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, 164 (2b), 704-18

Agmatine in the Hypothalamic Paraventricular Nucleus Stimulates Feeding in Rats: Involvement of Neuropeptide Y

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Agmatine in the Hypothalamic Paraventricular Nucleus Stimulates Feeding in Rats: Involvement of Neuropeptide Y

B G Taksande et al. Br J Pharmacol.

Abstract

Background and purpose: Agmatine, a multifaceted neurotransmitter, is abundantly expressed in the hypothalamic paraventricular nucleus (PVN). Our aim was to assess (i) the effect of agmatine on feeding behaviour and (ii) its association, if any, with neuropeptide Y (NPY).

Experimental approach: Satiated rats fitted with intra-PVN cannulae were administered agmatine, alone or jointly with (i) α₂-adrenoceptor agonist, clonidine, or antagonist, yohimbine; (ii) NPY, NPY Y₁ receptor agonist, [Leu³¹, Pro³⁴]-NPY, or antagonist, BIBP3226; or (iii) yohimbine and NPY. Cumulative food intake was monitored at different post-injection time points. Furthermore, the expression of hypothalamic NPY following i.p. treatment with agmatine, alone or in combination with yohimbine (i.p.), was evaluated by immunocytochemistry.

Key results: Agmatine robustly increased feeding in a dose-dependent manner. While pretreatment with clonidine augmented, yohimbine attenuated the orexigenic response to agmatine. Similarly, NPY and [Leu³¹, Pro³⁴]-NPY potentiated the agmatine-induced hyperphagia, whereas BIBP3226 inhibited it. Moreover, yohimbine attenuated the synergistic orexigenic effect induced by the combination of NPY and agmatine. Agmatine increased NPY immunoreactivity in the PVN fibres and in the cells of the hypothalamic arcuate nucleus (ARC) and this effect was prevented by pretreatment with yohimbine. NPY immunoreactivity in the fibres of the ARC, dorsomedial, ventromedial and lateral nuclei of the hypothalamus was not affected by any of the above treatments.

Conclusions and implications: The orexigenic effect of agmatine is coupled to increased NPY activity mediated by stimulation of α₂-adrenoceptors within the PVN. This signifies the importance of agmatine or α₂-adrenoceptor modulators in the development of novel therapeutic agents to treat feeding-related disorders.

Figures

Figure 1
Figure 1
Effect of intra-PVN injections of agmatine on food intake. Different group of rats were administered agmatine or aCSF just prior to lights out, and food intake (g) was measured at 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents the food consumption (g) ± SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B) Each column and bar represents the cumulative food consumption (g) ± SEM over 24 h post injection (n = 6–8 per group). Data were analysed by one-way anova followed by post hoc Dunnett's test. *P < 0.01 and **P < 0.001 versus aCSF-treated controls.
Figure 2
Figure 2
Effect of i.p. injections of agmatine on food intake. Different group of rats were administered agmatine or saline just prior to lights out, and food intake (g) was measured at 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents the food consumption (g) ± SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B) Each column and bar represents the cumulative food consumption (g) ± SEM over 24 h post injection (n = 6–8 per group). Data were analysed by one-way anova followed by post hoc Dunnett's test. *P < 0.05 and **P < 0.001 versus saline-treated controls.
Figure 3
Figure 3
Effect of clonidine on the agmatine-induced feeding. Separate groups of rats were injected by intra-PVN route with (1) aCSF + aCSF, (2) aCSF + agmatine (5 nmol·per rat), (3) clonidine (5 nmol·per rat) + aCSF or (4) clonidine (5 nmol·per rat) + agmatine (5 nmol·per rat) at the onset of the dark phase. Clonidine was injected 10 min prior to agmatine. Food intake (g) was measured at 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents the food consumption (g) ± SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B) Each column and bar represents the cumulative food consumption (g) ± SEM over 24 h post injection (n = 6–8 per group). Data were analysed by one-way anova followed by post hoc Newman–Keuls mean comparison test. *P < 0.001 versus agmatine- and $P < 0.001 versus clonidine-treated animals.
Figure 4
Figure 4
Effect of yohimbine on the orexigenic effect of agmatine. Different groups of rats received intra-PVN injections of (1) aCSF + aCSF, (2) aCSF + agmatine (10 nmol·per rat), (3) yohimbine (2.5 nmol·per rat) + aCSF or (4) yohimbine (2.5 nmol·per rat) + agmatine (10 nmol·per rat) at the onset of the dark phase. Yohimbine was injected 10 min prior to agmatine. Food intake (g) was measured at 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents the food consumption (g) ± SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B) Each column and bar represents the cumulative food consumption (g) ± SEM over 24 h post injection (n = 6–8 per group). Data were analysed by one-way anova followed by post hoc Newman–Keuls mean comparison test. $P < 0.01, $$P < 0.001 versus saline treatment, *P < 0.01, **P < 0.001 versus agmatine-treated animals.
Figure 5
Figure 5
Effect of NPY and [Leu31, Pro34]-NPY on the hyperphagic action of agmatine. Separate groups of rats were administered, by the intra-PVN route, (1) aCSF + aCSF, (2) aCSF + agmatine (5 nmol·per rat), (3) NPY (0.1 nmol·per rat) or [Leu31, Pro34]-NPY (10 pmol·per rat) + aCSF or (4) NPY (0.1 nmol·per rat) or [Leu31, Pro34]-NPY (10 pmol·per rat) + agmatine (5 nmol·per rat) at the onset of the dark phase. NPY or [Leu31, Pro34]-NPY were injected 10 min prior to agmatine. Food intake (g) was measured at 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents the food consumption (g) ± SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B) Each column and bar represents the cumulative food consumption (g) ± SEM over 24 h post injection (n = 6–8 per group). Data were analysed by one-way anova followed by post hoc Newman–Keuls mean comparison test. *P < 0.01, **P < 0.001 versus agmatine treatment, $P < 0.01, $$P < 0.001 versus NPY treatment, #P < 0.001 versus [Leu31, Pro34]-NPY-treated animals.
Figure 6
Figure 6
Effect of BIBP3226 on the agmatine-induced hyperphagia. Different groups of rats received intra-PVN injections of (1) aCSF + aCSF, (2) BIBP3226 (0.5 nmol·per rat) + aCSF, (3) aCSF + agmatine (10 nmol·per rat) or (4) BIBP3226 (0.5 nmol·per rat) + agmatine (10 nmol·per rat) at the onset of the dark phase. BIBP3226 was injected 10 min prior to agmatine. Food intake (g) was measured at 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents the food consumption (g) ± SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B) Each column and bar represents the cumulative food consumption (g) ± SEM over 24 h post injection (n = 6–8 per group). Data were analysed by one-way anova followed by post hoc Newman–Keuls test. $P < 0.001 versus aCSF- and *P < 0.05, **P < 0.001 versus agmatine-treated animals.
Figure 7
Figure 7
Effect of yohimbine on the orexigenic response induced by intra-PVN co-administration of NPY and agmatine. Rats received injections of yohimbine (0.15 mg·kg−1, i.p.) or saline (1 mL·kg−1, i.p.) 30 min before the administration of NPY (0.1 nmol·per rat, intra-PVN) or aCSF and agmatine (5 nmol·per rat, intra-PVN) or aCSF at the onset of the dark phase. Food intake (g) was determined at 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents the food consumption (g) ± SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B) Each column and bar represents the cumulative food consumption (g) ± SEM over 24 h post injection (n = 6–8 per group). Data were analysed by one-way anova followed by post hoc Newman–Keuls test. $P < 0.001 versus agmatine, #P < 0.001 versus NPY, *P < 0.001 versus agmatine + NPY.
Figure 8
Figure 8
Photomicrographs showing NPY-immunoreactive fibres (arrows) and/or cells (arrowheads) in the PVN (paraventricular nucleus of hypothalamus) and ARC (arcuate nucleus of hypothalamus) of rats following administrations of saline (1 mL·kg−1, i.p.; A and D), agmatine (40 mg·kg−1, i.p.; B and E) and yohimbine (0.15 mg·kg−1, i.p.) + agmatine (40 mg·kg−1, i.p.; C and F). Note a significant increase in NPY immunoreactivity in the PVN fibres and ARC cells following agmatine treatment. These changes were prevented by pretreatment with yohimbine. 3V, third ventricle. Scale bar = 200 µm.
Figure 9
Figure 9
Representation of the semiquantitative morphometric analysis of NPY immunoreactivity in fibres and/or cells of the paraventricular nucleus of hypothalamus (PVN; A) and arcuate nucleus of hypothalamus (ARC; B) of rats following injections of saline (SAL;1 mL·kg−1, i.p.), agmatine (AGM; 40 mg·kg−1, i.p.) and yohimbine (YOH; 0.15 mg·kg−1, i.p.) + AGM (40 mg·kg−1, i.p.). The outline of the transverse section through the brain indicates the regions of the PVN and the ARC at the co-ordinates −1.80 mm and −2.56 mm with reference to bregma, respectively (Paxinos and Watson, 1998), from which the measurements were collated (square, not to scale). AHC, central part of the anterior hypothalamus; DMH, dorsomedial nucleus of hypothalamus; ME, median eminence; Rch, retrochiasmatic nucleus; VMH, ventromedial nucleus of hypothalamus. The values in the histograms are shown as the mean ± SEM of five measurements from predetermined fields of the PVN and ARC on both the sides of each brain (n = 6). Data were analysed by one-way anova followed by post hoc Newman–Keuls multiple comparison test. *P < 0.01 versus SAL + SAL-treated animals.
Figure 10
Figure 10
Photomicrographs showing NPY-immunoreactive fibres (arrows) in the dorsomedial nucleus of hypothalamus (DMH), ventromedial nucleus of hypothalamus (VMH) and lateral hypothalamus (LH) of rats following saline (SAL; A, E and I), agmatine (AGM; B, F and J) and yohimbine (YOH) + AGM (C, G and K). Moreover, the semiquantitative morphometric analysis of NPY immunoreactivity in fibres of the DMH, VMH and LH is represented in panels D, H and I respectively. The outlines of the transverse sections through brain indicate the regions of the DMH or LH at the coordinates −2.56 mm and the VMH at the coordinates −2.80 mm with reference to bregma, respectively (Paxinos and Watson, 1998), from which the measurements were collated (not to scale). 3V, third ventricle; ARC, arcuate nucleus of hypothalamus; F, fornix; ME, median eminence. The values in the histograms are shown as the mean ± SEM of five measurements from predetermined fields of the DMH, VMH and LH on both the sides of each brain (n = 6). Data were analysed by one-way anova followed by post hoc Newman–Keuls multiple comparison test. Scale bar = 200 µm (DMH) and 100 µm (VMH and LH).

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