Serum biomarkers from cell-based assays for AhRL and MIS strongly predicted the future development of diabetes in a large community-based prospective study in Korea

Exposure to environment-polluting chemicals (EPC) is associated with the development of diabetes. Many EPCs exert toxic effects via aryl hydrocarbon receptor (AhR) and/or mitochondrial inhibition. Here we investigated if the levels of human exposure to a mixture of EPC and/or mitochondrial inhibitors could predict the development of diabetes in a prospective study, the Korean Genome and Epidemiological Study (KoGES). We analysed AhR ligands (AhRL) and mitochondria-inhibiting substances (MIS) in serum samples (n = 1,537), collected during the 2008 Ansung KoGES survey with a 4-year-follow-up. Serum AhRL, determined by the AhR-dependent luciferase reporter assay, represents the contamination level of AhR ligand mixture in serum. Serum levels of MIS, analysed indirectly by MIS-ATP or MIS-ROS, are the serum MIS-induced mitochondria inhibiting effects on ATP content or reactive oxygen species (ROS) production in the cultured cells. Among 919 normal subjects at baseline, 7.1% developed impaired glucose tolerance (IGT) and 1.6% diabetes after 4 years. At the baseline, diabetic and IGT sera displayed higher AhRL and MIS than normal sera, which correlated with indices of insulin resistance. When the subjects were classified according to ROC cut-off values, fully adjusted relative risks of diabetes development within 4 years were 7.60 (95% CI, 4.23–13.64), 4.27 (95% CI, 2.38–7.64), and 21.11 (95% CI, 8.46–52.67) for AhRL ≥ 2.70 pM, MIS-ATP ≤ 88.1%, and both, respectively. Gender analysis revealed that male subjects with AhRL ≥ 2.70 pM or MIS-ATP ≤ 88.1% showed higher risk than female subjects. High serum levels of AhRL and/or MIS strongly predict the future development of diabetes, suggesting that the accumulation of AhR ligands and/or mitochondrial inhibitors in body may play an important role in the pathogenesis of diabetes.

chemicals 10 , with the nature of these mixtures differing between populations and over time. Experimental studies have shown that EPC mixtures have substantial toxic effects in vivo at concentrations 100-fold or more below their no-observed-adverse-effect-levels (NOAELs) 11 , but little is known about the health risks of the EPC mixture in humans 10 . Understanding the link between EPC mixtures and the diseases requires large prospective studies with a series of measurements of a wide range of EPCs.
Having successfully determined the levels of human exposure to EPC mixtures by in vitro incubation of the cultured aryl hydrocarbon receptor (AhR)-dependent luciferase reporter cells with human serum samples 12,13 , we applied these bioassays to the prospective epidemiologic study. Since many of these EPC, EDC, and MDC chemicals are ligands of AhR transcription factor, serum AhR ligand (AhRL)-mediated luciferase bioactivity can be used as a biomarker for exposure level to EPC mixtures composed of various AhR ligands 14 in humans. Our previous studies showed that serum AhRL, which was linearly correlated with the toxic equivalency (TEQ) value of the tested persistent organic pollutant (POP) mixtures, was higher in Koreans with impaired glucose tolerance (IGT) and diabetes than those with normal glucose tolerance (NGT) 13 , and was associated with components of metabolic syndrome and insulin resistance 15 . AhRL had a positive correlation with serum insulin and HOMA-IR, and a negative correlation with adiponectin 15 . There is also strong evidence that exposure to EPC can cause mitochondrial dysfunction in cells and animals 16,17 .
Human studies of individuals with insulin resistance, both those with established diabetes and IGT, have consistently demonstrated structural or functional defects in the mitochondria 18,19 . Although it is not clear whether mitochondrial defects are the primary cause or secondary to subtle defects in glucose metabolism, insulin resistance, or impaired insulin secretion in the early stages of disease development 20 , mitochondrial dysfunction is believed to be involved in the pathogenesis of diabetes. Regardless of whether the chemicals in serum were AhR ligands, some of them inhibited mitochondrial activity in cells 12 . Incubating cells with mitochondria-inhibiting substances (MIS) decreases intracellular ATP content and increases DCF-DA-labelled reactive oxygen species (ROS) levels 12 . Thus, the levels of MIS in a serum sample could be indirectly determined by other cell-based assays measuring intracellular ATP concentration and/or ROS production in the cultured cells treated with the human serum sample. The resulting levels of ATP and ROS represent indirectly how much MIS are present in serum samples. To avoid confusion from endogenous ATP and ROS contents in the sample, we defined the outputs of the ATP and ROS assays as MIS-ATP and MIS-ROS, respectively. The resulting MIS-ATP and MIS-ROS levels also significantly correlated with TEQ of the tested POP mixtures 12,13 .
In the current study, we investigated whether serum AhRL, MIS-ATP, and MIS-ROS could predict the future development of IGT and diabetes using serum samples collected from a large, well-characterized, community-based prospective epidemiologic study, the Korean Genome and Epidemiology Study (KoGES) 21 . We report evidence that serum AhRL and MIS are important predictive factors of diabetes. These data suggest the inhibition of mitochondrial function by EPC mixtures might be a key mechanism leading to diabetes.

Methods
Detailed descriptions of the methods are presented in the Supplementary Methods. Study participants. The Ansung cohort of KoGES was established to investigate the genetic and environmental aetiology of common, complex diseases in Koreans. The results of KoGES are open to the public, and a summary of this study was published 22 . The data used in this study were downloaded from the KoGES depository with permission (KoGES; 4851-302). We analysed all the available samples, 1,537 sera collected during oral glucose tolerance test (OGTT) in 2008, to maximize the statistical power. The definitions of NGT, IGT, and diabetes were based on the results of the 75 g OGTT and the WHO criteria 23,24 . We then compared data obtained from samples collected during the 2012 follow-up study.
AhR ligand (AhRL) bioactivity assay. The pGL4-DRE-luc(puromycin+)/pRL-mTK double-positive stable cells and heat-inactivated serum samples were prepared as described previously 12 . The AhRL assay is similar to the CALUX assay 25 , except utilizing the different recombinant cell lines and organic solvent extraction-free sample preparation method. All cell-based assays were performed in duplicate on blinded samples. The intra-and inter-assay coefficients of variation for AhRL were less than 5.0%.

MiS-Atp and MiS-RoS assays for serum-induced mitochondria inhibition. Levels of MIS
in serum samples were evaluated by measuring intracellular ATP content (MIS-ATP) and ROS generation (MIS-ROS) as described 12,13 . pRL-mTK-transfected mouse Hepa1c1c7 cells (5 × 10 4 /well) in a 96-well plate were treated with 10 μL heat-inactivated-serum samples for 48 h. The ATP content was determined using the CellTiter-Glo luciferase kit (Promega, Madison, WI, USA), with the output being normalized to Renilla luciferase activity. ROS level was determined using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate and acetyl ester (CM-H 2 DCFDA; Molecular Probes, Eugene, OR, USA). Both MIS-ATP and MIS-ROS were expressed as % of charcoal stripped serum (CSS)-treated control. The intra-and inter-assay coefficients of variation for these methods were less than 6.0%.

Statistical analysis.
Results are presented as means ± SD, as numbers and percentages, or as RR with 95% CI. Student's t-tests or ANOVA were used to compare the means of continuous variables among the groups. Tukey post hoc tests were performed to assess which pairs of groups showed significant differences. Comparisons of the proportions of categorical variables among the groups were performed using the χ 2 test or logistic regression. Optimal cut-off values of AhRL and MIS-ATP for the prediction of diabetes development were estimated using receiver-operating characteristics (ROCs) and the Youden method 26 . RRs represented by AhRL or MIS-ATP for diabetes development within 4 years were analysed with logistic regression, with or without adjustment Results clinical characteristics and serum biomarkers of the participants. When the participants (n = 1,537) were classified according to glucose tolerance, 919 (59.8%) had NGT, 244 (15.9%) had IGT, and 374 (24.3%) had diabetes. The clinical characteristics of the participants are shown in Supplementary Table S1. The average age of the study subjects was 61.4 ± 8.2 years (men = 45.1%). Most of the parameters related to insulin resistance and inflammation, including BMI, blood pressure, HbA1c, HOMA-β, HOMA-IR, triglycerides, ALT/ AST, and hsCRP, of participants in the NGT group were significantly different from those in the IGT or diabetes groups. Smoking status and alcohol intake were slightly higher in diabetes group. Exercise rate was not different among three groups. Table 1 shows the mean levels of three serum biomarkers for AhRL (TCDDeq, pM), MIS-ATP (% of CSS-treated control), and MIS-ROS (% of CSS-treated control) according to glucose tolerance state at 2008. IGT and diabetes groups showed 1.8-2.8-fold increase of AhRL compared to the NGT group (p < 0.001, Supplementary Fig. S1). MIS-ATP of the IGT and diabetes groups were 12-15% lower than the NGT group (p < 0.001). MIS-ROS in the IGT and diabetes groups were 10-16% higher than the NGT group (p < 0.001). No gender differences were observed in mean levels of AhRL, MIS-ATP, and MIS-ROS ( Fig. 1a,b, Supplementary  Fig. S1).

correlations of AhRL, MiS-Atp and MiS-RoS with clinical parameters in the baseline survey.
AhRL across the whole group showed significant positive correlations with age, obesity, systolic blood pressure, blood glucose concentrations, insulin resistance, triglycerides, liver dysfunction, and inflammation, while it showed negative correlations with HOMA β-cell function and HDL ( Table 2). Correlation of AhRL with blood glucose levels was moderately strong (r = 0.41~0.51, p < 0.001). MIS-ATP significantly, but negatively correlated with these clinical parameters (r = −0.28 ~ −0.36, p < 0.001). MIS-ROS was significantly and weakly correlated with most of the parameters analysed, but not with age, total cholesterol, or liver dysfunction. These results suggested that AhRL, MIS-ATP, and MIS-ROS may influence the status of obesity, hyperglycemia, insulin resistance, β-cell function, and inflammation of the subjects. It was noted that LDL was correlated with MIS-ATP and MIS-ROS, but not with AhRL. Differences with respect to gender were not observed for the correlations of most parameters, however, only in women, AhRL and MIS-ATP correlated with liver dysfunction (Supplementary  Table S2). Since ALT and AST were higher in men than in women, other factors such as smoking or alcohol may affect the increases of ALT and AST in men. AhRL, but not MIS-ATP, was significantly higher in ex-smokers and former alcohol drinkers (Supplementary Table S3). MIS-ROS was significantly higher in ex-smokers and in people that exercised regularly. All other clinical parameters showed weak or moderate correlation with the three biomarkers, implying that they are independent parameters.  Table 2, Supplementary Fig. S2). However, these relationships did not appear to be linear; when AhRL was plotted against MIS-ATP, many subjects had both low MIS-ATP and low AhRL. These findings raise the possibility that there are at least two groups of MIS in the serum: those acting through AhR (AhR ligands) and others acting through non-AhR-mediated mechanisms (non-AhR ligands) 12 . Using K-means clustering analysis, two clusters were identified: a cluster with high AhRB and MIS-ROS, but low MIS-ATP (black dots in Fig. 1c www.nature.com/scientificreports www.nature.com/scientificreports/  Fig. 1c). The proportion of diabetes within the black cluster in 2008 was significantly higher than in the red cluster (67.2% vs. 8.2%, p < 0.001). In 2012, the black cluster had also significantly more diabetes patients than the red cluster (74.6% vs. 11.7%, p < 0.001).

The influence of AhRL and MIS-ATP on the development of IGT or diabetes.
Since MIS-ATP is a better indicator than MIS-ROS, MIS-ATP was used as a parameter for mitochondria inhibiting activity in the following analyses. Low MIS-ATP means high levels of MIS exist in serum. The mean levels of AhRL and MIS-ATP are shown in Table 3, with participants classified according to whether they developed IGT or diabetes during the 4-year follow-up period (2008)(2009)(2010)(2011)(2012). Of the 919 NGT subjects in 2008, 109 cases (7.1%) and 25 cases (1.6%) in 2012 were progressed to IGT (NGT-IGT) and diabetes (NGT-DM), respectively. These newly-diagnosed IGT (NGT-IGT) and diabetes (NGT-DM) patients showed 60%~70% higher AhRL and 6%~12% lower MIS-ATP than NGT-NGT (p < 0.001).     Table 5). The subjects in group 3 (high AhRL and high MIS-ATP) were more at risk (RR = 13.45, 95% CI, 4.93-36.72 in Model A) than those in group 2 (low AhRL and low MIS-ATP) (RR = 5.08, 95% CI, 1.91-13.51 in model A). Thus, although MIS-ATP is also an independently important parameter, high-level exposure to AhR ligands may be more important than mitochondrial inhibition in diabetes incidence. Again, men showed higher RR than women in group 4 (model D), suggesting that gender-dependent effects on confounding factors may be present (Fig. 1e, Table 5). www.nature.com/scientificreports www.nature.com/scientificreports/ When the incidence of new-onset diabetes was analysed according to the quartiles of AhRL or MIS-ATP among the 1,163 non-diabetics, most subjects in the upper fourth quartile (Q4) of AhRL (≥2.62 pM) developed diabetes 4 years later (Fig. 1f). In contrast, few subjects in the lowest quartile (Q1) of AhRL (<1.08 pM) developed diabetes. The incidence of diabetes in the Q2 and Q3 quartiles of AhRL increased as the MIS-ATP decreased. As in the upper fourth quartile (Q4) of AhRL, almost all subjects in the lower fourth quartile (Q4) of MIS-ATP (≤84.1%) developed diabetes regardless of their AhRL levels. Again, these results indicate that AhRL and MIS-ATP are strong, independent but interacting, and predictive factors for diabetes development.

Discussion
In this study, human exposure to EPC mixture of AhR ligands (AhRL) and MIS (MIS-ATP or MIS-ROS) was measured using serum samples obtained from KoGES study to see if it could be used as a predictor of the incidence of diabetes. Among NGT participants, those who developed into diabetes (NGT-DM) or IGT (NGT-IGT) in 2012 had higher AhRL and lower MIS-ATP in serum than those who maintained NGT (NGT-NGT). People with diabetes were characterized by the highest AhRL and the lowest MIS-ATP in serum (Table 1). AhRL and MIS-ATP were significantly correlated with insulin resistance index and the metabolic syndrome components (Supplementary Table S2). For those with AhRL ≥ 2.70 pM (cutoff value), the RR of diabetes developed within 4 years was 7.60, and for those with MIS-ATP ≤ 88.1%, the RR was 4.27 after adjustment of confounding factors (Model D, Table 4). These results are in good agreement with the hypothesis that exposure to AhR ligands and/ or mitochondrial inhibiting substances are important risk factors for insulin resistance, metabolic syndrome, and diabetes.
The ligand-activated transcription factor AhR is multifunctional nuclear receptor, playing a pivotal role in mediating the toxic responses induced by POPs and in detoxifying xenobiotics 28 . The toxicities of POPs have been estimated relative to 2,3,7,8-tetrachlorodibenzodioxin (TCDD; toxic equivalency factor = 1) and total POP-induced toxicity is expressed as TEQ, the sum of each chemical's concentration multiplied by its toxic equivalency factor value, which reflects the ability to activate AhR 29,30 . Thus, serum concentrations of individual POPs should be measured by instrumental analysis to calculate TEQ. Since there are many known and unknown AhR ligands 31 , it is difficult to measure all AhR ligands present in serum samples. Previously, we had shown that the serum AhRL correlated linearly with the TEQ values calculated from the plasma concentrations of the plasma concentrations of dioxin congeners, suggesting that serum AhRL represents an entire circulating AhR ligand mixture 12,13 . Thus, our AhRL assay can be used as an alternative to TEQ assessment without measuring EPC individually, although serum AhRL was not exactly equivalent to serum TEQ values.
Cells cultured with diabetic serum containing high levels of AhR ligands caused mitochondrial dysfunction, i.e. inhibited mitochondrial oxygen consumption rate and intracellular ATP, and increased ROS and mitochondrial fragmentation 13 . Among polychlorinated biphenyls in serum, both AhR ligands and non-AhR ligands appeared to have contributed to the reduction in mitochondrial activity 12 . Non-AhR ligands, for example, tetrabromobisphenol A 32 and perfluorooctanoic acid 33 , induce mitochondrial dysfunction, ROS production, and inflammatory responses in pancreatic β-cells or osteoblasts. Indeed, there is a complex relationship among serum AhRL, MIS-ATP, and MIS-ROS. The correlation coefficients between AhRL with MIS-ATP or MIS-ROS were not very high (r = −0.34 for MIS-ATP; r = 0.36 for MIS-ROS, p < 0.001) ( Table 2). It suggests they are possibly independent contributors or variables. The relationships between AhRL, MIS-ATP, and MIS-ROS were non-linear, and the data-points appeared as two clusters (Fig. 1c, Supplementary Fig. S2). These results suggest the possibility of two groups of mitochondrial inhibitors, either AhR-dependent or AhR-independent, exist in serum samples. AhR ligands with mitochondrial inhibitory activity may be diabetogen or obesogen. Given the large number of AhR ligands and their biological effects 14 , further experimental studies are needed to account for these relationships.
Next, it should be noted that the combination of AhRL and MIS-ATP parameters strongly predicted the future development of diabetes (Fig. 1e, Supplementary Table S4). In subjects with AhRL≥2.70 pM and MIS-ATP ≤ 88.1% (group 4), the incidence of diabetes was 22.6% (38 out of 168), and RRs were 29.82 and 21.11 in Model A and in Model D, respectively (Table 5). In comparison, from a cross-sectional study in the U.S (the National Health and Nutrition Examination Survey), Lee et al. reported that among the participants with the highest exposure to 6 persistent organic pollutants (POPs; Σ6POPs), the risk of diabetes was 38 times higher than that among the least exposed group 34 . When four POPs were analysed in the Helsinki Birth Cohort Study, Airaksien et al. reported that the risk of developing diabetes in the highest exposed group was 1.64~2.24 times higher than in the least exposed group 35 . The ORs for diabetic risk genes were less than 1.7 in genome-wide association studies 36 . A 2-fold higher ceramide-24 level was associated with 1.45 times higher risk of incident impaired fasting glucose 37 . We believe that an important factor in explaining why RRs in this study were so high was the measurement of AhRL and MIS with the cell-based bioassays.
According to another study of the KoGES cohort, a progressive decline in β-cell function and insulin sensitivity is a crucial factor in the deterioration of glucose tolerance and the development of diabetes 21 . Table 2 shows that HOMA-β-cell function correlated negatively with AhRL and positively with MIS-ATP, whereas HOMA-IR  www.nature.com/scientificreports www.nature.com/scientificreports/ correlated positively with AhRL and negatively with MIS-ATP. This suggests that high AhRL and/or low MIS-ATP may be involved in the impairment of insulin secretion and insulin resistance. This agrees well with our previous observation that AhRL was negatively associated with serum adiponectin concentration 15 . Indeed, AhRL and MIS-ATP were also correlated with hsCRP and with serum ALT and AST. Both adiponectin and hsCRP are well-known risk factors for insulin resistance 38 . These correlations mean that AhR ligands and/or MIS may play an important role in the development of chronic inflammation and fatty liver disease, both of which are common in metabolic syndrome. Other experimental evidence also supports the hypothesis that dioxins impair β-cell function 39,40 or induces vascular inflammation 41 in mice. Proteomic analysis of mitochondria from skeletal muscles of diabetes patients showed that subunits of oxidative phosphorylation were downregulated 42 . Transcriptome profiling revealed that a number of genes responsible for mitochondria ATP synthesis, oxidative phosphorylation, or mitochondrial dynamics were downregulated in human diabetes islets or β-cells 43,44 . Inflammatory cytokines were elevated before the onset of diabetes 45 . However, triggering mechanisms of mitochondrial dysfunction and/ or inflammation in diabetes are still ill-understood. Chronic hyperglycaemia 46 was recently suggested to start a vicious cycle of progressive deterioration of β-cells by impairing insulin secretion and mitochondria. However, our study clearly demonstrated that non-diabetic subjects developed diabetes within 4 years when they showed high levels AhRL and/or MIS in serum (Fig. 1e, Table 4). Therefore, if "exposure to high EPCs" is inserted instead of "chronic hyperglycaemia", many unanswered questions about the pathogenic mechanism of diabetes can be explained, namely, EPCs (AhR ligands or MIS) impair mitochondria in β-cell, skeletal muscle, or liver, resulting in insulin resistance and metabolic dysregulation.
Finally, our data showed that AhRL and mitochondrial dysfunction increased with age. Lipophilic EPC tends to remain in the human body and bioaccumulate when entering through food, air, or other routes. Lifelong accumulation of EPCs in the human body could account for the high prevalence of metabolic syndrome and diabetes in the elderly. A comprehensive worldwide literature review has shown that age is positively associated with most dioxin congeners and their TEQs in the unexposed general adult population 47 . The increased levels of AhR ligands and MIS in circulation had temporal associations with clinical phenotypes, including behavioural factors such as smoking and lack of exercise.
In summary, we show people who have high serum AhRL and/or low MIS-ATP are at high risk of developing diabetes, and AhRL and MIS-ATP have more predictive power when combined. Serum AhRL and/or MIS are tightly associated with insulin resistance, metabolic syndrome, and diabetes and may play an important role in their pathogenesis. Further studies are needed to confirm essential findings of this study in other cohorts or in experimental models.