Adipokines are cytokines secreted by the white adipose tissue, able to influence a variety of physiological and pathophysiological processes through endocrine, paracrine, and autocrine mechanisms. ADPN, one of the most abundant adipokines secreted in the blood stream, regulates food intake by sending satiety signals at the central level, and exerts peripheral effects (Idrizaj et al., 2019). The observation that ADPN serum levels are correlated with body fat content and are lower in obese subjects (Bastard et al., 2006) has generated enormous interest within the scientific community (Henstridge and Febbraio, 2010). ADPN receptors, Adipo-R1 and Adipo-R2, have been originally identified in the hypothalamus (Scherer et al., 1995). Previous studies (Hoyda and Ferguson, 2010) showed that ADPN acts at a central level, controlling neuronal excitability of the hypothalamic paraventricular nucleus through the modulation of different K⁺ conductances and contributing to changes in membrane potential. Moreover, ADPN receptors have been found also in a variety of peripheral tissues (Yamauchi et al., 2003; Fasshauer et al., 2004; Kharroubi et al., 2004; Blüher et al., 2005; Liu et al., 2008; González et al., 2010; Hong et al., 2016) including the gastrointestinal tract (Idrizaj et al., 2018a). Acting through different signaling pathways, ADPN exerts antidiabetic, anti-inflammatory, antiatherogenic, and antiapoptotic effects (Idrizaj et al., 2019). Recently, other physiological roles of ADPN have emerged including the skeletal muscle sensitivity to this hormone (Krause et al., 2019), the prevention of cardiac dysfunctions (Francisco et al., 2016), and actions on the smooth muscle (AlSaif et al., 2015). Particularly, it has a proved vasorelaxant effect on vascular cells (Hong et al., 2016; Schinzari et al., 2017), and some of the mechanisms by which ADPN influences the contractile tone of small arteries have been clarified (Baylie et al., 2017). Besides vascular muscle activity, ADPN can influence that of the gastric one (Idrizaj et al., 2018a), but no effects of ADPN on the excitability of the SMCs of the gastrointestinal tract are reported at present. To this aim, we here intended to investigate the effect of this hormone on the bioelectric properties of SMC from the gastric fundus, focusing on the RMP, the ion currents responsible of the RMP control (Currò, 2014), and the voltage-dependent I_Ca, mainly responsible for triggering the mechanical activity.

The experimental procedure followed the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive of 24 November 1986; 86/609/EEC) and was approved by the Committee for Animal Care and Experimental Use of the University of Florence in conformity with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (Idrizaj et al., 2018a, b). C57BL/6 (8–12 weeks old) female mice (Charles River, Lecco, Italy) were used (Squecco et al., 2013).

A muscular strip from the gastric fundus was pinned in a recording chamber (Squecco et al., 2013) bathed with a Krebs–Henseleit solution (mM): 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂, and 10 glucose (pH 7.4). Intracellular recording was made by conventional microelectrode (resistance = 60–70 MΩ) inserted in a cell of the longitudinal smooth muscle layer and filled with the following internal solution (mM): 130 KCl, 10 NaH₂PO₄, 0.2 CaCl₂, 1 ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), 5 MgATP, and 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2), unless otherwise stated. We used the Krebs–Henseleit solution as Ctrl external solution to record RMP and passive properties of SMCs. In order to record K⁺ current (I_K) we used the Krebs–Henseleit as external solution with specific channel blockers such as Nifedipine (10 μM) for L-type I_Ca, BaCl₂ (0.4 mM) for eventual inward rectifier K⁺ current, 4-aminopyridine (4-AP, 2 mM) for eventual transient outward K⁺ current (Castle and Slawsky, 1993; Crescioli et al., 2008). According to Idrizaj et al. (2018a), to record only I_Ca we used a high-TEA external solution (mM): 10 CaCl₂, 145 tetraethylammonium bromide, 10 HEPES, and a suitable filling pipette solution (mM): 150 CsBr, 5 MgCl₂, 10 EGTA, and 10 HEPES (pH = 7.2). The current amplitude was normalized to cell capacitance, C_m, to properly compare the currents recorded from cells of different size.

Recombinant full-length mouse ADPN was tested from 2 × 10^–11 up to 10^–7 M. Heptanol (1 mM) was consistently used to block gap junctional currents of the functional syncytium (Squecco et al., 2013). Drugs were from Sigma Chemical (St. Louis, MO, United States).

We recorded RMP of the SMCs before and after chemical stimulation in current clamp mode, with a stimulus waveform: I = 0 pA (Squecco et al., 2015). The membrane passive properties were consistently estimated in voltage clamp starting from a HP of −70 mV. I_K activation was elicited by 1-s long voltage pulses ranging from −80 to 50 mV applied in 10-mV increments (HP = −60 mV). I_Ca kinetics was analyzed as in Idrizaj et al. (2018b). Mathematical analysis of data was performed by pClamp6 (Axon Instruments). Statistical analysis was done using Student’s t-test or one-way ANOVA followed by Bonferroni’s post hoc test when more than two groups of data were compared. n represents the number of SMCs analyzed. Results are mean ± SEM. P ≤ 0.05 was considered significant unless otherwise specified.

Some adipokines that act at the central level to influence feeding behavior seem to affect gastrointestinal motor phenomena, which represent peripheral signals involved in the regulation of food intake (Duca and Covasa, 2012). In this view, leptin (Yarandi et al., 2011) and more recently ADPN appear to influence gastrointestinal motility in addition to their central actions. Particularly, ADPN is able to induce a decrease of the gastric mechanical activity in mice (Idrizaj et al., 2018a). The present results indicate for the first time that the hormone can influence the gastric SMCs’ electrophysiological properties, which represent the first steps for the mechanical responses. Indeed, in keeping with our previous observation that ADPN induced gastric relaxation (Idrizaj et al., 2018a), we note that the hormone strongly influences SMCs’ excitability by inducing membrane hyperpolarization. This effect can be determined, at least in part, by the here-observed tendency toward the reduction of G_m, since this may hamper the aspecific entry of depolarizing ions, leading to a decreased SMC excitability. Moreover, we also noted that ADPN induces an increase of I_K, which is known to play an important role in RMP Ctrl. Although this effect was not extraordinarily broad, it may indeed contribute to the hyperpolarization. A more negative RMP definitely disturbs the related electromechanical coupling since a more intense stimulus than usual is required to activate high voltage threshold-operated ionic channels (Dwyer et al., 2011; Idrizaj et al., 2018a). Accordingly, it became remarkable to study ADPN effect also on I_Ca. In fact, this current represents a chief source for intracellular Ca²⁺ elevation useful for contractile activation and its eventual modifications may further affect the SMC mechanical activity. Interestingly, we found that ADPN reduced I_Ca amplitude exerting an inhibitory effect on Ca²⁺ influx through voltage-dependent Ca²⁺ channels, further supporting its influence in hindering the SMC electromechanical coupling.

However, our study raises several additional queries needing further investigation, such as the type of K⁺ channel mostly involved ADPN effects, and the possible signaling pathways through which ADPN modulates the gastric SMC excitability. To answer these questions, further studies are in progress in our laboratory. Several signaling paths have been reported in relation to non-gastric smooth muscle and other targets for ADPN such as AMP-activated protein kinase (AMPK), peroxisome proliferative-activated receptor (PPAR)-α expression, ceramidase activity, and sphingosine 1 phosphate (S1P) formation (Botta et al., 2019; Kim and Park, 2019). This and some other previous reports dealing with NO signaling (Chen et al., 2003; Grossini et al., 2016; Nour-Eldine et al., 2016) will provide a useful background for our future studies.

In conclusion, this preliminary study offers the first evidence that ADPN exerts a novel inhibitory function at the SMC plasma membrane level in gastric preparations that concurs to an actual weakened SMC excitability (Koh et al., 2012). ADPN seems to hinder the first steps of the excitation–contraction coupling, which is in perfect agreement with our previously published mechanical findings (Idrizaj et al., 2018a). Thus, ADPN seems to favor gastric muscle relaxation, which may lead to a consequent increase of organ capacity. Because gastric distension represents, from a physiological point of view, a peripheral satiety signal, we speculated that the here-observed peripheral effects are part of a control system designed to regulate food intake, which might concur to suppress feeding behavior. These observations provide a stimulating background to the challenging hypothesis that ADPN and/or its receptors could be a potential therapeutic tool in the treatment of obesity (Li et al., 2017) and eating disorders and, certainly, this issue deserves further investigation in a translational perspective.

This study was carried out in accordance with the recommendations of the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive of 24 November 1986; 86/609/EEC). The protocol was approved by the Committee for Animal Care and Experimental Use of the University of Florence in conformity with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health.

EI and RS performed the electrophysiological experiments. EI, RG, FF, and RS analyzed the data. EI, RS, and FF prepared the figures. RS, MB, GC, and VR designed the research study. RS wrote the manuscript. RS, EI, MB, FF, RG, GC, and VR critically revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.