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

Expression of proopiomelanocortin (POMC), glutamic acid decarboxylase 65 (GAD65) and neuropeptide Y (NPY) mRNA in the arcuate nucleus of the hypothalamus at 30 minutes after i.p. injection of saline or pancreatic polypeptide (PP).

<p>Data are means ± standard error of 5 wild type mice per group, with neuron labeling intensity expressed as a percentage of saline-injected controls. **, P<0.01; ***P<0.001 versus saline-injected controls.</p

Pancreatic polypeptide (PP) injection induces c-Fos immunoreactivity in neurons positive for alpha melanocyte stimulating hormone (α-MSH) and glutamic acid decarboxylase 65 (GAD65) in the arcuate nucleus of the hypothalamus (ARC).

<p>(A) c-Fos immunoreactivity, (B) α-MSH immunoreactivity, and (C) overlay of c-Fos and α-MSH immunoreactivity in neurons as indicated by white arrows at 30 minutes after i.p. injection of PP. Sale bar for A–C  = 25 µm. (D) Brightfield micrograph showing c-Fos and GAD65 immunoreactivity at 30 minutes after i.p. injection of PP. Scale bar  = 200 µm. (E) Higher magnification of the boxed area from D. Black arrows indicate neurons positive for c-Fos immunoreactivity only. These neurons are darkly stained. Black arrowheads indicate neurons positive for GAD65 immunoreactivity only. These neurons are lightly stained. Red arrows indicate neurons double-labeled for c-Fos and GAD65. The double staining on these neurons makes these neurons appear larger than the neurons positive only for c-Fos or CAG65 immunoreactvity. Scale bar  = 5 µm. 3V, third cerebral ventricle.</p

Number of c-Fos-like immunoreactive neurons in the brainstem and hypothalamus of wild type and Y4 receptor knockout (<i>Y4</i>^−/−) mice at 30 or 90 minutes after i.p. injection of saline or pancreatic polypeptide (PP).

<p>Data are means ± SEM of 4–6 mice per groups.* P<0.05 versus saline-injected wild type mice; # P<0.05 versus PP-injected wild type mice (90 minutes). NTS: nucleus tractus solitarus; AP: area postrema; ARC: arcuate nucleus of the hypothalamus; PVN: paraventricular nucleus of the hypothalamus; VMHDM: dorsomedial part of the ventromedial nucleus of the hypothalamus; LHA: lateral hypothalamic area.</p

Immunoreactivity for phosphorylated extracellular signal-regulated kinases 1 and 2 (P-Erk1/2) in the hypothalamus and co-expression of c-Fos and tyrosine hydroxylase (TH) immunoreactivity in the brainstem after PP injection.

<p>(A) Brightfield micrograph showing P-Erk1/2 immunoreactivity in the arcuate nucleus of the hypothalamus (ARC) at 30 minutes after i.p. injection of PP in wild type mice. Scale bar = 200 µm. (B) Higher magnification of the boxed area from A. Scale bar  = 5 µm. (C, E) Fluorescence micrographs of the nucleus tractus solitarius (NTS) and area postrema (AP) respectively, 30 minutes after i.p. injection of PP. Scale bar  = 40 µm. (D, F) Higher magnifications of the NTS and AP, respectively, from the boxed areas in C and E. Scale bar  = 25 µm. White arrows indicate neurons positive for c-Fos immunoreactivity only (red fluorescence); white arrowheads indicate neurons positive for TH immunoreactivity only (green fluorescence); blue arrows indicate neurons double-labeled for c-Fos and TH. 4V, fourth cerebral ventricle; cc, central canal.</p

Effects of pancreatic polypeptide (PP) on the expression of proopiomelanocortin (POMC) and GAD65 mRNA in the arcuate nucleus of the hypothalamus (ARC).

<p>Emulsion-dipped autoradiographs showing POMC mRNA at 30 minutes after i.p. injection of (A) saline or (C) PP, orGAD65 mRNA at 30 minutes after i.p. injection of (B) saline or (D) PP. Scale bar for all panels  = 25 µm. 3V, third cerebral ventricle.</p

Conditional deletion of Y4 receptors in the arcuate nucleus of the hypothalamus (ARC) alters pancreatic polypeptide (PP)-induced c-Fos immunoreactivity and POMC mRNA expression.

<p>(A) Schematic representation of primer positions used for PCR verification of Y4 receptor gene knockout.The un-deleted gene is 3.4 kilobases, and after Cre-mediated Y4 receptor gene deletion a PCR product of 290 base pairs is produced. (B) Confirmation of Y4 receptor deletion (indicated by production of the 290 base pair PCR product) from DNA isolated from the hypothalamus (Hypo) but not from the cerebellum (Cer) of Y4 receptor conditional knockout (Y4F) mice injected with AAV-Cre recombinase into the ARC, or from mice not injected with AAV-Cre(Y4F Hypo). (C) Photomicrograph showing c-Fos immunoreactivity and (D+E) emulsion-dipped autoradiograph of POMC mRNA expression in the brain of a conditional Y4 receptor knockout mouse, 30 minutes after i.p. injection of PP. Mice received unilateral injection of (D) AAV-Cre or (E) AAV-empty 28 days prior to PP injection in order to induce local deletion of Y4 receptors by virally-delivered Cre-recombinase, the contra-lateral side (at left in C, D and E) was used as control. Scale bar  = 100 µm in C and 25 µm in D and E. 3V, third cerebral ventricle.</p