Molecular and Metabolic Analysis of Arsenic-Exposed Humanized AS3MT Mice

Background: Chronic exposure to inorganic arsenic (iAs) has been associated with type 2 diabetes (T2D). However, potential sex divergence and the underlying mechanisms remain understudied. iAs is not metabolized uniformly across species, which is a limitation of typical exposure studies in rodent models. The development of a new “humanized” mouse model overcomes this limitation. In this study, we leveraged this model to study sex differences in the context of iAs exposure. Objectives: The aim of this study was to determine if males and females exhibit different liver and adipose molecular profiles and metabolic phenotypes in the context of iAs exposure. Methods: Our study was performed on wild-type (WT) 129S6/SvEvTac and humanized arsenic +3 methyl transferase (human AS3MT) 129S6/SvEvTac mice treated with 400 ppb of iAs via drinking water ad libitum. After 1 month, mice were sacrificed and the liver and gonadal adipose depots were harvested for iAs quantification and sequencing-based microRNA and gene expression analysis. Serum blood was collected for fasting blood glucose, fasting plasma insulin, and homeostatic model assessment for insulin resistance (HOMA-IR). Results: We detected sex divergence in liver and adipose markers of diabetes (e.g., miR-34a, insulin signaling pathways, fasting blood glucose, fasting plasma insulin, and HOMA-IR) only in humanized (not WT) mice. In humanized female mice, numerous genes that promote insulin sensitivity and glucose tolerance in both the liver and adipose are elevated compared to humanized male mice. We also identified Klf11 as a putative master regulator of the sex divergence in gene expression in humanized mice. Discussion: Our study underscored the importance of future studies leveraging the humanized mouse model to study iAs-associated metabolic disease. The findings suggested that humanized males are at increased risk for metabolic dysfunction relative to humanized females in the context of iAs exposure. Future investigations should focus on the detailed mechanisms that underlie the sex divergence. https://doi.org/10.1289/EHP12785


Table of Contents
Figure S1.Small RNA-seq read length distributions.Distributions shown sequencing in (A) liver and (B) adipose of mice exposed to iAs.Data reported in Table S2.S3.

Figure S3
. Assessing microRNA profiles in liver tissue of humanized and wild type mice exposed to iAs.A) PCA plot of rlog transformed miR expression in the liver of iAs exposed mice.Male only (left panel), female only (right panel).Two different sequencing batches are shown by circle and triangle, respectively.B) Volcano plots showing differentially expressed (DE) miRs in the liver of WT vs. humanized mice for males (left panel) and females (right panel).miRs upregulated in WT vs. humanized shown in red, down-regulated shown in blue (p-adjusted < 0.05, log2foldcahnge <-0.5 or >0.5, basemean > 500).Log2Fold change = humanized mice/ WT mice for either male or female groups.Data reported in Table S8.

Figure S4.
Assessing gene profiles in adipose tissue of humanized and wild type mice exposed to iAs.A) PCA plot of rlog transformed gene expression in the adipose of iAs exposed mice.Male only (top panel), female only (bottom panel).B) Volcano plots showing differentially expressed (DE) genes in the adipose of WT vs. humanized for males (top panel) and females (bottom panel).Genes up-regulated in WT vs. humanized shown in red, down-regulated shown in blue (padjusted < 0.05, log2foldcahnge <-0.5 or >0.5, basemean > 500).Log2Fold change = humanized mice/ WT mice for either male or female groups.Data reported in Table S10.S12.

Figure S6.
Analyzing pathway enrichment among genes up-regulated in the liver of male vs. female mice exposed to iAs.A) Results of KEGG pathway analysis for WT male vs. WT females (top panel) and humanized males vs. humanized females (bottom panel).B) Results of Elsevier pathway analysis for WT male vs. WT females (top panel) and humanized males vs. humanized females (bottom panel).Data reported in Table S13.

Figure S7.
Analyzing pathway enrichment among genes downregulated in the liver of humanized male vs. female mice exposed to iAs.A) Results of Elsevier pathway analysis for WT male vs. WT females (top panel) and humanized males vs. females (bottom panel).Pathways with an asterisk indicate those relevant to metabolism.B) Results of KEGG pathway analysis for WT male vs. WT females (top panel) and humanized males vs. females (bottom panel).Data reported in Table S14.

Figure S8
. Additional examples of gene expression differences between humanized males and females exposed to iAs.Expression (normalized counts) of genes that are associated with metabolic health in humanized (left panel) and WT (right panel) mice.DESeq2 performs an internal normalization where geometric mean is calculated for each gene across all samples.The counts for a gene in each sample is then divided by this mean.The median of these ratios in a sample is the size factor for that sample.Significance determined by DESeq2 (** p-adjusted value < 0.05).Whiskers represent the minimum and maximum values, the midline is the median and the limits are the 25 th and 75 th percentile.Data reported in Table S15.

Figure S9.
Correlating miR-34a expression with insulin resistance to iAs exposure WT and humanized mice.Correlation of (A) FPI, (B) HOMA-IR, and (C) FBG with expression levels of miR-34a in liver (left) and adipose (right) of male and female mice.(D) Correlation of FPI with adipose miR-34a shown separately for humanized males (left) and humanized females (right).(E) Correlation of HOMA-IR with adipose miR-34a shown separately for humanized males (left) and humanized females (right).(F) Correlation of FBG with liver miR-34a shown separately for humanized males (left) and humanized females (right).R, Pearson's correlation coefficient.Data reported in Table S17.

Figure S10.
Correlating miR-34a expression with arsenical levels in the liver of iAs exposure WT and humanized mice.Correlation analysis between miR-34a and percent arsenicals in the (A) liver or (B) adipose.R, Pearson's correlation coefficient.Data reported in Table S18.

Figure S2 .
Figure S2.Assessing microRNA profiles in adipose tissue of humanized and wild type mice exposed to iAs.A) PCA plot of rlog transformed miR expression in the adipose of iAs exposed mice.Male only (top panel), female only (bottom panel).Two different sequencing batches are shown by circle and triangle, respectively.B) Volcano plots showing differentially expressed (DE) miRs in the adipose of WT vs. humanized mice for males (top panel) and females (bottom panel).miRs up-regulated in WT vs. humanized shown in red, down-regulated shown in blue (padjusted < 0.05, log2foldcahnge <-1 or >1, basemean > 500).Log2Fold change = humanized mice/ WT mice for either male or female groups.Data reported in TableS3.

Figure S5 .
Figure S5.Assessing gene profiles in adipose tissue of humanized and wild type mice exposed to iAs.A) PCA plot of rlog transformed gene expression in the liver of iAs exposed mice.Male only (top panel), female only (bottom panel).B) Volcano plots showing differentially expressed (DE) genes in the liver of WT vs. humanized for males (top panel) and females (bottom panel).Genes up-regulated in WT vs. humanized shown in red, down-regulated shown in blue (p-adjusted < 0.05, log2foldcahnge <-0.5 or >0.5, basemean > 500).Log2Fold change = humanized mice/ WT mice for either male or female groups.C) Western blot of isolated protein from liver samples of WT mice.20 ug of protein was loaded with protein ladder in first and last lane to ensure gel ran evenly.ImageJ densitometry was used to quantify protein content.D) Western blots of WT and humanized showing Klf11 and b-actin on the same blot at 1 second exposure time.E) Western blots of WT and humanized showing KLF11 and b-actin on the same blot at 30 second exposure time.Data reported in TableS12.