NPY2 receptor activation in the dorsal vagal complex increases food intake and attenuates CCK-induced satiation in male rats

Abstract

Neuropeptide Y (NPY), peptide YY (PYY), and their cognate receptors (YR) are expressed by subpopulations of central and peripheral nervous system neurons. Intracerebroventricular injections of NPY or PYY increase food intake, and intrahypothalamic NPY1 or NPY5 receptor agonist injections also increase food intake. In contrast, injection of PYY in the periphery reduces food intake, apparently by activating peripheral Y2R. The dorsal vagal complex (DVC) of the hindbrain is the site where vagal afferents relay gut satiation signals to the brain. While contributions of the DVC are increasingly investigated, a role for DVC YR in control of food intake has not been examined systematically. We used in situ hybridization to confirm expression of Y1R and Y2R, but not Y5R, in the DVC and vagal afferent neurons. We found that nanoinjections of a Y2R agonist, PYY-(3-36), into the DVC significantly increased food intake over a 4-h period in satiated male rats. PYY-(3-36)-evoked food intake was prevented by injection of a selective Y2R antagonist. Injection of a Y1R/Y5R-preferring agonist into the DVC failed to increase food intake at doses reported to increase food intake following hypothalamic injection. Finally, injection of PYY-(3-36) into the DVC prevented reduction of 30-min food intake following intraperitoneal injection of cholecystokinin (CCK). Our results indicate that activation of DVC Y2R, unlike hypothalamic or peripheral Y2R, increases food intake. Furthermore, in the context of available electrophysiological observations, our results are consistent with the hypothesis that DVC Y2R control food intake by dampening vagally mediated satiation signals in the DVC.

Keywords: hindbrain; hunger; neuropeptide; satiation; vagus.

Fig. 1.

Images from dual-labeled fluorescence in situ hybridization of sections of nodose ganglion and dorsal vagal complex reveal NPY receptor (YR) and cholecystokinin type A receptor (CCK-AR) mRNAs. A: examples of neurons in the left nodose ganglion that express Y2R mRNA (red; open arrow) and neurons that express CCK-AR mRNA (green) together with Y2R mRNA (stick arrows). B: neurons in the dorsal motor nucleus of the vagus expressing CCK-AR mRNA (green) alone (open arrows) or CCK-AR and Y2R mRNA (red; stick arrows). C: examples of neurons in the nucleus tractus solitarius that express Y1R mRNA (red; open arrows). Low levels of Y5R mRNA (green) were sometimes coexpressed in Y1R mRNA-expressing cells (stick arrows). D: examples of neurons in the nucleus tractus solitarius expressing Y2R mRNA (red; open arrows). Blue fluorescence from DAPI staining in C and D reveals cell nuclei.

Fig. 2.

A: distribution of 18 cannula tip locations for 100-nl injections of 25 pmol of PYY-(3–36) or NaCl. ✪, Sites (n = 11) where PYY-(3–36) evoked ≥1 g more food intake than NaCl over 4 h; ⊙, sites (n = 7) where PYY-(3–36) did not evoke ≥1 g more food intake than NaCl over 4 h. B: dual-labeled fluorescence images of representative dorsal vagal complex (DVC) cannula placements from those diagramed in A. Vagal afferent distribution is revealed in green with Alexa 488-conjugated isolectin B4, and diffusion of 100 nl of 3,000-mol wt biotinylated dextran (BDA) infusions is revealed in red by Alexa 555-conjugated avidin. B1: BDA invades the vagal terminal field at the level of the area postrema, where PYY-(3–36) infusion evoked increased food intake. B2: BDA injected at this more-rostral site did not diffuse caudally to the vagal afferent terminal field adjacent to the area postrema. B2′: PYY-(3–36) infused at this site did not increase food intake. C: average food intake following PYY-(3–36) infusions (25 pmol/100 nl). Top: average increase of food intake following PYY-(3–36) infusions (25 pmol/100 nl) at sites comparable to those diagramed in A. Repeated-measures ANOVA followed by Bonferroni’s comparisons indicated significantly increased food intake after PYY-(3–36) compared with NaCl injection: ^aP < 0.05, ^bP < 0.01, ^cP < 0.0001. Bottom: average food intake was not increased at any time following PYY-(3–36) infusions at sites comparable to B or into the 4th ventricle.

Fig. 3.

Increased food intake following PYY-(3–36) injection (25, 50, and 125 pmol) into the dorsal vagal complex. Repeated-measures ANOVA and Bonferroni’s test for individual differences revealed significantly increased food intake 60, 120, and 240 min after injection of 50 and 125 pmol of PYY-(3–36) compared with NaCl: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Although 25 pmol of PYY-(3–36) consistently increased food intake, the increase did not achieve statistical significance. Data are from 6 rats, all of which had histologically verified cannula tip placements in the dorsal vagal complex.

Fig. 4.

BIIE-0246 (BIIE), a Y type 2 receptor (Y2R) antagonist, prevented increased food intake following PYY-(3–36) injection into the dorsal vagal complex (DVC). Six rats with verified cannula tip placement in the DVC near the rostral border of the area postrema increased food intake between 30 and 240 min after injection of PYY-(3–36) (25 pmol). Repeated-measures ANOVA and Bonferroni’s comparison revealed that while PYY-(3–36) significantly increased food intake 120 and 240 min postinjection (^aP < 0.05), DVC injection of BIIE prevented the PYY-(3–36)-evoked increase in food intake. Of the 9 rats that were initially included in the experiment, 3 did not increase food intake in response to PYY-(3–36) and were not included in the analysis. Subsequent histological analysis revealed cannula placement in the 4th ventricle in 2 of these rats and in the hindbrain rostral to the DVC in 1 rat.

Fig. 5.

Injection of Leu³,Pro³⁴-NPY (LP NPY), a Y type 1 receptor (Y1R) agonist, into the dorsal vagal complex failed to increase food intake. Results are from 8 rats, all of which increased their food intake in response to PYY-(3–36) injection (25 pmol). A 9th rat that did not increase its intake following PYY-(3–36) injection into the DVC and, subsequently, was found to have a cannula tip in the 4th ventricle, was not included in the analysis. Repeated-measures ANOVA indicated significantly increased food intake between 30 and 240 min in rats injected with PYY-(3–36) compared with NaCl. Bonferroni’s comparisons revealed that food intake following PYY-(3–36) differed significantly from that following NaCl 120 and 240 min postinjection: ^aP < 0.05, ^bP < 0.001. Intake following LP NPY injection did not differ from that following NaCl injection at any dose or time point.

Fig. 6.

Increased food intake following injection of [cPP-(1–7),NPY-(19–23),Ala³¹,Aib³²,Gln³⁴]-hpancreatic polypeptide (cPP), a Y5R agonist, into the dorsal vagal complex (DVC). In 9 rats, all with histologically verified DVC cannulas, food intake significantly increased 120 and 240 min after injection of PYY-(3–36) (25 pmol) into the DVC compared with NaCl. In the same rats, repeated-measures ANOVA and Bonferroni’s comparisons revealed that cPP (50 pmol) also increased food intake 240 min postinjection. Food intake after 25 and 100 pmol of cPP did not differ from that after NaCl at any time point: ^aP < 0.05, ^bP < 0.01.

Fig. 7.

Injection of BIIE-0246 (BIIE) into the dorsal vagal complex (DVC) prevented increased food intake following [cPP-(1–7),NPY-(19–23),Ala³¹,Aib³²,Gln³⁴]-hpancreatic polypeptide (cPP) injection into the DVC. Nine rats with histologically verified DVC cannula placements significantly increased their food intake in response to PYY-(3–36) (data not shown). Repeated-measures ANOVA with Bonferroni’s comparison revealed that cPP significantly increased food intake 120 and 240 min postinjection. Food intake after BIIE + cPP did not differ from that after vehicle control injection. However, BIIE + NaCl also significantly reduced food intake 240 min postinjection compared with control (DMSO + NaCl). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Fig. 8.

Injection of PYY-(3–36) (50 pmol/100 nl) into the dorsal vagal complex attenuated reduction of 30-min food intake following intraperitoneal injection of cholecystokinin octapeptide (CCK-8, 3 μg/kg) in 15-h food-deprived rats (n = 10 rats, all with histologically verified cannula tip placements in the dorsal vagal complex). Top: food intake for the first 30 min. Repeated-measures ANOVA and Bonferroni’s comparisons revealed that CCK significantly reduced 30-min food intake compared with control (NaCl + NaCl) (P < 0.05). While PYY-(3–36) alone did not significantly reduce 30-min food intake, reduction of food intake by CCK was prevented by PYY-(3–36). Intake after PYY-(3–36) + CCK was not different from intake after NaCl + NaCl. Bottom: brief 30-min time course of CCK action and the more prolonged effect of PYY-(3–36). At 120 and 240 min, PYY-(3–36) + NaCl and PYY-(3–36) + CCK significantly enhanced food deprivation-induced eating compared with both NaCl + NaCl and NaCl + CCK. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.