Environmental exposure to lead and cadmium are associated with triglyceride glucose index

The triglyceride glucose (TyG) index was suggested as a novel reliable surrogate marker for insulin resistance and related cardiovascular-metabolic diseases. We aimed to evaluate the association between the TyG index and environmental exposure to lead (Pb), mercury (Hg), and cadmium (Cd). A total of 9645 adults who enrolled in the Korea National Health and Nutrition Examination Survey in 2005, 2008–2013, and 2016 were included. Fasting plasma glucose and triglyceride levels were used to calculate the TyG index. Multivariate logistic regression model was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). We noted an increasing trend in the TyG index with increment of blood Pb and Cd concentrations. Participants in the highest quartile of blood Pb and Cd concentrations had higher TyG index values than those in the lowest quartile, with ORs (95% CIs) of 1.32 (1.07–1.63) and 1.29 (1.04–1.59) for Pb and Cd, respectively. Strong associations between blood Pb and Cd concentrations and the TyG index were found in men. Blood Hg concentrations did not show a significant association with the TyG index. Our study suggests that public health strategies for cardiovascular-metabolic disorder prevention should be directed toward individuals exposed to priority heavy metals.


Association between blood heavy metals and TyG index
The associations between blood Pb concentrations and the TyG index according to the six cut-off points are shown in Table 3 and Supplementary Table S1.Briefly, a positive association was observed between blood Pb concentrations and the TyG index in both the continuous and categorical models using the six cut-off points.For example, as blood Pb concentrations increased, the prevalence of high TyG index increased by 23% and 25% the cut-off points 1 (95% CI = 1.06-1.44)and 2 (95% CI = 1.04-1.51),respectively.Likewise, participants in the highest quartile of blood Pb concentration had a higher TyG index than those in the lowest quartile (odds ratio [OR] [95% CI] 1.32 [1.07-1.63]and 1.46 [1.13-1.90] for cut-off points 1 and 2, respectively), with a significant linear trend.Notably, a stronger association between blood Pb concentrations and the TyG index was identified in men, whereas a null association was found in women.We observed consistent results when further adjusting for blood Hg and Cd concentrations.
A null association was identified between blood Hg concentrations and the TyG index using cut-off points 1 and 2 (Table 3).However, we found the association to be inconsistent between the lowest and the highest cutoff points.The prevalence of high TyG index tended to decrease in women when the lowest cut-off point 3 was applied, whereas it tended to increase in men when applying the highest cut-off point 6 (Supplementary Table S2).
As shown in Table 3 and Supplementary Table S3, the TyG index was positively associated with blood Cd concentrations.For instance, when we applied cut-off point 1, the OR (95% CI) for the prevalence of high TyG index was 1.14 (1.01-1.30)for natural log-transformed blood Cd concentrations.Similarly, compared with participants in the lowest quartile of blood Cd concentrations, those in the highest quartile had a 29% higher prevalence of high TyG index (95% CI = 1.04-1.59).When separated by sex, the positive association persisted in men but not in women.
In addition, Supplementary Tables S4 and S5 present results of subgroup analysis for 3678 participants who with HOMA-IR values.There was no significant association between blood heavy metals and HOMA-IR (Supplementary Table S4).While blood Pb and Cd concentrations were significantly associated with TyG index (OR [95% CI]: for overall, 1.43 [1.08-1.89]for natural log-transformed blood Pb concentrations; for men, 1.50 [1.02-2.20]and 1.40 [1.04-1.89]for natural log-transformed blood concentrations of Pb and Cd, respectively) (Supplementary Table S5).

Discussion
We used a nationally representative data in Korea to identify the association of blood Pb, Hg, and Cd concentrations with the TyG index, a surrogate marker for IR and related cardiovascular-metabolic diseases.In summary, regardless of the cut-off points, the TyG index tended to increase with the increment of Pb and Cd blood concentrations.These positive associations were prominent in men.We also observed that the TyG index did not differ with blood Hg concentrations.The findings of this study indicate that management on priority heavy metals are imperative for prevention of IR and related cardiovascular-metabolic diseases.
Some previous studies have reported that concentrations of Pb and Cd measured in blood 16,17 , urine 16,18,19 , and adipose tissue 20 were not significantly associated with IR (HOMA-IR).Especially, a Korean study that used data from the 2009-2010 Korean National Health and Nutrition Examination Survey (KNHANES) demonstrated that the HOMA-IR was not associated with blood concentrations of Pb, Hg, and Cd 17 .Incidentally, the composition of the previous study population differed significantly from that of the present study (Supplementary Table S6).While Moon SS's study comprised adult participants aged 30 years and older, which might influence the HOMA-IR value, our study encompassed adult participants aged 19 years and older, excluding those with T2DM, hyperlipidemia, hypertension, stroke, myocardial infarction, and cancer.Consequently, the GMs of HOMA-IR were higher than that in our study (2.25 vs. 2.06 for Moon SS's study vs. our study), which resulted in a higher prevalence of HOMA-IR (36.5% vs. 28.5% for Moon SS' study vs. our study).Similarly, the prevalence of elevated TyG index in dataset of Moon SS's study was also higher than our study when applying all the 4 cut-off points referred in this study.The misclassification of outcomes in the Moon SS's study may underestimate the effect size of associations.
On the other hand, the present study, which included 3678 participants with HOMA-IR values from KNHANES 2008-2010, indicated no significant association between blood heavy metals and HOMA-IR, consistent with Moon SS's study.While significant associations of blood Pb and Cd with TyG index were observed among 3678 participants with HOMA-IR.In light of these findings, the significant associations between blood Pb and Cd concentrations and the TyG index in this study might to be due to the better performance of the TyG index than the HOMA-IR and other indices when predicting IR.According to a study on validation of TyG index, www.nature.com/scientificreports/ it had higher sensitivity and specificity than the HOMA-IR in identifying IR patients (area under the receiver operating characteristic curve: 0.79 for the TyG index and 0.77 for the HOMA-IR) 10 .Furthermore, in the TyG index equation, the fat distribution is corrected so that the patients with normal weight but displaying obesityrelated metabolic derangements can also be applied.However, the HOMA-IR model has poor detect power in cases other than insulin signaling pathway defects such as impaired hepatocyte function 21 .
On the other hand, concentrations of Pb and Cd have been reported to be associated with IR-related cardiovascular-metabolic disorders.Positive associations have also been observed between Pb and Cd exposures and prevalence 5 , incidence 22 , and mortality 23 of CVDs.A meta-analysis of 37 case-control and prospective studies concluded that the risk of CVDs increased by 43% and 33% with the increment of Pb and Cd levels, respectively 4 .Exposure to Pb and Cd may also be related to MetS 24 , especially in the Asian population 25 .Furthermore, several observational and meta-analyses have demonstrated the positive association between Pb exposure and dyslipidemia 26,27 and between Cd exposure and T2DM 19,28 .However, the literature shows inconsistent results on the association between environmental Hg exposure and IR 17,29,30 .A study in Taiwan reported that blood Hg concentrations were associated with elevated HOMA-IR 29 , whereas the positive association was observed only in men in a Korean population 30 .A systematic review reported that the inconsistent results of previous longitudinal studies make it difficult to infer causality between Hg exposure and T2DM or MetS 31 .Further evaluations are needed to explore the effects of heavy metal exposure on the risk of IR and related cardiovascular-metabolic disorders in large prospective studies.Table 3. Associations between blood Pb, Hg, and Cd concentrations and the TyG index according to sex (for the cut-off point 1).Pb, lead; Hg, mercury; Cd, cadmium; TyG index, triglyceride glucose index; OR, odds ratio; CI, confidence interval.The cut-off point 1: 8.84 for the highest quartile of the TyG index in this study.a Model 1, with no adjustment.b Model 2, adjusted for age, sex (for men and women combined), survey year, BMI, alcohol consumption, smoking status, educational level, occupation, physical activity, menopausal status, grain consumption, fish consumption, seaweed consumption, vegetable consumption, and mushroom consumption.c Model 3, further adjusted for natural log-transformed blood concentrations of Pb, Hg, or Cd.www.nature.com/scientificreports/

Overall
Several studies have suggested pathological mechanisms underlying the toxicity of heavy metals on IR progression.The heavy metals discussed in this study are considered hyperglycemic metals 32 .When these heavy metals accumulate in the body, they promote direct damage to pancreatic β-cells by interacting with the plasma calcium channel and altering intracellular calcium homeostasis 33 .Accordingly, the inhibited calcium-ATPase may further affect insulin biosynthesis and secretion 34 .As another potential mechanism of oxidative stress, heavy metals in the target tissues may stimulate the production of reactive oxygen species and depletion of the antioxidant pool 35 .Heavy metal-induced oxidative stress results in the reduction of insulin sensitivity and pancreatic β-cell function, which is further linked to the progression of T2DM 21 .It has also been suggested that chronic and sub-chronic exposure to Cd could reduce mRNA and protein expression of the insulin-regulated glucose transporter type 4 (GLUT4) in muscle and adipose tissues 36,37 .Consequently, most glucose uptake occurs in other insulin-independent tissues, such as the liver, kidney, and brain 38 .IR may be developed when insulin secretion from pancreatic β-cells increases to compensate for the hyperglycemic state 39 .Moreover, several in vivo and in vitro studies have reported genotoxicity of the heavy metals via stimulation of DNA breakage and modulation of mRNA expression 40 .Nevertheless, detailed evidence of the effect of heavy metal exposures on IR and its complications is limited, warranting further experimental and epidemiological investigations.
The Human Biomonitoring (HBM) Commission of the German Federal Environmental Agency has recently defined the reference values for heavy metal concentrations in blood: 9.00 µg/dL in men and 7.00 µg/dL in women for Pb; 2.00 µg/L for Hg; and 1.00 µg/L for Cd 41 (Supplementary Figure S1).The GMs of blood Pb and Cd concentrations among this study's participants (2.06 µg/dL and 0.94 µg/L, respectively) were lower than the HBM Commission's reference values.Likewise, the concentrations of Pb and Cd in other countries have also been found to fall below the reference values: 1.32 µg/dL Pb and 0.37 µg/L Cd (GMs in blood) in the US 42 ; 1.88 µg/dL Pb and 0.39 µg/L Cd (GMs in blood) in Northern France 43 ; 2.40 µg/dL Pb (GM in blood) and 0.28 µg/L Cd (GM in urine) in Spain 44,45 ; and 2.33 µg/dL Pb and 0.82 µg/L Cd (medians in blood) in China 46 .Given the significant positive association of blood Pb and Cd concentrations with the TyG index in this study, IR and related cardiovascularmetabolic disorders may be induced even at low-dose exposures of Pb and Cd 47 .By contrast, the GMs of blood Hg concentrations in this study participants (3.69 µg/L) and Spanish adults (6.35 µg/L) 48 were higher than the HBM Commission's reference value, whereas concentrations below the reference value were assessed among individuals in the US (0.93 µg/L [GM in blood]) 42 , Northern France (1.38 µg/L [GM in blood]) 43 , and China (1.17 µg/L [median in blood]) 46 .The null association between blood Hg concentrations and the TyG index in this study could be interpreted as a high distribution of Hg exposure, as a non-linear dose-response relationship between Hg concentrations and risk of hypertension was observed in previous studies 49 .
With this study, we are the first to evaluate the association between Pb, Hg, and Cd exposure and TyG index.The large sample size from nationally representative survey data supports the external validity of the results in this study.Additionally, the cut-off points of the TyG index determined in different populations were applied to minimize the probability of misclassification.However, several limitations remain.First, the findings of this cross-sectional study may not offer causal insights into the effect of heavy metal exposure on the risk of IR and related complications.Second, we did not evaluate the additive effect of Pb, Hg, and Cd 24 .However, the association between blood concentrations of heavy metals and the TyG index persisted after adjusting for each.Finally, even though we controlled for all confounding factors considered, residual bias cannot be ruled out.
In conclusion, this study's findings indicate that low dose environmental exposure to Pb and Cd-but not Hg-were associated with TyG index, a reliable surrogate marker for IR-related cardiovascular-metabolic diseases.Implementing of management policies for priority heavy metals to prevent IR and related cardiovascularmetabolic disorders is of great significance for public health.

Study population
The data used in this study were acquired from the KNHANES, a nationally representative health survey conducted by the Korea Disease Control and Prevention Agency (KDCA) 50 .Approximately 10000 individuals aged 1 year and older were selected annually via multi-stage clustered probability sampling.After a face-to-face interview and health examination, information on socioeconomic status, behavioral and nutritional factors, anthropometric indices, clinical profiles, and medical history was obtained from each participant.
In the KNHANES, blood heavy metal measurement was conducted in a randomly selected sample in 2005, 2008-2013, and 2016-2017.Participants except those who enrolled in 2017 were initially included due to the lack of information on food frequency questionnaire (FFQ).Of the 18686 participants, 1779 children and adolescents were excluded.We further excluded participants who had received a physician's diagnosis of or been treated with medication for hyperlipidemia (n = 1697), T2DM (n = 726), hypertension (n = 1673), stroke (n = 60), myocardial infarction (n = 20), or cancer (n = 250); those who did not complete fasting plasma glucose (FPG) (n = 10) or TG (n = 1) measurements; those who did not provide information on body mass index (BMI) (n = 36), alcohol consumption (n = 320), smoking status (n = 5), educational level (n = 94), occupation (n = 8), physical activity (n = 4), menopausal status in women (n = 228), or grain, fish, seaweed, vegetable, or mushroom consumption (n = 2128); and those with a missing sampling weight (n = 2).After exclusion, 9645 participants (4314 men and 5331 women) were included in the final analysis.A flow diagram of the inclusion and exclusion criteria for the study population is shown in Fig. 1.

Statistical analysis
Due to the skewed distribution, the blood concentrations of Pb, Hg, and Cd were converted to natural log-transformed values.Baseline descriptions of the study participants were presented as mean (standard error) or frequency (percentage).Differences in baseline characteristics according to quartiles of the TyG index were determined using ANOVA tests for continuous variables and Rao-Scott Chi-Square tests for categorical variables.We calculated the GMs and 95% CIs of the heavy metal concentrations using PROC SURVEYMEANS.
ORs and 95% CIs for the associations between blood Pb, Hg, and Cd concentrations and the TyG index with the six cut-off points were estimated using PROC SURVEYLOGISTIC.The natural log-transformed blood Pb, Hg, and Cd concentrations were used in the association analyses.Categorical models using the quartiles of blood concentrations of Pb, Hg, and Cd were also applied, with the lowest quartile used as the reference group.The median value of the blood heavy metal concentrations was assigned to each quartile group to evaluate the linear trend.
All analyses in this study were conducted with a sampling weight.Statistical analyses were conducted using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).P-values < 0.05 were considered to indicate significant differences.

Table 1 .
Baseline characteristics of the study participants according to the TyG index.TyG index, triglyceride glucose index; SE, standard error; BMI, body mass index; Pb, lead; Hg, mercury; Cd, cadmium.a p-value was calculated using ANOVA tests for continuous variables and Rao-Scott Chi-Square tests for categorical variables.