Estrogen Drives Brain Melanocortin to Increase Physical Activity in Females (original) (raw)

• Journal List
• HHS Author Manuscripts
• PMC9113400

Nature. Author manuscript; available in PMC 2022 May 17.

Published in final edited form as:

PMCID: PMC9113400

NIHMSID: NIHMS1799531

William C. Krause,1 Ruben Rodriguez,1 Bruno Gegenhuber,2,7 Navneet Matharu,3 Andreas N. Rodriguez,1 Adriana M. Padilla-Roger,4 Kenichi Toma,5 Candice B. Herber,1 Stephanie M. Correa,1,6 Xin Duan,5 Nadav Ahituv,3 Jessica Tollkuhn,7 and Holly A. Ingraham1

William C. Krause

1Department of Cellular and Molecular Pharmacology School of Medicine, University of California, San Francisco, CA 94143.

Ruben Rodriguez

1Department of Cellular and Molecular Pharmacology School of Medicine, University of California, San Francisco, CA 94143.

Bruno Gegenhuber

2Cold Spring Harbor Laboratory School of Biological Sciences, Cold Spring Harbor, NY 11724.

7Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

Navneet Matharu

3Department of Bioengineering and Therapeutic Sciences and Institute for Human Genetics, University of California, San Francisco, CA 94143.

Andreas N. Rodriguez

1Department of Cellular and Molecular Pharmacology School of Medicine, University of California, San Francisco, CA 94143.

Adriana M. Padilla-Roger

4Graduate Program in Neuroscience, UCSF.

Kenichi Toma

5Departments of Ophthalmology and Physiology, University of California San Francisco, San Francisco, CA, 94143

Candice B. Herber

1Department of Cellular and Molecular Pharmacology School of Medicine, University of California, San Francisco, CA 94143.

Stephanie M. Correa

1Department of Cellular and Molecular Pharmacology School of Medicine, University of California, San Francisco, CA 94143.

6Present Address: Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, California 90095, USA.

Xin Duan

5Departments of Ophthalmology and Physiology, University of California San Francisco, San Francisco, CA, 94143

Nadav Ahituv

3Department of Bioengineering and Therapeutic Sciences and Institute for Human Genetics, University of California, San Francisco, CA 94143.

Jessica Tollkuhn

7Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

Holly A. Ingraham

1Department of Cellular and Molecular Pharmacology School of Medicine, University of California, San Francisco, CA 94143.

1Department of Cellular and Molecular Pharmacology School of Medicine, University of California, San Francisco, CA 94143.

2Cold Spring Harbor Laboratory School of Biological Sciences, Cold Spring Harbor, NY 11724.

3Department of Bioengineering and Therapeutic Sciences and Institute for Human Genetics, University of California, San Francisco, CA 94143.

4Graduate Program in Neuroscience, UCSF.

5Departments of Ophthalmology and Physiology, University of California San Francisco, San Francisco, CA, 94143

6Present Address: Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, California 90095, USA.

7Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

Supplementary Materials

Extended Video Legend.

GUID: D07446BE-04B2-45A6-A5AC-7597C76E92F4

Extended Video.

GUID: A38C235A-D970-4DA3-BA1F-D22BB1543F32

Methods/Supplemental Figure Legends.

GUID: C339EF75-C864-40AB-9C1A-3A9036739BF9

Fig S1.

GUID: 7679774D-5BD3-4F81-B7E4-1F531E475422

Fig S3.

GUID: 086A35D7-B7E1-427D-A29D-E5BD418AFD3E

Fig S2.

GUID: D1AA1DE1-0EDA-4EC1-8C75-6689CC94D456

Fig 3_S1.

GUID: B5A5D432-DA3B-49AC-9DF4-CADFC79DAAC4

Fig 3_S2.

GUID: 001BC6B9-961B-4879-9E2B-F6FCE86D3B2D

Fig 3_S3.

GUID: DE8B16AD-2B0C-4BB6-9FCD-47152D4E2699

Fig 4_S1.

GUID: 8D46B87A-789B-4FA3-872E-D93A8E1F938A

Fig 2_S1.

GUID: 27549E51-E438-45A4-B563-9D3588E9C1E8

Fig 4_S2.

GUID: EB30BE64-D233-4EA7-A93C-4D3E6F95FB88

Abstract

Estrogen depletion in rodents and humans leads to inactivity, fat accumulation, and diabetes1,2, underscoring the conserved metabolic benefits of estrogen that inevitably decline with aging. In rodents, the preovulatory surge in 17β-estradiol (E2) temporarily increases energy expenditure to coordinate increased physical activity with peak sexual receptivity. Here we uncover a subset of estrogen-sensitive neurons in the ventrolateral ventromedial hypothalamic nucleus (VMHvl)3–7 that projects to arousal centers in the hippocampus and hindbrain and enables estrogen to rebalance energy allocation in females. Surges in E2 increase melanocortin-4 receptor (MC4R) signaling in these VMHvl neurons by directly recruiting estrogen receptor alpha (ERα) to the Mc4r gene. Sedentary behavior and obesity in estrogen-depleted females are reversed following chemogenetic stimulation of VMHvlERα/MC4R neurons. Similarly, long-term elevation in physical activity is observed following CRISPR-mediated activation of this node. These data extend the impact of MC4R signaling – the most common cause of monogenic human obesity8 – beyond the regulation of food intake and rationalize reported sex differences in melanocortin signaling, including greater disease severity of MC4R insufficiency in women9. This hormone-dependent node illuminates the power of estrogen during the reproductive cycle in motivating behavior and maintaining an active lifestyle, which are often diminished in postmenopausal women.

Keywords: Physical Activity, ERα, Mc4R, Estrogen, Menopause, Hormone Dependent Neurocircuits, Ventromedial Hypothalamus, CRISPRa

Estrogen Signaling In VMHvl Promotes Activity

To establish that females rely on VMHvl ERα signaling to maximize their spontaneous physical activity, we ablated ERα in the VMHvl or arcuate nucleus (ARC) of adult Esr1 fl/fl female mice using stereotaxic delivery of AAV-Cre-GFP (VMHvlERαKO, ARCERαKO). Control female littermates received similarly targeted AAV-GFP (VMHvlControl or ARCControl). Reduced ambulatory activity was observed in VMHvlERαKO females during the dark (active) cycle that corresponded with a modest increase in body weight, a reduction of Ucp1 in interscapular brown adipose tissue (iBAT), and unchanged food intake (Fig. 1a and Extended Data 1a-e). While we showed previously that ARCERαKO females exhibit a surprisingly high bone mass phenotype4, no changes in activity, body weights, or food intake were noted in this cohort (Fig. 1a and Extended Data 1b). Normal food consumption, particularly in ARCERαKO females, suggests that estrogen’s anorexigenic effects are mediated by extra-ARC sites10 or masked by mouse strains/institutional housing conditions used here. When considered together with other ERα knockout mouse models, our data demonstrate a requirement for ERα in the VMHvl to maximize physical activity levels in adult female mice.

VMHvl neurons are sensitive to estrogen and maintain energy expenditure in adult females.

a, Body weight (*_P_=0.0310), ambulatory activity (**_P_=0.0014), and food intake in VMHvlERαKO (n=10), ARCERαKO (n=12), and control (grey, n=7/5) females. b, ERa and pS6244/47 co-expression (arrows) in proestrus and estrus (representative of 5 mice). c, Number of pS6244/47-labeled VMHvl (***_P_=0.0005) and ARC cells in vehicle (n=4) or EB (n=5) treated females. d, Enrichment of peptide ligand-binding receptors (red) (Benjamini-Hochburg adjusted P<0.05, dashed line). e, VMHvl Mc4r expression in proestrus (n=5), male (♂, n=6, *_P_=0.0189) and estrus (n=5, *_P_=0.0163) mice. f, Mc4r and Esr1 expression in estrus and proestrus (representative of 5 mice). g, CPM-normalized coverage tracks of ERE-containing ERα binding sites (pink boxes) within Mc4r (2/3 biological replicates) and Nmur2 (2/3 biological replicates) loci (MACS2, q < 0.01) in sub-cortical nuclei from vehicle and EB treated gonadectomized mice. Data are mean ± SEM, scatter, or box plots (whiskers indicate minimum and maximum values, edges of box are 25th and 75th percentiles, and center line indicates mean). a, c, unpaired 2-tailed t Test; a, RM 2-way ANOVA; e, 1-way ANOVA. Holm-Šidák multiple comparisons.

Hormone responsiveness of VMHvlERα neurons was visualized across the estrous cycle by monitoring phosphorylated ribosomal protein S6 (pS6) during estrus (low E2) and proestrus (high E2). VMHvl pS6 signals rise substantially during proestrus or following an estradiol benzoate (EB) injection into ovariectomized (OVX) females (Fig. 1b, ​c and Extended Data 2a), but were negligible during estrus, in females lacking ERα, or in intact, untreated males (Extended Data 2b,c), underscoring a complete dependence of this pS6 response on both estrogen and ERα. Estrogen induction of pS6 in VMHvlERα neurons occurs via a classical genomic mechanism that begins slowly starting 2 hrs post-treatment. By contrast, no hormone-dependent pS6 induction was detected in adjacent ARCERα neurons (Fig. 1c and Extended Data 2a). That VMHvlERα neurons respond highly to estrogen, but not to fasting11, suggests that fluctuating hormones rather than hunger engage these neurons, setting the stage for behavioral changes across the reproductive cycle.

MC4R Levels Controlled by Estrogen

Candidate mediators of ERα signaling were identified after profiling the VMHvl transcriptome in OVX mice treated with vehicle or EB (Fig. 1d). Among differentially expressed genes we noted enrichment of peptidergic G-protein coupled receptors, Mc4r, Nmur2, Npy1r, and Ghsr, and known estrogen-dependent genes (Greb1, Pgr4), some of which are linked to locomotor activity (MP:0003313, adjusted _P_= 3.19E-4), (Extended Data 2d,e). We focused on Mc4r given its expression in the VMH12, its role in locomotor behavior13,14, and observed sex differences in Mc4r loss-of-function mutations in mice13,15 and humans9,16. Mc4r was induced in females during proestrus (P) but not in estrus (E) nor in intact males (Fig. 1e). We confirmed increased Mc4r expression in VMHvl neurons during proestrus or after EB treatment that colocalized with ERα (Esr1) and Rprm, a VMHvl female-specific marker7 (Fig. 1f and Extended Data 2f-i).

We further established that Mc4r is a direct transcriptional target of ERα using CUT&RUN (Cleavage Under Targets and Release Using Nuclease), a technique that detects transient in vivo binding events within heterogenous tissues17. As expected, hormone-dependent ERα-chromatin interactions were detected in Greb1 and Pgr (Extended Data 3a,b). High sensitivity afforded by CUT&RUN enabled detection of two conserved ERα binding sites within the Mc4r locus (Fig. 1g, and Extended Data 3c,d). The first, located - 210kb downstream of the transcript, contains a canonical estrogen response element (ERE) consensus sequence. The second, in the proximal promoter, consists of an ERE half-site and a site for the trans-acting transcription factor 1 (Sp1) that together coordinate estrogen-dependent global regulation of ERα target genes18. An ERE was detected +200kb upstream of Nmur2, consistent with its upregulation during proestrus (Fig. 1g and Extended Data 2e, 3e). These data establish a direct molecular link between ERα and MC4R and imply that estrogen dynamically regulates the responsiveness of VMHvlERα neurons to neuropeptides.

VMHvlMc4R Neurons Project to Arousal Centers

ERα and MC4R coexpression, assessed using a Cre-dependent reporter crossed with Mc4r-t2a-Cre (Ai14_Mc4r_), revealed that VMHvlERα/MC4R neurons are a subset of the VMHvlERα population (Fig. 2a). This near-perfect concordance of Ai14_Mc4r_ and ERα and stage-dependent Mc4r induction was not detected in the medial amygdala (MeA) (Fig. 2a and Extended Data 4a,b). ERα was undetected in the paraventricular hypothalamus (PVH), a primary site that couples MC4R with food intake (Fig. 2a).

VMHvlMC4R neurons are molecularly and anatomically distinct subset of VMHvlERα neurons.

a, ERα and Ai14 expression and quantification in the PVH (n=4), VMHvl (n=9, ****P<0.0001), and MeA (n=4, **P_=0.0057) of Ai14_Mc4r female mice. b, Labeling vector and map of major VMHvlMC4R projections. c, VMHvl projections to anterior (upper row) and posterior (lower row) regions. Images representative of bilateral VMHvl targeting (n=3 mice, scale bars=200μm). d, Semi-quantitative comparison of VMHvlMC4R and VMHvlERα projection19 intensities. Anatomical abbreviations in Extended Data Table 1. See Fig. 1c legend for box plot description. a, 1-way ANOVA Holm-Šidák multiple comparisons.

We then asked how afferent VMHvlMC4R neuron projections, labeled by Cre-dependent, membrane-targeted YFP (mYFP), compared to the broader VMHvlERα population19 (Fig. 2b). Overall, while many (~84%) of the same major projections reported for VMHvlERα neurons were identified, robust targeting to the ARC and medial amygdala (MeA) was not observed (Fig. 2c,​d Cluster I and Extended Data 4c,d). Unexpectedly, VMHvlMC4R neurons projected to the dorsal CA1 (CA1d) and the adjacent subiculum (SUBd) (Fig. 2c,​d Cluster II), a hippocampal region controlling locomotor speed in mice20 and containing “speed cells” whose firing rate correlates with velocity21. Expected projections from VMHvlMC4R neurons to the midbrain pre-motor periaqueductal grey (PAG) region showed a unique pattern restricted to the lateral and dorsolateral PAG columns (PAGdl/l) associated with escape behaviors22, while conspicuously avoiding the ventrolateral PAG (PAGvl), involved in freezing and defensive behaviors23 (Extended Data 4e). VMHvlMC4R neurons also projected to the hindbrain pontine region containing a cluster of nuclei that mediate sexual receptivity and locomotor arousal24,25.

Activating VMHvlMc4R Node Offsets Estrogen Loss

To assess the functional output of VMHvlERα/MC4R neurons we stimulated this population using Cre-dependent DREADDs (Designer Receptors Exclusively Activated by Designer Drugs, AAV-DIO-hM3Dq-mCherry) injected bilaterally into the VMHvl of Mc4r-t2a-Cre and control littermates (Fig. 3a). Administration of clozapine-n-oxide (CNO) during the inactive period (Light) significantly increased spontaneous physical activity in female and male VMHvlMC4R::hM3Dq mice, but not in VMHvlCre- controls (Fig. 3b and Extended Data 5a). Responses to a single injection of CNO lasted approximately five hours in VMHvlMC4R::hM3Dq mice, with the distance traveled jumping by 700% concomitant with a precipitous drop in immobile behavior (Extended Data 5b).

VMHvlMC4R neurons control physical activity levels and when stimulated reverse inactivity and hypometabolism in obese OVX females.

a, ERα and mCherry in VMHvlMC4R::hM3Dq female. b, Spontaneous activity in VMHvlMC4R::hM3Dq (female=5, ****P<0.0001, male=5, ****P<0.0001) and VMHvlCre- (female=5, male=4) mice ± CNO injection. c, Thermography of VMHvlCre- (left) and VMHvlMC4R::hM3Dq (right) females and iBAT surface temperatures 30 and 45 min after Sal, CNO, or CL injection (n=4/5 VMHvlCre- P<0.0001, VMHvlMC4R::hM3Dq _P_=0.0003). d, Body weight normalized food consumption in females (n=5/5) following Sal/CNO injection during light period (ZT4–9). e, 24-hour weight change in females (n=5/5) administered drinking water (H2O) or CNO-water (CNO, ****P<0.0001). f, g, Dark period (ZT12–24) activity in VMHvlMC4R::hM4Di (n=8) and VMHvlCre- (n=4) females administered water or DCZ-water. ****P<0.0001, **_P_=0.0068, *_P_=0.0213. h, Activity levels in intact (n=16), OVX (n=12, ****P<0.0001), and OVX+CNO VMHvlMC4R::hM3Dq (n=5, **_P_=0.0023) mice. i, j, Body weight and gonadal white adipocyte area following 8-day CNO treatment of OVX/HFD mice (n=5/5). Data are mean ± SEM, scatter, or box plots (see Fig. 1c legend). b, c, d, e, f, h, RM 2-way ANOVA; g, unpaired 2-tailed t Tests; h, 1-way ANOVA; i, nested t Test. Holm-Šidák multiple comparisons as appropriate.

Aside from increased movement, other metabolic functions were insensitive to VMHvlMC4R neuron stimulation. For example, compared to β−3 adrenergic agonist, CL (CL-316–243), CNO failed to elevate iBAT temperature or Ucp1 in VMHvlMC4R::hM3Dq mice (Fig. 3c and Extended Data 5c,d). Glucose homeostasis was unchanged in CNO-treated VMHvlMC4R::hM3Dq mice; albeit the higher body weights inherent to the Mc4r-t2a-Cre line increased fasting glucose (Extended Data 5e,f). Food intake was unaffected by stimulation during the light period and decreased modestly during the dark period (Fig. 3d and Extended Data 5g). Providing CNO in the drinking water over 24 hours led to a nearly 10% drop in body weight in VMHvlMC4R::hM3Dq females with a corresponding increase in activity (Fig. 3e, Supplementary Video, and Extended Data 5h-j) and resulted in a 13% drop in body weight when extended over eight days (Extended Data 5k). Conversely, targeting the VMHvl with inhibitory DREADDs (AAV-DIO-hM4Di-mCherry) increased sedentary behavior during the dark period following administration of the DREADD ligand, deschloroclozapine (DCZ) (Fig. 3f,​g and Extended Data 5l,m). Thus, the marked changes in physical activity following chemogenetic or genetic manipulation of VMHvlERα/MC4R cells imply that this neuron cluster is an essential generator for maximal physical activity in female mice and constitutes a potent node for promoting physical activity, which can be artificially engaged in both sexes.

Increased sedentary behavior and metabolic decline are hallmarks of declining estrogen during aging. We asked if DREADD-activation of VMHvlMC4R neurons overrides these deleterious features in estrogen-depleted OVX female mice. Stimulating VMHvlMC4R neurons over a short 24 hr period fully restored physical activity parameters and promoted significant weight loss in OVX females (Fig. 3h and Extended Data 6a,b). Stimulating this node in OVX females challenged with a high-fat diet (HFD) (Extended Data 6c) reversed the overt metabolic impairment due to estrogen depletion and chronic overnutrition. Fasting glucose and insulin tolerance improved notably after a single bout of CNO-induced activity in HFD-fed VMHvlMC4R::hM3Dq females (Extended Data 6d-f). Further, chronic stimulation of VMHvlMC4R neurons in obese, sedentary OVX females resulted in a rapid, dramatic weight loss, accompanied by lowered fasting blood glucose, a drop in cellular adiposity of gonadal fat, and reduced plasma cholesterol (Fig. 3i,​j and Extended Data 6g-i), all markers of improved metabolic health. Food intake was unaffected (Extended Data 6j). Hence, engagement of the VMHvlMC4R activity node reduces body weight in OVX females and improves metabolic health in the face of a dietary challenge and estrogen depletion.

MC4R Gene Editing in VMHvl Drives Activity Long-Term

To determine whether melanocortin signaling itself regulates this VMHvl activity node, we initially confirmed that MT-II, a synthetic MC4R agonist, evoked cFOS expression in female mice pretreated with EB but not with vehicle (Extended data 7a,b). We next used the Cre-dependent Mc4r loxTB allele26 in combination with the Sf1-Cre transgene, which only overlaps with Mc4r expression in the VMH (Extended data 7c) to restore Mc4r in the VMH of otherwise null mice (Mc4r Sf1-Cre). In response to EB, this rescue approach increased Mc4r expression in VMHvl_Esr1_ neurons, similar to wild type (Mc4r+/+) females (Fig. 4a). Body weights were equivalent at weaning (Extended Data 7d). Consistent with loss of PVH MC4R signaling, null Mc4r loxTB and rescued Mc4r Sf1-Cre females developed obesity, hyperphagia, and increased body-lengths26 compared to control littermates. However, restoring Mc4r in the VMHvl attenuated overt weight gain and sedentary behavior in female but not male Mc4r Sf1-Cre mice (Fig. 4b,​e and Extended Data 7d-g). Thus, our data solidify the role of MC4R signaling in the female VMHvl for promoting spontaneous activity.

Sex-specific role for MC4R signaling in the VMHvl can be bypassed using CRISPR-mediated activation.

a, Esr1 and Mc4r expression in Mc4r+/+, Mc4r loxTB, and Mc4r Sf1-Cre females. b, Body weights in 8-week-old female (**P_=0.0026) and male Mc4r+/+, Mc4r loxTB, and Mc4r Sf1-Cre mice. c, Food intake in female cohorts. d, Body length in female cohorts. e, Light and dark period activity in females (****P<0.0001, *P_=0.0153). e, CRISPRa_Mc4r targets Mc4r promotor. f, Esr1 and Mc4r expression in control and CRISPRa_MC4R female and male mice. g, Home-cage activity in CRISPRa_Mc4r_ (n=6) and control (n=5) female mice. h, Distances for three most active runs from CRISPRa_Mc4r_ and control female (n=6/5, ****P<0.0001) and male (n=4/3) mice. i, Cortical bone volume fraction for female mice 4 months post-infection (_P_=0.0129). j, VMHvlERα/MC4R neurons integrates estrogen and melanocortin signaling to generate a specialized hormone-dependent activity node in females. Data are mean ± SEM or box plots (see Fig. 1c legend). b, c, 1-way ANOVA; d, h, RM 2-way ANOVA; Holm-Šidák multiple comparisons. i, unpaired 2-tailed t Tests.

To verify that MC4R signaling is an integral component of the hormone-responsive VMHvl activity node, CRISPR-mediated activation (CRISPRa) was employed to increase Mc4r expression. Previously, in haploinsufficient Mc4r+/− mice, gene dosage and energy imbalance were normalized by CRISPRa targeting the PVH27. Here, wild type female and male mice were stereotaxically injected with a dual vector system containing guide RNA targeting the Mc4r promoter ERE half-site (AAV-Mc4r-Pr-sgRNA) and dCas9 tethered to the VP64 transcriptional activator (AAV-dCas9-VP64) to selectively upregulate Mc4r expression in the VMHvl (Fig. 4f). Control mice received dCas9-VP64 without sgRNA. Delivery of Mc4r-CRISPRa-viral vectors to the VMHvl was confirmed post-mortem and revealed moderate but long-lived induction of Mc4r in both sexes (Fig. 4g and Extended Data 8a-c). CRISPRa_Mc4r females traveled twice the distance in the dark compared to controls, with increased movement persisting for at least 17 weeks post-injection (Fig. 4h,​i). Activity in CRISPRa_Mc4r males also increased, and in both sexes the drop in sedentary behavior in CRISPRa_Mc4r mice was restricted to nighttime, thus preserving regular diurnal activity patterns (Fig. 4i and Extended Data 8d,e). The lack of weight loss in female CRISPRa_Mc4r mice may reflect the modest but significant increase in daily food intake in females. BAT activity was unchanged (Extended Data 8f-h). Weeks of elevated physical activity (and mechanical loading) in CRISPRa_Mc4r_ females, increased cortical bone thickness and bone volume (Fig. 4j and Extended Data 8i). Under these conditions, CRISPRa_Mc4r_ failed to restore normal activity in OVX females (Extended Data 8j-l). Hence, sidestepping ERα and directly increasing Mc4r dosage in the VMHvl permanently increases spontaneous activity behavior in both sexes.

Concluding Remarks

Here, we identify an estrogen sensitive VMHvlERα/MC4R node that maximizes daily patterns of spontaneous physical activity in female mice. MC4R is an essential intermediary component coupling estrogen and energy expenditure as a direct ERα transcriptional target (Fig. 4k). Thus, as Mc4r expression increases during the pre-ovulatory period, sensitivity to melanocortin rises in the VMHvl, resulting in spikes of estrogen-dependent activity first described in 1924ref28, thus illuminating how estrogen drives behavioral outputs during a critical point in the reproductive cycle.

As human gain-of-function MC4R variants reduce receptor turnover and protect against weight gain29, identifying endogenous signals that modulate MC4R expression becomes of interest. We identify estrogen as a potent inducer of Mc4r expression. The high degree of conservation in consensus ERE binding motifs in the mammalian Mc4r locus suggests that estrogen similarly upregulates human MC4R expression. MC4R agonists that elicit sexual behaviors in estrogen-primed female rodents30 and enhance libido in premenopausal women suffering from hypoactive sexual desire disorder31 may act by directly targeting the VMHvlERα/MC4R node. Once engaged, VMHvlMC4R neurons target CNS regions involved in reproductive behaviors, as well as sites in the hippocampal region that regulate speed and orientation of locomotion20,21, and in hindbrain regions associated with arousal and motor output32. It remains to be determined if these VMHvl outputs contribute to psychiatric disorders (e.g., postpartum depression, premenstrual dysphoric disorder) that coincide with periods of hormonal fluctuations. Conversely, curtailment of MC4R expression following estrogen depletion might underlie the increased sedentary lifestyle associated with menopause33.

Despite the pronounced rise in physical activity in CRISPRa_Mc4r_ females, body weights remained stubbornly constant in the face of small increases in daily food intake. Whereas DREADD activation of VMHvlMC4R neurons reduced body weight rapidly in estrogen depleted OVX females, this rate of weight loss was not sustainable (Extended Data 7g). Collectively, these results reinforce the notion that engagement of adaptive responses limits exercise-induced weight loss34. Nonetheless, decreasing sedentary behavior reduces the risk of metabolic- and age-related co-morbidities, including heart disease, frailty, cancer, and infectious diseases35. As such, the extremely durable increase in spontaneous physical activity achieved by the non-transgenic CRISPRa_Mc4r_ approach provides a unique preclinical model to explore the motivational aspects and health benefits of an active lifestyle. Our findings underscore the benefits of estrogen in minimizing sedentary behavior and provoke further discussion about hormone replacement therapies in postmenopausal women.

Extended Data

Extended Data Table 1:

Brain Region Abbreviations

Extended Data Table 2:

Statistical tests and results

Supplementary Material

Extended Video Legend

Extended Video

Methods/Supplemental Figure Legends

Fig S1

Fig S3

Fig S2

Fig 3_S1

Fig 3_S2

Fig 3_S3

Fig 4_S1

Fig 2_S1

Fig 4_S2

REFERENCES

1. Mauvais-Jarvis F, Clegg DJ & Hevener ALThe role of estrogens in control of energy balance and glucose homeostasis. Endocr Rev 34, 309–338 (2013). [PMC free article] [PubMed] [Google Scholar]

2. Carr MCThe emergence of the metabolic syndrome with menopause. J Clin Endocrinol Metab 88, 2404–2411 (2003). [PubMed] [Google Scholar]

3. Correa SM, et al. An estrogen-responsive module in the ventromedial hypothalamus selectively drives sex-specific activity in females. Cell Rep 10, 62–74 (2015). [PMC free article] [PubMed] [Google Scholar]

4. Herber CB, et al. Estrogen signaling in arcuate Kiss1 neurons suppresses a sex-dependent female circuit promoting dense strong bones. Nat Commun 10, 163 (2019). [PMC free article] [PubMed] [Google Scholar]

5. Martinez de Morentin PB, et al. Estradiol regulates brown adipose tissue thermogenesis via hypothalamic AMPK. Cell Metab 20, 41–53 (2014). [PMC free article] [PubMed] [Google Scholar]

6. Xu Y, et al. Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction. Cell Metab 14, 453–465 (2011). [PMC free article] [PubMed] [Google Scholar]

7. van Veen JE, et al. Hypothalamic estrogen receptor alpha establishes a sexually dimorphic regulatory node of energy expenditure. Nat Metab 2, 351–363 (2020). [PMC free article] [PubMed] [Google Scholar]

8. Farooqi IS, et al. Dominant and recessive inheritance of morbid obesity associated with melanocortin 4 receptor deficiency. J Clin Invest 106, 271–279 (2000). [PMC free article] [PubMed] [Google Scholar]

9. Qi L, Kraft P, Hunter DJ & Hu FBThe common obesity variant near MC4R gene is associated with higher intakes of total energy and dietary fat, weight change and diabetes risk in women. Hum Mol Genet 17, 3502–3508 (2008). [PMC free article] [PubMed] [Google Scholar]

10. Thammacharoen S, Lutz TA, Geary N & Asarian LHindbrain administration of estradiol inhibits feeding and activates estrogen receptor-alpha-expressing cells in the nucleus tractus solitarius of ovariectomized rats. Endocrinology 149, 1609–1617 (2008). [PMC free article] [PubMed] [Google Scholar]

11. Villanueva EC, et al. Complex regulation of mammalian target of rapamycin complex 1 in the basomedial hypothalamus by leptin and nutritional status. Endocrinology 150, 4541–4551 (2009). [PMC free article] [PubMed] [Google Scholar]

12. Mountjoy KG, Mortrud MT, Low MJ, Simerly RB & Cone RDLocalization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 8, 1298–1308 (1994). [PubMed] [Google Scholar]

13. Ste Marie L, Miura GI, Marsh DJ, Yagaloff K & Palmiter RDA metabolic defect promotes obesity in mice lacking melanocortin-4 receptors. Proc Natl Acad Sci U S A 97, 12339–12344 (2000). [PMC free article] [PubMed] [Google Scholar]

14. Chen AS, et al. Role of the melanocortin-4 receptor in metabolic rate and food intake in mice. Transgenic Res 9, 145–154 (2000). [PubMed] [Google Scholar]

15. Huszar D, et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88, 131–141 (1997). [PubMed] [Google Scholar]

16. Sina M, et al. Phenotypes in three pedigrees with autosomal dominant obesity caused by haploinsufficiency mutations in the melanocortin-4 receptor gene. Am J Hum Genet 65, 1501–1507 (1999). [PMC free article] [PubMed] [Google Scholar]

17. Skene PJ, Henikoff JG & Henikoff STargeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc 13, 1006–1019 (2018). [PubMed] [Google Scholar]

18. Lin CY, et al. Whole-genome cartography of estrogen receptor alpha binding sites. PLoS Genet 3, e87 (2007). [PMC free article] [PubMed] [Google Scholar]

19. Lo L, et al. Connectional architecture of a mouse hypothalamic circuit node controlling social behavior. Proc Natl Acad Sci U S A 116, 7503–7512 (2019). [PMC free article] [PubMed] [Google Scholar]

20. Fuhrmann F, et al. Locomotion, Theta Oscillations, and the Speed-Correlated Firing of Hippocampal Neurons Are Controlled by a Medial Septal Glutamatergic Circuit. Neuron 86, 1253–1264 (2015). [PubMed] [Google Scholar]

21. Gois Z & Tort ABLCharacterizing Speed Cells in the Rat Hippocampus. Cell Rep 25, 1872–1884 e1874 (2018). [PubMed] [Google Scholar]

22. Evans DA, et al. A synaptic threshold mechanism for computing escape decisions. Nature 558, 590–594 (2018). [PMC free article] [PubMed] [Google Scholar]

23. Tovote P, et al. Midbrain circuits for defensive behaviour. Nature 534, 206–212 (2016). [PubMed] [Google Scholar]

24. Carter ME, et al. Tuning arousal with optogenetic modulation of locus coeruleus neurons. Nat Neurosci 13, 1526–1533 (2010). [PMC free article] [PubMed] [Google Scholar]

25. Coimbra B, et al. Role of laterodorsal tegmentum projections to nucleus accumbens in reward-related behaviors. Nat Commun 10, 4138 (2019). [PMC free article] [PubMed] [Google Scholar]

26. Balthasar N, et al. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell 123, 493–505 (2005). [PubMed] [Google Scholar]

27. Matharu N, et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science 363(2019). [PMC free article] [PubMed] [Google Scholar]

28. Slonaker JRThe Effect of Pubescence, Oestruatlon and Menopause on the Voluntary Activity in the Albino Rat. Am. J. Physiol 68, 294–315 (1924). [Google Scholar]

29. Lotta LA, et al. Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity. Cell 177, 597–607 e599 (2019). [PMC free article] [PubMed] [Google Scholar]

30. Pfaus JG, Shadiack A, Van Soest T, Tse M & Molinoff PSelective facilitation of sexual solicitation in the female rat by a melanocortin receptor agonist. Proc Natl Acad Sci U S A 101, 10201–10204 (2004). [PMC free article] [PubMed] [Google Scholar]

31. Clayton AH, et al. Bremelanotide for female sexual dysfunctions in premenopausal women: a randomized, placebo-controlled dose-finding trial. Womens Health (Lond) 12, 325–337 (2016). [PMC free article] [PubMed] [Google Scholar]

32. Chandler DJ, et al. Redefining Noradrenergic Neuromodulation of Behavior: Impacts of a Modular Locus Coeruleus Architecture. J Neurosci 39, 8239–8249 (2019). [PMC free article] [PubMed] [Google Scholar]

33. Duval K, et al. Effects of the menopausal transition on energy expenditure: a MONET Group Study. Eur J Clin Nutr 67, 407–411 (2013). [PMC free article] [PubMed] [Google Scholar]

34. O’Neal TJ, Friend DM, Guo J, Hall KD & Kravitz AVIncreases in Physical Activity Result in Diminishing Increments in Daily Energy Expenditure in Mice. Curr Biol 27, 423–430 (2017). [PMC free article] [PubMed] [Google Scholar]

35. Duggal NA, Niemiro G, Harridge SDR, Simpson RJ & Lord JMCan physical activity ameliorate immunosenescence and thereby reduce age-related multi-morbidity? Nat Rev Immunol 19, 563–572 (2019). [PubMed] [Google Scholar]