Synergistic effects of Y2 and Y4 receptors on adiposity and bone mass revealed in double knockout mice

Abstract

Neuropeptide Y regulates numerous physiological processes via at least five different Y receptors, but the specific roles of each receptor are still unclear. We previously demonstrated that Y2 receptor knockout results in a lean phenotype, increased cancellous bone volume, and an increase in plasma pancreatic polypeptide (PP), a ligand for Y4 receptors. PP-overexpressing mice are also known to have a lean phenotype. Deletion of the Y4 receptor also produced a lean phenotype and increased plasma PP levels. We therefore hypothesized that part of the Y2 phenotype results from increased PP action on Y4 receptors and tested this in PP transgenic Y4(-/-) and Y2(-/-) Y4(-/-) double knockout mice. Bone mass was not altered in Y4 knockout mice. Surprisingly, despite significant hyperphagia, Y2(-/-) Y4(-/-) mice retained a markedly lean phenotype, with reduced body weight, white adipose tissue mass, leptinemia, and insulinemia. Furthermore, bone volume was also increased threefold in Y2(-/-) Y4(-/-) mice, and this was associated with enhanced osteoblastic activity. These changes were more pronounced than those observed in Y2(-/-) mice, suggesting synergy between Y2 and Y4 receptor pathways. The lack of bone changes in PP transgenic mice suggests that PP alone is not responsible for the bone mass increases but might play a major role in the lean phenotype. However, a synergistic interaction between Y2 and Y4 pathways seems to regulate bone volume and adiposity and could have important implications for possible interventions in obesity and for anabolic treatment of osteoporotic bone loss.

FIG. 1.

Body weight (A) and food intake (B) of male Y2^−/−, Y4^−/−, and Y2^−/− Y4^−/− double knockout mice compared to those of controls. Data are means ± standard errors of the means per group. The number of mice is indicated in parentheses. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (versus control or the comparison indicated by horizontal bars).

FIG. 2.

Adiposity and plasma hormone and metabolite concentrations in male Y2^−/−, Y4^−/−, and Y2^−/− Y4^−/− double knockout mice compared to those in controls. (A and B) Masses of the right inguinal, right epididymal, right retroperitoneal, and mesenteric WAT depots, expressed as a percentage of body weight (BWT) (A) or as absolute WAT weight (B). (C to F) Concentrations of leptin (C), insulin (D), glucose (E), and corticosterone (cortico) (F) in plasma. Data are means ± standard errors of the means for the number of mice indicated in parentheses. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus control or the comparison indicated by horizontal bars).

FIG. 3.

Mass of BAT, expressed as a percentage of body weight (BWT) (A) or as absolute weight (B). Data are means ± standard errors of the means for the number of mice indicated in parentheses. *, P < 0.05; **, P < 0.01 (versus wild-type control or the comparison indicated by horizontal bars).

FIG. 4.

Plasma PP concentrations in Y2^−/−, Y4^−/−, and Y2^−/− Y4^−/− double knockout mice compared to those in controls. Data are means ± standard errors of the means for the number of mice indicated in parentheses. *, P < 0.05 versus control mice.

FIG. 5.

Sagittal micrographs of distal femoral metaphyses of Y2^−/− Y4^−/− double knockout mice (D) compared to those of control (A), Y2^−/− (B), or Y4^−/− (C) male mice. The micrographs show darkly stained bone tissue and are representative of the respective groups. Bar, 1 mm.

FIG. 6.

Effect of Y2^−/− Y4^−/− double knockout on bone morphology compared with no knockout, Y2^−/− knockout, or Y4^−/− knockout in male mice. Trabecular bone volume (A), trabecular number (B), trabecular thickness (C), cortical area (D), and cortical thickness (E) were determined. Data are means ± standard deviations for 6 to 14 mice per group. *, P < 0.05 versus control mice.

FIG. 7.

Effect of Y2^−/− Y4^−/− double knockout on CRH and TRH mRNA levels in neurons of the paraventricular nucleus (upper panels); on NPY, AgRP, POMC, and CART mRNA levels in the arcuate nucleus (middle panels); and on GnRH mRNA levels in forebrain neurons (lower panels) in male mice. High-power bright-field photomicrographs of dipped sections obtained from control and Y2^−/− Y4^−/− double knockout mice after in situ hybridization for the respective mRNA are shown. Bar, 100 μm. The paraventricular nucleus is depicted at about −0.8 mm and the arcuate nucleus is depicted at about −1.8 to −1.9 mm from Bregma as low-magnification film autoradiographs for TRH and CART.