Remodeling of the arcuate nucleus energy-balance circuit is inhibited in obese mice (original) (raw)
The hypothalamic energy-balance circuit is dynamically remodeled in the adult mouse. We have recently shown that neurogenesis occurs in the adult mouse hypothalamus (19, 21). Since the generation of new neurons is not accompanied by an expansion of the hypothalamus with age, neuronal turnover is likely to occur to maintain the integrity of the tissue. To evaluate the postnatal turnover of neurons in the hypothalamic ARN, we developed a strategy to label neurons born during embryogenesis and then to quantify those remaining in adulthood.
During development, most ARN neurons are generated between E10.5 and E12.5 (22, 23). Energy-balance neurons were the earliest born, with substantial numbers present at E10.5 that express the neuropeptide POMC (Figure 1A) shortly after cell-cycle exit (22, 23). These immature POMC+ neurons do not solely become mature POMC+ neurons but also give rise to mature NPY+ neurons (24). We confirmed our previous findings that embryonic generation of these energy-balance neurons occurred around E10.5 by counting the number of POMC+ immature ARN neurons in developing embryos within the E10.5 gestational window (n = 6). The mean number of POMC neurons present was 1,032 ± 202, ranging from 544 POMC+ neurons in the most immature embryo to 1,780 neurons in the most mature embryo (Figure 1A and data not shown). By E12.5, the number of immature POMC neurons had risen to 3,039 ± 154 POMC+ neurons (n = 6), similar to that present in the adult (22, 25). These data indicate that the E10.5 gestational window coincides with the main phase of energy-balance neuron generation.
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).
To label a sample of energy-balance neurons across this window, FVB female mice were time mated with male mice expressing humanized Renilla green fluorescent protein from the Npy promoter (NPY-hrGFP mice) (FVB background), and the resulting embryos were treated with BrdU at E10.5 to label the neural progenitors giving rise to the ARN neurons (22). At 4 weeks of age, the resulting mice were sacrificed, and BrdU+ cells were revealed by immunofluorescence.
Using WT mice, BrdU+ ARN cells were exclusively neurons, as revealed by their immunoreactivity for the neuronal markers HuC/D+ and NeuN+ (Figure 1B). Next, to quantify adult ARN neuronal turnover, we assayed the replacement of these BrdU+ neurons by new unlabeled neurons between 4 weeks and 12 to 26 weeks of age (Figure 2). Between 4 and 12 weeks, there was no change in the total number of cells within the ARN (DAPI staining in Figure 2, A–D; P = 0.25). However, the number of embryo-born BrdU+ neurons was reduced by 63% (P = 0.0013). There was no further depletion of BrdU+ neurons between 12 and 26 weeks (P = 0.9), although the total number of ARN cells decreased by 23% (P = 0.0003). Similar data was obtained when the number of BrdU+ neurons was calculated as a percentage of ARN cells at each stage (data not shown).
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.
POMC neurons were turned over at a similar rate as those in the ARN as a whole (compare black bars in Figure 2, D and H). The number of BrdU+ POMC neurons was reduced by 55% between 4 and 12 weeks (P = 0.0196), with no further reduction between 12 and 26 weeks (P = 0.2; Figure 2, E–H). Likewise, the number of BrdU+ POMC neurons as a proportion of the total POMC population decreased from 9.9% at 4 weeks to 4.5% at 12 weeks (P = 0.0051) with no further change by 26 weeks (P = 0.4, data not shown).
Using NPY-hrGFP, the turnover of NPY neurons was assessed. Compared with that of POMC neurons, the total population of NPY neurons decreased to a lesser extent, by 34% (P = 0.0059) between 4 weeks and 12 weeks, with no further reduction by 26 weeks (P = 0.6; Figure 2, I–L). Similar to POMC neurons, however, the number of BrdU+ NPY neurons initially decreased by 79% between 4 and 12 weeks (P = 0.0094), with no further depletion between 12 and 26 weeks (P = 0.9). At 4 weeks, BrdU+ NPY neurons represented 4.6% of the total NPY population, while this number decreased to 1.4% by 12 weeks (P = 0.0147), with no further depletion by 26 weeks (P = 0.9; data not shown).
The turnover of ARN neurons between 4 and 12 weeks of age was confirmed in a second cohort of mice using WT C57BL/6 mice, which received 3 BrdU pulses, one each at E10.5, E11.5, and E12.5 (n = 4 each stage, data not shown). Like E10.5 labeled FVB mice, E10.5–E12.5 labeled C57BL/6 mice also showed a loss of labeled ARN neurons, with 51% of labeled neurons remaining at 12 weeks compared with those at 4 weeks (P = 0.0190; data not shown). At 4 weeks, BrdU+ neurons represented 11.9% of the total ARN population decreasing to 6.9% by 12 weeks (P = 0.0184; data not shown). This confirms that remodeling is not limited to the FVB strain or the E10.5 labeling window. Together, these data unexpectedly reveal that more than half of the POMC and NPY neurons present at 4 weeks of age are replaced by ongoing neurogenesis during the following 8 weeks. This represents a substantial and unanticipated remodeling of the energy-balance circuit in adulthood.
Although few forebrain neurons are generated at E10.5 outside the hypothalamus, a distinct population of neural progenitor cells within the amygdala was found to have entered the cell cycle by BrdU staining at E10.5 (data not shown). However, in contrast to that in the ARN, there was no significant variation in the number of BrdU+ amygdala neurons between 4 and 12 weeks (P = 0.3) or 12 and 26 weeks (P = 0.9; Figure 2, M–P), suggesting that postnatal neuronal turnover, as seen in the ARN, is not found uniformly in the brain but only in discrete regions.
DIO leads to decreased hypothalamic neurogenesis in the adult. Our previous results indicate that increasing hypothalamic neurogenesis via CNTF treatment leads to a long-term reduction in body weight (19). We, therefore, then decided to investigate whether DIO has an effect on hypothalamic neurogenesis in the adult mouse.
The generation of new cells in the adult hypothalamus was evaluated by i.c.v. infusion of BrdU in 16-week-old DIO mice compared with that in chow-fed controls (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI43134DS1). Mice were sacrificed 4 weeks after infusion, and BrdU+ cells were then detected by immunofluorescence. The number of newborn BrdU+ cells in adult ARN was substantial in mice fed chow: an average of 66 ± 5 BrdU+ cells were revealed in each 12-μm section. Strikingly, in DIO mice, the number of BrdU+ cells was half of that seen in chow-fed mice (P = 0.0001; Figure 3, A–C). The proportion of BrdU+ cells that differentiated into neurons (revealed by the neuronal marker HuC/D+) was similar between DIO and control mice (Figure 3D). Together, these data indicate that DIO mice have a decrease in the number of newly generated cells in the ARN, including neurons.
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.
DIO leads to the depletion of actively proliferating progenitor-like cells in the hypothalamus. In the adult CNS, neurons are generated from precursor glial cells. A simplified view of the multistep process leading to generation of neurons is that slowly proliferating multipotent stem cells give rise to highly proliferative progenitor cells that have limited capacity for self-renewal (neuroblasts) and ultimately give rise to neurons (for reviews, see refs. 26, 27). Stem-like cells capable of generating multipotent neurospheres ex vivo are present in the hypothalamus (28). Likewise, we have previously shown that highly proliferative progenitor cells are present in the hypothalamus and give rise to neurons (19, 21). Therefore, the decreased neurogenesis observed in the hypothalamus of DIO mice may result from decreased availability of neural stem-like cells and/or of highly proliferative progenitor-like cells.
To investigate the cause of reduced adult neurogenesis in the context of DIO, we first examined the presence of hypothalamic neural stem-like cells in 16-week-old DIO mice using a neurosphere assay (28). The ventricular lining of the hypothalamus was dissociated and grown in serum-free conditions containing EGF and bFGF. Neurospheres derived from DIO mice were slightly larger than those from lean controls (diameter: lean, 0.47 ± 0.02 mm [n = 100] vs. DIO, 0.55 ± 0.02 mm [n = 100]; P = 0.0137; Figure 4, A and B). Unexpectedly, the hypothalamus of DIO mice generated 3.5 times more neurospheres per 2,000 cells plated than that of lean controls (P = 0.0002; Figure 4C). In addition, the number of DIO-derived neurospheres after 1 passage remained higher than that in lean controls (P = 0.0008; Figure 4D). These data indicate that DIO does not result in the loss of hypothalamic neural stem-like cells.
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.
To then evaluate the number of highly proliferative progenitor-like cells, mice on chow or HFD were repeatedly injected with BrdU (i.p.) over a day to cumulatively label cells in S-phase. Mice were sacrificed 48 hours after the first injection. The number of hypothalamic BrdU+ cells was significantly reduced in DIO mice compared with that in lean controls (P = 0.0496; Figure 4E), indicating a lower number of highly proliferative progenitor-like cells in DIO mice. This result was confirmed by immunohistochemistry against the proliferating cell markers Ki67 and proliferating cell nuclear antigen (PCNA) (P = 0.0078 for Ki67, Figure 4F; P = 0.0089 for PCNA, data not shown).
All together, these results indicate that DIO leads to an expansion of the pool of hypothalamic neural stem-like cells while leading to a depletion of the pool of highly proliferative progenitor-like cells. This suggests that DIO inhibits the differentiation of stem-like cells into more proliferative progenitor-like cells and/or impairs the survival of these progenitor-like cells.
DIO selectively increases apoptosis of newly divided cells. Interestingly, the number of hypothalamic BrdU+ cells in DIO mice was only reduced by 28% compared with that in control mice at 48 hours after labeling (Figure 4E), while it was reduced by 50% at 4 weeks after labeling (Figure 3C). This discrepancy suggests that many newborn cells are lost after S-phase in the hypothalamus of DIO mice.
To investigate the loss of these newborn cells, the rate of apoptosis was determined using the TUNEL method at 48 hours after BrdU labeling. In lean controls, no TUNEL+BrdU+ cells could be detected, whereas 8.9% ± 3.3% of BrdU+ cells were TUNEL+ in DIO mice (Figure 5, A–C). Although, the apoptosis of newborn cells was increased, there was no overall difference in the rate of apoptosis in the hypothalamus of DIO mice (lean, 2.1% ± 0.1% vs. DIO, 2.4% ± 0.4%; P = 0.54; Figure 5D). This result indicates that the loss of newborn neurons in DIO mice is due in part to the selectively increased apoptosis of newly divided cells.
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.
A short-term period of calorie restriction rescues DIO-impaired hypothalamic neurogenesis. In order to investigate the permanence of the neurogenic defects seen in DIO mice, 16-week-old DIO mice were calorie restricted to 70% of the normal intake of chow-fed mice. After 4 weeks of restriction, body weight (32.5 ± 0.8 g) had reduced to similar levels as those of age-matched chow-fed mice (34.2 ± 0.6 g; P = 0.1). A second cohort of DIO mice was maintained on ad libitum HFD as a control group (45.3 ± 1.5 g). The availability of hypothalamic neural stem-like cells was examined using the neurosphere assay, while the number of actively proliferating progenitor-like cells was evaluated in vivo with repeated BrdU injections followed by sacrifice 48 hours later, as described above.
After calorie restriction, the number of hypothalamic neurosphere-forming cells (neural stem-like cells) was similar to that obtained with DIO control mice (P = 0.84; Figure 6A). In contrast, the number of actively proliferating progenitor-like cells was increased by 69% after calorie restriction, as measured by in vivo BrdU incorporation (P = 0.0122; Figure 6B).
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.
All together, these results suggest that the loss of highly proliferative progenitor-like cells caused by DIO is a dynamic phenotype that can be rescued by calorie restriction, while the DIO-induced expansion of the hypothalamic stem-like cell pool may be a more long-lasting phenotype. This supports the view that the loss of the newborn progenitor cells occurs via a process that is distinct from that causing an increase in the number of hypothalamic stem-like cells.
DIO alters the dynamic remodeling of the hypothalamic energy-balance circuit and leads to a relative ageing of the neuronal population. Since hypothalamic neurogenesis is altered by DIO with a selective loss of newborn cells, but the overall rate of cell death remains unchanged compared with that of lean controls, we investigated whether the survival of old neurons is increased in DIO to compensate for the reduced generation of new neurons, in order to maintain hypothalamic integrity. C57BL/6 mouse embryos were labeled with BrdU at E10.5 as described above, weaned on chow, and were either given a HFD at 6 weeks of age or kept on chow until sacrifice at 16 weeks of age (Figure 7). The retention of the neurons born during embryogenesis was then examined by BrdU immunofluorescence detection.
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.
At 16 weeks, the number of BrdU+ ARN cells was greater in DIO mice than that in chow-fed controls (Figure 7, A–C). This indicates that DIO reduces the normal postnatal depletion of old ARN neurons. Although the number of BrdU+ neurons was increased, there was no difference in the total number of ARN cells between DIO and control mice (P = 0.13; data not shown), suggesting that the increased retention of old neurons in DIO is balanced by the decreased generation of new neurons. Likewise, the survival of embryo-born, specific energy-balance neurons was enhanced by DIO, with an increase in both the number of BrdU+NPY+ neurons (Figure 7, D–F) and BrdU+POMC+ neurons (Figure 7, G–I) remaining in DIO mice compared with those in lean controls. Together, these results suggest that neurons forming the energy-balance circuit in the hypothalamus of DIO mice are older than those of control mice.
Leptin is required for maintenance of hypothalamic stem cells. To investigate whether the loss of hypothalamic neurogenesis occurs in another model of obesity with distinct molecular etiology, we examined neurogenesis and neural stem cell biology in a genetic model of obesity lacking leptin, the ob/ob mouse.
The generation of new cells in the adult hypothalamus was evaluated by i.c.v. infusion of BrdU in 16-week-old ob/ob mice compared with that in lean controls. Mice were sacrificed 4 weeks after infusion, and BrdU+ cells were then detected by immunofluorescence. The number of newborn BrdU+ cells in adult ARN was severely reduced in ob/ob mice compared with that in WT controls (n = 5 vs. n = 5; P = 0.0005; Figure 8, A–C). The proportion of BrdU+ cells that differentiated into neurons (revealed by the neuronal marker HuC/D+) was similar between ob/ob and lean controls (Figure 8D). This indicates that leptin-deficient mice have a severely reduced neurogenesis, more extensive than that seen in DIO mice.
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.
To investigate the cause of this reduced adult neurogenesis in ob/ob mice, the ventricular lining of the hypothalamus was dissociated and grown in serum-free conditions containing EGF and bFGF. In contrast to findings in the DIO mouse, the hypothalamus of ob/ob mice generated fewer neurosphere-forming cells compared with that of lean controls (P < 0.0001; Figure 8, E–G). Although _ob/ob_-derived neurospheres were of a similar size compared with WT neurospheres, few of the neurosphere-forming cells derived from ob/ob mice were neural stem cells, as they failed to expand after passage (P < 0.0001; Figure 8H). This was confirmed using immunohistochemistry, which showed a widespread loss of the neural stem cell markers GFAP, Vimentin, Nestin, and Sox2 in the hypothalamus of ob/ob mice (data not shown). The loss of hypothalamic stem cells in ob/ob mice could be detected as early as 4 weeks of age (data not shown). These data indicate that leptin is required for postnatal hypothalamic neurogenesis at the level of neural stem cells.
To further explore the ability of leptin to regulate hypothalamic neurogenesis, 8-week-old male C57BL/6 mice were infused with leptin using a subcutaneously implanted osmotic minipump. After 14 days of leptin infusion, mice lost 2.1 ± 0.2 g of body weight (n = 5, P = 0.0016; data not shown). Then, the ventricular lining of the hypothalamus was dissociated and grown in serum-free conditions containing EGF and bFGF. The hypothalamus of leptin-infused mice contained 68% more neurosphere-forming cells per 2,000 cells plated compared with that of vehicle-infused controls (P = 0.0002; Figure 9A). This indicates that leptin is not only required for the maintenance of hypothalamic neural stem cells but is also able to induce their expansion in vivo.
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.
A direct role for leptin in the generation of new neurons in the adult hypothalamus was evaluated by i.c.v. infusion of leptin and BrdU for 7 days in 8-week-old C57BL/6 mice. Mice were sacrificed 4 weeks after infusion, and BrdU+ cells were then detected by immunofluorescence. After 7 days of leptin infusion, mice lost 3.6 ± 0.3 g of body weight (n = 5, P = 0.0013; data not shown). However, the number of newborn BrdU+ cells in adult ARN was unchanged in leptin-infused mice compared with that in mice infused with BrdU alone (n = 5 vehicle vs. n = 4 leptin, P = 0.3204; Figure 9B). Likewise, the proportion of BrdU+ cells that differentiated into neurons (revealed by the neuronal marker HuC/D+) was similar between leptin- and mock-infused mice (P = 0.1199; data not shown). Together, these data suggests that leptin acts primarily at the stem cell level and does not regulate progenitor cell proliferation or specification.