Functional requirement of AgRP and NPY neurons in ovarian cycle-dependent regulation of food intake

Abstract

In female mammals including rodents and humans, feeding decreases during the periovulatory period of the ovarian cycle, which coincides with a surge in circulating estrogen levels. Ovariectomy increases food intake, which can be normalized by estrogen treatment at a dose and frequency mimicking those during the estrous cycle. Furthermore, administration of estrogen to rodents potently inhibits food intake. Despite these well-known effects of estrogen, neuronal subtypes that mediate estrogen's anorexigenic effects have not been identified. In this study, we show that changes in hypothalamic expression of agouti-related protein (Agrp) and neuropeptide Y (Npy) coincide with the cyclic changes in feeding across the estrous cycle. These cyclic changes in feeding are abolished in mice with degenerated AgRP neurons even though these mice cycle normally. Central administration of 17beta-estradiol (E2) decreases food intake in controls but not in mice lacking the AgRP neurons. Furthermore, E2 treatment suppresses fasting-induced c-Fos activation in AgRP and NPY neurons and blunts the refeeding response. Surprisingly, although estrogen receptor alpha (ERalpha) is the key mediator of estrogen's anorexigenic effects, we find that expression of ERalpha is completely excluded from AgRP and NPY neurons in the mouse hypothalamus, suggesting that estrogen may regulate these neurons indirectly via presynaptic neurons that express ERalpha. This study indicates that neurons coexpressing AgRP and NPY are functionally required for the cyclic changes in feeding across estrous cycle and that AgRP and NPY neurons are essential mediators of estrogen's anorexigenic function.

Fig. 1.

Agrp and Npy expression undergoes cyclic changes during the estrous cycle and such changes coincide with cyclic changes in food intake. (A, B) Phase of estrous cycle, food intake and body weight were determined daily in 12-week-old female mice (n = 12). Food intake and body weight measurements were normalized to measurements obtained in M-D for each mouse. **, P < 0.01 comparing food intake in M-D and P–E. ***, P < 0.001 comparing body weight in D and E using Student's paired t test. (C) Female mice were killed at 4 PM in different phases of the estrous cycle, and hypothalamic gene expression of Pomc, Lepr, Npy, and Agrp was analyzed by semiquantitative real time RT-PCR. β-actin was used as internal control. *, P < 0.05. Npy expression in M-D and P-E and Agrp expression in D-P and P-E were compared. n = 7 (M-D), 13 (D-P), 7 (P-E) and 9 (E-M). D, diestrus; P, proestrus; E, estrus; M, metestrus.

Fig. 2.

Estrous cycle dependent regulation of food intake and body weight is abolished in mice lacking AgRP/NPY neurons. (A, B) Body weight and 24-h food intake were measured continuously for 20 days in all female Agrp-Tfam control and mutant mice regardless of their cycling status. n = 15 for controls, n = 14 for mutants. (C, D) Twenty-four–hour food intake and body weight were measured daily in cycling female Agrp-Tfam controls and mutants and the data are presented in panel C and D, respectively. For each mouse, body weight measurements were normalized to measurements obtained in estrus. n = 8 controls, n = 11 mutants. D, diestrus; P, proestrus; E, estrus; M, metestrus. **, P < 0.01 comparing food intake/body weight in M-D/D with P-E/E or E-M/M in controls as analyzed by Student's paired t test.

Fig. 3.

E2 decreases Npy and Agrp expression in cultured hypothalamic explants. Coronal hypothalamic slices were prepared from adult C57BL/6 female mice killed 3–4 PM during the day of diestrus-proestrus. Each slice were cut into two identical halves and cultured. One half was treated with either 10 nM E2 (A) or 100 nM progesterone (B) and the other half was treated with the corresponding vehicle (water). After 24 h of treatment, RNA was extracted and semiquantitative RT-PCR was performed. Lepr, Npy, and Agrp expression was analyzed using β-actin as internal control. Gene expression in hormone treated explants was normalized to the vehicle values (broken line). **, P < 0.01 and ***, P < 0.001 by pair-wise comparison between hormone and vehicle treated samples. n = 5 for each treatment group.

Fig. 4.

Central administration of E2 decreases food intake in controls but not in mice lacking AgRP/NPY neurons. Agrp-Tfam controls and mutants were infused with aCSF for at least 6 days until they reached metestrus/diestrus. In this particular experiment, the mice were synchronous in cycling and most of them reached metestrus/diestrus on the seventh day, at which point the mice were infused icv with water-soluble E2 (2 μg in aCSF per mouse). Fourteen- and twenty-four–hour food intake after E2 treatment was compared with values obtained in the same phase (metestrus/diestrus) during vehicle treatment, such that each mouse served as its own control. All injections were made at 6 PM and at the same time vaginal smears were collected. Food intake was measured at 8 AM and 6 PM *, P < 0.05. n = 6 controls, n = 6 mutants.

Fig. 5.

Expression of ERα is abundant in the ARC of the hypothalamus, but completely excluded from AgRP/NPY neurons. Hypothalamic sections were prepared from transgenic mice expressing GFP in NPY neurons. (A and B) The GFP signal was strong in the ARC (white arrows) and showed an expression pattern characteristic for NPY in the hypothalamus. ERα positive cells were found in the ARC and the VMH in the hypothalamus indicated by white arrows (C and D). However, zero out of 2,449 GFP positive neurons was found to be positive for ERα immunoreactivity (E-F). A total of 24 sections (bregma −2.46 mm to bregma −1.06) from four female and two male mice were analyzed. The specificity of the ERα antibody was validated as no signal was detected in ERα-deficient mice.

Fig. 6.

E2 inhibits fasting-induced c-Fos activation in AgRP/NPY-neurons. (A–F) Fed and 25-h fasted female NPY-hrGFP transgenic mice were perfused. Immunofluorescence was performed to examine c-Fos expression in the basomedial ARC (Bregma –2.06 to −2.46 mm). NPY neurons were identified by expression of GFP in both nucleus and cytoplasm. c-Fos immunoreactivity was nuclear. In fasted mice, 97.0% of the c-Fos positive cells (800 out of 825 cells) within this region were AgRP/NPY neurons. Seven sections from four fed mice and 14 sections from four fasted mice were used. (G–I) Female mice were fasted for 25 h (9 AM to 10 AM) and injected with either vehicle (saline) or E2 (150 μg water soluble E2) at three time points during this period (9 AM, 6 PM on day 1 and 9 AM on day 2). Mice were perfused 1 h after the last injection and immunofluorescence was performed to examine c-Fos expression in the basomedial ARC (bregma −2.06 to −2.46 mm). Number of c-Fos positive cells were quantified in panel I. ***, P < 0.001. Thirteen to sixteen sections from four saline- and four E2-injected mice were used.