Hypothalamic NPY-Y1R Interacts with Gonadal Hormones in Protecting Female Mice against Obesity and Neuroinflammation

Abstract

We previously demonstrated that Npy1r^rfb mice, which carry the conditional inactivation of the Npy1r gene in forebrain principal neurons, display a sexually dimorphic phenotype, with male mice showing metabolic, hormonal and behavioral effects and females being only marginally affected. Moreover, exposure of Npy1r^rfb male mice to a high-fat diet (HFD) increased body weight growth, adipose tissue, blood glucose levels and caloric intake compared to Npy1r^2lox male controls. We used conditional knockout Npy1r^rfb and Npy1r^2lox control mice to examine whether forebrain disruption of the Npy1r gene affects susceptibility to obesity and associated disorders of cycling and ovariectomized (ovx) female mice in a standard diet (SD) regimen or exposed to an HFD for 3 months. The conditional deletion of the Npy1r gene increased body weight and subcutaneous white adipose tissue weight in both SD- and HFD-fed ovx females but not in cycling females. Moreover, compared with ovx control females on the same diet regimen, Npy1r^rfb females displayed increased microglia number and activation, increased expression of Neuropeptide Y (NPY)-immunoreactivity (IR) and decreased expression of proopiomelanocortin-IR in the hypothalamic arcuate nucleus (ARC). These results suggest that in the ARC NPY-Y1R reduces the susceptibility to obesity of female mice with low levels of gonadal hormones and that this effect may be mediated via NPY-Y1R ability to protect the brain against neuroinflammation.

Keywords: high-fat diet; hypothalamic Y1R receptors; neuroinflammation; obesity; ovariectomy.

Figure 1

Body weight of standard diet (SD)- and high-fat diet (HFD)-fed cycling (A) and ovx (B) Npy1r^2lox control and Npy1r^rfb knockout female mice. No significant differences in body weight growth were observed between cycling Npy1r^2lox and Npy1r^rfb female mice during postnatal days (P) 57–156, independently of diet regimen. SD- and HFD-fed ovx Npy1r^rfb females displayed a significantly greater body weight compared with their control littermates on the same diet regimen. Data are the mean ± SEM; n = 17–26 from 6 litters. * p < 0.05 versus the corresponding control females. (C) Adiposity of standard diet (SD)- and high-fat diet (HFD)-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice. Significantly larger depots of subcutaneous fat were observed in ovx Npy1r^rfb females compared with their control littermates on the same diet regimen. Data are the mean ± SEM; n = 10–15 from 3 litters. ** p < 0.01 and * p < 0.05 versus SD-fed and HFD-fed ovx Npy1r^2lox female mice, respectively. (D) Leptin plasma levels of standard diet (SD)- and high-fat diet (HFD)-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice. Both ovariectomy and HFD increased leptin plasma levels of female mice. Data are the mean ± SEM; n = 10–15 from 3 litters. &&& p < 0.001.

Figure 2

(A) Spontaneous locomotor activity of standard diet (SD)- and high-fat diet (HFD)-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice. Both HFD and ovariectomy significantly decreased locomotor activity (distance traveled, cm) of Npy1r^2lox and Npy1r^rfb female mice. HFD-fed cycling and SD-fed ovx Npy1r^rfb females showed significantly lower locomotor activity compared with their corresponding controls. Data are the mean ± SEM; n = 8–12 from 2 litters. # p < 0.05 versus SD-fed cycling controls; ### p < 0.001 versus SD-fed cycling Npy1r^rfb female mice; § p < 0.05 versus SD-fed cycling controls; §§§ versus SD-fed cycling Npy1r^rfb female mice; * p < 0.05 versus SD-fed ovx controls. (B) Correlation between locomotor activity and body weight of 156 days old SD- and HFD-fed cycling and ovx Npy1r^2lox and Npy1r^rfb female mice. Data are the mean ± SEM; n = 6 from 3 litters. Food and energy intake of standard diet (SD)- and high-fat diet (HFD)-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice (C–E). (C) No significant differences of cumulative food intake (g) were observed among the different experimental groups. Data are the mean ± SEM; n = 4 from 3 litters. (D) HFD increased cumulative energy intake (Kcal) of cycling and ovx female mice, independently of the genotype. Data are the mean ± SEM; n = 6 from 3 litters. &&& p < 0.001 versus SD-females. (E) Nine-day cumulative energy intake (kcal) of SD- and HFD-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice during the metabolic challenge procedure (P66–156). Data are the mean ± SEM; n = 6 from 3 litters.

Figure 3

Glucose homeostasis and metabolism of standard diet (SD)- and high-fat diet (HFD)-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice (A–F). (A) Glucose tolerance tests and (B) area under the curve (AUC) of SD- and HFD-fed cycling and ovx Npy1r^2lox and Npy1r^rfb females. Data are the mean ± SEM; n = 11–20 from 3 litters. ## p < 0.01 HFD-fed versus SD-fed cycling controls; ### p < 0.001 HFD-fed versus SD-fed cycling Npy1r^2lox female mice. (C) Insulin tolerance test and (D) area under the curve (AUC) of SD- and HFD-fed cycling and ovx Npy1r^2lox and Npy1r^rfb females. Data are the mean ± SEM; n = 7–15 from 2 litters. (E) Pyruvate tolerance test and (F) area under the curve (AUC) of SD- and HFD-fed cycling and ovx Npy1r^2lox and Npy1r^rfb females. Data are the mean ± SEM; n = 7–16 from 2 litters. # p < 0.05 HFD-fed versus SD-fed cycling Npy1r^rfb females. (G) Heart rate and (H) blood pressure of standard diet (SD)- and high-fat diet (HFD)-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice. &&& p < 0.001 versus cycling females.

Figure 4

Expression of neuropeptides in the ARC and PVN of standard diet (SD)- and high-fat diet (HFD)-fed cycling and ovx Npy1r^2lox control and Npy1r^rfb knockout female mice. Agouti-related protein immunoreactivity (AgRP-IR) in the ARC (A) and in the PVN (B) of SD- and HFD-fed cycling and ovx Npy1r^2lox and Npy1r^rfb females. HFD and ovx significantly decreased AgRP expression in the ARC of female mice. SD-fed cycling Npy1r^rfb conditional mutants showed a significant decrease of AgRP-IR in the ARC compared to SD-fed cycling control females. Data are the mean ± SEM; n = 8–9 (ARC) and n = 7–10 (PVN) from 2 litters. ** p < 0.01 and ### p < 0.001 versus SD-fed cycling Npy1r^2lox females; §§ p < 0.01 versus SD-fed cycling Npy1r^2lox; mice; ## p < 0.01 versus SD-fed ovx Npy1r^2lox female mice. (C) Representative images of the ARC of ovx females expressing AgRP-IR immunoreactivity (scale bar: 150 μm). Neuropeptide Y immunoreactivity (NPY-IR) in the ARC (D) and in the PVN (E) of SD- and HFD-fed cycling and ovx Npy1r^2lox and Npy1r^rfb females. A significant decrease of NPY-IR was observed in the ARC of ovx Npy1r^2lox mice compared with cycling Npy1r^2lox mice. A significant increase of NPY-IR was observed in the ARC of ovariectomized Npy1r^rfb mice compared with their control littermates on the same diet regimen. § p < 0.05 versus cycling Npy1r^2lox females on the same diet regimen. ** p < 0.01 versus ovx Npy1r^2lox on the same diet regimen. Data are the mean ± SEM; n = 5–8 from 2 litters. (F) Representative images of the ARC of ovx females expressing NPY-IR immunoreactivity (scale bar: 150 μm). Proopiomelanocortin immunoreactivity (POMC-IR) in the ARC (G) and in the PVN (H) of SD- and HFD-fed cycling and ovx Npy1r^2lox and Npy1r^rfb females. A significant decrease of POMC-IR was observed in the ARC of SD-fed ovx Npy1r^rfb mice compared with SD-fed ovx Npy1r^2lox mice. Data are the mean ± SEM; n = 7–11 from 2 litters. *** p < 0.001 versus SD-fed ovx Npy1r^2lox mice. (I) Representative images of the ARC of ovx females expressing POMC-IR (scale bar: 150 μm).

Figure 5

Effect of HFD and Npy1r gene deletion on microglial activation and microglia number. The cell body to cell size ratio (cb/c) of microglia (A,B) and the number of microglia/mm² (C,D). HFD significantly increased cb/c (B) and microglia number (D) in the ARC of ovx females. Conditional inactivation of Npy1r gene increased cb/c (B) and microglia number (D) in the ARC of SD- and HFD-fed ovx mice. Data are the mean ± SEM; n = 4–6 from 2 litters. # p < 0.05 versus SD-fed ovx females. * p < 0.05 and ** p < 0.01 versus Npy1r^2lox ovx females on the same diet regimen. (E) Representative images of the ARC of ovx females expressing ionized calcium-binding adaptor protein-1 (IBA-1) immunoreactivity (scale bar: 150 μm). (F) Image analysis used to quantify the morphological characteristics of microglia in Iba-1 staining in ovx females. Upper panels: the unprocessed pictures. Middle panels: pixels darker than the background are traced (red) to determine the total cell size of microglia. Lower panels: pixel-clusters that are above an applied staining threshold and size-filter are traced (red) to determine the total cell body size of all microglia, as well as the number of microglia.

Figure 6

Interactions between estradiol and the NPY-Y1 receptor system in the ARC. (A) Estrogens (E2) directly inhibit NPY neurons. Microglia activation and proliferation is increased by HFD and inhibited by E2 and the NPY-Y1 receptor system. (B) In cycling females, the HFD-induced microglia activation is inhibited by E2 also in the absence of Y1 receptors on microglia. (C) In ovx Npy1r^2lox females, NPY inhibits microglia through activation of Y1 receptors. (D) In Npy1r^rfb ovx females, on both SD and HFD regimen, the deletion of Y1 receptors in microglia increases microglia activation and, in turn, body and WAT weight.