferred from the fasting metabolite profile . gluconeogenesis plays a pivotal role in glucose homeostasis ( 27 ) . in this study , three precursors of gluconeogenesis , alanine , lactate , and pyruvate , were predictors of postchallenge glucose after 6.5-year follow - up ( table 3 ) . the molecular mechanisms of how these gluconeogenic substrates are related to future glucose tolerance remain elusive . lactate has been associated with the prevalence of diabetes in older adults , and decreased oxidative capacity has been proposed to underpin this association ( 28 ) . fasting levels of alanine and pyruvate are highly correlated ( r = 0.59 ) , which could suggest that the metabolites are reporters of the same pathophysiological effect . on the other hand , pyruvate is involved in multiple glycolytic pathways , including pyruvate cycling pathways in the mitochondria that might underlie the prospective association ( 25,29 ) . the interconversion between pyruvate and alanine is catalyzed by alanine amino aminotransferase , a liver enzyme for which the plasma levels have been linked with obesity , liver fat extent , and the risk for diabetes ( 21,30 ) ; however , the correlations between alanine aminotransferase and alanine and pyruvate were modest ( r = 0.12 for both metabolites ) . alanine , lactate , and pyruvate were predictive of postchallenge glucose , but not fasting glycemia , at the follow - up survey . elevation of postload glucose is characterized by peripheral insulin resistance , whereas individuals with isolated high fasting glucose have been linked with attenuated insulin secretion and hepatic insulin resistance ( 1,3,812 ) . the prominent associations of the gluconeogenesis substrates with the peripheral insulin sensitivity index support this connection ( supplementary fig . on the other hand , essentially all the metabolites linked with glycemia were associated with impaired insulin sensitivity rather than insulin secretion . these results therefore do not fully elucidate the differences between fasting and postload glycemia observed in the longitudinal analyses . despite the cross - sectional associations with insulin sensitivity , the gluconeogenesis substrates remained predictors of postload glucose even when adjusting for baseline glucose and insulin . because glucose regulation is essential in metabolic fuel homeostasis , the circulating levels of gluconeogenesis precursors could potentially reflect impaired insulin sensitivity prior to elevation of fasting glucose and insulin levels . analyses of the changes in metabolites during follow - up indicated that alanine , lactate , and pyruvate levels were following changes in fasting but not postload glucose ( fig . 1 ) . these findings support that the gluconeogenic substrates contain a component of information that is not only related to immediate glucose levels but also reflects future glucose tolerance . in a nested case - control setting , alanine was predictive of incident diabetes more than a decade later ( p = 0.04 ) ( 7 ) . together with our population - based results , this suggests that metabolic perturbations causing elevated circulating alanine , lactate , and pyruvate could be early markers of attenuated glucose tolerance that may eventually progress to diabetes . circulating levels of five branched - chain and aromatic amino acids have been associated with insulin resistance ( 6,23,31,32 ) and reported to predict the development of future diabetes ( 7 ) . leucine , isoleucine , valine , phenylalanine , and tyrosine were all associated with glycemia and insulin sensitivity at baseline and predictors of fasting and/or postchallenge glucose in this study . these findings indicate that the amino acids are not only markers of the risk for overt diabetes but also predictors of future glycemia in the general population . importantly , the changes in branched - chain amino acids and phenylalanine concentrations did not parallel changes in fasting glucose during the follow - up . thus , whereas fasting glucose changes are accompanied by altered levels of fatty acids and gluconeogenic substrates , the regulation of branched - chain amino acids appears to be less tightly connected to changes in glycemia ( fig . 1 ) . these results suggest that abnormalities in branched - chain amino acid metabolism precede the development of hyperglycemia , and this may partly explain how the amino acids mediate the risk for future diabetes . experimental animal studies have suggested a mechanistic role of branched - chain amino acids in the development of insulin resistance ( 32 ) ; however , it remains unsettled whether the amino acids are causally implicated in humans . although our results are compatible with a functional role of branched - chain and aromatic amino acids in the pathogenesis of hyperglycemia , the elevated amino acid concentrations could also potentially be secondary markers of metabolic abnormalities that may not be fully observed in baseline glucose and insulin levels from a single time point . in this relation , we have recently shown that branched - chain amino acids as well as alanine , pyruvate , and 1-acid glycoprotein all are regulated by a variant in the gene encoding glucokinase regulatory protein ( gckr ) , which is suspected to affect the glucose sensory mechanism and indirectly affect lipid and amino acid metabolism ( 6,20,21,33 ) . in a similar manner , the prospective associations of amino acids and gluconeogenesis substrates with glycemia could reflect perturbations in glucose sensing , which eventually may lead to hyperglycemia . low - grade , systemic inflammation has been associated with the risk for diabetes ( 34,35 ) . the acute - phase protein 1-acid glycoprotein ( orosomucoid ) is an abundant immunomodulator protein in circulation , which is induced by stressful conditions such as infections and inflammation , and elevated levels have been found in diabetic patients ( 36,37 ) . expression of 1-acid glycoprotein has been linked with metabolic signaling , including hyperglycemia , and has been suggested to modulate immune responses to protect adipose tissue from inflammation and metabolic dysfunction ( 37 ) . in this study , 1-acid glycoprotein was prospectively associated with both fasting and postchallenge glucose ( table 3 ) . 1-acid glycoprotein has previously been linked with the risk for incidence of diabetes in the atherosclerosis risk in communities ( aric ) study ; however , the association was not independent of baseline bmi , insulin , and glucose ( 35 ) . our results suggest that 1-acid glycoprotein is not only related to metabolic dysfunction cross - sectionally but also a predictor of future glycemia , and thus highlight the role of prolonged inflammation as a risk marker for attenuating glucose tolerance . the role of metabolic dysfunction in the pathogenesis of diabetes has primarily focused on lipoproteins and lipid - induced mechanisms ( 5,38,39 ) . in contrast to this notion , the metabolites predictive of glycemia in this study were amino acids and gluconeogenesis substrates rather than lipid composition measures . comprehensive lipid profiling was recently used to identify a pattern of triacylglycerides associated with insulin resistance and the risk for future diabetes ( 8) . our results are somewhat surprising in this respect ; whereas several fatty acid species and saturation measures were cross - sectionally associated with glycemia and impaired insulin sensitivity ( table 2 and supplementary fig . however , the triacylglycerol signature linked with the risk for diabetes was composed of lipid species not quantifiable by the high - throughput metabolic profiling platform . these results may suggest that information on the molecular character of lipid components , including specific chain length and saturation content , is beneficial for elucidating the etiology of lipid - mediated hyperglycemia . as a limitation , we note that our study was inadequately powered to assess the risk for incidence of diabetes . although the cross - sectional associations were mutually replicated in two cohorts , the prospective associations merit further validation in independent populations . insulin action was approximated by ogtt - based surrogates , which have limited ability to separate contributions from peripheral and hepatic insulin resistance ( 1 ) . our study was conducted in a homogenous finnish population and care must be taken before generalization to other age groups and ethnicities . in conclusion , high - throughput profiling revealed a diverse metabolic footprint of glycemia in middle - aged men and women . alanine , lactate , and pyruvate were predictors of postchallenge glucose after a 6.5-year follow - up , indicating that early signs of attenuating glucose tolerance may be observed in gluconeogenesis precursors quantified from fasting serum samples . 1-acid glycoprotein as well as branched - chain and aromatic amino acids predicted both fasting and postload glycemia , but the changes in amino acid levels did not follow glucose changes over time . these findings support the notion that alterations in amino acid metabolism precede perturbations in glucose homeostasis , and the circulating amino acid levels may serve as predictors of future glycemia in a general population setting .
objectivemetabolite predictors of deteriorating glucose tolerance may elucidate the pathogenesis of type 2 diabetes . 
 we investigated associations of circulating metabolites from high - throughput profiling with fasting and postload glycemia cross - sectionally and prospectively on the population level.research design and methodsoral glucose tolerance was assessed in two finnish , population - based studies consisting of 1,873 individuals ( mean age 52 years , 58% women ) and reexamined after 6.5 years for 618 individuals in one of the cohorts . 
 metabolites were quantified by nuclear magnetic resonance spectroscopy from fasting serum samples . 
 associations were studied by linear regression models adjusted for established risk factors.resultsnineteen circulating metabolites , including amino acids , gluconeogenic substrates , and fatty acid measures , were cross - sectionally associated with fasting and/or postload glucose ( p < 0.001 ) . among these metabolic intermediates , branched - chain amino acids , phenylalanine , and 1-acid glycoprotein 
 were predictors of both fasting and 2-h glucose at 6.5-year follow - up ( p < 0.05 ) , whereas alanine , lactate , pyruvate , and tyrosine were uniquely associated with 6.5-year postload glucose ( p = 0.0030.04 ) . 
 none of the fatty acid measures were prospectively associated with glycemia . 
 changes in fatty acid concentrations were associated with changes in fasting and postload glycemia during follow - up ; however , changes in branched - chain amino acids did not follow glucose dynamics , and gluconeogenic substrates only paralleled changes in fasting glucose.conclusionsalterations in branched - chain and aromatic amino acid metabolism precede hyperglycemia in the general population . 
 further , alanine , lactate , and pyruvate were predictive of postchallenge glucose exclusively . 
 these gluconeogenic precursors are potential markers of long - term impaired insulin sensitivity that may relate to attenuated glucose tolerance later in life .