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

Highlights

• BPA exposure during fetal period and in vitro BPA treatment alters neurogenesis.

• BPA increases hypothalamic neuroprogenitor cell proliferation and differentiation.

• BPA increases protein expression of hypothalamic neurogenic bHLH factors.

• BPA promotes protein expression of hypothalamic appetite versus satiety neuropeptide.

• BPA promotes neuronal versus astroglial lineage.

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.

Introduction

Bisphenol A (BPA) is a monomer plasticizer used in the manufacture of common house-hold goods including polycarbonate plastics (e.g. food and drink containers), paints and adhesives (Vandenberg et al., 2007). As an estrogen endocrine disrupter chemical, BPA has been associated with a range of adverse perinatal, childhood, and adult health outcomes (Rochester, 2013), including reproductive and developmental effects (Kim et al., 2011), neurogenesis (Kim et al., 2009), neurological behaviour (Palanza et al., 2008), and metabolic disease (Teppala et al., 2012). BPA exposure has been linked to childhood and adult obesity and likely contributes to the on-going obesity epidemic (Di Ciaula and Portincasa, 2017, Janesick and Blumberg, 2012; Savastano et al, 2015). In rodents, maternal BPA exposure increases postnatal body weights and growth rates, with some studies showing greater susceptibility to BPA-increased adiposity in female as compared to male offspring (Richter et al., 2007, Rubin and Soto, 2009, Somm et al., 2009). Critically, fetal exposures to BPA at levels equivalent to, or below the established daily human safe-dose (50 µg BPA/kg body weight/day) not only increase body weight and postnatal growth rate, but also alter body composition in later life (Alonso-Magdalena et al., 2006, Alonso-Magdalena et al., 2010, Richter et al., 2007, Rubin and Soto, 2009, vom Saal et al., 2012)

One of the critical determinants of energy balance include energy (calorie) intake (Hill et al., 2012). The arcuate nucleus (ARC) of the hypothalamus is the key regulator of appetite, containing both orexigenic (neuropeptide Y, NPY; agouti-related peptide, AgRP) and anorexigenic (pro-opiomelanocortin, POMC) neurons involved in central regulation of food intake (Blevins et al., 2002). Orexigenic and anorexigenic neurons develop before birth, in preparation for extra-uterine life, (Kagotani et al., 1989) however functional projections are established during the early postnatal period in rodents (Grove et al., 2001, Nilsson et al., 2005, Padilla et al., 2010, Walsh and Brawer, 1979). Studies including those by our laboratory have shown prenatal nutrition-mediated effects on ARC neurogenesis resulting in a shift from satiety to appetite neurons in association with offspring hyperphagia and obesity (Staples et al., 2017, Val-Laillet et al., 2017). We have further shown that hypothalamic neuroprogenitor cell (NPC) proliferation (self-renewal) and differentiation (generation of neurons/glial cells) are vulnerable to endocrine disruption, with potential long-term consequences for appetite regulation and energy balance (Desai et al., 2011a, Desai et al., 2011b). Notably, BPA has been shown to influence neurogenesis in humans (Preciados et al., 2016) and animal models (Kim et al., 2009). In mice, prenatal exposure to BPA increases neurogenesis and neuronal migration (Nakamura et al., 2006) resulting in altered brain structure (Nakamura et al., 2007) and function (Nakamura et al., 2012).

Neurogenesis is regulated, in part, by basic-helix-loop-helix (bHLH) genes including differentiation repressor genes (e.g., Hes1) that maintain the NPC population, and activator genes (e.g. Math3; Mash1; Neurogenin, Ngn), which accelerate neurogenesis and differentiation (Kageyama et al., 2008, Masica et al., 1971, Ohtsuka et al., 2001). Maternal BPA up-regulates Math3 and Ngn2 in mouse embryos, (Nakamura et al., 2006) and accelerated neurogenesis due to BPA exposure may reduce the population of NPCs in fetal (e14.5) mice (Komada et al., 2012, Nakamura et al., 2006). However, the effects of perinatal BPA on rat hypothalamic NPC cell proliferation and differentiation have not been determined.

We studied the effects of maternal BPA exposure during pregnancy on cultured hypothalamic NPCs from 1 day old newborns and examined development of appetite/satiety neurons (Desai et al., 2014). To more fully explore the mechanisms of BPA-mediated effects, we then utilized established models of newborn rat primary hypothalamic NPCs (which ultimately form appetite/satiety neurons), exploring both proliferative (i.e., trophic) and differentiation effects of BPA (Desai et al., 2011a, Desai et al., 2011b). We further explored putative signal factors which explain, in part, NPC responses, and underlying epigenetic mechanisms mediated by BPA. Our results demonstrate marked effects of BPA on hypothalamic progenitor cell proliferation as well as differentiation. These findings emphasize the vulnerability of stem-cell populations that are involved in life-long regulation of metabolic homeostasis to endocrine disruption by BPA during early life.