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. Author manuscript; available in PMC: 2023 Jan 13.
Published in final edited form as: J Endocrinol. 2022 Jan 13;252(3):167–177. doi: 10.1530/JOE-21-0250

Exercise increases NPY/AgRP and TH neuron activity in the hypothalamus of female mice

Taylor Landry 1,2,3, Daniel Shookster 1,2,3, Alec Chaves 1,2,3, Katrina Free 1,2,3, Tony Nguyen 1,2,3, Hu Huang 1,2,3,4
PMCID: PMC9039839  NIHMSID: NIHMS1796581  PMID: 34854381

Abstract

Recent evidence identifies a potent role for aerobic exercise to modulate the activity of hypothalamic neurons related to appetite; however, these studies have been primarily performed in male rodents. Since females have markedly different neuronal mechanisms regulating food intake, the current study aimed to determine the effects of acute treadmill exercise on hypothalamic neuron populations involved in regulating appetite in female mice. Mature, untrained female mice were exposed to acute sedentary, low- (10 m/min), moderate- (14 m/min), and high (18 m/min)-intensity treadmill exercise in a randomized crossover design. Mice were fasted 10 h before exercise, and food intake was monitored for 48 h after bouts. Immunohistochemical detection of cFOS was performed 3 h post-exercise to determine the changes in hypothalamic neuropeptide Y (NPY)/agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), tyrosine hydroxylase (TH), and SIM1-expressing neuron activity concurrent with the changes in food intake. Additionally, stains for pSTAT3tyr705 and pERKthr202/tyr204 were performed to detect exercise-mediated changes in intracellular signaling. Briefly, moderate- and high-intensity exercises increased 24-h food intake by 5.9 and 19%, respectively, while low-intensity exercise had no effects. Furthermore, increases in NPY/AgRPARC, SIM1PVN, and TH neuron activity were observed 3 h after high-intensity exercise, with no effects on POMCARC neurons. While no effects of exercise on pERKthr202/tyr204 were observed, pSTAT3tyr705 was elevated specifically in NPY/AgRP neurons 3 h post-exercise. Overall, aerobic exercise increased the activity of several appetite-stimulating neuron populations in the hypothalamus of female mice, which may provide insight into previously reported sexual dimorphisms in post-exercise feeding.

Keywords: exercise, food intake, hypothalamus, POMC neuron, NPY/AgRP neuron, tyrosine hydroxylase, SIM1 neuron, females

Introduction

The effects of exercise on food intake have been studied extensively, yet the reasons for frequently conflicting results are still incompletely understood (Durrant et al. 1982, Staten 1991, King et al. 1996, Pomerleau et al. 2004, Church et al. 2009, Ebrahimi et al. 2013, Hagobian et al. 2013, Douglas et al. 2015). Briefly, human studies have demonstrated exercise to decrease (Hagobian et al. 2013), increase (Staten 1991), or have no effects (Douglas et al. 2015) on food intake, and several reviews have systematically analyzed the factors that may be responsible for these mixed findings (Melzer et al. 2005, Thackray et al. 2016, Dorling et al. 2018). These factors include biological sex (Applegate et al. 1982, Staten 1991, King et al. 1996), BMI (Durrant et al. 1982, Kissileff et al. 1990), exercise intensity (Pomerleau et al. 2004, Church et al. 2009), exercise mode (Durrant et al. 1982, Kissileff et al. 1990), and exercise duration (Staten 1991, Pomerleau et al. 2004). Particularly, the existence of sexual dimorphism in human feeding behavior post-exercise is also debated (Applegate et al. 1982, Staten 1991, King et al. 1996, Ebrahimi et al. 2013, Hagobian et al. 2013), possibly due to menstrual cycle-dependent fluctuations in metabolic hormones in females. Specifically, plasma glucagon-like peptide 1 (GLP-1) and insulin concentrations are lower and peptide YY (PYY) concentrations are higher during the follicular phase compared to the luteal phase, resulting in an increased food intake (Lissner et al. 1988, Brennan et al. 2009).

More consistently in the literature, female humans and rodents are more resistant to weight loss during exercise programs (Ballor & Keesey 1991, Jackson et al. 2018), and rodent studies observe females to eat more after exercise than their male counterparts (Oscai et al. 1971, Nance et al. 1977). This has resulted in the hypothesis that females possess mechanisms that favor weight maintenance, possibly via increased food intake in times of elevated energy expenditure. Physiologically, in both humans and rodents, females exhibit significantly different mechanisms in regulating their appetite, in part, due to lack of exposure to testosterone, which programs neural circuits involved in metabolic regulation at an early age (Nohara et al. 2011). Rodent studies examining the arcuate nucleus (ARC) of the hypothalamus have demonstrated that females have increased satiety-inducing pro-opiomelanocortin (POMCARC) neurons (Nohara et al. 2011), reduced hunger-inducing neuropeptide Y (NPY) expression (Urban et al. 1993), decreased sensitivity to central injection of agouti-related peptide (AgRP) and insulin (Goodin et al. 2008), and improved sensitivity to leptin (Clegg et al. 2003). Estrogen receptor α in the hypothalamus, specifically in POMC and steroidogenic factor-1 neurons, is also critical to negatively regulating food intake and positively regulating energy expenditure (Xu et al. 2011). Moreover, exercise-mediated changes in metabolic hormones differ between sexes, with human females exhibiting augmented acylated ghrelin, as well as reduced PYY and GLP-1 responses to exercise compared to male counterparts (Alajmi et al. 2016, Hazell et al. 2017). Notably, these differences are not consistently observed (Unick et al. 2010, Larson-Meyer et al. 2012, Hallworth et al. 2017). Overall, insight into the sex-specific physiological mechanisms driving variability in post-exercise feeding is lacking and would be valuable to prescribing optimal exercise programs.

Emerging evidence in mice identifies a potent ability for aerobic exercise to modulate the activity of hypothalamic neurons involved in appetite regulation, especially in the ARC (He et al. 2018, Jeong et al. 2018, Bunner et al. 2020, Lieu et al. 2020, Miletta et al. 2020, Landry et al. 2021a). The ARC’s proximity to the cerebrospinal fluid in the third ventricle and less selective blood–brain barrier in the median eminence allows for convenient responsiveness to substrate and hormone changes during exercise; thus, the ARC provides a promising direction for research into physiological mechanisms regulating post-exercise feeding behavior. However, like the effects of exercise on feeding, the effects of exercise on the activity of specific ARC neurons produce mixed results (He et al. 2018, Jeong et al. 2018, Bunner et al. 2020, Landry et al. 2021a).

Activation of NPY/AgRPARC neurons drives food intake (Krashes et al. 2011), and activity of this neuron population is elevated immediately after moderate- (MIE) and high-intensity treadmill exercise (HIE) (Bunner et al. 2020, Miletta et al. 2020, Landry et al. 2021a). NPY/AgRPARC activation occurs regardless of pre-exercise energy status, likely due to their critical role in post-exercise refeeding (Bunner et al. 2020, Landry et al. 2021a). Conversely, POMCARC neurons suppress food intake when activated (Zhan et al. 2013), and their responsiveness to exercise has recently been demonstrated to vary depending on pre-exercise feeding (Landry et al. 2021a). More specifically, POMCARC neurons are activated after fasted HIE but are not affected by fed HIE (Jeong et al. 2018, Bunner et al. 2020, Landry et al. 2021a). Lastly, tyrosine hydroxylase (TH)-expressing neurons in the ARC (THARC) and paraventricular nucleus (THPVN) have recently been shown to be involved in the regulation of food intake and thermogenesis, respectively (Shi et al. 2013, Zhang & van den Pol 2016). While no effects of exercise on THARC neurons have been observed, THPVN neuron activity is elevated 1 h after HIE regardless of pre-exercise energy status (Landry et al. 2021a). To date, the role of THPVN neurons in feeding behavior is unknown.

Contrary to other studies, He and colleagues observed NPY/AgRPARC neuron activity to be suppressed and POMCARC neuron activity to be elevated immediately after fed high-intensity interval training (HIIT) (He et al. 2018). These results may have been a result of using electric shock to motivate the mice to run, since electric shock has been shown to activate satiety neurocircuits in the hypothalamus (Lin et al. 2018) and acute stress stimulates POMCARC-mediated hypophagia (Calvez et al. 2011, Qu et al. 2020). Alternatively, He and colleagues may have identified a unique NPY/AgRPARC neuron-inhibiting effect specific to HIIT (He et al. 2018).

While the aforementioned studies provide valuable information regarding post-exercise changes in hypothalamic activity and subsequent feeding behavior, they focus primarily on male mice and lack sex-specific insight (He et al. 2018, Jeong et al. 2018, Bunner et al. 2020, Landry et al. 2021a). Considering the many sex-specific mechanisms regulating appetite and the drastic variability in studies investigating post-exercise feeding behavior, insight into exercise-mediated changes in hypothalamic neuron activity in female mice is needed. Thus, the current study aimed to utilize a translational, well-controlled, within-subject, treadmill exercise protocol to determine the effects of acute exercise on hypothalamic neuron activity and subsequent feeding behavior in female mice.

Materials and methods

Animals

Female B6.Tg(NPY-hrGFP)1Lowl/J (NPY-GFP reporter; JAX: 006417) mice were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and experimental protocols were approved by the Institutional Animal Care and Use Committee of East Carolina University. Mice were fed standard chow ad libitum (LabDiet Catalog Number 5P76) (3.16 kcal/g; 26% protein, 14% fat, 60% carbohydrate) and housed at 20–22°C with a 12 h light:12 h darkness cycle. Eight-week-old, single-housed mice were used for all experiments, and littermates were used as controls for between-mouse comparisons.

Acute treadmill exercise

Exercise protocols were adapted from previous studies performed in our lab (Bunner et al. 2020), and all mice were able to complete all exercise trials. Briefly, using a randomized crossover design and commonly used treadmill speeds, untrained mice performed 1-h sedentary, low- (10 m/min) (Bobinski et al. 2018), moderate- (14 m/min) (Kim et al. 2019), and high-intensity (18 m/min) (Jeong et al. 2018) exercise with 1 week between bouts. On the day before the experiments, mice were familiarized by running for 5 min at 5 m/min followed by 5 min at 10 m/min. On the day of exercise trials, mice ran at 5 m/min for 2 min, 10 m/min for 2 min, and then, when applicable, the speed was increased to 4 m/min every 2 min until the target speed was reached. To minimize stress, a soft bristle brush or gentle puff of air was used to motivate the mice, when needed. During sedentary trials, mice were placed in empty cages on top of the running treadmill for 1 h to simulate stress during exercise bouts. All bouts were performed between 18:30 and 19:30 h, immediately before the dark phase (lights off at 19:30 h and on at 7:30 h).

Food intake and body weight measurements

Eleven mice were used for food intake experiments. To prevent confounding post-prandial effects, mice were fasted by removing the food from cages 10 h before exercise. Immediately after sedentary and exercise bouts, food was weighed and added to individually housed cages (14–15 g). Measurements were made at 1, 2, 3, 6, 12, 24, and 48 h post-exercise by subtracting the remaining food from the total food. Bedding was inspected thoroughly for residual bits of food, which were included in measurements. All food intakes were normalized to body weight, which was measured immediately before bouts.

Immunohistochemistry

Three hours post-exercise, when differences in food intake were first observed, 4–6 mice/group were intracardially perfused with PBS followed by 10% formalin. Brains were incubated at 4°C in 10% formalin for 24 h before being stored in 30% sucrose until slicing into 20-μm coronal sections using a freezing microtome (VT1000 S; Leica). Slices were stored in anti-freeze solution at −20°C before immunohistochemistry was performed as described previously (Landry et al. 2021b). Briefly, brains were incubated at room temperature overnight in antibody to cFOS (goat; 1:250; Santa Cruz Biotechnology), POMC (rabbit; 1:6000; Phoenix Pharmaceuticals, Burlingame, CA, USA), TH (rabbit; 1:100,000; Millipore), phosphorylated ERKthr202/tyr204 (rabbit; 1:500; Cell Signaling Technology), and/or SIM1 (rabbit; 1:250; Millipore) followed by room temperature incubation with Alexa-fluorophore secondary antibody for 1 h (Abcam). To probe two proteins with samesource antibodies, 3,3′-diaminobenzidine (DAB) staining was performed prior to the immunohistochemistry protocols described above (Bunner et al. 2020). Briefly, brain sections were incubated overnight in antibody to phosphorylated STAT3tyr705 (rabbit; 1:500; Cell Signaling Technology) followed by biotinylated donkey anti-rabbit IgG (Vector; 1:1000) for 2 h. Sections were then incubated in the avidin–biotin complex (Vector Elite Kit, VectorLabs, Burlingam, CA, USA; 1:500) and incubated in 0.04% DAB, 0.02% cobalt chloride (Fisher Scientific), and 0.01% hydrogen peroxide. Note, pSTAT3tyr705 images were inverted (to white) for colocalization. All stains were photographed using an optical microscope (DM6000; Leica), followed by both blind manual analysis and automated colocalization analysis using ImageJ. The mean of at least three anatomically matched images per mouse was used for statistical analysis.

Statistical analysis

For time course food intake experiments, differences among sedentary or exercise bouts were determined using two-way ANOVA with repeated measures for time and treatment. Bonferroni corrections were used for multiple comparisons. For comparisons in neuronal activity between sedentary and high-intensity exercise, Student’s t-tests were used. Statistical outliers were determined as >2 s.d. from the mean; however, no outliers were identified. All analyses were performed using GraphPad Prism statistics software, and P < 0.05 was considered statistically significant.

Data availability statement

The data sets generated during the current study are available from the corresponding author on reasonable request.

Results

Moderate- and high-intensity treadmill exercise increase food intake in female mice

To investigate the effects of varying aerobic exercise intensities on post-exercise food intake, 11 fasted female mice underwent acute sedentary, LIE, MIE, and HIE bouts for 1 h on the treadmill. There were no effects of LIE on food intake; however, MIE resulted in increased cumulative 12- and 24-h food intake compared to sedentary trials (10.1 and 5.9%, respectively) (Fig. 1A and B). HIE elicited even greater increases in 12- and 24-h food intake (21.5 and 19.0%, respectively) (Fig. 1A and B), which trended to persist for at least 48 h (P = 0.12) (Fig. 1C). These increases in food intake in response to MIE and HIE occurred primarily 6–12 h post-exercise (Fig. 1D), highlighting the need for investigation into the effects of exercise on CNS-mediated control of food intake in female mice.

Figure 1. Acute moderate and high intensity treadmill exercise increases food intake in female mice.

Figure 1.

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(A) Timeline of cumulative food intake, (B) 24-hour cumulative food intake, (C) 48-hour cumulative food intake, and (D) Food intake by time intervals in fasted female mice in response to different acute treadmill exercise intensities (n=11). Data represented as mean ± SEM. * indicates p<0.05 high intensity vs. sedentary; $ indicates p<0.05 moderate intensity vs. sedentary.

High-intensity treadmill exercise increases the activity of NPY/AgRPARC and SIM1PVN neurons in female mice

Since HIE elicited the most robust effects on food intake in female mice, brains were removed 3 h after HIE, and immunohistochemical detection for cFOS (a marker of neuronal activity) was performed in the hypothalamus to determine the changes in neuronal activity concurrent with the changes in food intake. Compared to sedentary controls, HIE significantly increased cFOS expression in the ARC (2.0-fold) and trended to increase cFOS in the dorsomedial hypothalamus (1.3-fold; P = 0.09) (Fig. 2). Further analysis revealed 4.4-fold higher cFOS colocalization with the hunger-inducing NPY/AgRPARC neuron population 3 h post-exercise (Fig. 3A, B and C) and no effects on the satiety-inducing POMCARC neuron population (Fig. 3D, E and F). Additionally, HIE increased cFOS expression in SIM1PVN neurons (3.0-fold) (Fig. 3G, H and I), which exhibit synaptic connections to NPY/AgRP data suggest that the SIM1PVN→NPY/AgRPARC neurocircuit neurons to regulate food intake (Krashes et al. 2014). These may by activated by HIE to promote increases in food intake.

Figure 2. High intensity treadmill exercise increases neuronal activity in the ARC of female mice.

Figure 2.

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Representative images of cFOS (green) in the hypothalamus of fasted female mice 3 hours after (A) sedentary trials or (B) high intensity treadmill exercise. (C) Total cFOS-expressing cells in each region of the hypothalamus (n=9–10/group) DMH=dorsomedial hypothalamus; VMH=ventromedial hypothalamus; ARC=arcuate nucleus; 3V=third ventricle;. Scale bar = 100um. Data represented as mean ± SEM. * indicates p<0.05 vs. sedentary trials.

Figure 3. NPY/AgRPARC and SIM1PVN neuron activity is elevated 3 hours after high intensity treadmill exercise in female mice.

Figure 3.

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(A) Representative images of cFOS (magenta) colocalized with NPY/AgRPARC neurons (green) in fasted female mice 3 hours after sedentary trials or high intensity treadmill exercise. (B) Total and cFOS-expressing NPY/AgRPARC neurons and (C) Percent active NPY/AgRPARC neurons (n=5–6/group). (D) Representative images of cFOS (green) colocalized with POMCARC neurons (magenta) in fasted female mice 3 hours after sedentary trials or high intensity treadmill exercise. (E) Total and cFOS-expressing POMCARC neurons and (I) Percent active POMCARC neurons (n=5–6/group). (G) Representative images of cFOS (magenta) colocalized with SIM1PVN neurons (green) in fasted female mice 3 hours after sedentary trials or high intensity treadmill exercise. (H) Total and cFOS-expressing SIM1PVN neurons and (I) Percent active SIM1PVN neurons (n=4–5/group). 3V = Third ventricle; Scale bar = 50um. White arrows indicate examples of colocalization. Data represented as mean ± SEM. * indicates p<0.05 vs. sedentary trials.

High-intensity treadmill exercise increases the activity of TH neurons in female mice

Recent evidence identifies novel roles for hypothalamic TH neurons to regulate food intake and energy expenditure (Shi et al. 2013, Zhang & van den Pol 2016). We observed both THARC and THPVN neuron activity to be elevated 3 h post-exercise by 2.2- and 3.7-fold, respectively (Fig. 4). Considering THARC activation promotes food intake, these data may identify THARC neurons as additional neuronal mediators of post-exercise increases in food intake in female mice. To date, the role of THPVN neuron activity on food intake is unknown.

Figure 4. Tyrosine hydroxylase neuron activity is elevated 3 hours after high intensity treadmill exercise in female mice.

Figure 4.

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(A) Representative images of cFOS (green) colocalized with THARC neurons (magenta) in fasted female mice 3 hours after sedentary trials or high intensity treadmill exercise. (B) Total and cFOS-expressing THARC neurons and (C) Percent active THARC neurons (n=5/group). (D) Representative images of cFOS (green) colocalized with THPVN neurons (magenta) in fasted female mice 3 hours after sedentary trials or high intensity treadmill exercise. (E) Total and cFOS-expressing THPVN neurons and (F) Percent active THPVN neurons (n=4/group). 3V = Third ventricle; Scale bar = 50um. White arrows indicate examples of colocalization. Data represented as mean ± SEM. * indicates p<0.05 vs. sedentary trials.

High-intensity treadmill exercise increases ARC pSTAT3tyr705 in female mice

To investigate the intracellular changes concurrent with exercise-mediated increases in NPY/AgRPARC and THARC activity, immunohistochemical detection of pSTAT3tyr705 and pERKthr202/tyr204 was performed. pSTAT3tyr705 and pERKthr202/tyr204 are downstream mediators of anorexigenic hormones leptin and IL6, and they elicit inhibitory signals in NPY/AgRPARC neurons (Rahmouni et al. 2009, Ropelle et al. 2010, Varela & Horvath 2012). While no effects of exercise on pSTAT3tyr705 expression in THARC neurons were observed, pSTAT3tyr705 was surprisingly elevated in NPY/AgRPARC neurons 3 h post-exercise (Fig. 5A, B and C). Furthermore, no effects of exercise on pERKthr202/tyr204 were observed (Fig. 5D and E).

Figure 5. ARC pSTAT3 tyr705 is elevated 3 hours after high intensity treadmill exercise in female mice.

Figure 5.

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(A) Representative inverted DAB images of pSTAT3tyr705 (white) colocalized with NPY/AgRPARC (green) and THARC neurons (magenta) in fasted female mice 3 hours after sedentary trials or high intensity treadmill exercise. (B) Colocalization with NPY/AgRPARC neurons and (C) Colocalization with THARC neurons. (n=5/group). (D) Representative images of pERKthr202/tyr204 (magenta) colocalized with NPY/AgRPARC neurons (green) in fasted female mice 3 hours after sedentary trials or high intensity treadmill exercise. (E) Colocalization with NPY/AgRPARC neurons. (n=5–6/group). White arrows indicate examples of colocalization with NPY/AgRPARC neurons; green arrows indicate examples of colocalization with THARC neurons. 3V = Third ventricle; Scale bar = 50um. Data represented as mean ± SEM.

Discussion

To our knowledge, the current study is the first to investigate the effects of acute treadmill exercise on the activity of hypothalamic neuron populations involved in appetite regulation, specifically in female mice. It was determined that MIE and HIE significantly increased 24-h food intake, while LIE had no effects. Notably, these changes in food intake first occurred 3 h post-exercise and were concurrent with increases in the activity of appetite-stimulating NPY/AgRPARC and THARC neurons. These results may have identified novel sex-specific changes in the hypothalamic neuron activity post-exercise.

Our results demonstrating an absence of compensatory increases in food intake after LIE may suggest that this exercise protocol is optimal for creating caloric deficit in females. On the contrary, observed appetite-stimulating effects of higher intensity treadmill exercise are consistent with past studies in female mice (Oscai et al. 1971, Nance et al. 1977), which is unique compared to the appetite-suppressing effects of HIE in fasted male mice (Jeong et al. 2018, Landry et al. 2021a). This female-specific phenomenon may possibly explain why female rodents are frequently more resistant to weight loss during exercise programs (Ballor & Keesey 1991, Jackson et al. 2018) and has resulted in the hypothesis that females possess mechanisms that favor weight maintenance. These possible mechanisms remain incompletely understood; however, recent studies identifying specific neuron populations in the hypothalamus that respond to exercise and regulate appetite may provide insight into sexually dimorphic feeding behavior (He et al. 2018, Jeong et al. 2018, Bunner et al. 2020, Lieu et al. 2020, Miletta et al. 2020, Landry et al. 2021a).

Emerging evidence identifies a novel ability for acute aerobic exercise to modulate the activity of NPY/AgRPARC, POMCARC, THPVN, and single-minded 1 (SIMPVN) neurons in male mice (He et al. 2018, Jeong et al. 2018, Bunner et al. 2020, Lieu et al. 2020, Miletta et al. 2020, Landry et al. 2021a). For example, POMCARC neurons, which decrease food intake by releasing α-melanocyte-stimulating hormone and activating melanocortin 4 receptors in the paraventricular nucleus (PVN) (Zhan et al. 2013), are activated 1 h after fasted HIE in male mice (Jeong et al. 2018, Landry et al. 2021a). Interestingly, these effects on POMCARC neuron activity were not observed 3 h post-HIE in female mice. This potentially explains why fasted HIE decreases food intake in male mice (Jeong et al. 2018, Landry et al. 2021a), but has opposite effects in females; however, direct comparisons between sexes at the same timepoints are needed to validate this hypothesis. Furthermore, HIE-mediated POMCARC neuron activation and appetite suppression in male mice only occurs in the fasted status, while fed HIE has no effects on POMCARC neurons and increases food intake (Bunner et al. 2020, Landry et al. 2021a). Consequently, it is hypothesized that pre-exercise energy status differentially modulates POMCARC neuron activity and feeding behavior after exercise, at least in male mice; however, it remains to be seen if a similar phenomenon is observed in female mice.

Another novel hypothalamic response to HIE observed in female mice is an increase in THARC neuron activity 3 h post-exercise. THARC neurons increase food intake by co-releasing dopamine and gamma aminobutyric acid (GABA), which directly stimulate and inhibits NPY/AgRP and POMC neurons, respectively (Zhang & van den Pol 2016, Skov et al. 2019). Although investigation into THARC neurons 3 h post-exercise has not been performed in male mice, males do not exhibit any changes in THARC neuron activity 1 h after HIE (Landry et al. 2021a). These data may identify THARC neurons as another potential neuron population mediating exercise-induced hyperphagia specifically in female mice. This hypothesis is supported by past reports demonstrating female rats to have accelerated dopamine release and uptake in other brain regions in response to electrical stimulation compared to males (Walker et al. 2000). Additionally, THPVN neuron activity is elevated after HIE regardless of biological sex or pre-exercise energy status (Landry et al. 2021a). While this neuron population has recently been implicated in the regulation of thermogenesis (Shi et al. 2013), the importance of THARC neuron activation post-exercise and their role in feeding behavior remains unclear.

When making comparisons to studies in male mice, it should be noted that those studies examined exercise-mediated neuronal changes immediately or 1 h after exercise (He et al. 2018, Jeong et al. 2018, Bunner et al. 2020, Miletta et al. 2020, Landry et al. 2021a), while ours investigated 3 h after exercise in female mice, when changes in food intake first occurred. While analyzing only one timepoint is a limitation, the lasting neuronal changes observed in the current study after 3 h are striking. Past reports have demonstrated changes in hypothalamic neuron activity after exercise to be very rapid and transient (He et al. 2018, Lieu et al. 2020, Miletta et al. 2020), and future studies should investigate the sexual dimorphisms in the timelines of hypothalamic neuron responses to exercise.

Both NPY/AgRPARC and SIM1PVN neurons were also activated 3 h post-exercise in female mice, which is consistent with previous observations immediately and 1 h after exercise in males, regardless of pre-exercise feeding status (Bunner et al. 2020, Landry et al. 2021a). The consistent activation of NPY/AgRPARC neurons after exercise, regardless of biological sex or pre-exercise energy status, highlights the critical role of this neuron population in post-exercise refeeding. By co-releasing NPY, AgRP, and GABA, inhibiting satiety-inducing MC4R-expressing neurons in the PVN, and stimulating food intake, NPY/AgRP neurons may be part of an evolutionarily preserved mechanism to ensure adequate refueling post-exercise (Tong et al. 2008, Krashes et al. 2011, Shi et al. 2013). Interestingly, the current role of SIM1PVN neurons in post-exercise feeding behavior is unclear, but activation of this neuron population is also consistent across sex and energy status (Bunner et al. 2020, Landry et al. 2021a). Bunner and colleagues determined that SIM1PVN activation after MIE was independent from presynaptic NPY/AgRP neuron inputs (Bunner et al. 2020); however, recently a SIM1PVN→NPY/AgRPARC neurocircuit promoting food intake was identified (Krashes et al. 2014). It is plausible that this SIM1PVN→NPY/AgRPARC neurocircuit is activated by exercise to ensure homeostatic refeeding.

Overall, the current study observed acute HIE in female mice to increase the activity of NPY/AgRPARC, THARC, THPVN, and SIM1PVN neurons, with no effects on POMCARC neurons. Although direct comparisons to male mice are needed, it seems that increases in hunger-inducing THARC neuron activity after exercise may be specific to female mice, while increases in POMCARC neuron activity may be specific to males (Jeong et al. 2018, Landry et al. 2021a), thus, potentially identifying neuronal mechanisms for sexual dimorphism in post-exercise feeding behavior. However, the molecular mechanisms underlying these sex-specific differences remain incompletely understood. In response to exercise, concentrations of glucose and metabolic hormones like leptin, ghrelin, insulin, and PYY, fluctuate, depending on the exercise intensity and duration (Borghouts & Keizer 2000, Bouassida et al. 2006, Mani et al. 2018, Bunner et al. 2020). Furthermore, human females have been shown to exhibit increased hunger-inducing acylated ghrelin, as well as reduced satiety-inducing PYY and GLP-1 responses to exercise compared to male counterparts (Alajmi et al. 2016, Hazell et al. 2017). These female-specific differences in metabolic hormone response to exercise could directly modulate the activity of hypothalamic neurons, promoting food-seeking behavior (Mayer & Belsham 2009, Varela & Horvath 2012, Qiu et al. 2014, Chen et al. 2017); however, sexual dimorphisms in exercise-mediated changes in these factors are debated (Unick et al. 2010, Larson-Meyer et al. 2012, Hallworth et al. 2017).

Our data indicate that changes in hypothalamic neuron activity post-exercise are independent from changes in intracellular extracellular signal regulated kinase (ERK) signaling, but surprisingly, phosphorylated STAT3 was increased specifically in NPY/AgRPARC neurons 3 h after exercise. The canonical leptin→STAT3 signaling pathway conflicts with the increases in food intake observed after HIE in female mice. Despite the well-established anorexigenic role of leptin→STAT3 signaling, the functions of STAT3 in ARC control of energy balance are complex. For example, STAT3 null mice exhibit normal leptin sensitivity (Münzberg et al. 2007), while constitutively active STAT3 specifically in NPY/AgRPARC neurons increases energy expenditure but has no effects on food intake or AgRP expression (Mesaros et al. 2008). Alternatively, exercise has been shown to increase sensitivity to leptin (Bouassida et al. 2006), and it is possible that leptin→STAT3 signaling is acting as a negative feedback mechanism to prolonged NPY/AgRPARC neuron activation.

In summary, the current study identified a novel ability for exercise to promote the activity of hypothalamic neurons involved in stimulating food intake in female mice. While exercise-mediated reorganizing of appetite-related neurocircuitries has been an emerging focus of investigation, previous studies have focused primarily on males. Our results suggest that exercise-induced hyperphagia in female mice may be due to increases in the activity of hunger-inducing NPY/AgRPARC, SIM1PVN, and THARC neurons in the hypothalamus. These results may provide critical insight into the physiological mechanisms regulating energy balance in females and could have implications when tailoring exercise programs to individual goals.

Acknowledgments

Funding

The funding for this project was provided by East Carolina University start up, the National Institute of Diabetes and Digestive and Kidney Disease (DK121215) to H H.

Footnotes

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Guarantor statement

Dr. Hu Huang is the guarantor of this work and, as such, has full access to all the data in the study and takes responsibility for its integrity and the accuracy of the analysis.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data sets generated during the current study are available from the corresponding author on reasonable request.

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