Loss of CNS ERα impairs multiple aspects of energy homeostasis
To determine if CNS ERα is required for body weight control, we crossed mice carrying loxP-flanked ERα alleles (ERαlox/lox
) to the Nestin-Cre transgenic mice. These crosses produced mice lacking ERα in most of brain regions (ERαlox/lox
/Nestin-Cre) and their control littermates (ERαlox/lox
). Using immunohistochemistry, we demonstrated almost complete absence of ERα in the hypothalamus (and other brain regions) in the ERαlox/lox
/Nestin-Cre mice (Supple Fig. 1
Increased body weight, adiposity, and visceral fat distribution
Compared to controls, both male and female ERαlox/lox/Nestin-Cre mice displayed significant increases in body weight (). Further characterizations in female mice revealed that increases in body weight were mainly reflected by increased body fat mass (). We further demonstrated that ERαlox/lox/Nestin-Cre female mice had significantly higher visceral fat distribution (% of the whole body fat), but lower subcutaneous fat distribution (). These data indicate that CNS ERα is required to regulate body weight, adiposity and fat distribution.
Figure 1 CNS ERα regulates energy homeostasis. (A) Weekly body weight in male mice weaned on regular chow (n=8/genotype). (B) Weekly body weight in female mice weaned on regular chow (n=8/genotype). (C) Body composition in 15-week old female mice fed with (more ...) Hyperphagia, and decreased energy expenditure and physical activity
ERαlox/lox/Nestin-Cre mice displayed hyperphagia () and decreased heat production (). The lower energy expenditure may be partly caused by decreased physical activity, as the ambulatory movements and rearing activities in ERαlox/lox/Nestin- Cre mice were significantly reduced (). These results demonstrate that CNS ERα is required to mediate estrogenic effects on feeding, energy expenditure, and physical activity.
Elevated plasma estradiol-17β
Notably, we found that circulating estradiol- 17β was significantly elevated in ERαlox/lox/Nestin-Cre mice (). Our observations that mice lacking ERα only in the CNS develop obesity despite the elevated levels of estradiol- 17β in the circulation suggest that compared to ERα expressed in the peripheral tissues, CNS ERα appears to play more predominant roles in the regulation of energy balance.
Loss of ERα in VMH SF1 neurons affects energy expenditure and fat distribution
The ventromedial hypothalamic nucleus (VMH) is important for regulation of body weight (King, 2006
). We hypothesized that ERα in the VMH is required to mediate the estrogenic effects on energy balance. Because steroidogenic factor-1 (SF1), a transcription factor, is expressed exclusively in the VMH within the brain (Ikeda et al., 1995
), we used a SF1-Cre transgenic mice (line 7) (Dhillon et al., 2006
) to generate mice lacking ERα in the VMH (ERαlox/lox
/SF1-Cre). In control (SF1-Cre/rosa26GFP) mice, 48.3±5.7% of ERαneurons in the VMH co-express SF1 and 12.0±1.9% of SF1 neurons express ERα. In ERαlox/lox
/SF1-Cre/rosa26GFP mice, the majority of ERα was selectively deleted from the VMH SF1 neurons (Supple Fig. 2
). Importantly, we found that the numbers of SF1neurons in ERαlox/lox
/SF1-Cre/rosa26GFP mice and in control mice were comparable (Supple Fig. 2
), suggesting that deletion of ERα did not cause loss of SF1 population in the VMH.
Increased body weight, adiposity, and visceral fat distribution
When fed on regular chow, male ERαlox/lox/SF1-Cre and control (ERαlox/lox) littermates had comparable body weight and body composition (). The chow-fed ERαlox/lox/SF1-Cre females had modest but significant increases in body weight () and trended increases in both fat mass and lean mass (). When fed with HFD, male ERαlox/lox/SF1-Cre and control mice showed comparable body weight () and fat/lean mass (). On the other hand, HFD-fed ERαlox/lox/SF1-Cre females had significant increases in body weight (), which were reflected by increases in both fat mass and lean mass (). Collectively, these results indicate that ERα expressed by SF1 cells is required to maintain body weight homeostasis in females, but not in males.
Figure 2 ERα expressed by SF1 neurons regulates body weight and fat distribution. (A) Weekly body weight in male mice weaned on regular chow (n=6 or 12/genotype). (B) Body composition in 11-week old male mice fed with regular chow (n=6 or 12/genotype). (more ...)
/SF1-Cre females displayed significantly higher visceral fat distribution but lower subcutaneous fat distribution than controls (). Interestingly, 30% of adult ERαlox/lox
/SF1-Cre females had a massive accumulation of gonadal adipose tissue as demonstrated by appearance () and weight (). Although it is unclear why the gonadal fat pads in subsets of mutant mice form this peculiar shape, this phenomenon is consistent with the abdominal obese phenotype. Given that increased visceral fat is commonly associated with impaired glucose homeostasis (Lafontan and Girard, 2008
), we performed glucose tolerance tests, and found that ERαlox/lox
/SF1-Cre females were glucose intolerant (). Collectively, these results indicate that ERα in SF1 neurons is required for the regulation of fat distribution. Loss of ERα in SF1 neurons leads to abdominal obesity, which subsequently causes glucose dysregulation.
We further characterized the white adipose tissue (WAT) in these mutant females. We found that the average adipocyte size of gonadal WAT was larger in ERαlox/lox/SF1-Cre mice (). Importantly, compared to controls, ERαlox/lox/SF1-Cre females stored more energy in the gonadal WAT, demonstrated by significantly increased triglyceride content (normalized by fat weight) (). Consistently, mRNA levels of genes promoting lipogenesis and triglyceride storage, including stearoyl-CoA desaturase-1 (SCD1), lipoprotein lipase (LPL) and acetyl-CoA carboxylase α (ACCα), were significantly elevated in gonadal WAT of ERαlox/lox/SF1-Cre females (). These findings indicate that ERα in SF1 neurons is required for the regulation of energy partitioning in the gonadal WAT. On the other hand, the inguinal WAT of ERαlox/lox/SF1-Cre mice and controls had comparable adipocyte size (), triglyceride content (), and gene expression profile (). No significant difference was observed in the expression of fatty acid synthase (FAS), ERα, and ERβ in all the fat pads ().
Decreased energy expenditure
The increased body weight and adiposity in ERαlox/lox
/SF1-Cre females was not due to differences in energy intake, because young or adult female ERαlox/lox
/SF1-Cre and control mice consumed comparable levels of calories (). In contrast, ERαlox/lox
/SF1-Cre females were hypometabolic, as demonstrated by significant decreases in heat production (). Components of total energy expenditure include energy required for physical activities, basal metabolism and diet-induced thermogenesis (Castaneda et al., 2005
). In particular, ERαlox/lox
/SF1-Cre females showed normal ambulatory movements and rearing activities (), indicating that ERα in SF1 neurons is not required to regulate physical activity. We further assessed the basal metabolic rate by measuring the minimal heat production during the light cycle (Kaiyala et al., 2010
), and found that ERαlox/lox
/SF1-Cre females had significant reductions in basal metabolic rate (). To assessed diet-induced thermogenesis, we monitored energy expenditure in response to a fasting-re-feeding paradigm. The increased heat production in ERαlox/lox
/SF1-Cre mice was significantly reduced (). Thus, our results suggest that ERα expressed by VMH SF1 neurons is required to regulate basal metabolic rate and to mediate appropriate thermogenic responses to feeding.
Figure 3 ERα expressed by SF1 neurons regulates energy expenditure. (A) Daily food intake in 5-week old female mice fed wit regular chow (n=7 or 23/genotype), 11-week old female mice fed with regular chow (n=9/genotype) and in 12-week old female mice fed (more ...) Impaired BAT thermogenesis
Brown adipose tissue (BAT) plays crucial roles in mediating thermogenesis (Dulloo, 2002
). Although the weight of BAT was not different between genotypes (ERαlox/lox
: 86.67±7.68 mg vs ERαlox/lox
/SF1-Cre: 97.86±7.16 mg, P>0.05, N=12 or 15/genotype), histological analyses revealed a large amount of lipid deposition in the ERαlox/lox
/SF1-Cre BAT (). Consistently, UCP1 mRNA levels in the ERαlox/lox
/SF1-Cre BAT were significantly reduced (). In addition, mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ), PPARγ co-activator-1α (PGC-1α) and β3 adrenergic receptor, factors known to stimulate UCP1 expression (Seale et al., 2007
), were significantly lower in ERαlox/lox
/SF1-Cre BAT (). The levels of PRDM16 and ERα in BAT were not altered (). Taken together, these findings suggest that reduced ERα signals in VMH SF1 neurons led to a reduction in thermogenic functions of BAT by suppressing UCP1 expression.
Decreased sympathetic outflow
ERαlox/lox/SF1-Cre females had significantly lower plasma norepinephrine levels than controls; plasma epinephrine levels were not significantly different (). These findings suggest that ERα in VMH SF1 neurons is required to maintain normal central sympathetic outflow to the peripheral tissues. The decreased sympathetic tone in ERαlox/lox/SF1-Cre mice could contribute to decreased BAT thermogenesis and the increased lipid accumulation in gonadal WAT.
Decreased expression of leptin receptors
Messenger RNA of leptin receptors was significantly reduced in the VMH of ERαlox/lox/SF1-Cre females (). These findings support the possibility that ERα signals in SF1 neurons are required to maintain normal leptin sensitivity by regulating transcription of leptin receptors. No significant changes were found in the mRNA levels of SF1, ERβ, thyroid hormone receptor-α (THRα) and growth hormone secretagogue receptor (GHSR, ghrelin receptors) in the VMH of ERαlox/lox/SF1-Cre females ().
Responses in the pituitary, adrenal gland and gonads
In addition to the VMH in the brain, SF1 cells are also found in the pituitary, adrenal gland and gonads (Zhao et al., 2001
). Therefore, ERα may be deleted in these endocrine organs which may potentially confound the metabolic phenotypes. We found that ERα mRNA levels were comparable in these organs from the two genotypes (Supple Fig.3A
). These results suggest that ERα may not be expressed in SF1 cells in these peripheral organs in wildtype mice, and therefore SF1-Cre-induced recombination did not affect ERα expression. Alternatively, the loss of ERα from these peripheral SF1 cells, if any, may be compensated by elevated ERα expressed by non-SF1 cells in the same organs. We further examined the impact of the possible ERα deletion on the functions of these organs. We found that there were no significant changes in the plasma estradiol-17β, corticosterone, T3/T4, progesterone, FSH or LH (Supple Table 1
). No major abnormalities were observed in the morphology of the pituitary and adrenal gland (Supple Fig. 3B
). However, ERαlox/lox
/SF1-Cre ovaries mice showed increased number of antral follicles and lack of corpus luteum (Supple Fig. 3B
), consistent with our findings that ERαlox/lox
/SF1-Cre females were infertile (data not shown). Nevertheless, because the hormones secreted from the pituitary, adrenal gland and ovary were not significantly altered, it is unlikely that the metabolic phenotypes outlined above are due to the possible deletion of ERα in these peripheral organs, although this possibility cannot be fully excluded.
Loss of ERα in POMC neurons directly affects feeding and negative feedback
Pro-opiomelanocortin (POMC) neurons secrete α-melanocyte-stimulating hormone (α-MSH) to reduce food intake and increase energy expenditure (Morton et al., 2006
). Estrogens activate POMC neurons (Gao et al., 2007
; Malyala et al., 2008
). However, the physiological significance of ERα expressed by POMC neurons has not been directly tested. We crossed ERαlox/lox
mice to the POMC-Cre transgenic mice to generate mice lacking ERα in POMC neurons (ERαlox/lox
/POMC-Cre). In control (POMC-Cre/rosa26GFP) mice, 21.5±2.5% of POMC neurons in the ARH and 20.9±4.8% of POMC neurons in the NTS co-expressed ERα (Supple Fig. 4
). In ERαlox/lox
/POMC-Cre/rosa26GFP mice, only 1.9±0.4% of POMC neurons in the ARH and none in the NTS co-expressed ERα, confirming that the majority of ERα was selectively deleted from POMC neurons (Supple Fig. 4
). Importantly, deletion of ERα from POMC neurons did not cause loss of POMC neurons (Supple Fig. 4
Increased body weight
Male ERαlox/lox/POMC-Cre and control (ERαlox/lox) littermates showed comparable body weight (). On the other hand, ERαlox/lox/POMC-Cre females had significant increases in body weight (), mainly reflected by increases in lean mass (). No significant difference were observed in the visceral/subcutaneous fat distribution (), the weight of gonadal fat pads () and the adipocyte size in gonadal and inguinal WAT () between the ERαlox/lox/POMC-Cre females and controls.
Figure 4 Deletion of ERα in POMC neurons leads to increased body weight and lean mass. (A) Weekly body weight in male mice weaned on regular chow (n=33 or 34/genotype). (B) Weekly body weight in female mice weaned on regular chow (n=25 or 32/genotype). (more ...) Hyperphagia and blunted anorexigenic responses to leptin
ERαlox/lox/POMC-Cre females displayed a chronic hyperphagia. This is demonstrated by significant increases in food intake in chow-fed ERαlox/lox/POMC-Cre females at 4 and 6 months of age (). When normalized by lean mass, food intake was significantly increased at 6 months (). We further examined the effects of anorexigenic hormones in these females. While leptin (5 mg/kg, i.p.) significantly reduced food intake in controls, these anorexigenic effects were blunted in ERαlox/lox/POMC-Cre females (). In contrast, anorexia induced by MTII, a melanocortin agonist, was indistinguishable in ERαlox/lox/POMC-Cre and controls (). Collectively, these findings demonstrated that ERα in POMC neurons is required to maintain normal feeding behavior, at least partly through interacting with the anorexigenic leptin signals.
Figure 5 Deletion of ERα in POMC neurons leads to hyperphagic and hypermetabolic phenotypes. (A) Daily food intake in 4-month old and 6-month old female mice fed with regular chow (n=12 or 13/genotype). (B) The same food intake data in (A) presented as (more ...) Increased energy expenditure, sympathetic outflow and plasma estradiol- 17β
Unexpectedly, at libitum ERαlox/lox/POMC-Cre females showed increased heat production (). In addition, the ambulatory movements during the dark cycle were significantly elevated in the ERαlox/lox/POMC-Cre females (), which may at least partly contribute to the increased energy expenditure. The rearing activities were comparable between the two genotypes (). To further confirm the unexpected hypermetabolic phenotypes, we examined the energy expenditure during fasting. Consistent with results from at libitum mice, ERαlox/lox/POMC-Cre mice showed increased heat production during 24 hr fasting (), which led to a significantly greater weight loss (). Interestingly, ERαlox/lox/POMC-Cre females had significantly higher plasma norepinephrine than control mice, while plasma epinephrine levels were comparable (). The elevated sympathetic tone may be the underlying mechanisms for elevated metabolism seen in these ERαlox/lox/POMC-Cre mice.
Although we cannot fully exclude the possibility that the increases in sympathetic outflow and energy expenditure in ERαlox/lox/POMC-Cre females are directly due to ERα deletion from POMC neurons, it is more likely that these phenotypes may result from compensatory actions of ERα in other CNS sites. Supporting this notion, we found that ERαlox/lox/POMC-Cre females have significantly higher plasma estradiol-17β levels (). Presumably, the elevated estradiol- 17β would act on other CNS ERα sites (including SF1 neurons) to increase sympathetic outflow, energy expenditure and physical activity.
Figure 6 Deletion of ERα in POMC neurons leads to fertility phenotypes. (A–B) Plasma estradiol-17β (A) and progesterone (B) at diestrus in 6-month old female mice fed with HFD (n=6 or 8/genotype). (C–D) Relative mRNA levels of FSHβ (more ...) Impaired negative feedback and fertility
Plasma progesterone in ERαlox/lox
/POMC-Cre females trended to increase but failed to reach statistical significance (). Although the basal FSH and LH levels in gonad intact females were not significantly altered (Supple Table 2
), the estrogen-induced suppressions of FSH and LH were blunted in ERαlox/lox
/POMC-Cre females. Specifically, estradiol-17β replacement in OVX control females significantly suppressed expression of FSH and LH subunits in the pituitary, whereas these negative feedback effects were blunted in ERαlox/lox
/POMC-Cre females (). Similar patterns were also observed in plasma FSH and LH (). These results suggest that ERα in POMC neurons is required to mediate the negative feedback regulation. Of note, the plasma FSH/LH levels after OVX appeared to be lower in ERαlox/lox
/POMC-Cre compared to control mice (), while the pituitary mRNA levels of FSH/LH subunits in the same mice were comparable (). This discrepancy suggests that the secretion of FSH/LH or the stability/degradation of plasma FSH/LH may be differently regulated in the OVX ERαlox/lox
In addition, we found that the gonad intact ERαlox/lox
/POMC-Cre females had abnormal estrous cycles. The length of estrous phase was significantly reduced, while the diestrus phase trended to be elongated in ERαlox/lox
/POMC-Cre mice (). No differences were observed in the length of proestrus phase (). Further, female ERαlox/lox
/POMC-Cre mice had impaired reproductive capacity. In particular, only 30% of ERαlox/lox
/POMC-Cre females (6 out of 20) were able to conceive and deliver, while 100% of the age-matched control females gave birth (). In addition, it took significantly longer for the 6 ERαlox/lox
/POMC-Cre dams to conceive than the controls (). The average litter size from these dams was significantly reduced (). No major abnormalities were observed in the morphology of ovaries from ERαlox/lox
/POMC-Cre females (Supple Fig. 5C
). Collectively, these findings indicate that ERα in POMC neurons is required to maintain normal estrous cyclicity and female fertility.
Expression of hypothalamic genes
In an attempt to further characterize the effects of estrogen/ERα on the POMC neurons, we examined the gene expression profile in the hypothalamus of ERαlox/lox/POMC-Cre females. However, there was no significant difference in the expression of POMC, AgRP, NPY, CART, leptin receptors, SF1, GnRH, Kiss1 and GPR54 between ERαlox/lox/POMC-Cre females and controls ().
Responses in pituitary functions
In addition to the POMC neurons in the brain, POMC (ACTH) cells also exist in the pituitary. We found that ERα mRNA was significantly reduced in the ERαlox/lox
/POMC-Cre pituitary (Supple Fig. 5A
), indicating that ERα is also deleted from these pituitary ACTH cells. However, we did not find any significant changes in POMC (ACTH) expression in the pituitary (Supple Fig. 5B
). In addition, plasma levels of corticosterone at either basal or stressed conditions were comparable between ERαlox/lox
/POMC-Cre females and controls (Supple Table 2
). Morphologic analysis did not reveal any alterations in the pituitary (Supple Fig. 5B
). In addition, we did not detect any significant changes in plasma T3/T4 levels (Supple Table 2
). Therefore, it is unlikely that the metabolic and reproductive phenotypes observed in ERαlox/lox
/POMC-Cre females are due to loss of ERα from the pituitary ACTH cells, although we cannot fully rule out this possibility.
Double deletion of ERα from SF1/POMC neurons affects feeding, energy expenditure and fat distribution
Our data indicated that ERα in SF1 neurons is required to regulate energy expenditure and fat distribution, while ERα in POMC neurons is required for the regulation of feeding. To further confirm this model, we generated mice lacking ERα in both SF1 and POMC neurons (ERαlox/lox/SF1-Cre/POMC-Cre) and the control (ERαlox/lox) littermates.
Chow-fed male ERαlox/lox/SF1-Cre/POMC-Cre mice and controls showed comparable body weight and body composition (). Female ERαlox/lox/SF1-Cre/POMC-Cre mice had significantly increased body weight (), which was mainly reflected by increases in lean mass (). Although the whole body fat mass was not different (), the average weight of gonadal fat was significantly increased in ERαlox/lox/SF1-Cre/POMC-Cre females (). Similar to ERαlox/lox/SF1-Cre females, subsets of ERαlox/lox/SF1-Cre/POMC-Cre females displayed the massive gonadal fat expansion (). While 30% of ERαlox/lox/SF1-Cre females developed this phenotype during adulthood (2–3 months), 50% of ERαlox/lox/SF1-Cre/POMC-Cre females developed the same phenotype at earlier ages (as early as 2 weeks of age) (). Further, ERαlox/lox/SF1-Cre/POMC-Cre females were not only hyperphagic (), but also showed decreased heat production (). No significant difference was observed in ambulatory movements and rearing activities in female mice (). Finally, ERαlox/lox/SF1-Cre/POMC-Cre females had significantly lower plasma norepinephrine levels than controls; epinephrine levels were not significantly different (). Collectively, these findings indicate that ERα expressed by SF1 and POMC neurons provides the coordinated control of food intake, energy expenditure and fat distribution.
Figure 7 Deletion of ERα in both SF1 and POMC neurons produces hyperphagia and decreased energy expenditure. (A) Weekly body weight in male mice weaned on regular chow (n=6 or 15/genotype). (B) Body composition in 12-week old male mice fed with regular (more ...)
Cre transgenes do not affect body weight
To rule out the possibility that the Cre transgenes used in the present study may have independently contributed to the metabolic phenotypes, we generated parallel cohorts of transgenic mice carrying only the Nestin-Cre, SF1-Cre or POMC-Cre transgene and their respective wildtype littermates. These mice were produced at the same genetic background as those conditional knock-out mice. No significant change in body weight was observed in these transgenic Cre mice (Supple Fig. 6