Control of energy balance by hypothalamic gene circuitry involving two nuclear receptors, neuron-derived orphan receptor 1 and glucocorticoid receptor

Abstract

Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

Fig 1

NOR1 as a regulator of AgRP/NPY in AgRP neurons. (A) ISH for NOR1 expression in wild-type male mice under fed conditions. (B) Double-immunofluorescence ISH with the ARC samples of fed wild-type male mice reveals colocalization of NOR1 with NPY. Similar colocalization results were also obtained with AgRP (data not shown). Bar, 100 μm. (C) Representative images of ISH for AgRP, NPY, and αMSH expression in wild-type (WT) and NOR1-null female mice with or without 24 h of fasting (3 mice per each group). (D) Quantification of the results in panel C using ImageJ software. *, P < 0.05; **, P < 0.001.

Fig 2

Increased food intake with NOR1-null mice. For male (A to D) and female (E to H) wild-type and NOR1-null mice (5 to 8 mice per each group), we measured daily food intake for 6 days and calculated the percent food intake over body weight per day (A and E), finding that NOR1-null mice consume more food. (B and F) We also measured body weights during fasting (0 and 24 h) and refeeding (24 and 72 h). (C and G) We measured the loss in body weight upon 24 h of fasting relative to the beginning body weight. (D and H) Accumulated food intake (percent) at 24- and 72-h refeeding points. *, P < 0.05.

Fig 3

NOR1 interferes with Gc induction of AgRP. (A and B) Quantification of AgRP levels using qRT-PCR in P19 cells treated with either vehicle or 10 nM Dex for 4 h under the indicated conditions. (C) Luciferase (LUC) reporter assays in HEK293 cells for a reporter driven by two copies of the AgRP-GRE in the absence or presence of increasing amounts of the NOR1 expression vector. RLU, relative light units. Representative results from two to four independent experiments are shown (A to C). (D) ChIP with hypothalamus lysates of wild-type mice either fed or fasted overnight. (E) ChIP with P19 cells treated with either vehicle or 50 nM Dex for 4 h under the indicated conditions. P19 cells express endogenous GR but very little NOR1, and thus, we also expressed exogenous NOR1 before carrying out the ChIP experiments. The difference in GR recruitment between cells transiently transfected with either vector alone or the NOR1 expression vector was quantified. *, P < 0.05.

Fig 4

Gc-mediated repression of NOR1 expression. (A) Representative ISH images for AgRP, αMSH, NOR1, Nurr1, and Nur77 expression in wild-type male mice either fed or fasted for 24 h (3 mice per each group) as well as the quantification of the NOR1 results using ImageJ software. (B) Quantification of AgRP/NOR1 levels by qRT-PCR on the hypothalamic samples of wild-type male mice either fed or fasted for 24 h (5 mice per each group). (C) Representative coimmunostaining images of the ARC region of wild-type male mice either fed or fasted for 24 h (3 mice per each group) as well as the quantification of the NOR1 results using ImageJ. (D) Representative ISH images for AgRP, NPY, and NOR1 in fed ADX male mice sacrificed 6 h after intraperitoneal injection of either vehicle or Dex (5 mg/kg of body weight) (3 mice per each group) as well as the quantification of the NOR1 results using ImageJ. (E) RT-PCR analysis of the hypothalamic samples of fed ADX male mice sacrificed 6 h after intraperitoneal injection of either vehicle or Dex (5 mg/kg) (4 mice per each group). *, P < 0.05.

Fig 5

Regulation of NOR1 expression by leptin. (A) Representative ISH images for AgRP, αMSH, and NOR1 expression in fed wild-type and Ob/Ob male mice (2 mice per each group), as well as quantification of the NOR1 results using ImageJ software. (B) Representative ISH images for AgRP, αMSH, and NOR1 expression in wild-type male mice fasted for 24 h, followed by intraperitoneal injection of either vehicle or leptin (3 mg/kg) and perfusion 3 h after leptin injection (2 mice per each group). The NOR1 results were quantified using ImageJ. (C) Representative ISH images for AgRP, αMSH, and NOR1 expression in fed Ob/Ob male mice sacrificed 3 h after intraperitoneal injection of either vehicle or leptin (3 mg/kg) (2 mice per each group) as well as the quantification of the NOR1 results using ImageJ. *, P < 0.05; ***, P < 0.0001.

Fig 6

Involvement of CREB in the regulation of NOR1 expression by leptin. (A, B) ChIP and qRT-PCR to assess the levels of CREB and GR recruited to the NOR1-CRE region in fed Ob/Ob male mice 2 h after intraperitoneal injection of either vehicle or leptin (3 mg/kg) (A) and in wild-type male mice either fed or fasted for 24 h (B). Six mice were used per each treatment group, and their hypothalamus samples were pooled and subjected to ChIP with IgG, α-CREB, and α-GR. Significance was determined by one-way ANOVA, followed by the Bonferroni post hoc test. (C) Immunofluorescence microscopy was carried out on the ARC samples prepared from fed Ob/Ob male mice 2 h after intraperitoneal injection of either vehicle or leptin (3 mg/kg). (D) Quantification of the signal intensity for the results in panel C using ImageJ software. (E) Quantification by qRT-PCR of NOR1 levels in N42 cells treated with vehicle, leptin (60 nM), and/or Dex (10 nM) for 1 h (left) and in N42 cells treated with leptin (60 nM) for 1 h with the lentiviral particles for control siRNA or si-CREB from Santa Cruz (right). (F) Luciferase reporter assays in HEK293 cells for a reporter driven by a 1.7-kb NOR1 promoter fragment in the absence or presence of vehicle, forskolin (Fsk; 10 μM), and/or Dex (10 nM). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Fig 7

Leptin resistance of NOR1-null mice. (A, B) The ISH signal intensity for AgRP and NPY in wild-type and NOR1-null female mice sacrificed 3 h after intraperitoneal injection of leptin (3 mg/kg) following 24 h of fasting was quantified using ImageJ software. (C) Acute effects of leptin on fasting-induced changes in food intake and body weight were tested in wild-type and NOR1-null male mice (3 to 4 mice per each group). Prior to intraperitoneal injection of either vehicle or leptin (3 mg/kg), mice were fasted for 12 h, and percent body weight changes and percent food intake per body weight were measured 6 h after leptin injection. (D) Nonfasted wild-type and NOR1-null female mice (3 to 4 mice per each group) were injected intraperitoneally with either vehicle or leptin (3 mg/kg). Percent body weight changes and percent food intake per body weight were measured 15 h after leptin injection. *, P < 0.05; **, P < 0.001; ***, P < 0.0001; NS, not significant.

Fig 8

Working model (see the text).