Crosstalk between the aryl hydrocarbon receptor and hypoxia-inducible factor 1α pathways in human islet models

Abstract

Background: We previously showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD - a persistent organic pollutant) activates the aryl hydrocarbon receptor (AHR) in pancreatic islets. The AHR is known to crosstalk with hypoxia-inducible factor 1α (HIF1α) in other cell types but AHR-HIF1α crosstalk has not been previously examined in islet cells. Islet cell function is sensitive to hypoxia; we hypothesize that AHR activation by environmental pollutant(s) will interfere with the HIF1α pathway response in islets, which may be detrimental to islet cell function and survival during periods of hypoxia.

Methods: We assessed AHR-HIF1α crosstalk by treating human donor islets and stem cell-derived islets (SC-islets) with 10 nM TCDD ± 1% O₂ and measuring gene expression of downstream targets of AHR (e.g. CYP1A1) and HIF1α (e.g. HMOX1).

Results: In SC-islets, co-treatment with TCDD + hypoxia consistently suppressed CYP1A1 induction compared with TCDD treatment alone. In human islets, TCDD + hypoxia co-treatment suppressed CYP1A1 induction, but only in 2 of 6 donors. Both SC-islets and human donor islets displayed hypoxia-mediated suppression of glucose-6-phosphate catalytic subunit 2 (G6PC2) expression. Glucose-stimulated insulin secretion (GSIS) in human donor islets was impaired by hypoxia exposure, but unaffected by TCDD exposure.

Conclusion: Our study shows consistent AHR-HIF1α crosstalk in SC-islets and variable crosstalk in primary human islets, depending on the donor. In both cell models, hypoxia exposure interfered with activation of the AHR pathway by TCDD but there was no evidence that AHR activation interfered with the HIF1α pathway. In summary, our data show that co-exposure to an environmental pollutant and hypoxia results in molecular crosstalk in islets.

Keywords: CYP1A1; G6PC2; dioxin; human donor islets; hypoxia; stem cell-derived islets (SC-islets).

Graphical abstract

Figure 1.

Summary of treatment conditions applied to SC-islets and human donor islets. (A) SC-islets were exposed to TCDD (10 nM) ± hypoxia (1% O₂) for 48 hours continuously or to TCDD or hypoxia pre-treatment for 24 hours followed by TCDD + hypoxia co-treatment for 24 hours. (B) SC-islets or human donor islets were exposed to TCDD ± hypoxia co-treatment continuously for 48 hours. Made with BioRender.com.

Figure 2.

Hypoxia interfered with CYP1A1 upregulation by TCDD in SC-islets. (A) CYP1A1 and (B) HMOX1 gene expression in SC-islets co-treated with TCDD (10 nM) ± hypoxia (1% O₂) for 48 hours or following either TCDD or hypoxia pre-treatment for 24 hours and TCDD + hypoxia co-treatment for 24 hours. Gene expression is relative to DMSO-treated SC-islets. Data are mean ± SEM and individual data points represent biological replicates (i.e. different differentiations). Significance was determined by a one-way ANOVA and Tukey post-hoc test. Multiple comparison analysis showing treatment groups that are significantly different from each other are indicated by different letters (p < 0.05).

Figure 3.

TCDD + hypoxia co-treatment causes interactive effect for CYP1A1 and G6PC2 expression in SC-islets. SC-islets were exposed to TCDD (10 nM) ± hypoxia (1% O₂) for 48 hours and gene expression was measured for (A) CYP1A1, (B) AHR, (C) AHRR, (D) HMOX1, (E) VEGFA, (F) ARNT, (G) MNSOD, (H) SLC2A1, (I) MAFA, and (J) G6PC2. Gene expression is relative to DMSO-treated SC-islets. Data are mean ± SEM and individual data points represent biological replicates (i.e. different differentiations). Significance was determined by a two-way ANOVA and Tukey post-hoc test. Overall treatment and interaction effects are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Multiple comparison analysis showing treatment groups that are significantly different from each other are indicated by different letters (p < 0.05).

Figure 4.

Co-treatment of human islets to TCDD + hypoxia impairs induction of CYP1A1 by TCDD in 2 of 6 donors. SC-islets and human islets were exposed to TCDD (10 nM) ± hypoxia (1% O₂) for 48 hours. Gene expression was measured in SC-islets and human islets for (A,C) CYP1A1 and (B,D) HMOX1, and in human islets for (E) AHRR, (F) ARNT, and (G) G6PC2. Gene expression is relative to DMSO-treated islets. (A,B) Separate SC-islet biological replicates (i.e., different differentiations) and (C-G) separate human islet donors are shown for each gene. Data are mean ± SEM and individual data points represent technical replicates. Significance within each biological replicate was determined by a two-way ANOVA and Dunnett post-doc test. Overall treatment and interaction effects are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Multiple comparison analysis showing treatment groups that are significantly different from each other are indicated by different letters (p < 0.05). (H) Human islet characteristics are summarized for each individual donor.

Figure 5.

Hypoxia exposure negatively impacts glucose stimulated insulin secretion. Human islets were exposed to TCDD (10 nM) ± hypoxia (1% O₂) for 48 hours and (A) insulin secretion was measured following 1 hour in low (2.8 mM) and high (16.7 mM) glucose concentrations. (B) Stimulation index is expressed as secreted insulin concentration following high glucose relative to low glucose stimulation. (C) Total insulin content in islets following acid ethanol lysis. (D) Insulin secretion following low and high glucose stimulation normalized by total insulin content. Data are mean ± SEM and individual data points represent biological replicates (i.e. different islet donors). Significance was determined with (A, D) a two-way ANOVA and Šidák post-hoc test, (B) a brown-forsythe ANOVA and Dunnett post-hoc test, and (C) a one-way ANOVA and Dunnett post-hoc test. Multiple comparison analysis showing treatment groups that are significantly different from each other are indicated by different letters (*p < 0.05).