Reduced CCK signaling in obese-prone rats fed a high fat diet

Hormones and Behavior 64 (2013) 812–817

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Reduced CCK signaling in obese-prone rats fed a high fat diet Frank A. Duca a,b,c, Li Zhong d,e, Mihai Covasa a,b,d,f,⁎ a

UMR1913-MICALIS, INRA, Domaine de Vilvert, Jouy-en-Josas 78352, France UMR1913-MICALIS, AgroParisTech, Domaine de Vilvert, Jouy-en-Josas, 78352, France c Doctoral School of Physiology and Pathophysiology, University Pierre and Marie Currie, 15 rue de l'Ecole de Médecine, Paris 75006, France d Department of Basic Medical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, 309 E. Second Street, Pomona, CA 91766, USA e College of Life Sciences, Hebei University, Baoding, Hebei 071002, China f Department of Human Health and Development, University of Suceava, Universitatii 13, Suceava 720229, Romania b

a r t i c l e

i n f o

Article history: Received 21 June 2013 Revised 26 September 2013 Accepted 29 September 2013 Available online 5 October 2013 Keywords: Obesity High-fat diet CCK-1R Devazepide DIO

a b s t r a c t Deficits in satiation signaling during obesogenic feeding have been proposed to play a role in hyperphagia and weight gain in animals prone to become obese. However, whether this impaired signaling is due to high fat (HF) feeding or to their obese phenotype is still unknown. Therefore, in the current study, we examined the effects of CCK-8 (0.5, 1.0, 2.0, and 4.0 μg/kg) on suppression of food intake of HF-fed obese prone (OP) and resistant (OR) rats. Additionally, we determined the role of endogenous CCK in lipid-induced satiation by measuring plasma CCK levels following a lipid gavage, and tested the effect of pretreatment with devazepide, a CCK-1R antagonist on intragastric lipid-induced satiation. Finally, we examined CCK-1R mRNA levels in the nodose ganglia. We show that OP rats have reduced feeding responses to the low doses of exogenous CCK-8 compared to OR rats. Furthermore, OP rats exhibit deficits in endogenous CCK signaling, as pretreatment with devazepide failed to abolish the reduction in food intake following lipid gavage. These effects were associated with reduced plasma CCK after intragastric lipid in OP but not OR rats. Furthermore, HF feeding resulted in downregulation of CCK-1Rs in the nodose ganglia of OP rats. Collectively, these results demonstrate that HF feeding leads to impairments in lipidinduced CCK satiation signaling in obese-prone rats, potentially contributing to hyperphagia and weight gain. © 2013 Elsevier Inc. All rights reserved.

Introduction Human obesity is associated with overconsumption of palatable foods, especially those high in fats (Bray et al., 2004; Golay and Bobbioni, 1997). Similarly, obese rodents are hyperphagic on a high-fat (HF) diet, resulting in increased meal size (Farley et al., 2003; Moran et al., 1998; Savastano and Covasa, 2005). Despite deficits in hypothalamic systems regulating energy homeostasis and alterations in central reward systems (Berthoud and Morrison, 2008), it is now recognized that diminished sensitivity to the inhibitory effects of intestinal nutrients plays a role in the passive overconsumption of fats and subsequent development of obesity (Covasa, 2010). Under normal physiological conditions, luminal exposure to fat results in suppression of food intake and slowing of gastric emptying, an effect partially attributable to lipid-mediated release of gut peptides, such as cholecystokinin (CCK), peptide YY (PYY), and glucagon-like peptide-1 (GLP-1) (Little and Feinle-Bisset, 2011). However, we and others have demonstrated that chronic over-exposure to a HF diet results in reduced responsiveness to intestinal lipids in both rodents and humans (Boyd et al., 2003; Covasa and Ritter, 1999; Duca et al., 2012; ⁎ Corresponding author at: INRA, Centre de Recherche de Jouy-en-Josas, UMR 1319, MICALIS, Neurobiology of Ingestive Behavior, Domaine de Vilvert, 78350 Jouy-en-Josas, France. Fax: +33 1 34 65 24 92. E-mail address: [email protected] (M. Covasa). 0018-506X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yhbeh.2013.09.004

Lucas and Sclafani, 1996). Furthermore, HF feeding diminishes sensitivity to exogenous gut peptides, as has been documented for CCK and GLP-1 (Covasa and Ritter, 1998; Duca et al., 2013; Williams et al., 2011), leading to caloric overconsumption and development of obesity. Indeed, obese humans have decreased circulating levels of CCK, GLP-1, and PYY (le Roux et al., 2006; Verdich et al., 2001; Zwirska-Korczala et al., 2007). Interestingly, we have recently demonstrated that outbred HF fed diet-induced obese rats exhibit reduction in lipid-induced satiation compared to diet-resistant rats, which was associated with decreased intestinal protein content of CCK, GLP-1, and PYY (Duca et al., 2012). Inbred diet-induced obese rats, termed obese-prone (OP), are a good representation of human obesity, where most obesity results from multiple genes and environmental interactions, and as such, when placed on a HF diet, OP, but not obese-resistant (OR) rats, become hyperphagic and obese (Levin, 2010; Levin et al., 1997). Therefore, we hypothesize that the interaction of HF feeding with the polygenetic susceptibility to obesity promotes deficits in intestinal nutrient sensing and signaling via alterations in gut peptides, specifically CCK. CCK is released from I-cells predominately located in the proximal intestine in response to fats and proteins. CCK binds to CCK-1 receptor (CCK-1R) located on vagal afferents synapsing from the celiac, and intestinal branches of the vagus nerve and acts in a paracrine fashion as: 1) CCK-1R are localized on vagal afferents; 2) isolated nodose ganglia neurons are activated by CCK during ex-vivo preparations; 3) chemical

F.A. Duca et al. / Hormones and Behavior 64 (2013) 812–817

and or surgical vagal deafferentation attenuates the suppressive effect of CCK, and; 4) antagonism of CCK-1R with peripheral administration attenuates CCK-induced suppression during both real and sham feeding (Ritter, 2004). As such, there is accumulating evidence showing that decreased sensitivity to intestinal lipids during obesity or HF feeding is a function of diminished vagal CCK signaling (Brenner and Ritter, 1998; Covasa and Ritter, 1998; Covasa et al., 2000; Swartz et al., 2010b). First, CCK plays a major role in suppression of food intake elicited by fatty acids, as peripheral blockade of the CCK-1 receptor (CCK-1R) attenuates these lipid-mediated effects (Brenner and Ritter, 1998). Secondly, OLETF rats, which lack the CCK-1R, are obese and hyperphagic, and more specifically, have a reduced response to the suppressive effects of lipids (Swartz et al., 2010b). Third, HF fed obese rats are less sensitive to the satiating effect of CCK, through reductions in neural activation of the hindbrain, where vagal afferents terminate (Covasa and Ritter, 1998; Covasa et al., 2000). Interestingly, when examining sensitivity to CCK in OP and OR rats, prior to HF feeding, OP rats are more sensitive to exogenous CCK than OR rats, indicating that deficits in CCK signaling is not a genetic determinant (Swartz et al., 2010a). Therefore, here we aimed to examine the role of CCK signaling in reduced responsiveness to lipids in OP and OR animals during HF feeding. To determine this, we first tested sensitivity to exogenous CCK-8 in OP and OR rats maintained on a HF diet. Second, we examined the effect of pretreatment with devazepide, a CCK-1R antagonist, prior to a gastric load of lipid. Finally, we measured post-prandial circulating levels of CCK following a lipid load, and determined CCK-1R mRNA levels in the nodose ganglia. Material and methods Animals Twelve, 6-week old OP and OR male rats (n=6 per phenotype) from Charles River Laboratories (Wilimington, MA), originally weighing 145.50 ± 3 g and 133.80 ± 2 g, respectively, were used in all behavioral experiments. Rats were housed individually in a temperature controlled vivarium with 12:12 light/dark cycle in hanging wire mesh bottom cages. Upon arrival, rats were handled and acclimated to the environment for one week before being switched to a high fat, high calorie diet (Research Diets, NJ, D12451; 45% kcal from fat, 4.7 kcal/g). Rats were maintained on this HF-diet for a minimum of 4weeks prior to testing and thereafter throughout the experiments. All experimental protocols were approved by the Western University's Institutional Animal Care and Use Committee. Feeding responses to CCK-8 Ten week old OP and OR rats maintained on the HF diet for 4 weeks, weighing 368.00 ± 12 g and 301.20 ± 4 g, respectively at the start of experiment, were used to examine sensitivity to the satiating effect of CCK. Following a 16 h fast (17:00–9:00), rats were given an IP injection of either CCK-8 (American Peptides, Sunnyvale, CA) or saline vehicle. Five minutes following injection, rats were presented with pre-weighed solid-pelleted HF diet and intake was measured at 1 h post injection. Rats were acclimated to testing procedures with IP saline injections until a stable baseline of food intake was established. They were then tested for response to CCK-8 (0.5, 1.0, 2.0, 4.0 μg/kg) that was administered in a random order, with each dose being tested twice. Each CCK test was bracketed by a saline vehicle injection, with a minimum of 48 h between each test. Feeding response to devazepide After CCK-8 tests, rats were acclimated to a similar protocol to examine the effect of CCK-1R antagonism on the suppressive effect of gastric lipid loads. On test days, food-deprived rats (17:00–9:00) received an

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IP injection of either saline or devazepide (2 mg/kg; Sigma Aldrich, Milwaukee, WI) followed 5 min later by a 5 mL intragastric gavage (IG) of either saline or 25% corn oil emulsion (prepared with 0.75 mL of Tween-80 for every 100 mL mixture of oil and tap water), and a pre-weighed amount of HF diet was presented immediately, and intake was measured at 2 h post gavage. Devazepide dose was selected based on its efficacy to attenuate inhibition of food intake following intestinal intralipid infusion (Reidelberger et al., 2003). Rats were acclimated to the protocol by administering IP and IG saline for three consecutive trials. The order of the testing series was as follows (IP/IG): saline/ saline, saline/corn oil, saline/saline, devazepide/corn oil, saline/saline, devazepide/saline. Plasma CCK following oil gavage Following devazepide experiment, rats were subject to the blood collection protocol. Following an overnight fast, OP and OR rats were given a 5mL intragastric gavage of 25% corn oil emulsion. Blood was collected, via tail vein in EDTA-coated capillary tubes (Microvettes 200, Braintree Scientific, Braintree, MA) containing protease inhibitors, immediately before gavage (0 min), and at 30, 60, and 120 min thereafter. Blood was centrifuged at 3500 ×g at 4 °C for 15 min, plasma aliquoted and stored at −80 °C. Plasma CCK was determined by extraction-free EIA kit (Phoenix Peptides), with all samples run in duplicate. CCK-1R mRNA expression Bilateral nodose ganglia were removed from a separate set of OP and OR rats (n = 5 per phenotype) maintained on chow and HF diet (Duca et al., 2013). RNA was extracted with RNEasy Fibrous Tissue Mini-kit (Qiagen), reverse transcribed to cDNA, and qPCR was run as previously described (Duca et al., 2012). Taqman® Gene Expression Assay for CCK1R (Applied Biosystems, Courtaboeuf, France) was used to determine gene expression. Statistical analyses All statistics were analyzed by Statistical Analysis Software (SAS, version 9.1.3 Cary, NC). Bi-weekly average body weights were analyzed with repeated measures Analysis of Variance (rmANOVA), with post hoc Bonferroni adjustment. Raw food intake data for CCK and devazepide tests was analyzed by two-way (phenotype, treatment) rmANOVA with significant main effects and reported, and further analyzed with post hoc Bonferroni adjustment. Effect size estimates were provided by calculating eta squared (η2) for ANOVA main effects. Furthermore, for both tests, percent suppression of food intake from baseline at each time point was calculated by the formula: [(average of intake after saline prior and post test) − (intake after test)] / (average of intake after saline before and after test) ∗ 100, and analyzed by rmANOVA with post hoc Bonferroni adjustment. Plasma CCK was analyzed by two-way (phenotype, time) ANOVA, and CCK-1R qPCR was analyzed by twoway ANOVA (diet × phenotype) with Bonferroni post-hoc tests. Significance was considered at α b 0.05 for all tests. Results Reduced sensitivity to CCK-8 in OP rats OP rats weighed significantly more than OR rats before HF feeding (P b 0.05), an effect that was enhanced by HF feeding (P b 0.0001) and remained significant throughout the study (Fig. 1). Repeated measures ANOVA revealed a significant main effect of treatment [F(4, 121) = 55.15, P b 0.0001, η2 = 0.54] and strain × treatment interaction [F(4, 121) = 8.94, P b 0.0001, η2 = 0.09] on 1 h food intake following CCK administration. Compared to baseline saline intake, the two lowest doses of CCK, 0.5 μg/kg and 1.0μg/kg failed to decrease food intake in OP

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rats (P N 0.05), but significantly reduced food intake in OR rats (P b 0.0001; Fig. 2). However, 2.0 μg/kg and 4.0 μg/kg CCK reduced 1-h food intake equally in both OP and OR rats (P b 0.0001; Fig. 2). Furthermore, suppression of food intake was reduced in OP compared to OR rats at 0.5 μg/kg (OP: 4.57 ± 3.9% vs. OR: 40.93 ± 2.5%; P b 0.0001), 1.0 μg/kg (OP: 18.43 ± 4.9% vs. OR: 42.64 ± 3.7%; P b 0.0001), and 2.0 μg/kg (OP: 21.64 ± 2.1% vs. OR: 45.52 ± 4.9%; Pb0.01), but not 4μg/kg (OP:35.92±5.0% vs. OR: 53.11±3.9%; P b 0.19).

Abolished feeding response to devazepide in OP rats Analysis of 2 h food intake following intragastric corn oil and pretreatment with devazepide injections revealed a significant main effect of strain [F(1, 82) = 16.79, P b 0.0001, η2 = 0.09], treatment [F(3, 82) = 25.76, P b 0.0001, η2 = 0.40] and strain × treatment interaction [F(3, 82) = 5.98, P b 0.0001, η2 = 0.09]. Both OP and OR rats decreased food intake significantly from baseline after corn oil gavage (P b 0.0001). However, pretreatment with devazepide returned food intake to saline level in OR rats (P b 0.0001) but had no effect in OP rats (P = 1.0). Devazepide alone had no effect on food intake in either OP and OR (P = 1.0 for both; Fig. 3A). When expressed as percent suppression, OP rats suppressed food intake less than OR rats after the lipid load (P b 0.05; Fig. 3B). Furthermore, pretreatment with devazepide completely abolished the suppression of food intake by oil in OR (P b 0.0001), but not in OP rats (P = 1.0; Fig. 3B).

B 2-h % Suppression

Fig. 1. Average body weight of OP and OR rats prior and after HF feeding. OP rats weighed slightly more before HF feeding, and significantly more throughout the experiment. Data are expressed as means ± SEM, * denotes statistical difference between OP and OR, *P b 0.05, **P b 0.01, ***P b 0.001.

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Fig. 3. Two-hour food intake (A) and percent suppression (B) following 25% corn oil gavage (5 mL) and IP administration of devazepide (2 mg/kg). (A) Corn oil gavage decreased food intake in both OP and OR rats, but pre-treatment with devazepide abolished the effect only in OR rats. (B) OP rats had reduced percent suppression of food intake following corn oil gavage compared to OR rats, however, prior treatment with devazepide abolished the oil-induced percent suppression of food intake in OR but not OP rats. Data are expressed as means ± SEM, * denotes statistical difference from saline (or corn oil gavage/IP saline for 3B) within phenotype, ***P b 0.001. # denotes difference between oil gavage/IP saline vs. oil gavage/IP devazepide, ###P b 0.0001. † denotes difference between phenotypes within treatment, †P b 0.05, ††P b 0.01.

OP rats are less responsive to lipid-induced CCK release OP rats exhibited increased fasting plasma CCK circulating levels compared to OR rats during HF feeding (OP: 0.24 ± 0.1 ng/mL vs. OR: 0.21 ± 0.1 ng/mL; P b 0.05). Administration of oil load significantly increased plasma CCK in both groups compared to baseline. However, this increase was significantly less in OP compared to OR rats at 30 and 60 min post gavage (P b 0.05 for both), (Fig. 4), with OP rats exhibiting significantly lower circulating plasma levels at both

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Fig. 2. Sixty-minute HF diet intake following IP injection of CCK-8 (0.5, 1.0, 2.0, and 4.0 μg/kg). OP rats failed to significantly decreased food intake at 0.5 and 1.0 μg/kg doses of CCK, while OR rats significantly decreased food intake at all doses tested. Suppression of food intake was significantly decreased in OP compared to OR rats at 0.5, 1.0 and 2.0 μg/kg doses of CCK. Data are expressed as means ± SEM, * denotes statistical difference from saline, ***P b 0.001. † denotes difference between phenotype within treatment †P b 0.05, †††P b 0.0001.

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Minutes after Oil Gavage Fig. 4. Change in plasma CCK levels after 25% corn oil gavage in HF fed OP and OR rats. Following corn oil gavage, OP rats have reduced plasma CCK levels at 30 and 60 min. Data are expressed as means ± SEM, * denotes statistical difference from saline (or corn oil gavage/ IP saline for 3B) within phenotype, ***P b 0.001.

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30 min (OP: 0.38 ± 0.02 ng/mL vs. OR: 0.46 ± 0.02 ng/mL; P b 0.05) and 60 min (OP: 0.49 ± 0.02 ng/mL vs. OR: 0.62 ± 0.03 ng/mL; P b 0.05). OP rats have decreased CCK-1R mRNA in the nodose ganglia HF feeding resulted in a significant downregulation of CCK-1R gene expression in the nodose ganglia of OP (P b 0.001) (Fig. 5), but not OR rats. Furthermore, there were no significant differences in CCK-1RmRNA between OP and OR rats during chow feeding. Discussion The current results demonstrate that high fat feeding leads to impairments in lipid-induced CCK satiation signaling in obese-prone rats. We show that OP rats have reduced sensitivity to low doses of exogenous CCK compared to OR rats. Furthermore, OP rats exhibit deficits in endogenous CCK signaling, as pretreatment with devazepide, a CCK1R antagonist abolished lipid-induced satiation in OR, but not OP rats which was accompanied by reduced plasma CCK response to an oil gavage. Finally, reductions in CCK signaling in OP rats during HF feeding was associated with downregulation of CCK-1Rs in the nodose ganglia. Reduced sensitivity to satiation signals has been reported in both humans and rodent models following HF feeding (Covasa and Ritter, 1998; Covasa et al., 2001; Duca et al., 2013; Nefti et al., 2009; Savastano and Covasa, 2005; Williams et al., 2011). Indeed animals maintained on HF diet are less sensitive to the suppressive effects of CCK and lipids (Covasa and Ritter, 1998; Covasa et al., 2001; Nefti et al., 2009; Savastano and Covasa, 2005). Whether these impairments are due to the diet, phenotype or a combination of both is not completely known. The obese-prone animal model, that closely mimics human obesity resultant from a product of gene and environment interactions, is characterized by a host of central and peripheral deficits leading to dysregulation of food intake and energy (Levin, 2010). Here we show that under HF feeding conditions, the sensitivity to several doses of CCK is markedly reduced in OP compared to OR animals, an effect not seen during chow feeding (Swartz et al., 2010a). This is in line with a recent study demonstrating that outbred diet-induced obese (DIO) rats were less sensitive to CCK at a low dose (.22 nmol/kg) but maintained sensitivity at a higher dose (de Lartigue et al., 2012). Thus, the current study extends on previous findings by showing that HF feeding in combination with obesity results in reduced responses to CCK. Exogenous CCK elicits satiation by acting on CCK-1R on vagal afferent neurons, resulting in neural activation in the NTS, as evidenced by Fos-like (Fos-Li) immunoreactivity, which is abolished following surgical or chemical ablation of the vagus (Sayegh and Ritter, 2000). As such, it is likely that reduced sensitivity to exogenous CCK in HF fed OP rats is

CCK-1R mRNA

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Fig. 5. CCK-1R gene expression in the nodose ganglia of OP and OR rats maintained on either chow or HF diet. HF fed OP rats exhibited decreased CCK-1R mRNA levels compared to chow fed OP rats. Data are expressed as means ± SEM, † denotes significant difference from chow-fed diet condition within phenotype, ††P b 0.01.

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from a diminution in vagal activation and subsequent signaling to the hindbrain. Indeed, rats exhibit reductions in hindbrain neuronal Fos-Li during HF feeding (Covasa et al., 2000), and HF fed mice develop an attenuated jejunal afferent response to CCK, as well as a 65% reduction in CCK-responsive nodose neurons (Daly et al., 2011). Interestingly, CCK1R gene expression is downregulated after two weeks of HF feeding in mice (Nefti et al., 2009), although not all studies demonstrate this effect (Broberger et al., 2001). In line with these findings, we observed a reduction in CCK-1R mRNA expression following HF feeding in OP but not OR rats. Although the cause of this is unknown, it has been hypothesized that increased CCK levels during HF feeding could lead to receptor desensitization (Covasa, 2010). Indeed, in-vitro desensitization occurs rapidly with CCK pretreatment (Abdelmoumene and Gardner, 1980), and in-vivo, rats fitted with osmotic minipumps chronically administering CCK, have reduced sensitivity to IP administration of CCK (Covasa et al., 2001). Although no study has directly examined the effect of circulating CCK levels on CCK-1R mRNA expression, our current results demonstrate that fasting levels of circulating CCK are increased in OP rats about 15% during HF feeding. This is in line with a previous study that found that outbred OP rats exhibited higher baseline plasma CCK levels compared to OR rats (Li et al., 2011). Thus, it is possible that an increase in fasting plasma CCK during HF feeding downregulates CCK-1Rs in the nodose ganglia of OP rats, ultimately resulting in reduced sensitivity to exogenous CCK as we have shown. In addition to reduction in vagal signaling by CCK-1R activation, leptin resistance may contribute to reduced sensitivity to CCK. The leptin receptor is co-expressed with CCK-1R in vagal afferent neurons (Burdyga et al., 2002). Furthermore, leptin and CCK act synergistically and can enhance vagal afferent signaling and the inhibition of food intake and gastric emptying induced by CCK (Barrachina et al., 1997; de Lartigue et al., 2010, 2012; Matson et al., 2000; Peters et al., 2004). It has been well documented that OP rats have defective central leptin signaling and become highly leptin resistant during HF feeding. Interestingly, they develop leptin resistance in vagal afferent neurons as early as 8 weeks on a HF diet, before the development of central leptin resistance (de Lartigue et al., 2011). As such, it is possible that peripheral leptin resistance in OP rats contributes to reduced CCK-induced vagal afferent activation. At the molecular level, leptin fails to increase EGR-1 protein expression in outbred DIO rats during HF feeding, while translocation of EGR-1 to the nucleus in vagal afferents is increased following leptin and CCK administration in diet-resistant (DR) but not DIO rats (de Lartigue et al., 2012). Behaviorally, when given a preload meal, which increases endogenous CCK, along with a subthreshold dose of leptin, only outbred DR rats reduced food intake, indicating that the synergistic effect of leptin with endogenous CCK is impaired in OP rats during HF feeding (de Lartigue et al., 2012). However, our data demonstrate that CCK release is reduced in OP rats during HF feeding, therefore the observed difference in de Lartigue et al. (2012) may be from a reduced release of endogenous CCK following the preload and not from vagal leptin resistance. Despite an increase in fasting levels of circulating CCK, we found that the CCK response to the corn oil load was diminished in OP rats. In fact, OP rats had approximately 60% and 90% less increase in circulating CCK than OR rats at 30 and 60min, respectively. To our knowledge, this is the first study to directly examine the release of CCK following a lipid load in OP and OR rats, an effect similar to that seen in obese Zucker rats, which have a markedly reduced CCK response following a meal (Guilmeau et al., 2003). Studies in obese humans have been contentious, with some findings showing no differences in postprandial plasma CCK levels between obese and lean (Brennan et al., 2012; Lieverse et al., 1994), while others have found a blunted response during obesity (Baranowska et al., 2000; Zwirska-Korczala et al., 2007). A reduction in meal-induced circulating CCK is likely a result of reduced capacity of enteroendocrine I-cells to release CCK following luminal nutrient exposure. This may be due to a decrease in intestinal peptide content or a reduction in CCK-containing EECs, as we have observed for GLP-1 cells

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in HF fed OP rats (Duca et al., 2013). Indeed, outbred DIO rats exhibit reduced intestinal CCK peptide content in proximal intestinal epithelial cells (Duca et al., 2012), although I-cell numbers were not examined. Furthermore, leptin has been shown to induce CCK release both invitro and in-vivo, and as previously mentioned, Zucker rats exhibit a blunted meal-induced release of CCK (Guilmeau et al., 2003). Therefore, the development of leptin resistance in OP rats may reduce the ability of leptin to initiate CCK release. HF fed OP rats have reduced postprandial CCK, and are also less sensitive to the suppressive effects of exogenous CCK. These findings led to the hypothesis that endogenous CCK signaling is diminished in OP rats, thus promoting hyperphagia. Indeed, in OR rats, devazepide completely abolished the reduction in food intake following a lipid load, as has been shown in normal lean animals and humans (Brenner and Ritter, 1996; Lieverse et al., 1994; Matzinger et al., 1999; Reidelberger et al., 2003; Yox et al., 1992). However, blockade of CCK-1R in OP rats had no effect on lipid-induced satiation. Therefore, our results demonstrate that satiation induced by endogenous CCK is severely impaired in HF fed OP rats. As such, we also observed that, similar to outbred DIO rats (Duca et al., 2012), these animals are less sensitive to lipid-induced satiation. Therefore, this strongly suggests that diminished responsiveness to intestinal fats in HF fed OP rats is a direct result of defective endogenous CCK signaling, resulting in increased food intake. However, devazepide can cross the blood–brain barrier, and IP administration increases food intake in vagotomized rats (Reidelberger et al., 2003). Although no study has directly examined central CCK signaling in OP rats, HF-fed mice exhibit reduced hypothalamic CCK concentrations (Morris et al., 2008), which may explain the lack of devazepide effect to increase food intake in OP rats. Nonetheless, our current results and others showing reduced sensitivity to exogenous IP CCK, along with reduced circulating concentrations of CCK and downregulation of nodose ganglia CCK1Rs, all provide evidence that lack of efficacy of devazepide is from altered peripheral CCK signaling (de Lartigue et al., 2012). However, impaired signaling of other gut peptides, like GLP-1, may also contribute to reduced sensitivity to intestinal lipids, as reduction in food intake by jejunal infusions of linoleic acid are completely abolished by administration of a GLP1R antagonist (Dailey et al., 2011). Furthermore, HF feeding leads to decreased plasma GLP-1 levels (Anini and Brubaker, 2003; Williams et al., 2011), and intestinal protein expression of GLP-1 is reduced in outbred DIO rats (Duca et al., 2012) compared to OR rats. Additionally, we have recently demonstrated that HF feeding diminishes the anorexic effects of GLP-1 receptor activation in OP rats (Duca et al., 2013), indicating that reduced GLP-1 signaling may be partly responsible for the decreased responsiveness to lipid-induced satiation in OP rats. It is important to note that this study does not determine the exact time point at which OP rats become less sensitive to CCK during HF feeding. As such, reduced sensitivity could be a reflection of the obese state, not an interaction between the diet and susceptibility to obesity, as has been demonstrated with previous obese models such as dietary-induced obese animals, the Zucker rat, and the ob/ob mouse (Covasa et al., 2001; de Lartigue et al., 2012; McLaughlin and Baile, 1981; Strohmayer and Smith, 1986). However, both the Zucker rat and ob/ob mouse lack leptin signaling, and diet-induced obese animals are leptin resistant. Therefore, the decreased response to CCK in these animals may be a reflection of a loss in the synergistic effect of leptin and CCK. As previously mentioned, OP rats rapidly develop leptin resistance in vagal afferent neurons, before significant differences in adiposity occur (de Lartigue et al., 2011, 2012). As such, it is possible that reduced CCK sensitivity is an early consequence of HF feeding in OP rats (de Lartigue et al., 2011, 2012). Therefore, a reduction in the potentiating effect of leptin on CCK signaling during the early stages of HF feeding could result in reduced lipid-induced satiation, increased fat intake, which would exacerbate the effect and ultimately leading to weight gain. In conclusion, this study demonstrates that during HF feeding, OP rats exhibit impaired CCK response to intestinal lipids compared to OR

rats. Decreased postprandial CCK levels, and diminished peripheral sensitivity to CCK in OP rats results in an inability to properly control fat intake, leading to overeating, and likely perpetuating weight gain and further obesity. Acknowledgments F.A.D. and M.C. designed the study, researched data, and wrote the manuscript. L.Z. researched data and reviewed the manuscript. The study was supported by the INRA through a scientific package awarded to M. C. and by the PN-II-ID-PCE grant (MC) from National Research Council, Romania. References Abdelmoumene, S., Gardner, J.D., 1980. Cholecystokinin-induced desensitization of enzyme secretion in dispersed acini from guinea pig pancreas. Am. J. Physiol. 239, G272–G279. Anini, Y., Brubaker, P.L., 2003. Role of leptin in the regulation of glucagon-like peptide-1 secretion. Diabetes 52, 252–259. Baranowska, B., Radzikowska, M., Wasilewska-Dziubinska, E., Roguski, K., Borowiec, M., 2000. 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