The additive effect of allopregnanolone on ghrelin's orexigenic effect in rats

Highlights

• Allopregnanolone (AlloP) further increases food-intake in the presence of ghrelin in rats.

• At physiological concentrations, AlloP potentiates hypothalamic inhibitory transmission in the lateral ARC and medial PVN.

• AlloP's effect on food-intake appears to depend on the level of activity in GABAergic circuits involved in eating behavior.

Abstract

The progesterone metabolite, allopregnanolone (AlloP), is a GABA_A receptor modulating steroid and is known to have orexigenic and pro-obesity effects. The neurobiological mechanisms underpinning these effects are most likely due to enhanced GABAergic signaling in the lateral arcuate nucleus (ARC) and medial paraventricular nucleus (PVN) of the hypothalamus. Inspired by the finding that GABAergic signaling is also important for the orexigenic effects of the circulating hormone, ghrelin, we sought to determine the extent to which AlloP (one of the most potent endogenous GABA_A-receptor modulators) operates alongside ghrelin to enhance food intake. Male rats with ad libitum access to standard chow were injected intravenously with AlloP and/or ghrelin, alone or in combination. The intake of the standard chow was greater after AlloP 1 mg/kg together with ghrelin 30 μg/kg than with 30 μg/kg ghrelin alone. Food intake was also increased for the combined treatment of AlloP 0.5 mg/kg + ghrelin 10 μg/kg, AlloP 1 mg/kg + ghrelin 10 μg/kg, and AlloP 0.5 mg/kg + ghrelin 30 μg/kg. There was no significant difference in food intake between the two ghrelin doses or between the two doses of AlloP and the vehicle. In electrophysiological studies, physiologically relevant concentrations of AlloP prolonged the current decay time of spontaneous inhibitory post-synaptic current of dissociated cells of the ARC and PVN. We conclude that AlloP enhances the hyperphagic effect of ghrelin, findings of potential relevance for the hyperphagia associated with the luteal phase of the reproductive cycle.

Introduction

It is well established that GABA_A receptor modulating steroids (GAMS) have orexigenic and pro-obesity effects, especially progestogens, and the metabolite allopregnanolone (AlloP). During the luteal phase of the menstrual cycle, that can be considered a preparatory phase for a forthcoming pregnancy, energy intake is higher and food cravings are more common (Barr et al., 1995; Cross et al., 2001; Dalvit, 1981; Hormes and Rozin, 2009; Johnson et al., 1994; Reed et al., 2008). This fact underscores the important role of progestogens in ensuring adequate nutrition during pregnancy and lactation (Beksinska et al., 2010; Butte and King, 2005). It appears that some factor formed by the corpus luteum is implicated in these effects since the fluctuation in energy intake is abolished in anovulatory cycles (Ottander et al., 2005). Indeed, some hormonal contraceptives have the side effect of increasing body weight (Bahamondes et al., 2001; Beksinska et al., 2010; Berenson and Rahman, 2009; Bonny et al., 2006; Risser et al., 1999), and the progestogen, medroxyprogesterone-acetate has even been used to improve appetite and hence, the body weight in malnourished patients (Simons et al., 1996). Notably, circulating AlloP levels have been associated with binge eating in women and obesity in both men and women (Menozzi et al., 2002; Monteleone et al., 2003; Predieri et al., 2007). Stress is a known risk factor for obesity (Vieweg et al., 2007), and stress also stimulates the production of AlloP and other GABA_A-receptor active steroids in male rats (Purdy et al., 1991) and humans of both sexes (Droogleever Fortuyn et al., 2004).

In rodents, hyperphagia can be induced by the administration of AlloP, and the effect is dose-dependent (Chen et al., 1996; Reddy and Kulkarni, 1998, Reddy and Kulkarni, 1999). Their meal size is increased, which correlates with obesity in rats (Farley et al., 2003; Furnes et al., 2009), and weight gain is also achieved after long term treatment with AlloP (Holmberg et al., 2015). In a choice situation, AlloP does also promote intake of more calorie-dense foods (Holmberg et al., 2014).

Another potent orexigenic substance is ghrelin, which is a stomach-derived peptide hormone (Kojima et al., 1999; Wren et al., 2000) with pro-obesity effects (Tschop et al., 2000). Circulating levels of ghrelin increase pre-prandial, and it has been suggested that ghrelin plays an important role in hunger and meal initiation (Cummings et al., 2001). Ghrelin activates circuits involved in energy homeostasis and appetitive behavior that includes the hypothalamus (Hewson and Dickson, 2000), brainstem (Bailey et al., 2000; Faulconbridge et al., 2008) and mesolimbic reward circuits (Abizaid et al., 2006; Dickson et al., 2010; Egecioglu et al., 2010; Jerlhag et al., 2007; Skibicka et al., 2011; Skibicka et al., 2012). In the arcuate nucleus (ARC), a key target for ghrelin is the orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing cells (Dickson and Luckman, 1997; Qi et al., 2015), which inhibit the anorexigenic pro-opiomelanocortin (POMC) population in the arcuate nucleus (ARC) by a mechanism involving increased GABAergic transmission (Cowley et al., 2003; Gao and Horvath, 2008; Jobst et al., 2004). Indeed, important roles for GABAergic transmission in feeding and ghrelin's orexigenic effects are highlighted by the finding that specific deletion of the vesicular GABA transporter in AgRP-expressing neurons leads to a strongly reduced orexigenic effect of ghrelin (Tong et al., 2008). Both NPY/AgRP- and POMC-expressing neurons project to “second-order neurons” located in, for example, the paraventricular nucleus (PVN), where anorexigenic peptides are released thereby activating catabolic circuits (Gil-Campos et al., 2006; Valassi et al., 2008). Even at this site, inhibitory GABAergic transmission appears to be of importance for food intake (Dos-Santos et al., 2018; Pu et al., 1999). AlloP potentiates the effect of GABA at the GABA_A-receptor (Majewska et al., 1986) in a manner similar to that of benzodiazepine, which has a well-documented hyperphagic effect (Cooper, 2005).

Inspired by the important role of GABAergic transmission for the orexigenic effect of ghrelin, we sought to determine what effect AlloP, perhaps the most potent positive endogenous modulator of the GABA_A-receptor, would have on ghrelin's orexigenic effects. Additionally, given the large fluctuations in AlloP in normal physiology (Hill et al., 2007; Holzbauer, 1975; Nyberg et al., 2007), together with the large differences in sensitivity thresholds for the GABA_A-receptor between different brain areas (Herd et al., 2007), we also investigated the sensitivity of GABA_A-receptors to AlloP on cells from the lateral ARC and the medial PVN, two important hypothalamic areas for food intake regulation.