The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance

Abstract

The function of the pancreatic beta-cell is the storage and release of insulin, the main hormone involved in blood glucose homeostasis. The results in this article show that the widespread environmental contaminant bisphenol-A (BPA) imitates 17beta-estradiol (E2) effects in vivo on blood glucose homeostasis through genomic and nongenomic pathways. The exposure of adult mice to a single low dose (10 microg/kg) of either E2 or BPA induces a rapid decrease in glycemia that correlates with a rise of plasma insulin. Longer exposures to E2 and BPA induce an increase in pancreatic beta-cell insulin content in an estrogen-receptor-dependent manner. This effect is visible after 2 days of treatment and starting at doses as low as 10 microg/kg/day. After 4 days of treatment with either E2 or BPA, these mice developed chronic hyperinsulinemia, and their glucose and insulin tolerance tests were altered. These experiments unveil the link between environmental estrogens and insulin resistance. Therefore, either abnormal levels of endogenous estrogens or environmental estrogen exposure enhances the risk of developing type 2 diabetes mellitus, hypertension, and dyslipidemia.

Figure 1

Rapid change in blood glucose levels with E₂ and BPA compared with tocopherol-free corn oil (vehicle). (A) Measurement of blood glucose concentration in animals fasted for 12 hr, injected with 100 μL vehicle or 10 μg/kg body weight E₂ (n = 6–14 mice); *p < 0.05. (B) Increment of glycemia 30 min after the injection of vehicle or E₂ (n = 7–16); *p < 0.05 compared with vehicle. (C) Increment of glycemia 30 min after the injection of vehicle or BPA (n = 4–10); *p < 0.05 compared with vehicle. (D) Circulating plasma insulin in fasted (12 hr) animals 30 min after the injection of vehicle, 10 μg/kg E₂ or 10 μg/kg BPA (n = 8–16); *p = 0.024, and **p = 0.004 compared with vehicle. Error bars indicate SE.

Figure 2

Increment of glycemia (A) and plasma insulin (B) 30 min after the injection of vehicle, 10 μg/kg E₂, or 10 μg/kg BPA in animals with or without treatment with ICI (500 μg/kg/day) for 3 days. In (A), n = 4–12; *p < 0.002 compared with ICI or vehicle. In (B), n = 4–7; *p < 0.035 compared with vehicle.

Figure 3

Insulin content in β-cells from E₂- and BPA-treated mice. (A) Immunofluorescent staining of insulin in cells from mice treated with vehicle, 100 μg/kg/day E₂, or 100 μg/kg/day BPA for 4 days. Bar = 50 μm; blue indicates low fluorescence intensity, and red indicates high intensity. (B) Three-dimensional graphs of cells in (A), showing the pixel intensity [0 (low) to 256 pixels (high)]. (C) Quantification of insulin content using confocal microscopy of β-cells from mice treated with vehicle, E₂, or BPA for 4 days at either 10 or 100 μg/kg/day, shown as normalized pixel intensity. Each point represents the mean of at least 1,000 cells from three mice; *p < 0.003 compared with vehicle. (D) Time course indicating E₂ and BPA action in β-cells. Each point represents the mean of at least 1,000 single cells obtained from two mice; *p < 10^–5 compared with vehicle. (E) Insulin content of islets obtained from mice treated with vehicle, 100 μg/kg/day E₂ (n = 6; *p = 0.014), or 100 μg/kg/day BPA for 4 days (n = 6; **p = 0.04). All error bars indicate SE.

Figure 4

Insulin content in β-cells from E₂- and BPA-treated mice with and without treatment with the pure antiestrogen ICI. (A) Immunofluorescent staining for insulin in mice treated with vehicle, 100 μg/kg/day E₂, or E₂ plus 500 μg/kg/day ICI for 4 days. Bar = 50 μm; blue indicates low fluorescence, and red indicates high fluorescence. (B) Quantification of insulin content using confocal microscopy of β-cells from mice treated with vehicle, 100 μg/kg/day E₂ or BPA, or 100 μg/kg/day E₂ or BPA plus 500 μg/kg/day ICI. Each point represents the mean of at least 2,000 individual cells from four mice; *p < 10^–10. (C) Insulin content obtained by radioimmunoassay (n = 6); *p < 0.05, comparing E₂ with E₂+ICI and BPA with BPA+ICI. All error bars indicate SE.

Figure 5

Insulin secretion in vitro and in vivo. (A) Glucose-induced insulin secretion from isolated islets at 3, 7, and 16 mM glucose, from mice treated with vehicle, 100 μg/kg/day E₂, or 100 μg/kg/day BPA for 4 days (n = 4–6 animals per group); *p < 0.005, compared with vehicle. (B) Normalized plasma insulin with respect to the plasma concentration in mice treated with vehicle, 100 μg/kg/day E₂ or 100 μg/kg/day BPA for 4 days (n = 5–10 mice per group); *p < 0.0075 compared with vehicle. In the E₂ group, circulating insulin levels were 1.53 ± 0.25 ng/mL for vehicle-treated mice and 2.58 ± 0.42 ng/mL for E₂-treated mice (n = 5; p = 0.038); in the BPA group, circulating insulin levels were 1.02 ± 0.14 ng/mL for the vehicle-treated mice and 1.56 ± 0.11 ng/mL for those treated with BPA (n = 7; p = 0.005). All error bars indicate SE. Both vehicle groups were combined; the data are normalized with respect to the vehicle value from each group.

Figure 6

E₂ and BPA alter glucose tolerance and induce insulin resistance. (A) Glucose tolerance test in mice treated with vehicle or 100 μg/kg/day E₂ for 4 days; (n = 16); *p = 0.02, and **p = 0.003. (B) Same experiment as in (A) but with animals treated with vehicle or 100 μg/kg/day BPA (n = 8); *p = 0.017, and **p = 0.009. (C) Insulin tolerance test in awake, fed mice previously treated with vehicle, 100 μg/kg/day E₂, or 100 μg/kg/day BPA (n = 9); *p < 0.04, **p = 0.007, and ***p = 0.0002 compared with vehicle. (D) Experiment as in (C) but using an oral intake of either vehicle or 100 μg/kg/day BPA (n = 5); *p = 0.026, and **p = 0.0012. All error bars indicate SE.