Leptin Resistance in Vagal Afferent Neurons Inhibits Cholecystokinin Signaling and Satiation in Diet Induced Obese Rats

Raybould HE (2012) Leptin Resistance in Vagal Afferent Neurons Inhibits Cholecystokinin Signaling and Satiation in Diet Induced Obese Rats. PLoS ONE 7(3): e32967. doi:10.1371/journal.pone.0032967 Leptin Resistance in Vagal Afferent Neurons Inhibits Cholecystokinin Signaling and Satiation in Diet Induced Obese Rats Guillaume de Lartigue 0 Claire Barbier de la Serre 0 Elvis Espero 0 Jennifer Lee 0 Helen E. Raybould 0 Silvana Gaetani, Sapienza University of Rome, Italy 0 Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California Davis , Davis, California , United States of America Background and Aims: The gastrointestinal hormone cholecystokinin (CCK) plays an important role in regulating meal size and duration by activating CCK1 receptors on vagal afferent neurons (VAN). Leptin enhances CCK signaling in VAN via an early growth response 1 (EGR1) dependent pathway thereby increasing their sensitivity to CCK. In response to a chronic ingestion of a high fat diet, VAN develop leptin resistance and the satiating effects of CCK are reduced. We tested the hypothesis that leptin resistance in VAN is responsible for reducing CCK signaling and satiation. Results: Lean Zucker rats sensitive to leptin signaling, significantly reduced their food intake following administration of CCK8S (0.22 nmol/kg, i.p.), while obese Zucker rats, insensitive to leptin, did not. CCK signaling in VAN of obese Zucker rats was reduced, preventing CCK-induced up-regulation of Y2 receptor and down-regulation of melanin concentrating hormone 1 receptor (MCH1R) and cannabinoid receptor (CB1). In VAN from diet-induced obese (DIO) Sprague Dawley rats, previously shown to become leptin resistant, we demonstrated that the reduction in EGR1 expression resulted in decreased sensitivity of VAN to CCK and reduced CCK-induced inhibition of food intake. The lowered sensitivity of VAN to CCK in DIO rats resulted in a decrease in Y2 expression and increased CB1 and MCH1R expression. These effects coincided with the onset of hyperphagia in DIO rats. Conclusions: Leptin signaling in VAN is required for appropriate CCK signaling and satiation. In response to high fat feeding, the onset of leptin resistance reduces the sensitivity of VAN to CCK thus reducing the satiating effects of CCK. - Funding: Work funded by National Institutes of Health DK41004. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Under normal physiological conditions, the presence of fat in the small intestine stimulates the release of gut hormones, including cholecystokinin (CCK) and peptide YY (PYY) [1,2], thereby slowing gastric emptying [3] and suppressing food intake [1]. Paradoxically, chronic consumption of a high fat diet (HFD) results in hyperphagia and obesity [4,5]. There is evidence that obesity in both humans [6] and rats [7] results in reduced sensitivity to the satiating effect of lipids compared to their lean counterparts. This does not appear to occur as a result of reduced release of CCK [2], but rather to a reduction in sensitivity to the satiating effects of CCK [810]. The mechanisms leading to the decrease in response to CCK remain unknown, although it seems to occur independently of CCK1 receptor (CCK1R) expression [11]. CCK, released from the small intestine in response to fatty acids and proteins, activates CCK1R located on vagal afferent nerve terminals in the gut to inhibit gastric emptying and food intake, and stimulating pancreatic enzyme secretion and gall bladder contraction. In addition to stimulating the discharge of vagal afferent neurons (VAN), CCK also changes the neurochemical phenotype of VAN. The change in neurochemical phenotype is characterized by altered expression levels of G-protein coupled receptors and neuropeptide transmitters. Following feeding or administration of exogenous CCK, the peptide YY type 2 (Y2) receptor [12] and the anorexigenic neuropeptide transmitter cocaine and amphetamine regulated transcript (CART) [13] are upregulated; synthesis of the orexigenic neuropeptide transmitter melanin-concentrating hormone (MCH) [13,14] as well as the MCH1 receptor (MCH1R) [14] and CB1 receptor (CB1) [15] are inhibited. Conversely, fasting, or feeding in the presence of a CCK1R antagonist, decreases expression of CART and Y2, and increases MCH, MCH1R and CB1 expression in VAN. This suggests that CCK acts as the gatekeeper in the control of the neurochemical phenotype in VAN and can influence food intake and GI function both rapidly, by stimulating action potentials, or induce prolonged effects, by regulating the expression of proteins known to play a role in the control of feeding behavior. The long form of the leptin receptor has been found to be coexpressed with CCK1R in a subset of VAN [1618]. Leptin alone rapidly increases VAN discharge [19,20] and increases cytosolic calcium in culture [21]. However, leptin also plays a crucial role in modulating the sensitivity of these neurons to CCK. Leptin markedly enhances vagal afferent discharge [20], cytosolic calcium [21,22], and translocation of the early gene EGR-1 to the nucleus [23] in response to low doses of CCK. Leptin regulates EGR-1 levels while CCK activates EGR-1 in VAN. Inhibition of EGR-1 abolishes leptin-induced sensitization of VAN to CCK and reduces CCK-induced expression of CART in VAN [23]. Vagallymediated changes in function, such as inhibition of food intake and gastric emptying in response to CCK, are also enhanced by leptin [23,24]. We have recently demonstrated that chronic ingestion of a high fat diet leads to the development of leptin resistance in VAN of dietinduced obese rats, compared to low fat fed control rats or rats resistant to the obesigenic effects of a high fat diet [4]. In the present study, we hypothesize that leptin resistance in VAN of DIO rats is responsible for the reduced sensitivity of these neurons to CCK, resulting in an altered neurochemical phenotype in VAN and hyperphagia. Firstly, Zucker rats which have a genetic deletion of the leptin receptor were used to determine the effect of altered leptin function on VAN. Secondly, Sprague-Dawley rats with a predisposition to diet-induced obesity (DIO), that develop leptin resistance in VAN when fed a HF diet [4], were used to determine changes in CCK-induced inhibition of food intake and signaling in VAN in response to long term ingestion of a high fat diet. Reduced sensitivity of VAN to CCK in obese Zucker rats Initial studies using Zucker rats demonstrated that leptin signaling is required for CCK-induced activation of VAN and its subsequent anorexigenic effects. Zucker rats are a genetic model of leptin receptor deficiency [25,26]. As previously reported, homozygous Zucker rats (LepRfa/LepRfa), weighed significantly more than lean Zucker rats when fed on (...truncated)