pRb is an obesity suppressor in hypothalamus and high-fat diet inhibits pRb in this location

Abstract

pRb is frequently inactivated in tumours by mutations or phosphorylation. Here, we investigated whether pRb plays a role in obesity. The Arcuate nucleus (ARC) in hypothalamus contains antagonizing POMC and AGRP/NPY neurons for negative and positive energy balance, respectively. Various aspects of ARC neurons are affected in high-fat diet (HFD)-induced obesity mouse model. Using this model, we show that HFD, as well as pharmacological activation of AMPK, induces pRb phosphorylation and E2F target gene de-repression in ARC neurons. Some affected neurons express POMC; and deleting Rb1 in POMC neurons induces E2F target gene de-repression, cell-cycle re-entry, apoptosis, and a hyperphagia-obesity-diabetes syndrome. These defects can be corrected by combined deletion of E2f1. In contrast, deleting Rb1 in the antagonizing AGRP/NPY neurons shows no effects. Thus, pRb-E2F1 is an obesity suppression mechanism in ARC POMC neurons and HFD-AMPK inhibits this mechanism by phosphorylating pRb in this location.

Figure 1

Phosphorylation of pRb and de-repression of cyclin A and E2F1 in ARC neurons in response to HFD or AICAR. (A, B) ARC from C57/Bl wild-type mice fed on HFD for 11 months and matched mice fed on Chow co-stained with antibodies to pRbS608p and NeuN, as marked. (C) ARC from wild-type mice at 2 and 6 h after third ventricle injection of ACSF or AICAR stained with antibodies to pRbS608p, E2F1, or cyclin A, as marked. (D) ARC from Pomc-Cre;RosaYFP mice fed on HFD or Chow for 8 weeks following their 1 month birthday were stained with the indicated antibodies. Direct YFP was photographed in green. (E) Quantification of stain-positive cells versus YFP-positive cells in ARC presented as %, from samples in (D) and Supplementary Figure S2C (n=3). Data are expressed as average±s.e.m. Unpaired t-tests were performed, *P<0.05, **P<0.005 and ***P<0.0005. Scale bars: 100 μm.

Figure 2

Rb1 is required for POMC neuron maintenance. (A) ARC POMC neurons were visualized by anti-POMC stain in indicated mice; ARC sections were from Bregma −1.75 to −1.8 mm. (B) POMC neuron numbers were counted on sections at comparable Bregma positions. ARC1 at about −1.34, ARC2 about −1.58, ARC3 about −1.75 (n=3). (C) Soma areas of ARC POMC neurons were measured in 1-, 4-, 8-week-old mice (n=3). (D) POMC immunoreactive axon fibres in PVH of 8-week-old mice and their quantification based on POMC stain density (n=3). (E) Control mice densities were set as 1.0. (F) POMC expression was determined by RT–qPCR of hypothalamus (n=6). POMC/Actin ratios in control mice were set as 1.0. (G) Hypothalamus content of POMC pre-peptide, α-MSH, and β-endorphin (n=6). Data are expressed as average±s.e.m. Unpaired t-test was performed, *P<0.05, **P<0.005, ***P<0.0005. Scale bars: 200 μm.

Figure 3

Pomc-Cre;Rb1^lox/lox mice developed a hyperphagia-obesity-diabetes syndrome, and lost melanocortinergic tone in the hypothalamus. (A) Body weights of Rb1^lox/lox and Pomc-Cre;Rb1^lox/lox mice (n=5–12 for various age points). (B) Fat % of the mice in (A). (C) Nasoanal length. (D) Daily food intake during light and dark cycles (n=4). (E) Following an overnight fasting, tail vein blood was collected to measure leptin levels (n=5). (F) Fasting serum insulin concentration (n=4–8). (G) Fasting blood glucose concentration (n=6–8). (H) Glucose tolerance test was performed at 4 months of age (n=3). (I) Cumulative food intake measured for 6 h following i.c.v. administration of ACSF or SHU9119 (n=5–6). (J) Cumulative food intake measured for 3.5 h following i.c.v. administration of ACSF or PYY (n=5–6). (K) Cumulative food intake in response to i.c.v. administration of ACSF or MTII (n=5–6). Data are expressed as average±s.e.m. Unpaired t-tests were performed, *P<0.05, **P<0.005 and ***P<0.0005. For (I–K), # refers to differences in the same genotypes performed with paired t-test, ^#P<0.05 and ^##P<0.005.

Figure 4

Proliferation, nuclear morphology, and apoptosis in ARC and DG of Pomc-Cre;RosaYFP;Rb1^lox/lox mice. (A–H) ARC sections with native YFP (green) and anti-Ki67 stain at the indicated age (P0=newborn). (I) Quantification of Ki67-positive cells as ratio to YFP-positive cells (n=3). (J, K) Sections containing DG stained with DAPI and anti-Ki67. (L, M) ARC sections with native YFP (green) and DAPI stain. White arrowheads point to the same cells in each green-blue pairs. (N–Q) ARC sections with native YFP (green) and anti-Casp3a stain. (R) Quantification of Casp3a-positive cells in ARC (n=3). Data are expressed as average±s.e.m. Unpaired t-test was performed, *P<0.05, **P<0.01. Scale bars: 100 μm (10 μm for L and M).

Figure 5

pRb maintained POMC neurons postmitotic postnatally by repressing E2F1. ARC sections with YFP (pictured in green) and anti-MCM3 (A–C) or anti-cyclin A (E–G) stain of the indicated mice at 1 week of age. Consecutive sections of the same brains were stained with anti-Ki67 (I–K). (D, H, L) Quantification of stain-positive cells as ratio of YFP-positive cells (n=3). (M–P) ARC POMC neurons of the indicated mice were visualized by POMC stain and counted (n=3) (Q). Scale bar: 100 μm. Data are expressed as average±s.e.m. Unpaired t-test was performed *P<0.05, ***P<0.0005.

Figure 6

Rb1 is dispensable in AGRP/NPY neurons. The role of Rb1 in AGRP/NPY neurons was studied in three lines of mice. (A–C) NPY immunoreactive axons in PVH of 4-month-old Pomc-Cre;Rb1^lox/lox and control mice, and quantification of density of NPY stained axon fibres in PVH and DMH (n=3). (D) AGRP expression in hypothalamus was measured by RT–qPCR in 1- and 5-week-old Pomc-Cre;Rb1^lox/lox and control mice (n=3). Values in control mice were set as 1.0. (E, F) ARC sections of the indicated genotypes. AGRP/NPY neurons were visualized with direct YFP (pictured in green). (G–I) NPY immunoreactive axons in PVH of 6-month-old mice of the indicated genotypes and quantification of density of NPY stained axon fibres in PVH and DMH (n=3). (J) AGRP and NPY expression was determined by RT–qPCR (n=6). Values in control mice were set as 1.0. (K) Body weight and fat % of 2-month-old male mice on Chow. (L) Body weight and fat % of 3-month-old male mice that were on HFD for the last 2 months. (M) Body weight and fat % of 7-week-old female mice of the indicated genotypes (n=3–6). (N) Single cell RT–PCR detected Rb1 expression in POMC neurons and AGRP/NPY neurons. Data are expressed as average±s.e.m. **P<0.005, ***P<0.0005. Scale bars: 100 μm.