Altered gastric vagal mechanosensitivity in diet-induced obesity persists on return to normal chow and is accompanied by increased food intake

International Journal of Obesity volume 38, pages636–642(2014)Cite this article

Subjects

Abstract

Background and aims:

Gastric vagal afferents convey satiety signals in response to mechanical stimuli. The sensitivity of these afferents is decreased in diet-induced obesity. Leptin, secreted from gastric epithelial cells, potentiates the response of vagal afferents to mechanical stimuli in lean mice, but has an inhibitory effect in high-fat diet (HFD)-induced obese mice. We sought to determine whether changes in vagal afferent function and response to leptin in obesity were reversible by returning obese mice consuming a HFD to standard laboratory chow diet (SLD).

Methods:

Eight-week-old female C57BL/6 mice were either fed a SLD (N=20) or HFD (N=20) for 24 weeks. A third group was fed a HFD for 12 weeks and then a SLD for a further 12 weeks (RFD, N=18). An in vitro gastro-oesophageal vagal afferent preparation was used to determine the mechanosensitivity of gastric vagal afferents and the modulatory effect of leptin (0.1–10 nM) was examined. Retrograde tracing and quantitative RT–PCR were used to determine the expression of leptin receptor (LepR) messenger RNA (mRNA) in whole nodose and specific cell bodies traced from the stomach.

Results:

After 24 weeks, both the HFD and RFD mice had increased body weight, gonadal fat mass, plasma leptin, plasma insulin and daily energy consumption compared with the SLD mice. The HFD and RFD mice had reduced tension receptor mechanosensitivity and leptin further inhibited responses to tension in HFD, RFD but not SLD mice. Mucosal receptors from both the SLD and RFD mice were potentiated by leptin, an effect not seen in HFD mice. LepR expression was unchanged in the whole nodose, but was reduced in the mucosal afferents of the HFD and RFD mice.

Conclusion:

Disruption of gastric vagal afferent function by HFD-induced obesity is only partially reversible by dietary change, which provides a potential mechanism preventing maintenance of weight loss.

Introduction

The experience of most obese people is that following diet-induced weight loss, they return at least to their previous weight within two years.1, 2, 3, 4 There is evidence from both animal5 and human studies6 that fat mass is permitted to increase to what is presumably a genetically determined upper limit. At any particular point along that trajectory, the fat mass reached is defended such that if it decreases, mechanisms are activated to ensure that it is restored. The mechanism responsible for this defence of body fat has been suggested to be mediated by prolonged changes in the level of appetite-regulating gastrointestinal hormones, known to be altered following weight loss and changed in such a way as to promote weight regain.7

Neurally mediated signals from the gastrointestinal tract have a role in the regulation of food intake.8, 9, 10 For example, cognitive perception of fullness following food intake is reliant on two vagally mediated mechanisms. One pathway relies on the presence of nutrients, which triggers gastrointestinal hormone endocrine and paracrine secretions from the stomach and small intestine.11, 12 The other pathway is via mechanical distension of the stomach, which in conjunction with small intestinal nutrient signals can induce satiety.13, 14 Two classes of vagal afferent mechanoreceptors have been identified in the stomach.15 The first are tension receptors, which respond to distension and contraction of the stomach. The second class is mucosal receptors, which are located close to the lumen, are responsive to contact by food particles with the mucosa and are believed to participate in the control of gastric motor function.15, 16, 17, 18 Previously it has been shown that diet-induced obesity caused a reduction in the ability of gastric and intestinal vagal afferents to respond to stretch/distension, suggesting in obesity there is reduced gastrointestinal signalling in response to food intake.19, 20

Leptin is a peptide released from both adipocytes21 and gastric epithelial cells.22 Ordinarily, leptin reduces food intake, which can be largely attributed to actions within the arcuate nucleus.23 However, leptin receptor (LepR) is also present in gastric vagal afferent neurons24 and leptin has been shown to have a role in regulating food intake through a vagally mediated mechanism.25 In diet-induced obesity, this effect is lost.26, 27 Previously, we have demonstrated that leptin has a potentiating effect on gastric vagal mucosal receptors.28 Furthermore, we have shown that obesity causes leptin to lose its potentiating effect and instead have an inhibitory effect on vagal tension receptors.28 This indicates that leptin may act within the stomach to modulate peripheral appetite signals and its ability to do so is disrupted by obesity to a degree that defends the obese state. We sought to determine the effect of returning these mice to a standard laboratory diet (SLD) for 12 weeks on their body weight, food intake, gastric mechanosensitivity and the vagal afferent response to leptin. To establish whether any change in the effect of leptin was associated with receptor expression changes, we determined the expression of LepR in both whole nodose and mucosal-traced gastric vagal afferents.

Materials and methods

Ethical approval

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.

Animals

All mice in these studies were obtained from Animal Resource Centre (Canning Vale, WA, Australia) and group housed under a 12 h light–dark cycle (lights on at 06:00) with free access to food and water. Female C57BL/6 mice aged 7 weeks entered the experimental protocol in groups such that a maximum of four mice completed their respective diet each week. They were randomly assigned to either a 24-week SLD group (7% energy from fat; N=20), a 24-week high-fat diet (HFD) group (60% energy from fat; N=20) or a group that was fed a HFD for 12 weeks and then returned to a SLD for a further 12 weeks (RFD; N=18). All mice were allowed to acclimatize for 1 week before being started on their respective diet. The mice were weighed weekly and had food intake monitored over the 24-week diet period. Blood glucose level and gonadal fat pad mass were determined from all mice on the day they were used for the in vitro mouse gastro-oesophageal afferent preparation.

In vitro mouse gastro-oesophageal afferent preparation

This preparation has been described in detail previously.15 In short, female C57BL/6 mice on the SLD, HFD or RFD diets were anaesthetised with isoflurane and killed via exsanguination. The stomach and oesophagus, with intact vagal nerves, were removed and placed mucosa side up in an organ bath containing a modified Krebs solution comprising (in mM): 118.1 NaCl, 4.7 KCl, 25.1 NaHCO3, 1.3 NaH2PO4, 1.2 MgSO4.7 H2O, 1.5 CaCl2, 1.0 citric acid, 11.1 glucose and 0.001 nifidipine, bubbled with 95% O2–5% CO2. The dissection process was carried out at 4 °C to prevent metabolic breakdown.

Characterization of gastric vagal afferent properties

In mice, two types of mechanosensitive gastric vagal afferent have been reported,15 those that respond to mucosal stroking, but not to circular tension (mucosal receptors) and those that respond to both mucosal stroking and circular tension (tension receptors). Receptive fields of these receptors were first located using mechanical stimulation with a brush in the mouse gastro-oesophageal preparation. Once located, specific stimuli were then applied. Mucosal stroking was performed using calibrated von Frey hairs (10–1000 mg), which were stroked across the mucosa at a rate of 5 mm s−1. Each receptive field was stroked 10 times and mechanical responses from the middle eight strokes taken for analysis. Circular tension was applied using a threaded hook attached to an underpinned point adjacent to the receptive field. The threaded hook was attached to a cantilever via a pulley close to the preparation. Standard weights (0.5–5 g) were then placed on the opposite end of the cantilever. Each weight was applied for 1 min with a break of another minute between removing one weight and applying the next. After analysing the two stimulus-response curves, we would classify a receptive field as either a mucosal or tension receptor.

Effect of leptin on the mechanosensitivity of vagal afferents

After the mechanosensitivity of a receptive field was determined, the effect of leptin was assessed. Leptin (0.1 nM, Sigma-Aldrich, Castle Hill, NSW, Australia) was added to the superfusing Krebs solution and allowed to equilibrate for 20 min after which, the stimulus response curves were re-determined. This procedure was repeated for leptin 1 and 10 nM.

Data recording

Afferent impulses were amplified with a biological amplifier (DAM 50, World Precision Instruments, Sarasota, FL, USA), and filtered (band-pass filter 932, CWE, Ardmore, PA, USA). Single units were discriminated on the basis of action potential shape, duration and amplitude by use of Spike 2 software (Cambridge Electronic Design, Cambridge, UK).

RNA Extraction

Nodose ganglia were removed bilaterally from mice from all three experimental groups. Total RNA was extracted using an RNeasy Micro Kit (Qiagen, Doncaster, VIC, Australia). Total RNA was also extracted from the gonadal fat deposits of all groups of mice using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was quantified by measuring the absorbance at 260 nm (A260) using a NanoDrop ND 1000 spectrophotometer (Thermo Fisher Scientific, Scoresby, VIC, Australia) and RNA purity was estimated via the 260/280 absorbance ratio.

Quantitative RT-PCR

Quantitative RT-PCR reactions were performed as described in detail previously.29 In short, reactions were performed using a Chromo4 (MJ Research, Bio-Rad, Gladesville, NSW, Australia) real-time instrument attached to a PTC-200 Peltier thermal cycler (MJ Research) and analysed with Opticon Monitor software (MJ Research). Quantitative RT-PCR reactions were performed using a QuantiTect SYBR green RT-PCR one-step RT-PCR kit (Qiagen) according to the manufacturer’s instructions. All primers used were pre-designed validated QuantiTect Primer assays (Qiagen) targeting leptin, LepR, β-actin and β-tubulin. RT-PCR reactions were carried out under the following conditions: reverse transcription, 50 °C for 30 min; initial PCR activation, 95 °C for 15 min; PCR cycles 94 °C for 15 sec, 55 °C for 30 sec and 72 °C for 30 sec repeated for 50 cycles. A melt curve was obtained to confirm the specificity of the products produced. Each assay was run in triplicate and repeated on separate days. Control PCRs were carried out substituting RNase-free water for template RNA. Relative RNA levels were calculated using the comparative CT method as described previously.30

Retrograde tracing

This procedure has been detailed previously.28 In short, to identify specific gastric mucosal afferents SLD (N=4), HFD (N=4) and RFD (N=4) mice were anaesthetised with isoflurane (1–1.5% in oxygen), a laparotomy performed, and a mucolytic (10% N-acetylcysteine; 200 μl) was injected into the stomach lumen then removed via syringe after 5 min, this was followed by two saline rinses (200 μl each). Subsequently, 10 μl of 0.5% Alexa Fluor 555 conjugate of cholera toxin β-subunit ((CTB-AF555); Invitrogen, Mulgrave, VIC, Australia) was injected into the proximal gastric lumen via a 30 ga Hamilton syringe (Hamilton Company, Reno, NV, USA) and the proximal stomach walls gently opposed to expose the dorsal and ventral surfaces to the tracer. The injection site was dried and skin incision closed. Antibiotic (Baytril; 50 μl of 50 mg ml−1) and analgesic (butorphanol; 5 mg kg−1) were administered subcutaneously. Food and water were withheld for 2 h postoperatively to maximize exposure of tracer. All mice recovered well from surgery and were routinely monitored. Isolation of tension afferents is possible; however, we have previously determined that diet-induced obesity has no effect on the expression of LepR mRNA in this sub-population.28

Laser capture microdissection

After 2 days, retrogradely traced mice were anaesthetised with pentobarbitone (60 mg kg−1, intraperitoneal), their left and right nodose ganglia were then removed and dissociated before being cultured on a duplex dish for 2 h at 37 °C in 5% CO2.28 Cells were then subjected to laser-capture microdissection, performed on a PALM Microbeam microdissection system (Carl Zeiss, Jena, Germany). Fluorescent labelled nodose neurons were microdissected and catapulted directly into a lysis and stabilization buffer (Buffer RLT, RNeasy Micro RNA extraction Kit, Qiagen) containing 0.14 M β-mercaptoethanol (Sigma-Aldrich). RNA was extracted and quantitative RT-PCR was performed on these cells using the same protocol as for whole nodose ganglia.

Plasma hormone measurements

Blood samples from all groups of mice were collected on the day of the experiment from the abdominal aorta, under anaesthesia. Blood was placed into K2EDTA-coated tubes and spun for 15 min at 1000 g to separate the plasma, which was stored at −80 °C until needed. Plasma leptin and insulin were determined using commercially available ELISA assay kits (Millipore, Billerica, MA, USA) according to manufacturers’ instructions. The sensitivities of the assays were 0.05 ng ml−1 (leptin) and 0.2 ng ml−1 (insulin); the intra-assay variations were 1.4% (leptin), 4.65% (insulin).

Statistical analysis

All data in graphs are expressed as mean values±s.e.m. with N=number of animals studied and n=number of individual afferents analysed. Vagal afferent stimulus-response curves and weight change were analysed using two-way analysis of variance and Bonferroni post hoc tests. RNA levels, fat mass, food/energy intake and plasma peptide levels were analysed using one-way analysis of variance with Tukey post hoc tests. Significance was defined at P<0.05.

Results

Diet-induced changes to mouse weight, fat mass, food consumption and plasma peptide levels

The HFD mice gained more weight than both the SLD and RFD mice (Figure 1a, both P<0.05). The RFD mice initially lost some weight upon return to the SLD, but by the end of the 24th week they had gained back all lost weight and on average weighed more than at the point they were removed from the HFD. The weight gain from the 22- to the 24-week time point was significantly greater in the RFD mice (6.1±2.7%) compared with the SLD mice (0.5±0.95%; P<0.05: unpaired t-test). The HFD and RFD mice consumed less food (in grams) than the SLD mice during the first 10 weeks (Figure 1b, P<0.05 SLD vs HFD and RFD). After the diet change, the RFD mice consumed more food than the SLD and HFD mice from week 14 through to week 24 (Figure 1b, P<0.05 SLD vs RFD and HFD vs RFD). Both the HFD and RFD mice were consuming more calories than the SLD mice during the first 12 weeks when both groups were on the high-fat diet (Figure 1c, P<0.05 SLD vs HFD and RFD). The RFD mice initially reduced their energy consumption after switching to the standard chow diet (P<0.05, weeks 14–18, HFD vs RFD); however, by week 20, they were consuming equivalent calories to the HFD mice, both of which were greater than the calorie consumption of the SLD mice (Figure 1c, P<0.05 SLD vs HFD and RFD).

Figure 1
figure1

Diet-dependent changes to mouse weight and food consumption. (a) The weight gain of mice that were either fed a chow diet for 24 weeks (SLD, N=16) (•), a high-fat diet for 24 weeks (HFD, N= 16) (▪) or a HFD for 12 weeks followed by 12 weeks on a chow diet (RFD, N=14) (○). Average food (b) and energy (c) consumption per mouse per day over the 24-week diet period • SLD, N=4, ▪ HFD, N=4 and ○ RFD, N=4, *P<0.05 SLD vs HFD, #P<0.05 SLD vs RFD, †P<0.05 HFD vs RFD.

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Gonadal fat mass of the HFD (2.71±0.37 g) mice was greater than that of both the RFD (0.62±0.10 g) and SLD (0.34±0.02 g) mice (Figure 2a, both P<0.05); however, the RFD fat mass was also greater than the SLD mice (Figure 2a, P<0.05 SLD vs RFD). Plasma leptin levels (Figure 2b) were also higher in the HFD (23.09±1.27 ng ml−1, P<0.05 vs SLD and RFD) and RFD (16.33±1.83 ng ml−1, P<0.05 vs SLD) mice compared with the SLD mice (5.01±0.31 ng ml−1). Leptin mRNA content of gonadal fat was higher in the HFD mice compared with the SLD and RFD mice (Figure 2c, both P<0.05). There was a significant positive correlation between fat mass and both leptin mRNA levels in the gonadal fat pads (r2=0.59, P<0.05 linear regression analysis) and plasma leptin levels (r2=0.65, P<0.05). Blood glucose levels were higher in both the HFD and RFD mice (12.78±0.82 mM and 11.07±0.79 mM, respectively) compared with the SLD mice (6.45±0.73 mM) (Figure 2d, both P<0.05 vs SLD). Plasma insulin (Figure 2e) was elevated in the HFD (0.97±0.08 ng ml−1 P<0.05 vs SLD and RFD) and RFD (0.69±0.03 ng ml−1 P<0.05 vs SLD) relative to SLD mice (0.55±0.13 ng ml−1).

Figure 2
figure2

Diet-dependent changes to mouse body parameters. The gonadal fat deposit weight (a), plasma leptin (b), leptin mRNA content of gonadal fat (c), blood glucose levels (d), plasma insulin (e) of SLD, HFD and RFD mice at the end of the 24-week diet regime. *P<0.05 SLD vs HFD, #P<0.05 SLD vs RFD, †P<0.05 HFD vs RFD.

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High-fat diet-induced changes in gastric mechanosensitivity are not altered by reverting to ‘normal’ chow feed

Gastric tension mechanosensitivity of the HFD and RFD mice was reduced by 50% (at 5 g) compared with the SLD mice (Figure 3a, both P<0.05 vs SLD). There was no significant difference between the responses of the gastric tension receptors of the HFD mice compared with the RFD mice (P>0.05). There was no difference in the response to mucosal stroking by mucosal receptors between any of the groups of mice (Figure 3b, P>0.05).

Figure 3
figure3

Selective high-fat diet suppression of gastric tension receptor mechanosensitivity is maintained after chow-diet feeding. Stimulus response functions of tension-sensitive (a) and mucosal (b) gastric vagal afferents from mice fed a SLD (•, a: n=16 b: n=18), HFD (▪, a: n=25, b: n=29) or RFD (○ a: n=18, b: n=22). *P<0.05 SLD vs HFD, #P<0.05 SLD vs RFD.

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Leptin’s effects on gastric vagal afferent mechanosensitivity are dependent on diet

Leptin (0.1–10 nM) potentiated gastric mucosal mechanosensitivity (Figures 4a and e, P<0.05 vs Control) in SLD mice, but had no effect in HFD mice (Figures 4b and f, P>0.05). In the RFD mice, leptin increased mucosal receptor mechanosensitivity (Figures 4c and g, P<0.05 vs Control). The level of potentiation induced by leptin (0.1–10 nM) in response to 50 mg stroking was different in all three groups of mice (Figure 4d, P<0.05 SLD vs HFD, SLD vs RFD and HFD vs RFD).

Figure 4
figure4

Mucosal receptor sensitivity to leptin is partially restored upon reverting to a chow diet. The responses of gastric mucosal receptors to stroking with calibrated von Frey hairs (10–1000 mg) in the absence (•) and presence of leptin 0.1 (○), 1 (□) and 10 nM (Δ) from mice fed either a SLD for 24 weeks (a, N=8), HFD for 24 weeks (b, N=9) or a HFD for 12 weeks followed by 12 weeks of SLD (c, N=6). *P<0.05 vs afferents prior to leptin exposure. (d) The effect of leptin on response to mucosal stroking with a 50 mg von Frey hair in SLD (•), HFD (▪) and RFD (○) mice. Diet significantly modulated the potentiating ability of leptin. *P<0.05 SLD vs HFD, #P<0.05 SLD vs RFD, †P<0.05 HFD vs RFD. Original recordings of a mucosal receptor in response to 50 mg stroking before (i) and after addition of leptin 10 nM (ii) from SLD (e), HFD (f) and RFD (g) mice.

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Leptin had no effect on the mechanosensitivity of gastric tension receptors (1–5 g) in SLD mice (Figures 5a and e, P>0.05), however, caused a reduction in HFD (Figures 5b and f, P<0.05 vs Control) and RFD (Figures 5c and g, P<0.05 vs Control) mice. The level of inhibition caused by leptin in response to 3-g stretch was highest in the HFD mice followed by the RFD and SLD mice (Figure 5d, P<0.05 SLD vs HFD, SLD vs RFD and HFD vs RFD).

Figure 5
figure5

Tension receptor inhibition by leptin is reduced by subsequent chow feeding. The responses of gastric tension receptors to circular stretch (1–5 g) in the absence (•) and presence of leptin 0.1 (○), 1 (□) and 10 nM (Δ) from mice fed either a SLD for 24 weeks (a, N=8), HFD for 24 weeks (b, N=9) or a HFD for 12 weeks followed by 12 weeks of SLD (c, N=10). *P<0.05 vs afferents prior to leptin exposure. (d) The effect of leptin on the response to 3 g circular tension in SLD (•), HFD (▪) and RFD (○) mice. Diet significantly modulated the inhibiting ability of leptin. *P<0.05 SLD vs HFD, #P<0.05 SLD vs RFD, †P<0.05 HFD vs RFD. Original recordings of a tension receptor in response to 3 g tension before (i) and after addition of leptin 10 nM (ii) from SLD (e), HFD (f) and RFD (g) mice.

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Diet-induced changes in the expression of LepR in vagal afferents

There was no difference in LepR expression in whole nodose ganglia between any of the groups of mice (Figure 6a, P>0.05). However, when LepR mRNA expression was quantified specifically in mucosal afferents, there was a 99% reduction in LepR mRNA in the HFD mice compared with the SLD mice (Figure 6b, P<0.05 SLD vs HFD). The RFD mucosal afferents had a 68% reduction in LepR mRNA relative to the SLD mice (P<0.05 SLD vs RFD). The RFD LepR mRNA content was greater than that observed in the HFD mice (P<0.05 HFD vs RFD).

Figure 6
figure6

Diet-induced changes in leptin receptor mRNA expression in (a) whole nodose ganglia and (b) mucosal afferent cell bodies. Leptin receptor mRNA in whole nodose ganglia was no different in any of the groups of mice (all groups N=8). In mucosal-traced afferents, leptin receptor mRNA was reduced in afferents from HFD and RFD mice (N=4, all groups). *P<0.05 SLD vs HFD, #P<0.05 SLD vs RFD, †P<0.05 HFD vs RFD.

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Discussion

These data show when HFD-induced obese mice received a standard chow diet, brief weight loss was followed by a return to their previously established weight, associated with an increase in food intake. The reduced mechanosensitivity of gastric vagal afferent tension receptors, in HFD obesity, was maintained. However, the switch in the effect of leptin, from excitatory on gastric mucosal receptors in SLD mice to inhibitory on gastric tension receptors in HFD mice, is partially reversed upon return to a standard chow diet.

The RFD mice followed the same weight gain trajectory as the HFD mice up to the 12-week time point when placed back on to the SLD. In the first 2 weeks on the comparatively unpalatable standard mouse chow, the RFD mice lost a significant amount of weight. Presumably mechanisms, required to maintain the higher level of adiposity achieved on the HFD, were then activated to increase food intake. Although food intake was elevated compared with SLD and HFD after only 4 weeks on the SLD, it took more than 8 weeks on the SLD before energy intake in the RFD mice was equivalent to that of HFD mice. Thus, the reduced adiposity observed at the 24-week time point is a reflection of the weight loss that occurred when the RFD mice were initially placed back on the SLD. Further studies are needed to establish whether the increase in food intake in RFD mice is maintained and to determine the full trajectory of the RFD mice to their new set level of adiposity.

The reduction in tension receptor mechanosensitivity is consistent with the reduced neural activation seen in the hypothalamus of obese humans in response to gastric distension.31 The increase in food intake, observed in the RFD mice upon return to a SLD, may be a consequence of this large maintained reduction in gastric vagal afferent mechanosensitivity in conjunction with a composite of additional factors driving increased food intake including persistent compensatory changes in both gut peptides7 and hypothalamic regulation of food intake.32 Although the amount of food consumed increased, the absolute caloric intake matched that consumed on the HFD. It is well established that chronic feeding of a palatable, high-fat, energy-dense diet, induces obesity,33 which once obtained, is defended against perturbations in body weight.34, 35 These data demonstrate that after 12 weeks on a high-fat diet, there appears to be changes in gastric mechanosensitivity, which serve to protect an increased body weight, but it remains to be determined whether the increased food intake will see the RFD mice obtain the same level of adiposity and weight as the HFD mice or settle at a point between the HFD and SLD mice. The failure of gastric tension receptors to revert back to the lean phenotype is also observed in neuronal responses to food intake in the brain of post-obese individuals.36 The persistence of obese phenotype neuronal responses may, at least in part, explain the high failure rate of diet regimes in humans.

The partial restoration of leptin sensitivity, on mucosal receptors, is of interest, as it may preclude the ongoing increase in weight occurring in mice that continue on the HFD, and facilitate maintenance of the weight reached at the time the switch occurred. Leptin sensitivity has previously been shown to be restored both centrally and peripherally in diet-induced obese rats resuming a chow diet.37 However, we only observed a partial restoration in the potentiating effect of leptin. This may represent either a new level of leptin sensitivity or a transient effect with restoration to normal after a longer period back on the standard chow diet. The mechanisms responsible for the varying levels of potentiation of mucosal receptors by leptin still need to be elucidated, but we speculate that it may, at least, involve differential expression of the LepR, as we observed a 99% reduction in LepR mRNA in mucosal afferents from HFD mice, and a 68% reduction of LepR mRNA in the RFD mice. It remains to be determined whether these changes at the transcript level reflect changes in functional protein at the cell surface. The partial reversal in leptin sensitivity appears insufficient to overcome other compounding factors, such as the maintained decrease in gastric tension receptor mechanosensitivity, that facilitate food intake.

Similar to the effect of leptin on mucosal receptors, the RFD mice exhibited a partial restoration of the effect of leptin on tension receptors to the lean phenotype. Previously, we have shown that LepR expression in gastric muscular vagal afferents is unchanged in obese mice;28 however, changes in LepR protein levels have been observed in cultured Caco-2 cells in the absence of any changes in mRNA,38 and it remains to be determined whether such a change in LepR protein exists in these afferents. Alternatively, there may be a change in the ability for leptin to activate a downstream signalling protein, which previously has been suggested to involve activation of the large conductance calcium-activated potassium channel.28

It has been demonstrated in humans that following diet-induced weight loss, there are persistent long-term changes to gut peptides, with increased levels of the orexigenic peptide ghrelin, and decreased levels of the anorexigenic peptides, cholecystokinin, glucagon-like peptide 1 and peptide YY present 1 year later.7 The lack of adaptation of gastric tension receptors upon return to a normal diet could therefore exacerbate the situation making it more difficult to lose weight. Whether the reduced mechanosensitivity of gastric tension receptors are also part of the mechanism favouring weight regain after diet-induced weight loss in obese humans and the extent to which the remarkable efficacy of bariatric surgery, even in individuals with hypothalamic obesity,39, 40 depends on circumventing this decrease, remains to be determined. Nevertheless, HFD-induced obesity in mice, rather than transgenic models, mimics more accurately the pathogenesis of human obesity and as such provides a powerful tool for obesity research.41

In conclusion, we have established that changes in regards to leptin’s vagal afferent modulatory action are not fully reversible by placing obese mice back on a standard chow diet for a period of 12 weeks. Furthermore, they continue to exhibit decreased response to stretch, which could indicate a mechanism that promotes the overconsumption of food in an attempt to maintain the obese state and thus combat successful weight loss. Further studies including a longer time course and pair-feeding experiments are required to determine whether the observed changes are driven by the macronutrient content of the diet, the inducement of obesity or a combination of the two, as well the timing of the onset of the irreversibility, and potential for reversibility over time.

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Affiliations

  1. Nerve-Gut Research Laboratory, Hanson Institute, Discipline of Medicine, University of Adelaide, Adelaide, SA, Australia
    • S J Kentish
    • , H Li
    • , G A Wittert
    •  & A J Page
  2. Royal Adelaide Hospital, Adelaide, SA, Australia
    • T A O'Donnell
    • , C L Frisby
    •  & A J Page
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Correspondence to A J Page.

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Author Contributions

SJK was responsible for performing electrophysiology experiments, ELISA assays and writing the manuscript. TAO’D monitored the weight and food intake of the mice. CLF performed the QRT-PCR experiments. HL performed the retrograde tracing. GAW provided intellectual input into the design, and interpretation of the experiments and assisted with the preparation of the manuscript. AJP collected and processed the tissue samples, co-designed experiments and aided in the preparation of the manuscript.

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Kentish, S., O'Donnell, T., Frisby, C. et al. Altered gastric vagal mechanosensitivity in diet-induced obesity persists on return to normal chow and is accompanied by increased food intake. Int J Obes 38, 636–642 (2014) doi:10.1038/ijo.2013.138

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Keywords

  • leptin
  • vagal afferents
  • high fat diet

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