Remodeling of the arcuate nucleus energy-balance circuit is inhibited in obese mice

Abstract

In the CNS, the hypothalamic arcuate nucleus (ARN) energy-balance circuit plays a key role in regulating body weight. Recent studies have shown that neurogenesis occurs in the adult hypothalamus, revealing that the ARN energy-balance circuit is more plastic than originally believed. Changes in diet result in altered gene expression and neuronal activity in the ARN, some of which may reflect hypothalamic plasticity. To explore this possibility, we examined the turnover of hypothalamic neurons in mice with obesity secondary to either high-fat diet (HFD) consumption or leptin deficiency. We found substantial turnover of neurons in the ARN that resulted in ongoing cellular remodeling. Feeding mice HFD suppressed neurogenesis, as demonstrated by the observation that these mice both generated fewer new neurons and retained more old neurons. This suppression of neuronal turnover was associated with increased apoptosis of newborn neurons. Leptin-deficient mice also generated fewer new neurons, an observation that was explained in part by a loss of hypothalamic neural stem cells. These data demonstrate that there is substantial postnatal turnover of the arcuate neuronal circuitry in the mouse and reveal the unexpected capacity of diet and leptin deficiency to inhibit this neuronal remodeling. This insight has important implications for our understanding of nutritional regulation of energy balance and brain function.

Figure 1. BrdU administration at E10.5 labels embryo-born neurons in the adult hypothalamus.

(A) Coronal sections of ARN at E10.5, showing proliferative neural stem cells (labeled with BrdU [red]) within the hypothalamic neuroepithelium giving rise to energy-balance neurons (POMC⁺ [green] counterstained with DAPI [blue]). (B) By 4 weeks of age, all strongly BrdU-labeled (red) cells within the parenchyma (white arrowheads) had differentiated into neurons (NeuN⁺HuC/D⁺ [green] counterstained with DAPI [blue]). The inset shows a high-magnification view of a labeled ARN neuron. Rare ependymal glia retain strong BrdU label (asterisk) and may represent adult neural stem cells. Scale bar: 50 μm. Original magnification, ×2 (inset).

Figure 2. ARN energy-balance neurons are turned over by ongoing neurogenesis.

(A–C) Nuclei labeled with BrdU (red) at E10.5 remaining in the ARN at 4, 12, and 26 weeks, counterstained with DAPI (blue). (D) Quantification of ARN neuron survival, showing that labeled ARN neurons were lost between 4 and 12 weeks of age but not between 12 and 26 weeks of age. (E–G) POMC neurons (green) labeled with BrdU (red) at E10.5 remaining in the ARN at 4, 12, and 26 weeks. (H) Quantification of POMC neuron survival, showing that labeled POMC neurons were lost between 4 and 12 weeks of age but not between 12 and 26 weeks of age. (I–K) NPY neurons (green) labeled with BrdU (red) at E10.5 remaining in the ARN at 4, 12, and 26 weeks. We made use of NPY-hrGFP mice to reveal NPY neurons. (L) Quantification of NPY neuron survival, showing that labeled NPY neurons were lost between 4 and 12 weeks of age but not between 12 and 26 weeks of age. (M–O) Nuclei labeled with BrdU (red) at E10.5 remaining in the amygdala at 4, 12, and 26 weeks, counterstained with DAPI (blue). (P) Quantification of amygdala neuron survival, showing that labeled amygdala neurons were not lost between 4 and 12 weeks of age or between 12 and 26 weeks of age. (Columns indicate total cell counts as a percentage of cell count at 4 weeks.) *P < 0.05 compared with 4 weeks; ^#P < 0.05 compared with 12 weeks. (Q) Schematic of analysis. Mean ± SEM; n = 10–19 at 4 weeks, 7–11 at 12 weeks, 4–9 at 26 weeks. Scale bar: 100 μm.

Figure 3. DIO inhibits adult hypothalamic neurogenesis.

Sixteen-week-old mice were i.c.v. infused with BrdU for 7 days and harvested 4 weeks later. (A–C) The number of newborn (BrdU-labeled) cells, including neurons, was significantly reduced in the hypothalamus of DIO mice compared with that in lean controls. (D) However, there was no difference in the percentage of cells adopting a neuronal fate between the 2 groups, as indicated by an equal proportion of BrdU-labeled cells adopting a HuC/D⁺ fate. Scale bar: 100 μm. Data are mean ± SEM. n = 5 chow; n = 6 DIO.

Figure 4. Obese mice do not lack hypothalamic stem cells but have a reduced number of actively proliferating cells.

The number of hypothalamic neural stem/progenitor cells in 16-week-old mice was examined using the neurosphere assay. (A and B) Neurospheres derived from DIO mice were of a similar size and appearance as those derived from lean controls. (C) However, DIO mice contain a higher number of hypothalamic neurosphere-forming cells than lean controls (n = 4 mice each; 2,000 cells plated per well). (D) Neurospheres from both DIO mice and lean controls expanded after passage. However, a greater number of secondary neurospheres were formed in DIO mice than in lean controls. (E) Sixteen-week-old mice were i.p. injected with BrdU and harvested 48 hours later. The number of newborn BrdU⁺ cells was significantly decreased in DIO mice compared with that in lean controls, although this decrease appeared mild compared with the loss observed 4 weeks after BrdU administration (see Figure 1). (F) This decrease in BrdU-labeled cells was mirrored by a reduction in Ki67-expressing glia in DIO mice compared with that in lean controls. Data are mean ± SEM. n = 4–7 chow; n = 4–5 DIO. Scale bar: 1 mm.

Figure 5. DIO results in the apoptosis of newly dividing cells.

Proliferating cells in 16-week-old DIO mice and lean controls were cumulatively labeled with 5 i.p. injections of BrdU and assayed for apoptosis 48 hours later using the TUNEL method. (A–C) Unlike lean mice in which no newly divided cells were apoptotic, several newly divided cells were apoptotic in DIO mice, indicating that the failure of hypothalamic neurogenesis is partially due to the apoptosis of newborn cells. (D) There was no difference in the overall rate of apoptosis in the ARN between DIO mice and lean controls. Data are mean ± SEM. n = 5 chow; n = 5 DIO. Scale bar: 100 μm.

Figure 6. Calorie restriction partially restores neurogenesis in DIO mice.

Sixteen-week-old DIO mice were either maintained on HFD or calorie restricted. (A) Four weeks of calorie restriction did not affect the number of hypothalamic neurosphere-forming cells observed with DIO mice. (B) However, calorie restriction restored the proliferation of neuronal progenitor cells in DIO mice. Data are mean ± SEM. n = 3–5 chow; n = 4–5 DIO. ad lib, ad libitum; CR, calorie restriction.

Figure 7. HFD feeding inhibits remodeling of the ARN energy-balance circuit.

ARN neurons were labeled with BrdU during embryogenesis. Mice were divided into a HFD-fed and a chow-fed cohort at 6 weeks, and BrdU-labeled embryonic neurons were assayed after 10 weeks of differential feeding. (A–C) In mice continually fed a chow diet, few labeled ARN neurons (BrdU [red] and DAPI [blue]) remained at 16 weeks, but a significantly greater number of labeled ARN neurons remained in littermates placed on HFD at 6 weeks of age. (D–F) A similar retention of labeled NPY neurons (yellow arrowheads) was found in HFD-fed mice (BrdU [red] and NPY [green]). (G–I) A similar retention of labeled POMC neurons (yellow arrowheads) was found in HFD-fed mice (BrdU [red] and POMC [green]). (J) Schematic of analysis. Mean ± SEM. Scale bar: 100 μm.

Figure 8. Loss of neural stem cells and neurogenesis in mice lacking leptin.

(A–C) Hypothalamic neurogenesis is almost completely abolished in 16-week-old obese ob/ob mice lacking leptin compared with that in lean littermates (WT/WT or WT/ob [WT/?]). (D) However, there was no difference in the percentage of cells adopting a neuronal fate (HuC/D⁺) between the 2 groups. (E–G) The number of neurosphere-generating cells is reduced in the hypothalamus of ob/ob mice compared with that in lean littermates (2,000 cells plated per well). (H) The majority of primary neurosphere-forming cells present in ob/ob mice are not stem cells, as they fail to generate new neurospheres after passage. Mean ± SEM. Scale bar: 1 mm.

Figure 9. Leptin infusion increases the number of neurosphere-forming neural stem/progenitor cells in vivo but does not directly increase neurogenesis acutely.

(A) Sixteen-week-old mice were infused with leptin peripherally for 14 days. Leptin-treated mice contained higher numbers of neurosphere-forming cells than vehicle-infused control mice. (B) Acute central infusion of leptin and BrdU did not alter the rate of hypothalamic neurogenesis compared with that in mice infused with BrdU alone. Mean ± SEM.