TRPV1 Channels and Gastric Vagal Afferent Signalling in Lean and High Fat Diet Induced Obese Mice

Transient receptor potential (TRP) channels comprise a superfamily of nonselective cation channels [1]. Initially described as “Thermo TRPs” [2], more recent investigations have shown that they respond to a number of different stimuli. For example, it has been demonstrated that several TRP vanilloid (V) channels, including TRPV1, are also mechano- and osmo-sensitive [3]. The TRPV1 channel is best known as a mediator of noxious or painful stimuli [4]. However, since its discovery, physiological roles of TRPV1, not associated with pain are becoming apparent, including roles in the regulation of energy balance and intermediary metabolism [5, 6]. It has been suggested that activation of TRPV1 channels might be a target for the management of obesity [7].

Gastrointestinal (GI) vagal afferent nerves play an important role in the control of food intake and satiety signalling. An important component of these satiety signals involves gastric vagal afferents which respond to mechanical distension of the stomach [8, 9]. Within the wall of the stomach there are two classes of mechanically sensitive gastric vagal afferents. As mentioned, in the muscular layer, tension receptors detect fullness by responding to distension and contraction of the stomach wall [8, 10]. In contrast, mucosal receptors are excited by mechanical forces associated with contact of larger food particles with the epithelium, and thus are likely to be involved in the discrimination of particle size. The net effect of activating mucosal afferents is to trigger vagal reflexes which slow gastric emptying and facilitate mechanical digestion in the stomach [11]. In high fat diet (HFD) induced obesity there is a dampened response of gastric tension receptors to stretch [12] which may contribute to the facilitation of ongoing food intake despite energy sufficiency and an expanded adipose tissue store.

TRPV1 channels are expressed in the nodose ganglia [13] and more specifically in cell bodies of vagal afferents innervating the upper GI tract [14]. Gastro-oesophageal vagal afferent responses to distension are attenuated in TRPV1-/- mice [15] indicating a role in gastric tension receptor mechanosensitivity. It is plausible that the HFD induced reduction in gastric vagal afferent mechanosensitivity is due to changes in TRPV1 mediated signalling. Therefore we aimed to determine whether this was the case, by using an in vitro gastro-oesophageal vagal afferent preparation from TRPV1 intact and deficient mice fed either a standard laboratory or high fat diet.

All studies were approved and performed in accordance with the guidelines of the Animal Ethics Committees of the University of Adelaide and Institute for Medical and Veterinary Science, Adelaide, Australia. Every attempt was made to limit the number of animals used and minimize their suffering.

These data provide the first evidence that the reduced activity of gastric vagal afferents to distension in HFD conditions is likely due to disruption of TRPV1 channel signalling. The reduction in gastric tension receptor mechanosensitivity in HFD-induced obesity was not observed in TRPV1-/- mice. In addition, pharmacological studies revealed that the TRPV1 agonist OLDA potentiates responses of gastric tension receptors to circular tension in mice fed standard mouse chow but not in mice fed a HFD. The mechanosensitivity of gastric mucosal receptors was unaffected by HFD-induced obesity and/or TRPV1 channel disruption.

Satiety signals from the stomach and small intestine, mediated by the vagus, are reduced in HFD conditions [12, 20]. In addition, consistent with previous reports on colonic [21], jejunal [22] and gastro-oesophageal vagal afferent mechanosensitivity [15], the response of gastric tension receptors to stretch are also reduced in TRPV1-/- mice. However, there was no further reduction in gastric tension receptor mechanosensitivity in TRPV1-/- mice fed a HFD. This suggests that the reduction in mechanosensitivity, observed in HFD mice in this study and by others [12, 20], may be due to disruption of the TRPV1 signalling pathway. This is supported by the observation that OLDA, a TRPV1 agonist, potentiated the response of gastric tension receptors to stretch in mice fed a SLD but not in mice fed a HFD. An alternative hypothesis is that TRPV1-/- or HFD feeding reduces gastric tension receptor mechanosensitivity to its lowest point so that both interventions fail to have an additive effect. However, we know that we can further reduce gastric tension receptor mechanosensitivity in HFD mice by exposing the afferent endings to leptin [23] and thus these afferents are capable of further reduction. In this study, only the maximum response to stretch was reduced in TRPV1-/- mice, and not the threshold. In contrast, a previous report [15] indicated the threshold for activation of gastroesophageal vagal afferents is increased in TRPV1-/-, with no change in maximum response. Therefore, a more detailed evaluation of the mechanisms behind TRPV1 effects on gastric vagal afferents is required. In adipose tissue expression of TRPV1 mRNA and protein is reduced in obesity [5], however, in the whole nodose ganglia TRPV1 mRNA content was not significantly altered in HFD mice. Thus the change in mechanosensitivity in HFD conditions cannot be simply explained by changes in TPRV1 channel expression. It should be noted, in the current study TRPV1 mRNA content was determined in the whole nodose ganglia and this may not reflect receptor expression in specific vagal afferent populations. Gastric afferents represent only about 10% of neurones in the nodose ganglia [24] and therefore it is feasible that changes in TRPV1 channels within select subpopulations of gastric afferents may not be apparent especially considering that about half of the cells within the whole nodose ganglia are TRPV1 positive [25]. In addition to direct gating mechanisms TRP channels are known to be modulated downstream of G protein-coupled receptors via a variety of second messenger pathways [26, 27]. Thus in neurons TRP channels can serve as cellular sensors to a variety of chemical stimuli. For example, endocannabinoids can modulate the activity of TRPV1 channels either directly or via G protein-coupled cannabinoid receptors [28]. In GI vagal afferent neurons this modulation of TRPV1 channels may play an important role in vagal afferent satiety signalling. In HFD conditions, where the circulating levels of appetite regulating hormones are known to change considerably [29–31], it is likely that the modulation of TRPV1 channels is altered. An understanding of how TRPV1 channels, in gastric vagal afferents, are modulated and how these interactions change in HFD induced obesity may lead to more targeted treatments of obesity. An anatomical study, by Ward et al., indicates that only a proportion of gastric vagal afferent endings are TRPV1 positive [32]. Therefore, the observed effect of global TRPV1 knockout on gastric vagal afferents may be secondary to knockout of TRPV1 channels on gastric epithelial or intrinsic mesenteric nerves [32, 33] potentially involved in modulation of gastric vagal afferent mechanosensitivity. This requires further investigation.

In the stomach the role of TRPV1 in gastric vagal afferent mechanosensitivity appears to be specific to gastric tension receptors as the mechanosensitivity of gastric mucosal receptors was unaffected by TRPV1 deletion. Determination of gastric mucosal receptor TRPV1 mRNA content remains to be established. However, considering the lack of effect of disrupting TRPV1 on mucosal receptor mechanosensitivity, perhaps it is not surprising that there is no change in gastric mucosal receptor mechanosensitivity in HFD conditions observed previously [12] and confirmed in this study.

Consistent with previous reports [12, 20, 34, 35] the mice fed a HFD gained more weight than the mice fed a SLD. The effect of disrupting TRPV1 channels on weight gain appears to depend on both the quantity and type of fat in the diet. In response to a diet with 26% energy from fat (lard based diet), TRPV1-/- mice gained less weight than their wild type counterparts [36]. However, in another study [34] and the current study TRPV1+/+ and-/- mice gained an equivalent amount of weight on a diet containing ~ 60% energy from fat (lard based diet). Further, a recent study reports TRPV1-/- mice on a 55% energy from fat diet (vegetable fat based diet) gain more weight than TRPV1+/+ mice [37]. Mice fed a diet with 49% energy from fat (ingredients unreported) and treated with the TRPV1 agonist capsaicin were found to be resistant to obesity [5]. In all of these studies there were no differences in weight gain between TRPV1+/+ and-/- mice fed a SLD, although one study has reported a lesser weight gain in TRPV1-/- mice fed a SLD [38]. TRPV1 channels are increasingly implicated in various metabolic processes or syndromes, related to high saturated fat intake, such as adipogenesis, lipid metabolism and type 2 diabetes [39], therefore, the observed changes in body weight under specific dietary conditions may be a result of a multitude of TRPV1 mediated metabolic effects.

The reduction in mechanosensitivity of gastric tension receptors in TRPV1-/- mice was associated with an increase in daily food intake in mice fed a SLD. From the data obtained it is difficult to determine whether this is due to an increase in meal size or the number of meals. The stomach content, measured at the end of the diet period, provides a snapshot of food intake with the limitation that the time between the meal and the measurement of food content is unknown. In TRPV1-/- mice fed a SLD stomach content was significantly greater than TRPV1+/+ mice suggesting meal size was increased. Although this is consistent with a role for the vagus in the short-term regulation of food intake and meal termination[40], it is recognised that the association between reduced mechanosensitivity of gastric vagal afferents and food intake does not imply a direct cause and effect relationship and further investigation is required. Although little is known about the role of TRPV1 channels in the nucleus of the solitary tract [41, 42] and the dorsal motor nucleus of the vagus (DMV) [43] in terms of food intake there is some evidence for a possible interaction between TRPV1 channels and leptin signalling [44] in the DMV. Therefore the observed effects on food intake in TRPV1-/- mice in the current study could be due to disruption of TRPV1 channels in central regions associated with food intake. Further studies, for example, using mice with a conditional knockout of TRPV1 channels in vagal afferents, are required. In addition to food intake gastric emptying rate was also measured. In TRPV1 +/+ and-/- mice fed a SLD there appears to be a slight delay in gastric emptying, however, this did not reach significance and thus the increase in stomach content was not due to alterations in the rate of gastric emptying further supporting the hypothesis that meal size is increased. The observation that a reduction in gastric tension receptor mechanosensitivity does not result in reduced gastric emptying is unpredicted and highlights the current limited knowledge of the physiological role of specific subtypes of vagal afferent. An alternative hypothesis for reduced gastric vagal afferent mechanosensitivity in TRPV1-/- mice is that increased food intake stretches the stomach which subsequently leads to decreased sensitivity of the gastric tension sensitive afferents. A more detailed analysis of stomach compliance and gastric vagal afferent mechanosensitivity in TRPV1-/- mice needs to be undertaken to determine the importance of this alternate hypothesis. Although there was an increase in daily food intake in TRPV1-/- mice, in line with the reduction in gastric vagal afferent mechanosensitivity, there was no associated increase in body mass compared to TRPV1+/+ mice. This discrepancy is possibly due to the increased energy expenditure necessary for the hyperactivity observed in young TRPV1-/- mice [45]. Consistent with the lack of difference, between TRPV1+/+ and-/- mice fed a HFD, in gastric tension receptor mechanosensitivity there was no difference in food intake over the 20 week diet period or stomach content at the end of the diet period. In the first day of the HFD, presumably due to the high palatability of the diet, there was a significant increase in food intake. This was followed by a dramatic drop in food intake possibly due to the satiety effects of fat mediated in the small intestine [46]. There was no difference in the rate of gastric emptying between HFD fed TRPV1+/+ and-/- mice, however, the rate of gastric emptying was significantly delayed compared to the SLD mice. Again, this is probably due to dietary fat induced small intestinal gut peptide inhibition of gastric motility [47, 48].

Previous studies have shown that the TRPV1 agonist capsaicin can acutely sensitize humans to gastric distension [49] and reduces food intake [50]. In addition, the lipid messenger oleoylethanolamide reduces short term food intake in a TRPV1-dependent manner [51]. The observation, albeit in mice, that TRPV1 channels modulate gastric vagal afferent tension receptor mechanosensitivity and may mediate the reduction in gastric vagal afferent mechanosensitivity in response to HFD induced obesity suggests that modulation of TRPV1 channels may be an important target for the regulation of food intake.

All relevant data are within the paper.

This work was supported by Royal Adelaide Hospital Gum Bequest. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.