In vivo maternal and in vitro BPA exposure effects on hypothalamic neurogenesis and appetite regulators

Abstract

In utero exposure to the ubiquitous plasticizer, bisphenol A (BPA) is associated with offspring obesity. As food intake/appetite is one of the critical elements contributing to obesity, we determined the effects of in vivo maternal BPA and in vitro BPA exposure on newborn hypothalamic stem cells which form the arcuate nucleus appetite center. For in vivo studies, female rats received BPA prior to and during pregnancy via drinking water, and newborn offspring primary hypothalamic neuroprogenitor (NPCs) were obtained and cultured. For in vitro BPA exposure, primary hypothalamic NPCs from healthy newborns were utilized. In both cases, we studied the effects of BPA on NPC proliferation and differentiation, including putative signal and appetite factors. Maternal BPA increased hypothalamic NPC proliferation and differentiation in newborns, in conjunction with increased neuroproliferative (Hes1) and proneurogenic (Ngn3) protein expression. With NPC differentiation, BPA exposure increased appetite peptide and reduced satiety peptide expression. In vitro BPA-treated control NPCs showed results that were consistent with in vivo data (increase appetite vs satiety peptide expression) and further showed a shift towards neuronal versus glial fate as well as an increase in the epigenetic regulator lysine-specific histone demethylase1 (LSD1). These findings emphasize the vulnerability of stem-cell populations that are involved in life-long regulation of metabolic homeostasis to epigenetically-mediated endocrine disruption by BPA during early life.

Keywords: Epigenetic; Neuroprogenitor cells; Obesity; Perinatal exposures; Proliferation, differentiation.

Figure 1:. Maternal BPA Effects on Offspring NPC Proliferation

Hypothalamic NPCs from Control and BPA 1 day old male newborns were cultured in complete media. (A) Double immunostained images of (x40; scale bar = 50μm) of DAPI (blue, nuclear stain) and Nestin (red, NPC marker). (B) Proliferative index measured at 570 OD. (C) Protein expression of NPC marker (Nestin) and neuroproliferative factor (Hes1). Values are fold change (mean±SE) of n=6 of pooled hypothalami from each of 6 litters per group. * P< 0.05 BPA (□) vs. Control (■).

Figure 2:. Maternal BPA Effects on Offspring NPC Differentiation and Neuropeptides

Hypothalamic NPCs from Control and BPA 1 day old male newborns were cultured in differentiation media. (A) Double immunostained images of (x40; scale bar = 50μm) of DAPI (blue, nuclear stain) and Ngn3 (red, proneurogenic factor). (B) Protein expression of Ngn3. (C) Protein expression of appetite (AgRP) and satiety (POMC) neuropeptides. Values are fold change (mean±SE) of n=6 of pooled hypothalami from each of 6 litters per group. * P< 0.05 BPA (□) vs. Control (■).

Figure 3:. In Vitro BPA Effects on NPC Proliferation

Hypothalamic NPCs from 1 day old Control newborns were cultured in complete media and treated with DMSO (Control) or BPA (10 μM unless otherwise specified) for 5 days. (A) Images of NPCs (x20; scale bar = 50μm) and proliferative index measured at 570 OD of NPCs treated with BPA (1, 10, 20 μM). (B) Protein expression of Nestin (NPC marker) and bHLH proliferative (Hes1). (C) Immunostained images of (x40) Nestin (red), Hes1 (green) and DAPI (blue, nuclear stain). Values are fold change (mean±SE) of n=4 of pooled NPCs from each of 4 litters. * P< 0.05 BPA (□) vs. Control (■).

Figure 4:. In Vitro BPA Effects on NPC Differentiation

Hypothalamic NPCs from 1 day old Control newborns were cultured in differentiation media and treated with DMSO (Control) or BPA (10 μM,) for 5 days. (A) Protein expression of bHLH proneurogenic factors (Mash1, Ngn3) and neuronal (Tuj1) and astrocyte (GFAP) markers. (B) Protein expression of appetite (AgRP, NPY) and satiety (POMC) neuropeptides .Values are fold change (mean±SE) of n=4 of pooled NPCs from each of 4 litters; * P< 0.05 BPA (□) vs. Control (■).

Figure 5:. In Vitro BPA Effects on NPC Epigenetic Factors

Hypothalamic NPCs from 1 day old Control newborns were cultured in complete medium and treated with DMSO (Control) or BPA (10 μM,) for 5 days and protein expression of DNMT3a and LSD1 analyzed. Values are fold change (mean±SE) of of pooled cells n=4 from each of 4 litters; * P< 0.05 BPA (□) vs. Control (■).