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Arsenic is more potent than cadmium or manganese in disrupting the INS-1 beta cell microRNA landscape

ABSTRACT: Diabetes is a metabolic disorder characterized by fasting hyperglycemia and impaired glucose tolerance. Laboratory and population studies have shown that inorganic arsenic (iAs) can impair these pathways. Other metals including cadmium (Cd) and manganese (Mn) have also been linked to diabetes phenotypes. MicroRNAs, short non-coding RNAs that regulate gene expression, have emerged as potential drivers of metabolic dysfunction. MicroRNAs responsive to metal exposures in vitro have also been reported in independent studies to regulate insulin secretion in vivo. We hypothesize that microRNA dysregulation may associate with and possibly contribute to insulin secretion impairment upon exposure to iAs, Cd, or Mn. We exposed insulin secreting rat insulinoma cells to non-cytotoxic concentrations of iAs (1 µM), Cd (5 µM), and Mn (25 µM) for 24 h followed by small RNA sequencing to identify dysregulated microRNAs. RNA sequencing was then performed to further investigate changes in gene expression caused by iAs exposure. While all three metals significantly inhibited glucose-stimulated insulin secretion, high-throughput sequencing revealed distinct microRNA profiles specific to each exposure. One of the most significantly upregulated microRNAs post-iAs treatment is miR-146a (~ + 2-fold), which is known to be activated by nuclear factor κB (NF-κB) signaling. Accordingly, we found by RNA-seq analysis that genes upregulated by iAs exposure are enriched in the NF-κB signaling pathway and genes down-regulated by iAs exposure are enriched in miR-146a binding sites and are involved in regulating beta cell function. Notably, iAs exposure caused a significant decrease in the expression of Camk2a, a calcium-dependent protein kinase that regulates insulin secretion, has been implicated in type 2 diabetes, and is a likely target of miR-146a. Further studies are needed to elucidate potential interactions among NF-kB, miR-146a, and Camk2a in the context of iAs exposure.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE131544 | GEO | 2020/05/21

REPOSITORIES: GEO

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Project description: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 leverage this model to study sex differences in the context of iAs exposure. Objectives: The aim of this study iwas 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 epididymales gonadal adipose depot were harvested for iAs quantification as well as sequencing-based microRNA and gene expression analysis. Serum blood was collected for fasting blood glucose, fasting plasma insulin, and HOMA-IR. Results: We detected sex divergence in liver and adipose markers of diabetes (e.g., insulin signaling pathways, fasting blood glucose, fasting plasma insulin, and HOMA-IR) only in humanized (not WT) male 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 underscoreds the importance of future studies leveraging the humanized mouse model to study iAs-associated metabolic disease. The findings also suggest suggested that humanized females are protected from metabolic dysfunction relative to humanized males in the context of iAs exposure. Future investigations should focus on the detailed mechanisms that underlie the sex divergence, including the potential role of miR-34a and/or Klf11.

Project description:Inorganic arsenic, a major environmental contaminant, has risen as an important health problem worldwide. More detailed identification of the molecular mechanisms associated with iAs exposure would help to establish better strategies for prevention and treatment. Although chronic iAs exposures have been previously studied there is little to no information regarding the early events of exposure to iAs. To better characterize the early mechanisms of iAs exposure we conducted gene expression studies using sublethal doses of iAs at two different time-points. The major transcripts differentially regulated at 2 hrs of iAs exposure included antioxidants, detoxificants and chaperones. Moreover, after 12 hrs of exposure many of the down-regulated genes were associated with DNA replication and S phase cell cycle progression. Interestingly, the most affected biological pathway by both 2 or 12 hrs of iAs exposure were the Nrf2-Keap1 pathway, represented by the highly up-regulated HMOX1 transcript, which is transcriptionally regulated by the transcription factor Nrf2. Additional Nrf2 targets included SQSTM1 and ABCB6, which were not previously associated with acute iAs exposure. Signalling pathways such as interferon, B cell receptor and AhR route were also responsive to acute iAs exposure. Since HMOX1 expression increased early (20 min) and was responsive to low iAs concentrations (0.1 M-oM-^AM--M), this gene could be a suitable early biomarker for iAs exposure. In addition, the novel Nrf2 targets SQSTM1 and ABCB6 could play an important and previously unrecognized role in cellular protection against iAs We used human lymphoblastoid cell line (CL-1), immortalized with Epstein-Barr virus. Cell cultures were propagated in RPMI media supplemented with 10% fetal bovine serum (FBS), L-glutammine 2 mM and antiobiotics penicillin and 10 mg/ml streptomycin) at 37M-BM-:C in an atmosphere of 5% CO2. In the case of primary cultures, 1% phytohemaglutinine were added. Cell cultures (1x106) were treated with 5M-BM-5M sodium arsenite for 2 and 12 hrs while control cultures were incubated with PBS. Nine biological replicates for each condition were used for microarray analysis. After iAs treatment, total RNA from each biological replicate was isolated with Trizol. From the 9 biological replicates performed on each condition, we generated 3 pools at a concentration of 300 ng/M-BM-5L, where each pool was processed to a single microarray platform.

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Arsenic is more potent than cadmium or manganese in disrupting the INS-1 beta cell microRNA landscape

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