NPY Neuron-Specific Y2 Receptors Regulate Adipose Tissue and Trabecular Bone but Not Cortical Bone Homeostasis in Mice

BACKGROUND: Y2 receptor signalling is known to be important in neuropeptide Y (NPY)-mediated effects on energy homeostasis and bone physiology. Y2 receptors are located post-synaptically as well as acting as auto receptors on NPY-expressing neurons, and the different roles of these two populations of Y2 receptors in the regulation of energy homeostasis and body composition are unclear. METHODOLOGY/PRINCIPAL FINDINGS: We thus generated two conditional knockout mouse models, Y2(lox/lox) and NPYCre/+;Y2(lox/lox), in which Y2 receptors can be selectively ablated either in the hypothalamus or specifically in hypothalamic NPY-producing neurons of adult mice. Specific deletion of hypothalamic Y2 receptors increases food intake and body weight compared to controls. Importantly, specific ablation of hypothalamic Y2 receptors on NPY-containing neurons results in a significantly greater adiposity in female but not male mice, accompanied by increased hepatic triglyceride levels, decreased expression of liver carnitine palmitoyltransferase (CPT1) and increased expression of muscle phosphorylated acetyl-CoA carboxylase (ACC). While food intake, body weight, femur length, bone mineral content, density and cortical bone volume and thickness are not significantly altered, trabecular bone volume and number were significantly increased by hypothalamic Y2 deletion on NPY-expressing neurons. Interestingly, in situ hybridisation reveals increased NPY and decreased proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus of mice with hypothalamus-specific deletion of Y2 receptors in NPY neurons, consistent with a negative feedback mechanism between NPY expression and Y2 receptors on NPY-ergic neurons. CONCLUSIONS/SIGNIFICANCE: Taken together these data demonstrate the anti-obesogenic role of Y2 receptors in the brain, notably on NPY-ergic neurons, possibly via inhibition of NPY neurons and concomitant stimulation of POMC-expressing neurons in the arcuate nucleus of the hypothalamus, reducing lipogenic pathways in liver and/or skeletal muscle in females. These data also reveal as an anti-osteogenic effect of Y2 receptors on hypothalamic NPY-expressing neurons on trabecular but not on cortical bone

Effects of deleting Y2 receptors from hypothalamic NPY-expressing neurons on body composition in female and male mice.

<p>(A, B) Total weight of dissected white adipose tissue (WAT) depots, expressed as a percent of body weight, in wild type or NPYCre/+;Y2^lox/lox mice injected into the hypothalamus with either saline or doxycyline (Dox). (C, D) Total WAT weight of Dox-injected NPYCre/+;Y2^lox/lox mice relative to that of Dox-injected wild type mice (black columns) versus saline-injected NPYCre/+;Y2^lox/lox mice relative to saline-injected wild type mice (open column). (E, F) whole body fat mass (expressed as a percent of body weight), (G, H) whole body fat mass of Dox-injected NPYCre/+;Y2^lox/lox mice relative to that of Dox-injected wild type mice (black columns) versus saline-injected NPYCre/+;Y2^lox/lox mice relative to saline-injected wild type mice (open column). (I, J) whole body lean mass (expressed as a percent of body weight) of wild type or NPYCre/+;Y2^lox/lox mice injected into the hypothalamus with either saline or doxycyline, as determined by dual energy X-ray absorptiometry (DXA). Data are mean ± SEM of 5 or more mice per group. *: p<0.05 for the comparison indicated by horizontal bar.</p

Schematic diagram of NPY-Cre-GFP knock-in strategy and confirmation of Y2 receptor deletion.

<p>(A) Conditional NPY neuron-specific Y2 receptor knockout mice (NPYCre/+;Y2^lox/lox mice) were generated using a knock-in strategy. The inducible NPY neuron-specific expression of the Cre gene is achieved by fusion of the initiation codon of the NPY gene with that of the tetR gene, enabling the endogenous NPY promoter to drive the expression of this regulatory protein upon addition of doxycycline (Dox). (B) Schematic drawing of position of oligonucleotide primers (Oligo A & B) used for PCR verification of Y2 receptor knockout. (C) Using genomic DNA extracted from the hypothalamus of NPYCre/+;Y2^lox/lox mice, Oligos A and B produced a 250 base pair (bp) product from a Dox-injected mouse, demonstrating deletion of the Y2 receptor gene, but no product was produced using DNA from a saline-injected control mouse. (D) Y2 receptor mRNA extracted from the hypothalamus of male NPYCre/+;Y2^−/− mice was significantly reduced compared to that of saline-injected mice.</p

Hypothalamus-specific Y2 deletion increases food intake and body weight.

<p>(A) Marked reduction in Y2 receptor mRNA in the Arc of male Y2^lox/lox mice – as detected by <i>in situ</i> hybridization – after injection of Cre-expressing adenovirus into the hypothalamus (Y2^hyp KO mice, Figure A (b)) compared to that in male Y2^lox/lox mice (Figure A (a), scale bar 40 µm). Food intake (B) and body weight (C) in male Y2^hypKO mice compared to that of Y2^lox/lox-empty vector-injected control mice. Values are means ± SEM of 8 male mice per group. * p<0.05 versus Y2^lox/lox control mice. Arc: Arcuate nucleus of the hypothalamus; 3V: third cerebral ventricle.</p

NPY neuron-specific Y2 deletion in the hypothalamus increases NPY and decreases POMC expression in the arcuate nucleus with no significant effects on body weight, spontaneous or fasting-induced food intake.

<p>(A, C) Bright-field photomicrographs of coronal brain sections, including the Arc, obtained from saline-injected control and doxycycline (Dox)-injected male NPYCre/+;Y2^−/− mice after <i>in situ</i> hybridization for NPY and proopiomelanocortin (POMC) mRNA. Scale bar, 40 µm. (B, D) Quantification of mean labeling intensity of neurons from <i>in situ</i> hybridization, given as percentage coverage of neuronal surface by silvergrains ± SEM of 5 male mice per group. *: p<0.05 versus saline-injected control mice. Arc: Arcuate nucleus of the hypothalamus; 3V: third cerebral ventricle. Spontaneous food intake (E, F), cumulative fasting-induced food intake (G, H) and body weight (I, J) are not altered by NPY neuron-specific Y2 receptor deletion in the hypothalamus. Data are mean ± SEM of 5 or more mice per group.</p

Summary of the effects of deletion of Y2 receptors on non-NPY-ergic or NPY-ergic neurons in the brain.

<p>In normal mice, Y2 receptors on non-NPY-ergic neurons in the hypothalamus (e.g. in the arcuate nucleus) reduce cortical and trabecular bone mass, as evidenced by the fact that hypothalamic ablation of Y2 receptors on non-NPY-ergic as well as on NPY-ergic neurons increases cortical and trabecular bone mass <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011361#pone.0011361-Baldock3" target="_blank">[31]</a>, whereas ablation of Y2 receptors specifically from NPY-ergic neurons in the current study only partially increases trabecular bone mass and has no effect on cortical bone mass. Additionally in normal mice, Y2 receptors on non-NPY-ergic neurons in brain centres regulating stress responses (such as the amygdala) are not likely involved in mediating the anxiolytic effects of NPY, which are likely mediated by Y1 and Y5 receptors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011361#pone.0011361-Heilig1" target="_blank">[56]</a>. However, Y2 receptors on NPY-ergic neurons in the amygdala induce anxiogenic effects <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011361#pone.0011361-Tasan1" target="_blank">[50]</a>, likely due to decreased endogenous NPY-ergic action <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011361#pone.0011361-Heilig1" target="_blank">[56]</a>. Our current work shows that when Y2 receptors are deleted specifically from NPY-ergic neurons in the brain, there is a resultant increase in NPY and a decrease in POMC mRNA expression levels in the arcuate nucleus of the hypothalamus, and this – in conjunction with likely sex hormone interactions – could contribute to the increased adiposity and hepatic fat content observed in these female knockouts. In addition to effects mediated by the hypothalamus, Y2 receptor deletion on NPY-ergic neurons in centres controlling stress responses may increase NPY-ergic actions in brain regions such as the amygdala, which can thereby reduce stress responses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011361#pone.0011361-Tasan1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011361#pone.0011361-Heilig1" target="_blank">[56]</a> and probably contribute to the absence of stress-induced weight loss in male and female mice lacking Y2 receptors specifically from NPY-ergic neurons. An additional mechanism by which central Y2 deletion can promote positive energy balance could be via blockade of PYY-induced catabolic effects <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011361#pone.0011361-Boey2" target="_blank">[43]</a> on non-NPY-ergic or NPY-ergic neurons or both.</p

Effect of deleting Y2 receptors from hypothalamic NPY-expressing neurons on body weight, feeding parameters, body composition and glucose metabolism.

<p>Values are means ± SEM of 5–12 wild type or NPYCre/+;Y2^lox/lox mice injected into the hypothalamus with either saline or doxycyline (Dox), as indicated in parentheses.</p><p>*p<0.05 versus sex-matched NPYCre/+;Y2^lox/lox mice.</p

Effect of deleting Y2 receptors from hypothalamic NPY-expressing neurons on body weight change, feeding parameters, rectal temperature, oxygen consumption (VO2), respiratory exchange ratio (RER) and physical activity as determined during indirect calorimetry.

<p>Values are means ± SEM of 5–12 wild type or NPYCre/+;Y2^lox/lox mice injected into the hypothalamus with either saline or doxycyline (Dox). The number of mice in each group is indicated in parentheses.</p>#<p>p<0.05 versus sex-matched saline-injected WT mice.</p><p>*p<0.05 versus sex-matched saline-injected NPYCre/+;Y2^lox/lox mice. Body weight change is expressed as a percentage of initial body weight after 2 days in the indirect calorimetry chambers.</p

Effects of NPY neuron-specific Y2 receptor deletion on bone parameters determined by dual energy X-ray absorptiometry (DXA).

<p>Whole body bone mineral density (BMD), bone mineral content (BMC) and isolated femur BMD and BMC were measured by DXA. Values are means ± SEM of 5–12 mice per group as shown in parentheses.</p