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

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

Diet-induced obesity leads to the development of leptin resistance in vagal afferent neurons

Ingestion of high-fat, high-calorie diets is associated with hyperphagia, increased body fat, and obesity. The mechanisms responsible are currently unclear; however, altered leptin signaling may be an important factor. Vagal afferent neurons (VAN) integrate signals from the gut in response to ingestion of nutrients and express leptin receptors. Therefore, we tested the hypothesis that leptin resistance occurs in VAN in response to a high-fat diet. Sprague-Dawley rats, which exhibit a bimodal distribution of body weight gain, were used after ingestion of a high-fat diet for 8 wk. Body weight, food intake, and plasma leptin levels were measured. Leptin signaling was determined by immunohistochemical localization of phosphorylated STAT3 (pSTAT3) in cultured VAN and by quantifaction of pSTAT3 protein levels by Western blot analysis in nodose ganglia and arcuate nucleus in vivo. To determine the mechanism of leptin resistance in nodose ganglia, cultured VAN were stimulated with leptin alone or with lipopolysaccharide (LPS) and SOCS-3 expression measured. SOCS-3 protein levels in VAN were measured by Western blot following leptin administration in vivo. Leptin resulted in appearance of pSTAT3 in VAN of low-fat-fed rats and rats resistant to diet-induced obesity but not diet-induced obese (DIO) rats. However, leptin signaling was normal in arcuate neurons. SOCS-3 expression was increased in VAN of DIO rats. In cultured VAN, LPS increased SOCS-3 expression and inhibited leptin-induced pSTAT3 in vivo. We conclude that VAN of diet-induced obese rats become leptin resistant; LPS and SOCS-3 may play a role in the development of leptin resistance

Altered neurochemical phenotype in DIO rats.

<p>Photomicrographs of sections of nodose ganglia to show immunoreactivity for Y2 (A), MCH1R (B), and CB1 (C) from fed and fasted Sprague Dawley rats after 8 weeks on respective diets. A) Y2 is barely detectable in nodose neurons of LF, DR, and DIO rats fasted 24 hours. Refeeding for 1 hour increased Y2 expression in LF and DR but not DIO rats. Quantification of positive Y2 cells as a percent of total cells. B) MCH1R and C) CB1 expression is elevated in fasted rats and is decreased by refeeding for 1 hour in LF and DR but not DIO rats. Quantification of positive MCH1R and CB1 cells as a percent of total cells. Representative images from experiments with four to six rats in each group are shown. Significant differences were represented as ^a,b,c between groups.</p

Reduced satiation to exogenous and endogenous CCK in DIO rats.

<p>A) CCK feeding study (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032967#s4" target="_blank">methods</a>) in Sprague Dawley rats after 8 weeks on respective diets. CCK8S (0.22 nmol/kg; i.p.) significantly inhibited food intake compared to vehicle in LF and DR, but not DIO rats. N = 6. B) CCK8S (2.19 nmol/kg; i.p.) significantly inhibited food intake in all rats. N = 6. C) Protocol for leptin feeding study in which endogenous CCK was upregulated D) Endogenous CCK reduced food intake in LF fed and DR rats at 4, 6 and 8 weeks. Endogenous CCK reduced food intake in DIO rats at 4 weeks but not at 6 and 8 weeks. N = 6; data expressed as mean ± SEM. Significant differences were represented as ** for p<0.01.</p

Altered CCK induced signaling in obese Zucker rats.

<p>Photomicrographs of sections of nodose ganglia to show immunoreactivity for Y2 (A), MCH1R (B), and CB1 (C) from lean and obese Zucker rats fasted 24 hr or refed for 1 hr. A) Y2 expression is elevated by feeding in lean Zucker rats, but not in obese Zucker rats. N = 4. B) MCH1R is decreased by feeding in lean Zucker rats but not in obese Zucker rats. N = 4. C) CB1 is decreased by feeding in lean and obese Zucker rats, but CB1 expression remains significantly higher in fed obese Zucker rats compared to lean Zucker rats. Significant differences were represented as ^a,b,c between groups.</p

Altered CCK induced satiety in obese Zucker rats.

<p>A) Obese Zucker rats weigh significantly more than lean Zucker rats (N = 12; t-test). B) Immunoblot of pSTAT3 in VAN of lean (top) and obese (bottom) Zucker rats treated with vehicle or leptin. C) Densitometry analysis of <i>B</i> showing that STAT3 is phosphorylated in response to leptin (i.p) in VAN of lean Zucker rats but not in obese Zucker rats. N = 4 D) Immunoblot of pSTAT3 in hypothalamus of lean and obese Zucker rats treated with leptin. E) Densitometry analysis of <i>D</i> showing that STAT3 is phosphorylated in response to leptin in the arcuate nucleus of the hypothalamus of lean Zucker rats, but not obese Zucker rats. (N = 4).F) CCK8S (i.p., 0.22 nmol/kg) significantly inhibited food intake in lean Zucker rats but not obese Zucker rats. N = 6. Data expressed as mean ± SEM. Significant differences were represented as ^a,b,c between groups in one-way ANOVA. Significant differences were represented as * for p<0.05; ** for p<0.01; and *** for p<0.001 in Student's t-test.</p

Effect of a high-fat (HF) diet on body weight and food intake <i>A</i>) Body weight of rats ingesting either a LF diet or HF diet (45% kcal) for 8 weeks.

<p>There was a significant increase in body weight in diet-induced obesity (DIO) rats compared with diet-induced obesity-resistant (DR) and LF animals. <i>B</i>) The increase in body weight of LF, DR, DIO rats expressed as a percent of weight gain from initial weight. DIO rats became significantly heavier than LF fed animals after 4 weeks of HF diet, and after 4 weeks of a HF diet compared to the DR rats. <i>C</i>) Average daily food intake (kcal) per week over the 8 weeks on the diets. In the first week, all rats on the HF diet had a significantly higher energy intake than LF fed rats but this initial hyperphagia only lasted for one week after which there was no significant difference in caloric intake between the groups. At <i>week 5</i>, the DIO group had a significantly higher energy intake than LF rats and DR rats. N = 12 per group; data expressed as mean ± SEM. Significant differences were represented as ^a,b,c between groups.</p

EGR-1 expression in VAN of DIO rats.

<p>A) Protein expression of EGR-1 in VANs of LF, DR and DIO rats treated with leptin. B) Densitometry analysis of <i>A</i> showing that EGR-1 levels in VAN are significantly reduced in DIO rats compared to LF and DR rats. N = 3. C) and D) Quantification of the number of EGR-1 immunopositive neurons in cultured VAN from DR rats (C) and DIO rats (D) fed a high fat diet for 8 weeks. C) Leptin reduced the concentration of CCK required to induce EGR-1 translocation in cultured VAN. D) Leptin had no effect on CCK induced EGR-1 translocation in cultured VAN. N = 6. Data expressed as mean ± SEM. Significant differences were represented as ^a,b,c between groups.</p