Screening of Relevant Metabolism-Disrupting Chemicals on Pancreatic β-Cells: Evaluation of Murine and Human In Vitro Models

Abstract

Endocrine-disrupting chemicals (EDCs) are chemical substances that can interfere with the normal function of the endocrine system. EDCs are ubiquitous and can be found in a variety of consumer products such as food packaging materials, personal care and household products, plastic additives, and flame retardants. Over the last decade, the impact of EDCs on human health has been widely acknowledged as they have been associated with different endocrine diseases. Among them, a subset called metabolism-disrupting chemicals (MDCs) is able to promote metabolic changes that can lead to the development of metabolic disorders such as diabetes, obesity, hepatic steatosis, and metabolic syndrome, among others. Despite this, today, there are still no definitive and standardized in vitro tools to support the metabolic risk assessment of existing and emerging MDCs for regulatory purposes. Here, we evaluated the following two different pancreatic cell-based in vitro systems: the murine pancreatic β-cell line MIN6 as well as the human pancreatic β-cell line EndoC-βH1. Both were challenged with the following range of relevant concentrations of seven well-known EDCs: (bisphenol-A (BPA), bisphenol-S (BPS), bisphenol-F (BPF), perfluorooctanesulfonic acid (PFOS), di(2-ethylhexyl) phthalate (DEHP), cadmium chloride (CdCl₂), and dichlorodiphenyldichloroethylene (DDE)). The screening revealed that most of the tested chemicals have detectable, deleterious effects on glucose-stimulated insulin release, insulin content, electrical activity, gene expression, and/or viability. Our data provide new molecular information on the direct effects of the selected chemicals on key aspects of pancreatic β-cell function, such as the stimulus-secretion coupling and ion channel activity. In addition, we found that, in general, the sensitivity and responses were comparable to those from other in vivo studies reported in the literature. Overall, our results suggest that both systems can serve as effective tools for the rapid screening of potential MDC effects on pancreatic β-cell physiology as well as for deciphering and better understanding the molecular mechanisms that underlie their action.

Keywords: diabetes; electrical activity; insulin secretion; metabolic disorders; metabolism-disrupting chemicals; pancreatic β-cell.

Figure 1

BPA effects on pancreatic β-cells. (A) Viability of MIN6 cells treated for 24 h with different BPA concentrations (100 pM–10 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = five independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. (B) mRNA expression of Ins, Pdx1, Hnf4α, MafA, Kir6.2, Sur1, Glut2, and Gck in MIN6 cells treated for 24 h with different BPA concentrations (100 pM–10 μM). n = three independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. # vs. Control; Student’s t-test. (C) Effects of BPA (100 pM–10 μM) on GSIS in MIN6 cells treated for 24 h. n = six independent experiments. * vs. Control 16.7 mM G; two-way ANOVA. $ vs. Control 1.67 mM G and + vs. Control 16.7 mM G; Kruskal-Wallis. (D) MIN6 insulin content after 24 h BPA treatment. n = six independent experiments. * vs. Control; Kruskal-Wallis. # vs. Control; Student’s t-test. (E) Viability of EndoC-βH1 cells treated for 72 h with different BPA concentrations (1 nM–1 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = four independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. (F) mRNA expression of INS, PDX1, HNF4α, MAFA, MAFB, KIR6.2, SUR1, SNAP25, GLUT1, and GCK in EndoC-βH1 cells treated for 72 h with different BPA concentrations (1 nM–100 nM). n = three independent experiments. * vs. Control; Kruskal-Wallis. (G) Upper panel, representative recordings of K⁺ currents in response to depolarizing voltage pulses in Control or BPA (1 nM–100 nM) EndoC-βH1 treated-cells for 72 h. Lower panel, relationship between K⁺ current density and the voltage of the pulses. Control (n = 12) and BPA (n = 12 per condition) cells. * Control vs. 1 nM BPA and # Control vs. 100 nM BPA; two-way ANOVA. (H) Upper panel, representative recordings of Ca²⁺ currents in response to depolarizing voltage pulses in Control or BPA (1 nM–100 nM) EndoC-βH1 treated-cells for 72 h. Lower panel, relationship between Ca²⁺ current density and the voltage of the pulses. Control (n = 11) and BPA (n = 10 per condition) cells. * Control vs. 10 nM BPA and # Control vs. 100 nM BPA; two-way ANOVA. (I) Effects of BPA (1 nM–1 μM) on GSIS in EndoC-βH1 cells treated for 72 h. Left panel: GSIS in response to low glucose (2.8 mM G) and high glucose (20 mM G). Right panel is an inset graph that shows insulin release in response to 20 mM G. n = five independent experiments. * vs. Control 20 mM G; two-way ANOVA. $ vs. Control 2.8 mM G and + vs. Control 20 mM G; one-way ANOVA. (J) EndoC-βH1 insulin content after 72 h BPA treatment. n = five independent experiments. * vs. Control; one-way ANOVA. # vs. Control; Student’s t-test. All data are expressed as mean ± SEM. Significance * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # p < 0.05, ### p < 0.001, and #### p < 0.0001; $ p < 0.05, and $$ p < 0.01; + p < 0.05, ++ p < 0.01, and ++++ p < 0.0001.

Figure 2

BPS effects on pancreatic β-cells. (A) Viability of MIN6 cells treated for 24 h with different BPS concentrations (100 pM–10 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = four independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. (B) mRNA expression of Ins, Pdx1, Hnf4α, MafA, Kir6.2, Sur1, Glut2, and Gck in MIN6 cells treated for 24 h with different BPS concentrations (100 pM–10 μM). n = three independent experiments. # vs. Control; Student’s t-test. (C) Effects of BPS (100 pM–10 μM) on GSIS in MIN6 cells treated for 24 h. n = five independent experiments. * vs. Control 16.7 mM G; two-way ANOVA. + vs. Control 16.7 mM G; Kruskal-Wallis. (D) MIN6 insulin content after 24 h BPS treatment. n = five independent experiments. * vs. Control; one-way ANOVA. # vs. Control; Student’s t-test. (E) Viability of EndoC-βH1 cells treated for 48 h with different BPS concentrations (1 nM–1 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = four independent experiments. * vs. Control; one-way ANOVA. (F) mRNA expression of INS, PDX1, HNF4α, MAFA, MAFB, KIR6.2, SUR1, SNAP25, GLUT1, and GCK in EndoC-βH1 cells treated for 48 h with different BPS concentrations (1 nM–100 nM). n = three independent experiments. * vs. Control; Kruskal-Wallis. # vs. Control; Student’s t-test. (G) Upper panel, representative recordings of K⁺ currents in response to depolarizing voltage pulses in Control or BPS (1 nM–100 nM) EndoC-βH1 treated-cells for 48 h. Lower panel, relationship between K⁺ current density and the voltage of the pulses. Control (n = 27) and BPS (n = 13–20 per condition) cells. (H) Upper panel, representative recordings of Ca²⁺ currents in response to depolarizing voltage pulses in Control or BPS (1 nM–100 nM) EndoC-βH1 treated-cells for 48 h. Lower panel, relationship between Ca²⁺ current density and the voltage of the pulses. Control (n = 12) and BPS (n = 11–12 per condition) cells. * Control vs. 10 nM BPS and # Control vs. 100 nM BPS; two-way ANOVA. (I) Effects of BPS (1 nM–1 μM) on GSIS in EndoC-βH1 cells treated for 48 h. Left panel: GSIS in response to low glucose (2.8 mM G) and high glucose (20 mM G). Right panel is an inset graph that shows insulin release in response to 20 mM G. n = five independent experiments. * vs. Control 20 mM G; two-way ANOVA. + vs. Control 20 mM G; Kruskal-Wallis. (J) EndoC-βH1 insulin content after 48 h BPS treatment. n = five independent experiments. All data are expressed as mean ± SEM. Significance * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001; + p < 0.05, and ++++ p < 0.0001.

Figure 3

DEHP effects on pancreatic β-cells. (A) Viability of MIN6 cells treated for 24 h with different DEHP concentrations (100 pM–10 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = four independent experiments. * vs. Control; Kruskal-Wallis. (B) mRNA expression of Ins, Pdx1, Hnf4α, MafA, Kir6.2, Sur1, Glut2, and Gck in MIN6 cells treated for 24 h with different DEHP concentrations (100 pM–10 μM). n = five independent experiments. * vs. Control; Kruskal-Wallis. (C) Effects of DEHP (100 pM–10 μM) on GSIS in MIN6 cells treated for 24 h. n = four independent experiments. * vs. Control 16.7 mM G; two-way ANOVA. + vs. Control 16.7 mM G; one-way ANOVA. (D) MIN6 insulin content after 24 h DEHP treatment. n = four independent experiments. # vs. Control; Student’s t-test. (E) Viability of EndoC-βH1 cells treated for 7 d with different DEHP concentrations (1 nM–1 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = four independent experiments. * vs. Control; one-way ANOVA. (F) mRNA expression of INS, PDX1, HNF4α, MAFA, MAFB, KIR6.2, SUR1, SNAP25, GLUT1, and GCK in EndoC-βH1 cells treated for 7 d with different DEHP concentrations (1 nM–100 nM). n = four independent experiments. (G) Upper panel, representative recordings of K⁺ currents in response to depolarizing voltage pulses in Control or DEHP (1 nM–100 nM) EndoC-βH1 treated-cells for 7 d. Lower panel, relationship between K⁺ current density and the voltage of the pulses. Control (n = 15) and DEHP (n = 12–15 per condition) cells. (H) Upper panel, representative recordings of Ca²⁺ currents in response to depolarizing voltage pulses in Control or DEHP (1 nM–100 nM) EndoC-βH1 treated-cells for 7 d. Lower panel, relationship between Ca²⁺ current density and the voltage of the pulses. Control (n = 11) and DEHP (n = 11 per condition) cells. (I) Effects of DEHP (1 nM–1 μM) on GSIS in EndoC-βH1 cells treated for 7 d. Left panel: GSIS in response to low glucose (2.8 mM G) and high glucose (20 mM G). Right panel is an inset graph that shows insulin release in response to 20 mM G. n = five independent experiments. * vs. Control 20 mM G; two-way ANOVA. + vs. Control 20 mM G; Kruskal-Wallis. (J) EndoC-βH1 insulin content after 7 d DEHP treatment. n = five independent experiments. # vs. Control; Student’s t-test. All data are expressed as mean ± SEM. Significance * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # p < 0.05; + p < 0.05, and +++ p < 0.001.

Figure 4

PFOS effects on pancreatic β-cells. (A) Viability of MIN6 cells treated for 24 h with different PFOS concentrations (100 pM–10 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = four independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. (B) mRNA expression of Ins, Pdx1, Hnf4α, MafA, Kir6.2, Sur1, Glut2, and Gck in MIN6 cells treated for 24 h with different PFOS concentrations (100 pM–10 μM). n = three independent experiments. * vs. Control; one-way ANOVA. #vs. Control; Student’s t-test. (C) Effects of PFOS (100 pM–10 μM) on GSIS in MIN6 cells treated for 24 h. n =five independent experiments. * vs. Control 16.7 mM G; two-way ANOVA. + vs. Control 16.7 mM G; one-way ANOVA. (D) MIN6 insulin content after 24 h PFOS treatment. n = five independent experiments. * vs. Control; Kruskal-Wallis. (E) Viability of EndoC-βH1 cells treated for 72 h with different PFOS concentrations (1 nM–1 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = five independent experiments. * vs. Control; Kruskal-Wallis. (F) mRNA expression of INS, PDX1, HNF4α, MAFA, MAFB, KIR6.2, SUR1, SNAP25, GLUT1, and GCK in EndoC-βH1 cells treated for 72 h with different PFOS concentrations (1 nM–100 nM). n = three independent experiments. (G) Upper panel, representative recordings of K⁺ currents in response to depolarizing voltage pulses in Control or PFOS (1 nM–100 nM) EndoC-βH1 treated-cells for 72 h. Lower panel, relationship between K⁺ current density and the voltage of the pulses. Control (n = 11) and PFOS (n = 11–14 per condition) cells. * Control vs. 10 nM PFOS; two-way ANOVA. (H) Upper panel, representative recordings of Ca²⁺ currents in response to depolarizing voltage pulses in Control or PFOS (1 nM–100 nM) EndoC-βH1 treated-cells for 72 h. Lower panel, relationship between Ca²⁺ current density and the voltage of the pulses. Control (n = 10) and PFOS (n = 10–12 per condition) cells. * Control vs. 10 nM PFOS and # Control vs. 100 nM PFOS; two-way ANOVA. (I) Effects of PFOS (1 nM–1 μM) on GSIS in EndoC-βH1 cells treated for 72 h. Left panel: GSIS in response to low glucose (2.8 mM G) and high glucose (20 mM G). Right panel is an inset graph that shows insulin release in response to 20 mM G. n = five independent experiments. * vs. Control 20 mM G; two-way ANOVA. $ vs. Control 2.8 mM G and + vs. Control 20 mM G; Kruskal-Wallis. (J) EndoC-βH1 insulin content after 72 h PFOS treatment. n = five independent experiments. * vs. Control; Kruskal-Wallis. # vs. Control; Student’s t-test. All data are expressed as mean ± SEM. Significance * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001; $ p < 0.05, and $$ p < 0.01; + p < 0.05, ++ p < 0.01, and +++ p < 0.001.

Figure 5

CdCl₂ effects on pancreatic β-cells. (A) Viability of MIN6 cells treated for 24 h with different CdCl₂ concentrations (100 pM–10 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = four independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. (B) mRNA expression of Ins, Pdx1, Hnf4α, MafA, Kir6.2, Sur1, Glut2, and Gck in MIN6 cells treated for 24 h with different CdCl₂ concentrations (100 pM–100 nM). n = three independent experiments. * vs. Control; one-way ANOVA. (C) Effects of CdCl₂ (100 pM–1 μM) on GSIS in MIN6 cells treated for 24 h. n = three independent experiments. * vs. Control 1.67 mM G; two-way ANOVA. # vs. Control 16.7 mM G; Student’s t-test. (D) MIN6 insulin content after 24 h CdCl₂ treatment. n = three independent experiments. # vs. Control; Student’s t-test. (E) Viability of EndoC-βH1 cells treated for 72 h with different CdCl₂ concentrations (1 nM–1 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = five independent experiments. * vs. Control; Kruskal-Wallis. (F) mRNA expression of INS, PDX1, HNF4α, MAFA, MAFB, KIR6.2, SUR1, SNAP25, GLUT1, and GCK in EndoC-βH1 cells treated for 72 h with different CdCl₂ concentrations (10, 100 nM). n = three independent experiments. # vs. Control; Student’s t-test. (G) Upper panel, representative recordings of K⁺ currents in response to depolarizing voltage pulses in Control or CdCl₂ (1 nM–100 nM) EndoC-βH1 treated-cells for 72 h. Lower panel, relationship between K⁺ current density and the voltage of the pulses. Control (n = 10) and CdCl₂ (n = 10 per condition) cells. (H) Upper panel, representative recordings of Ca²⁺ currents in response to depolarizing voltage pulses in Control or CdCl₂ (1 nM–100 nM) EndoC-βH1 treated-cells for 72 h. Lower panel, relationship between Ca²⁺ current density and the voltage of the pulses. Control (n = 8) and CdCl₂ (n = 8–10 per condition) cells. * Control vs. 10 nM CdCl₂ and # Control vs. 100 nM CdCl₂; two-way ANOVA. (I) Effects of CdCl₂ (1 nM–1 μM) on GSIS in EndoC-βH1 cells treated for 72 h. Left panel: GSIS in response to low glucose (2.8 mM G) and high glucose (20 mM (G). Right panel is an inset graph that shows insulin release in response to 20 mM G. n = five independent experiments. * vs. Control 20 mM G; two-way ANOVA. + vs. Control 20 mM G; one-way ANOVA. (J) EndoC-βH1 insulin content after 72 h CdCl₂ treatment. n = five independent experiments. All data are expressed as mean ± SEM. Significance * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # p < 0.05; + p < 0.05, and ++++ p < 0.0001.

Figure 6

DDE effects on pancreatic β-cells. (A) Viability of MIN6 cells treated for 24 h with different DDE concentrations (100 pM–10 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = six-seven independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. (B) mRNA expression of Ins, Pdx1, Hnf4α, MafA, Kir6.2, Sur1, Glut2, and Gck in MIN6 cells treated for 24 h with different DDE concentrations (100 pM–10 μM). n = three independent experiments. # vs. Control; Student’s t-test. (C) Effects of DDE (100 pM–10 μM) on GSIS in MIN6 cells treated for 24 h. n = four independent experiments. # vs. Control; Student’s t-test. (D) MIN6 insulin content after 24 h DDE treatment. n = three independent experiments. # vs. Control; Student’s t-test. (E) Viability of EndoC-βH1 cells treated for 7 d with different DDE concentrations (1 nM–1 μM) as evaluated by RZ, NRU and CFDA-AM assays. n = eight independent experiments. * vs. Control; one-way ANOVA or Kruskal-Wallis. (F) mRNA expression of INS, PDX1, HNF4α, MAFA, MAFB, KIR6.2, SUR1, SNAP25, GLUT1, and GCK in EndoC-βH1 cells treated for 7 d with different DDE concentrations (1 nM–100 nM). n = four independent experiments. (G) Upper panel, representative recordings of K⁺ currents in response to depolarizing voltage pulses in Control or DDE (1 nM–100 nM) EndoC-βH1 treated-cells for 7 d. Lower panel, relationship between K⁺ current density and the voltage of the pulses. Control (n = 9) and DDE (n = 10 per condition) cells. (H) Upper panel, representative recordings of Ca²⁺ currents in response to depolarizing voltage pulses in Control or DDE (1 nM–100 nM) EndoC-βH1 treated-cells for 7 d. Lower panel, relationship between Ca²⁺ current density and the voltage of the pulses. Control (n = 8) and DDE (n = 9–10 per condition) cells. (I) Effects of DDE (1 nM–1 μM) on GSIS in EndoC-βH1 cells treated for 7 d. Left panel: GSIS in response to low glucose (2.8 mM G) and high glucose (20 mM G). Right panel is an inset graph that shows insulin release in response to 20 mM G. n = three independent experiments. +vs. Control 20 mM G; Kruskal-Wallis. (J) EndoC-βH1 insulin content after 7 d DDE treatment. n = three independent experiments. All data are expressed as mean ± SEM. Significance * p < 0.05, ** p < 0.01, and *** p < 0.001; # p < 0.05; + p < 0.05, and +++ p < 0.001.