| As one of the major non-communicable disease,diabetes has attracted extensive attention worldwide due to high mortality and disability burden.Type 2 diabetes is the most common type among diabetic patients and is characterized by persistently hyperglycemic status,insulin resistance,and/or insufficient insulin secretion.In addition to some wellestablished risk factors such as genetic susceptibility,obesity,and unhealthy lifestyle,environmental pollutants have been proved to be involved in glucose metabolic dysfunction,impaired insulin secretion,decreased insulin sensitivity,and the occurrence and development of type 2 diabetes.Zinc is an essential metal element for the human body.The functions of zinc include acting as a second messenger and as a component of over 300 proteins and participating in several metabolic processes such as glucose metabolism and synthesis,storage,and secretion of insulin.Zinc is also a heavy metal which is widely distributed in the environment and widespread use in industry.With the persistent progress of industrialization and urbanization and the more frequent appearance of zinc-based material in daily life,zinc contamination in our living circumstance has become deteriorating.Environmental zinc enters the human body mainly through respiratory inhalation and gastrointestinal ingestion,and differences in diet and lifestyles lead to different levels of zinc exposure.Increased zinc exposure can disrupt zinc dynamic balance,leading to cell physiological dysfunction and even multiple organs damage.Previous clinical trials have discovered the potential toxicity of elevated zinc supplementation for glycemia control.However,the relationship of zinc exposure with glucose metabolism and the risk of type 2diabetes is largely uncertain for general adult population.Furthermore,the potential mechanism underlying zinc-related glycometabolic disorder and increased risk of type 2 diabetes remains not fully understood.Published literatures have indicated that epigenetic factor,as links between the environment,genes,and health outcomes,plays an important role in disease progression associated with pollutant exposure.As one of the main regulatory factors of epigenetics,micro RNA(miRNA)negatively regulates target genes post-transcriptionally through binding to the 3’-untranslated region of messenger RNA(m RNA).Emerging evidence from epidemiological studies and in vivo and in vitro experiments has suggested that miRNAs can affect the progression of type 2 diabetes by regulating the key genes involved in glucose metabolism.In addition,altered miRNA expression has been proved to be associated with environmental pollutants exposure,and then participates in the development of diseases through regulating target m RNA.Nevertheless,the effects of zinc exposure on circulating miRNA expression profile and the specific role of miRNA in zinc-related glycometabolic dysfunction and type 2 diabetes are unclear.Therefore,the present study was conducted based on the Wuhan-Zhuhai cohort.We measured urinary zinc concentration to assess the level of zinc internal exposure.Fasting blood glucose and fasting blood insulin were detected to compute indexes of insulin resistance and β-cell function and further to investigate the associations of urinary zinc with glycemia traits and risk of type 2 diabetes cross-sectionally and longitudinally.Two-stage population-based studies were performed to screen and validate zinc exposure-and type 2diabetes-related plasma miRNA and to evaluate the potential role of candidate miRNA in zinc-related hyperglycemia and type 2 diabetes.Ultimately,in vitro experiment was conducted to verify the regulating role of candidate miRNA and to explore the underlying signaling pathway.This study includes three parts as follows:Part Ⅰ.Associations of urinary zinc with glucose homeostasis and type 2 diabetes in community residentsObjectives: To assess the level and distribution of urinary zinc among Chinese community residents,and to estimate the associations of urinary zinc with glucose homeostasis traits and type 2 diabetes in general community residents.Methods: Urinary zinc was measured with the method of inductively coupled plasma-mass spectrometry for 3550 community residents from the Wuhan-Zhuhai cohort.Homeostasis model assessment was adopted to assess insulin resistance(Homeostasis model assessment of insulin resistance,HOMA-IR)and β-cell function(Homeostasis model assessment of β cell function,HOMA-β).In the cross-sectional analyses,generalized linear model was performed to investigate the associations of urinary zinc with glucose homeostasis traits,including fasting blood glucose,HOMA-IR,and HOMA-β.Logistic regression model was performed for the dichotomous outcome variable of type 2 diabetes.We further included participants who completed both of 3-year and 6-year follow-ups to explore long-term effects of zinc exposure.According to the concentrations of urinary zinc at baseline and follow-up,participants were divided into three categories of low,inconsistent,and high zinc exposure.The effects of long-term zinc exposure on glucose homeostasis traits and type 2 diabetes were estimated with generalized linear model and logistic regression model,respectively.Results: The median urinary zinc concentration of community residents was 310.76 μg/L.In the cross-sectional analyses,significant associations of urinary zinc with disordered glucose metabolism and elevated risk of type 2 diabetes were observed.Each 2-fold increment in urinary zinc was associated with a 0.30 mmol/L,6.06%,and 106% increase in fasting blood glucose,HOMA-IR,and risk of type 2 diabetes,respectively,and an 8.72% decrease in HOMA-β(all P <0.05).Stratified analyses showed that age,gender,and body mass index significantly modified the positive relationship between urinary zinc and fasting blood glucose,which was stronger among subjects over 55 years,males,and overweight participants with body mass index over 24 kg/m2.Furthermore,the positive association between urinary zinc and the risk of type 2 diabetes was stronger among overweight subjects(Pinteraction <0.05).In the longitudinal analyses,zinc exposure showed significantly adverse effects on glucose homeostasis and type 2 diabetes.In the 3-year follow-up,a 0.09 decrease in HOMA-β,and a 0.49 mmol/L,0.06,and 157% increment in fasting blood glucose,HOMA-IR,and risk of type 2 diabetes,respectively,were observed among individuals with high urinary zinc levels,when compared with those had low levels of urinary zinc.Likely,in the 6-year follow-up,high zinc exposure was associated with a 0.36 mmol/L increment in fasting blood glucose,a 289% increase in the risk of type 2 diabetes,and a 0.08 decline in HOMA-β(all P <0.05).Conclusions: The level of urinary zinc among enrolled participants was higher than the guidance value for nutritional adequacy proposed by Institute of Medicine(Male: 206 μg/L;Female: 159 μg/L).Significant associations of zinc exposure with disordered glucose metabolism and elevated risk of type 2 diabetes were observed among Chinese general community residents.Part Ⅱ.Potential role of plasma microRNA in the associations of urinary zinc with fasting plasma glucose and type 2 diabetesObjectives: To screen and validate urinary zinc-and type 2 diabetes-related plasma miRNA,and to estimate the potential role of candidate miRNA in the relationship of urinary zinc with fasting blood glucose and type 2 diabetes.Methods: Two-stage population-based studies were conducted based on the second followup of Wuhan residents.In the discovery stage,87 subjects from the panel of the cohort were included for urinary zinc-related miRNA screening,and 9 pairs of age(±5 years)and gender-matched newly diagnosed type 2 diabetes cases and healthy controls were enrolled for type 2 diabetes-related miRNA screening.We profiled plasma miRNA expression related to urinary zinc and type 2 diabetes by next-generation sequencing and analyzed with generalized linear model and Fisher exact test,respectively.In the validating stage,plasma miRNA associated with both of urinary zinc and type 2 diabetes was measured by quantitative real-time polymerase chain reaction(q RT-PCR)among 161 subjects which were randomly selected from population of the second follow-up.The relationships among urinary zinc,candidate miRNA,and fasting blood glucose or type 2 diabetes were verified with restricted cubic spline model.Kyoto encyclopedia of genes and genomes(KEGG)analyses were performed for the candidate miRNA to explore the potential signaling pathway involved.Results: In the discovery stage,a total of 142 miRNAs were associated with increased urinary zinc at the criteria of P value <0.05.Among them,103 miRNAs were up-regulated while 39 miRNAs were down-regulated.Likewise,with the criteria of P value <0.05 and fold change >1.5,22 miRNAs significantly altered in patients with type 2 diabetes when compared with matched healthy controls with 20 increased miRNAs and 2 decreased miRNAs.Among differentially expressed miRNAs,increased miR-144-3p and increased miR-215-5p were significantly associated with both of elevated urinary zinc concentrations and type 2 diabetes risk.In the validating stage,we found significantly linear exposure-response relationship between urinary zinc and miR-144-3p.With increasing plasma miR-144-3p levels,monotonic increments in fasting blood glucose and risk of type 2 diabetes were observed(all Poverall <0.05 and Pnonlinear >0.05).Furthermore,plasma miR-144-3p showed significant mediation effects in the associations of urinary zinc with fasting blood glucose and type 2 diabetes,with the proportion mediated of 25.08% and 22.26%,respectively.The KEGG enrichment analysis indicated that a total of 88 pathways were significantly enriched including type 2 diabetes pathway and insulin resistance pathway.Conclusions: Up-regulated plasma miR-144-3p was associated with urinary zinc and type 2 diabetes,and miR-144-3p partially mediated increased urinary zinc-related hyperglycemia and type 2 diabetes risk elevation.The KEGG enrichment analysis suggested that miR-144-3p might be involved in type 2 diabetes by regulating insulin resistance pathway.Part Ⅲ.Potential mechanism of miR-144-3p/Nrf2 involved in zinc exposure-related insulin resistanceObjectives: To assess the effects of zinc exposure on levels of insulin resistance and miR-144-3p expression,and to explore the potential mechanism underlying zinc exposureinduced insulin resistance.Methods: Hep G2 cells were treated with various concentrations of zinc sulfate(60,80,100,120,and 140 μM)for 24 hours.The cell viability was detected with cell counting kit-8 assay.Cellular glucose consumption level was calculated by measuring glucose concentrations in the medium with the glucose oxidase-peroxidase assay kit.And the relative m RNA and protein levels of insulin resistance indicators were determined with q RT-PCR and western blotting,respectively.The relative expression levels of miR-144-3p were detected after 120 μM zinc treatment for 24 hours.Target genes of miR-144-3p were predicted with Target Scan and miRDB,and their binding relationship was confirmed by dual-luciferase reporter assay.The relative protein levels of nuclear factor erythroid 2-related factor 2(Nrf2),the predicted target gene of miR-144-3p,and the relative m RNA levels of Nrf2 and its downstream genes were measured after 120 μM zinc treatment for 24 hours.Furthermore,we interfered miR-144-3p expression levels with mimic or inhibitor to verify the regulating role of miR-144-3p.Results: Compared with the control group,significant decline in glucose consumption,increases in m RNA and protein levels of phosphoenolpyruvate carboxy kinase(PEPCK)and m RNA levels of glycogen synthase kinase 3β(GSK3β),and decrease in GSK3β phosphorylation at Ser 9 were observed in Hep G2 cells exposed to 120 and 140 μM zinc for 24 hours(all P <0.05).Dual-luciferase reporter assay indicated that miR-144-3p can bind to the mRNA of Nrf2 and play regulating role negatively.Mi R-144-3p expression level significantly increased with zinc exposure when compared with the control group.After 120 μM zinc treatment for 24 hours,the relative m RNA and protein levels of Nrf2 and the relative m RNA of downstream antioxidant enzymes significantly decreased,while cellular reactive oxygen species(ROS)and the relative m RNA levels of interleukin(IL)-6 and IL-1β significantly increased(all P <0.05).In zinc-induced cells transfected with miR-144-3p inhibitor,the m RNA and protein levels of Nrf2 significantly increased and zinc-induced Nrf2 signaling pathway alterations were alleviated.Compared with the group of zinc exposure with negative control(NC),the relative m RNA levels of antioxidant enzymes in the group of zinc exposure with inhibitor significantly elevated,while cellular ROS and the relative m RNA levels of IL-6 and IL-1β significantly decreased.In addition,miR-144-3p inhibitor transfection ameliorated insulin resistance with increased glucose consumption and phosphorylated protein level of GSK3β and decreased m RNA and protein levels of PEPCK and GSK3β m RNA expression(all P <0.05).On contrary,miR-144-3p mimic transfection inhibited Nrf2 expression,exacerbated zinc-induced Nrf2 signaling pathway alterations,and aggravated zinc-resulted insulin resistance.Conclusions: Zinc exposure might upregulate levels of miR-144-3p targeting Nrf2 to suppress downstream antioxidant genes and promote downstream pro-inflammatory factors expression,resulting in elevated ROS and inflammation and the occurrence of hepatic insulin resistance. |
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