Vinyl chloride enhances high-fat diet-induced proteome alterations in the mouse pancreas related to metabolic dysfunction

Abstract

Alterations in physiological processes in pancreas have been associated with various metabolic dysfunctions and can result from environmental exposures, such as chemicals and diet. It was reported that environmental vinyl chloride (VC) exposure, a common industrial organochlorine and environmental pollutant, significantly exacerbated metabolic-related phenotypes in mice fed concurrently with high-fat diet (HFD) but not low-fat diet (LFD). However, little is known about the role of the pancreas in this interplay, especially at a proteomic level. The present study was undertaken to examine the protein responses to VC exposure in pancreas tissues of C57BL/6J mice fed LFD or HFD, with focus on the investigation of protein expression and/or phosphorylation levels of key protein biomarkers of carbohydrate, lipid, and energy metabolism, oxidative stress and detoxification, insulin secretion and regulation, cell growth, development, and communication, immunological responses and inflammation, and biomarkers of pancreatic diseases and cancers. We found that the protein alterations may indicate diet-mediated susceptibility in mouse pancreas induced by HFD to concurrent exposure of low levels of inhaled VC. These proteome biomarkers may lead to a better understanding of pancreas-mediated adaptive or adverse response and susceptibility to metabolic disease.

Keywords: high-fat diet; metabolic dysfunction; mouse; pancreas; proteome; vinyl chloride.

Figure 1.

Changes at protein expression levels of the identified proteins involved in (A) immunity and immune responses; (B) metabolic events such as fatty acid oxidation, oxidative stress, and energy metabolism; (C) cell growth and development, cellular communications and signaling; (D) clinical biomarkers of metabolic syndromes, and pancreatic diseases and cancers. All the relative fold changes were obtained as compared to the expression levels of proteins in LFD mice. α means increased expression and β means decreased expression in the comparisons. These altered proteins were specific to pancreas-mediated metabolic processes and dysfunctions, and the alterations of pancreatic disease biomarkers suggested metabolic stresses and pathological processes existing in the exposed mice. Abbreviation: LFD: low-fat diet.

Figure 2.

Western blot analyses of phosphorylation levels of AKT and GSK3β in pancreas of LFD, HFD, LFD + VC, and HFD + VC mice. Phosphorylation status of AKT and GSK3β was determined by the ratio of phosphorylation/expression levels of AKT and GSK3β in mouse pancreas. α represents significantly higher and β significant lower in the comparisons (p < .05). β-actin was used as an internal control to normalize protein levels. Abbreviations: AKT: protein kinase B; GSK3β: glycogen synthase kinase 3 β; HFD: high-fat diet; LFD: low-fat diet; VC: vinyl chloride.

Figure 3.

Protein-protein interaction by IPA. Networks of proteins showing inter-relationships and pathway which was obtained using IPA in pancreas of VC, HFD, and HFD + VC mice. A, LFD + VC versus LFD. B, HFD + VC versus LFD + VC. Solid lines represent direct interactions between molecules, and dotted lines represent indirect interactions. Lines connecting the proteins in networks indicate known inter-relationships found in the IPA database. The shapes of nodes in the networks delineate a function. These protein interacting networks were centered by AKT, GSK3β, Stat5, and NFkB, suggesting that these proteins might play important roles in the potentiation of HFD-induced metabolic stresses and dysfunctions that occur due to treatment of VC, possibly through regulation of the associated proteins presented in the networks. Abbreviations: AKT: protein kinase B; GSK3β: glycogen synthase kinase 3 β; HFD: high-fat diet; IPA: Ingenuity Pathway Analysis; LFD: low-fat diet; VC: vinyl chloride.

Figure 4.

Pearson correlations of the differentially expressed or phosphorylated proteins with mouse body weights, and between the altered proteins. The first column was the correlations of the differentially expressed and phosphorylation proteins with mouse body weights. Shaded red color denotes negative correlation; light red denotes not significant correlation (p ≥ .05); and dark red (p < .05) denotes significant correlation. The remaining columns were the correlations among the altered proteins. Shaded blue color denotes positive correlation, light blue denotes probability of ≥0.05 (not significant), and dark blue (p < .05) denotes significant correlation. The hierarchical cluster analysis using Ward’s method applying squared Euclidean Distance as the distance or similarity measure was constructed using SAS software (SAS institute). Correlation values of >0.6 were significant (p < .05).

Figure 5.

A proposed scheme to decipher key toxicity pathways underlying metabolic syndromes mediated by VC and/or HFD. ADIPO played a central role in coordinating signaling proteins of pAKT and pGSK3β, and their associated proteins including multiple cytokines to regulate key proteomics pathways and underlying metabolic dysfunctions mediated by HFD and VC. The potentiation of HFD-induced proteome changes contributed to underlying metabolic dysfunction and was augmented by VC exposure. Our data suggested this could occur through regulation of ADIPO and downstream modulation of AKT, GSK3β, IGF-1, and NFkB. Given the known coordination of these pathways to influence important functions of cell growth, development, and death, insulin resistance, metabolism, and inflammation, the pancreas may therefore play a key part in mediating HFD + VC exposure contribution to metabolic disease. Abbreviations: ADIPO: adiponectin; AKT: protein kinase B; GSK3β: glycogen synthase kinase 3 β; HFD: high-fat diet; IGF-1: insulin-like growth factor 1; VC: vinyl chloride.