Growth Hormone Receptor in Lateral Hypothalamic Neurons Is Required for Increased Food-Seeking Behavior during Food Restriction in Male Mice

Abstract

Growth hormone (GH) action in the brain regulates neuroendocrine axes, energy and glucose homeostasis, and several neurological functions. The lateral hypothalamic area (LHA) contains numerous neurons that respond to a systemic GH injection by expressing the phosphorylated STAT5, a GH receptor (GHR) signaling marker. However, the potential role of GHR signaling in the LHA is unknown. In this study, we demonstrated that ∼70% of orexin- and leptin receptor (LepR)-expressing neurons in the LHA are responsive to GH. Male mice carrying inactivation of the Ghr gene in the LHA were generated via bilateral injections of an adeno-associated virus. In ad libitum-fed mice, GHR ablation in LHA neurons did not significantly change energy and glucose homeostasis. Subsequently, mice were subjected to 5 d of 40% food restriction. Food restriction decreased body weight, energy expenditure, and carbohydrate oxidation. These effects were similarly observed in control and LHA^ΔGHR mice. While food-deprived control mice progressively increased ambulatory/exploratory activity and food-seeking behavior, LHA^ΔGHR mice did not show hyperactivity induced by food restriction. GHR ablation in the LHA reduced the percentage of orexin neurons expressing c-Fos during food restriction. Additionally, an acute GH injection increased the expression of c-Fos in LHA^ORX neurons. Inactivation of Ghr in LepR-expressing cells did not prevent hyperactivity in food-deprived mice, whereas whole-brain Ghr knock-out mice showed reduced ambulatory activity during food restriction. Our findings indicate that GHR signaling in the LHA regulates the activity of orexin neurons and is necessary to increase food-seeking behavior in food-deprived male mice.

Significance Statement

Growth hormone (GH)-deficient patients frequently present problems in appetite, memory, mood, well-being, metabolism, and sleep. The mechanisms behind these alterations are unknown, but neurons in the lateral hypothalamic area (LHA) are involved in the regulation of all these functions. Here, we showed in male mice that orexin neurons in the LHA express GH receptors (GHRs), and GH increases the activity of these cells. Unlike control animals, mice carrying inactivation of GHRs in LHA neurons are unable to increase their ambulatory/exploratory activity when subjected to food restriction, which increases food-seeking behavior. Thus, our study revealed a new neuronal population affected by GH action that can regulate several neurological aspects, including feeding, arousal, reward, and motivated behaviors.

Introduction

Classical studies detected the expression of growth hormone (GH) receptor (GHR) mRNA in neuroendocrine hypothalamic areas, which includes the periventricular nucleus and the arcuate nucleus of the hypothalamus (ARH; Walsh et al., 1990; Burton et al., 1992; Chan et al., 1996; Kamegai et al., 1996; Pellegrini et al., 1996). These findings highlighted the potential of central GH action in regulating the activity of the somatotropic axis via negative feedback loops (Steyn et al., 2016). In the last years, our group and others have used the capacity of a systemic GH injection to induce the phosphorylation of signal transducer and activator of transcription-5 (pSTAT5) to allow a more precise visualization of GH-responsive neurons. Using this approach, additional neuronal populations that exhibit pSTAT5 after an acute GH injection were identified (Cady et al., 2017; Furigo et al., 2017; de Lima et al., 2021b; Quaresma et al., 2021; Wasinski et al., 2021a). Subsequently, several mouse models carrying neuron-specific GHR ablation were generated and provided evidence that GHR signaling in specific neuronal populations regulates food intake (Furigo et al., 2019b; Quaresma et al., 2019; Teixeira et al., 2019; Wasinski et al., 2021b; Dos Santos et al., 2022), glucose homeostasis (Cady et al., 2017; Furigo et al., 2019a; Teixeira et al., 2019; de Lima et al., 2021b), energy expenditure (Furigo et al., 2019b; Stilgenbauer et al., 2023), exercise performance (Pedroso et al., 2021), anxiety-like behavior, and fear memory (Dos Santos et al., 2023). The importance of GHR signaling in hypothalamic neurons for controlling GH secretion was also demonstrated (Wasinski et al., 2020; Chaves et al., 2022; Dos Santos et al., 2022). However, the potential physiological role of GHR signaling in several GH-responsive neuronal populations remains undetermined.

A large number of GH-induced pSTAT5 cells are found in the lateral hypothalamic area (LHA) (Cady et al., 2017; Furigo et al., 2017; de Lima et al., 2021b; Wasinski et al., 2021a). LHA neurons regulate different functions, such as feeding, arousal, sleep–wake cycle, stress, reward, pain perception, autonomic system, and motivated behavior (Stuber and Wise, 2016; Arrigoni et al., 2019). LHA contains several neurochemically defined neurons, including orexin- (ORX) and melanin-concentrating hormone (MCH)-expressing cells (Mickelsen et al., 2017). Previous studies indicated that some GH-induced pSTAT5 cells in the LHA express the leptin receptor (LepR) (Cady et al., 2017; Furigo et al., 2019b). LHA^LepR neurons regulate feeding, body weight, and locomotor activity via projections to the neighboring ORX neurons and mesolimbic dopaminergic system (Leinninger et al., 2009, 2011; Louis et al., 2010). However, a detailed identification of the neuronal populations in the LHA responsive to GH has not been performed.

Notably, GH-deficient patients have neurological and cognitive problems that are possibly associated with the function of LHA neurons because these individuals frequently display alterations in appetite, memory, mood, well-being, metabolism, and sleep (Nyberg, 2000; Nyberg and Hallberg, 2013; Karachaliou et al., 2021). Since the LHA contains numerous GH-responsive neurons (Cady et al., 2017; Furigo et al., 2017; de Lima et al., 2021b; Wasinski et al., 2021a), it is conceivable to hypothesize that reduced GHR signaling in LHA neurons is underlying some of the neurological and cognitive dysfunctions of GH-deficient patients. Thus, the present study aims to investigate possible physiological roles played by GHR expression in LHA neurons. For this purpose, the neurochemical identity of GH-responsive neurons in the LHA was characterized. Furthermore, mice carrying a deletion of the Ghr gene, specifically in the LHA, were generated via bilateral stereotaxic injections of an adeno-associated virus (AAV), and their phenotype was characterized. Additional experiments were performed in mice carrying inactivation of the Ghr gene in the entire brain or LepR-expressing cells. Our findings revealed a novel and essential role of GHR signaling in the LHA, regulating the activity of ORX neurons and food-seeking behavior during food deprivation.

Materials and Methods

Mice

Neurons that express the LepR or corticotropin-releasing hormone (CRH) were visualized by crossing the LepR^Cre (The Jackson Laboratory; RRID: IMSR_JAX:008320) or CRH^Cre strains (RRID: IMSR_JAX:012704) with mice that express a Cre-dependent tdTomato reporter protein (The Jackson Laboratory; RRID: IMSR_JAX: 007909). To induce GHR ablation, we used GHR^flox/flox mice (List et al., 2013) in the stereotaxic surgeries or crossed with the LepR^Cre or Nestin^Cre (RRID: IMSR_JAX:003771) strains until generating, respectively, LepR^ΔGHR and Brain^ΔGHR mice (both homozygous for the loxP-flanked Ghr alleles and carrying the Cre transgene). C57BL/6J mice were used in the experiments to investigate the effects of porcine GH (National Hormone and Pituitary Program) on pSTAT5 or c-Fos expression in the brain. Only adult male mice were used in the experiments. Mice were weaned and genotyped within 3–4 weeks of life. The mutations were confirmed by PCR using the REDExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich). The experiments were performed with ∼4-month-old mice. In the metabolic and behavioral experiments, the same animals were used in all experiments except for the buried food-seeking test, which used a different cohort of mice. The animal room was maintained in a 12 h light/dark cycle. A regular rodent diet (2.99 kcal/g; 9.4% kcal derived from fat; Nuvilab CR-1, Quimtia) was provided to the mice. The experimental procedures were approved by the Ethics Committee on the Use of Animals of the Instituto de Ciencias Biomedicas at the Universidade de Sao Paulo.

Identification of GH-responsive neurons

GH-responsive neurons were identified using an acute injection of GH to induce pSTAT5 (Furigo et al., 2017; Quaresma et al., 2021; Wasinski et al., 2021a). Thus, C57BL/6J, LepR-reporter, or CRH-reporter mice received an intraperitoneal injection of porcine GH (20 µg/g body weight; National Hormone and Pituitary Program) and were perfused 90 min later (n = 3–4/strain). Another group of C57BL/6 mice received a saline injection to demonstrate the low expression of pSTAT5 in the LHA of nonstimulated mice. The perfusion and brain sectioning were performed as previously described (Furigo et al., 2017; Quaresma et al., 2021; Wasinski et al., 2021a). In the immunofluorescence reactions, brain slices were rinsed in 0.02 M potassium PBS, pH 7.4 (KPBS), followed by pretreatment in a water solution containing 1% hydrogen peroxide and 1% sodium hydroxide for 20 min. After rinsing in KPBS, sections were incubated in 0.3% glycine and 0.03% lauryl sulfate for 10 min each. Next, slices were blocked in 3% normal serum for 1 h, followed by incubation in an anti-phospho^Tyr694-STAT5 antibody (1:1,000; Cell Signaling Technology; catalog #9351; RRID: AB_2315225). If pSTAT5 was colocalized with other proteins, the sections were coincubated with anti-ORX (1:8,000; Phoenix Pharmaceuticals; H-003-30; RRID: AB_2315019), anti-MCH (1:5,000; Phoenix Pharmaceuticals; H-070-47; RRID: AB_10013632), or anti-thyrotropin-releasing hormone (TRH; 1:1,000; Invitrogen; catalog #PA5-57331; RRID: AB_2648903) antibodies. Subsequently, sections were rinsed in KPBS and incubated for 90 min in Alexa Fluor-conjugated secondary antibodies (1:500, Jackson ImmunoResearch Laboratories). After rinsing in KPBS, sections were mounted onto gelatin-coated slides and covered with Fluoromount G (Electron Microscopic Sciences). Visualizing tdTomato or green fluorescent protein (GFP) reporter proteins did not require additional staining. In the peroxidase reaction, sections were incubated for 1 h in biotin-conjugated IgG donkey anti-rabbit (1:1,000, Jackson ImmunoResearch Laboratories) and for 1 h in avidin–biotin complex (1:500, Vector Laboratories). The peroxidase reaction to label pSTAT5 was performed using 0.05% 3,3′-diaminobenzidine, 0.25% nickel sulfate, and 0.03% hydrogen peroxide to produce black nuclear staining, while the subsequent reaction to label specific neuronal populations did not use nickel, producing cytoplasmic brownish staining. Sections were mounted onto gelatin-coated slides, dried overnight, dehydrated in ethanol, cleared in xylene, and coverslipped with DPX (Millipore Sigma).

Stereotaxic surgery

For bilateral injections into the LHA, GHR^flox/flox mice were anesthetized with ketamine (90 µg/g body weight) and xylazine (13.8 µg/g body weight). After the loss of reflexes, they were subjected to preoperative analgesia with ketoprofen (12 mg/kg, s.c.) and placed in a stereotaxic apparatus (Stoelting). Isoflurane (2%) was used for maintenance during surgery. To ablate the Ghr gene specifically in the LHA neurons, an AAV inducing the expression of Cre recombinase and GFP (AAV8-hSyn-GFP-Cre; Vector Core, University of North Carolina) was bilaterally injected (350 nl on each side; infusion rate, 70 nl/min) from a glass micropipette into the LHA using the following coordinates in relation to the bregma using the Allen Brain Atlas (https://mouse.brain-map.org/static/atlas) as the reference: −1.20 mm anteroposterior, ±1.00 mm mediolateral, and −5.20 mm depth. The control group received a bilateral injection of AAV5-hSyn-GFP (Vector Core, University of North Carolina). The sutures were performed using a surgical veterinary skin stapler. We kept the animals in recovery for 4 weeks before the experiments began.

Evaluation of energy and glucose homeostasis

Body weight was analyzed before and after 4 weeks of surgery. Body composition was analyzed using time–domain nuclear magnetic resonance (LF50 body composition mice analyzer; Bruker). Subsequently, mice were single-housed for acclimation. Then, daily food intake was recorded. To evaluate glucose homeostasis, food was removed from the cage 4 h before each test, and after determining basal glucose levels (time 0), mice received intraperitoneal injections of 2 g/kg of glucose or 0.75 IU/kg of insulin, followed by serial determinations of blood glucose levels using a glucose meter through samples collected from the tail tip.

Food restriction

After determining the average food intake, mice were housed in the Comprehensive Lab Animal Monitoring System (Columbus Instruments), which determines O₂ consumption (VO₂), CO₂ production, respiratory exchange ratio (RER; CO₂ production/O₂ consumption), and ambulatory activity. The ambulatory activity was determined using 39 pairs of infrared sensors that were positioned on opposite sides of the animal’s box. To consider ambulatory activity, a series of infrared beams need to be activated in sequence, characterizing ambulation inside the cage. In contrast, repeated interruptions of the same infrared beams do not incur ambulatory counts (which may be associated with other behaviors, like grooming, eating, scratching, etc.). The ambulatory activity data represent the number of infrared motion sensors activated by the mice in a given period of time. Basal metabolic measurements were performed after the acclimation period in ad libitum-fed mice, followed by 5 d of food restriction. During this period, each mouse received 40% of their basal food intake 2 h before lights off and body weight was recorded daily. We used this protocol of food restriction because it leads to increased basal GH levels (Zhao et al., 2010; Fang et al., 2019; Furigo et al., 2019b; de Sousa et al., 2023). In contrast, acute fasting blocks GH secretion in mice (de Sousa et al., 2023). Blood collections (~30 µl) were performed in ad libitum-fed mice and after 4 d of food restriction using heparinized capillaries (75 mm length and 1.5 mm diameter) through a small cut in the tail tip. After each collection, blood was centrifuged, and plasma was collected and stored to subsequently assess GH levels using an in-house enzyme–linked immunosorbent assay, as previously described (Wasinski et al., 2020, 2021b; Chaves et al., 2022; Dos Santos et al., 2022; Gusmao et al., 2022). After 5 d of food restriction, mice were perfused, and their brains processed to analyze c-Fos expression and the colocalization with agouti-related protein (AgRP) or ORX. These reactions were performed using the anti-c-Fos (1:20,000, Ab5, Millipore; RRID: AB_2314043), anti-ORX (1:10,000), and anti-AgRP (1:4,000, Phoenix Pharmaceuticals; catalog #H-003-53; RRID: AB_2313908) antibodies, following the peroxidase reaction protocol described above.

Buried food-seeking test

Another cohort of mice subjected to 4 d of food restriction as well as ad libitum-fed controls were evaluated in the buried food-seeking test. At 4 P.M. (the lights turn off at 7 P.M.), mice underwent a 10 min adaptation period in cages measuring 31 × 20 × 13 cm, lined with fresh corncob bedding. Then, the animals were transferred to a new cage (similar dimensions) in the same room containing a food pellet (∼1.3 g) buried 0.5 cm deep in the fresh bedding. The mice were placed in the opposite corner of the food pellet (∼30 cm away). The test was recorded, and the time it took for the mice to uncover the food pellet was considered latency (seconds), and the test was stopped as soon as the mouse touched the pellet. If a mouse was unable to find the food pellet within 10 min, the test was terminated, and the latency was recorded as 600 s. The experimenter changed gloves, and new cages were used for each animal to avoid olfactory cues.

GH-induced c-Fos expression

To investigate whether an acute GH injection induces c-Fos expression in the brain, C57BL/6J male mice received an intraperitoneal injection of GH (20 µg/g body weight) or saline and were perfused 2 h later. c-Fos was colocalized in ORX neurons using a peroxidase reaction, and the percentage of double-labeled neurons was determined.

Image analysis

Photomicrographs were obtained using an Axioimager A1 microscope (Carl Zeiss) connected with a Zeiss Axiocam 512 camera. Single- and double-labeled cells were manually analyzed in 100× (LHA) or 200× (ARH) magnification photomicrographs using the counting tool available in the Adobe Photoshop software. The average of two or three brain sections (one side) was calculated per each animal. The percentage of neurons that are responsive to GH in the LHA was analyzed in three or four mice per each marker (LepR, ORX, MCH, TRH, or CRH). The sample size of other immunostaining reactions is available in the figure legends.

Statistical analysis

Two-way repeated–measure ANOVA analyzed changes over time. Paired or unpaired two-tailed Student’s t test was used to analyze possible differences between the experimental groups. The Prism software (version 8.4.3; GraphPad) was used for the statistical analyses. The results are expressed as the mean ± standard error of the mean. The statistical tests and sample sizes are listed in each figure legend.

Results

LepR and ORX neurons in the LHA are highly responsive to GH

Previous studies have shown that the LHA contains numerous GH-responsive neurons (Cady et al., 2017; Furigo et al., 2017; de Lima et al., 2021b; Wasinski et al., 2021a). In accordance with these findings, GH-injected mice displayed a large number of neurons expressing pSTAT5 in the LHA (Fig. 1A). In contrast, saline-injected mice showed virtually no pSTAT5-positive cells in the LHA (Fig. 1B). To determine the neurochemical identity of GH-responsive neurons in the LHA, GH-induced pSTAT5 was colocalized with several markers of specific neuronal populations. Approximately 70% of LepR- and ORX-expressing neurons were responsive to GH in the LHA (Fig. 1C–E). Only 2, 7, and 10% of MCH, TRH, and CRH neurons, respectively, exhibited GH-induced pSTAT5 in the LHA (Fig. 1F–H). Thus, GH-responsive cells in the LHA are represented mainly by LepR and ORX neurons.

Figure 1.

LepR and ORX neurons in the LHA are highly responsive to GH. A, B, Distribution of pSTAT5 immunoreactive neurons in the LHA of C57BL/6J mice after an injection of porcine GH (pGH; A) or saline (B). C, The percentage of neurons responsive to GH in the LHA (n = 3–4 mice for each reaction). D–H, Epifluorescence photomicrographs showing the expression of different neuronal markers (magenta) and pSTAT5 (green) in the LHA of mice that received an intraperitoneal pGH injection. Double-labeled neurons may appear as white. Abbreviations: DMH, dorsomedial nucleus of the hypothalamus; fx, fornix. Scale bar, 100 µm.

Ablation of GHR in the LHA using bilateral stereotaxic injections of AAV-Cre

To investigate the importance of GHR signaling in LHA neurons, GHR^flox/flox mice received a bilateral injection of an AAV-Cre-GFP into the LHA. The expression of GFP initially confirmed the injection site (Fig. 2A). Mice were included in the LHA^ΔGHR group (successfully targeted the LHA) only if the distribution of GFP cells encompassed the entire LHA bilaterally, considering its rostrocaudal, mediolateral, and dorsoventral extension and did not significantly affect adjacent nuclei (Fig. 2A). Such injections abolished the capacity of an acute GH injection to induce pSTAT5 in the LHA (Fig. 2B,D). At the same time, GH-responsive cells were still observed in adjacent areas, like the ARH, ventromedial, and dorsomedial nuclei. In contrast, GHR^flox/flox mice that received a bilateral injection of AAV-GFP remained fully responsive to GH in the LHA, representing the control group (Fig. 2C,D). Thus, we induced the ablation of GHR in LHA neurons using a bilateral injection of AAV-Cre.

Figure 2.

Ablation of GHR in the LHA using bilateral stereotaxic injections of AAV-Cre-GFP. A, A representative mouse showing the injection site through the expression of GFP after a bilateral infusion of AAV-Cre-GFP into the LHA. B–D, Distribution of GH-induced pSTAT5 in the hypothalamus of GHR^flox/flox mice that received a bilateral injection of AAV-Cre-GFP (B) or AAV-GFP (C) in the LHA. Notice the absence of pSTAT5 in the LHA of AAV-Cre injected (LHA^ΔGHR) mice compared with the AAV-GFP (control) injection. The bar graphs show the quantification of the number of pSTAT5+ cells in the LHA of control (n = 4) and LHA^ΔGHR (n = 6) mice. Abbreviations: 3V, third ventricle; ARH, arcuate nucleus of the hypothalamus; fx, fornix; VMH, ventromedial nucleus of the hypothalamus. Scale bar, 500 µm. Differences between groups were analyzed by unpaired two-tailed Student’s t test. ***p < 0.001 significant difference between groups.

GHR ablation in the LHA does not affect energy and glucose homeostasis in ad libitum-fed mice

GHR signaling in the hypothalamus regulates food intake, body weight, energy expenditure, and glucose homeostasis (Bohlooly et al., 2005; Egecioglu et al., 2006; Cady et al., 2017; Furigo et al., 2019a,b; Quaresma et al., 2019; Teixeira et al., 2019; de Lima et al., 2021a,b; Donato et al., 2021; Pedroso et al., 2021; Wasinski et al., 2021b; Dos Santos et al., 2022; Stilgenbauer et al., 2023; Tavares et al., 2023). Thus, we started investigating whether GHR ablation in the LHA affects energy and glucose homeostasis of male mice. No differences in the body weight of control and LHA^ΔGHR mice were observed before surgery and after 4 weeks (Fig. 3A). Furthermore, no significant differences between the groups were observed in the weight gain after 4 weeks from surgery and in the fat and lean body mass (Fig. 3B,C). Control and LHA^ΔGHR mice also exhibited similar daily food intake (Fig. 3D), glucose tolerance (Fig. 3E), and insulin sensitivity (Fig. 3F). No differences in energy expenditure (VO₂; Fig. 4C,D), RER (Fig. 4E,F), and ambulatory activity (Fig. 4G,H) were observed between ad libitum-fed control and LHA^ΔGHR mice. Thus, GHR ablation in the LHA does not affect energy and glucose homeostasis in normal conditions.

Figure 3.

GHR ablation in the LHA does not affect energy and glucose homeostasis in ad libitum-fed mice. A–F, Body weight (n = 16–17/group), changes in body weight after surgery (4 weeks), fat mass (n = 4/group), lean body mass, daily food intake (n = 11–13/group), and glucose and insulin tolerance tests (n = 6–8/group) of control and LHA^ΔGHR mice. Possible differences between groups were analyzed by unpaired two-tailed Student’s t test or two-way repeated–measure ANOVA (glucose and insulin tolerance tests).

Figure 4.

GHR in the LHA is required for the increased activity and food-seeking behavior of food-deprived mice. A–H, Body weight (n = 9–10/group), weight loss, VO₂ (n = 7–9/group), RER, and ambulatory activity (n = 13–14/group) of control and LHA^ΔGHR mice in the basal (fed) state and during 5 d of food restriction (40% of basal intake). The recording time on the fifth day of food restriction ended ∼3–4 h before completing 24 h, leading to a lower cumulative ambulatory activity count than the previous days. The other metabolic parameters were not affected. The arrows indicate when the animals received the food (2 h before lights off). The representative figures are the average of all mice in each group. Gray and white backgrounds represent dark and light cycles, respectively. I, Latency to find food in the buried food-seeking test in ad libitum-fed mice and after 4 d of food restriction (n = 5–8/group). J, Plasma GH levels in ad libitum-fed mice and after 4 d of food restriction (n = 5–18/group). Differences between groups were analyzed by two-way repeated–measure ANOVA. *p < 0.05; **p < 0.01 significant differences between groups.

GHR in the LHA is required for the increased activity and food-seeking in food-deprived mice

There is evidence that GH action on hypothalamic neurons modulates metabolic responses to food deprivation (Furigo et al., 2019b; de Lima et al., 2021a). Thus, the metabolic responses to 5 d of food restriction (40% of basal intake) were analyzed in LHA^ΔGHR and control mice. As expected, food restriction produced a significant reduction in body weight (F_{(2, 37.1)}= 305.8; p < 0.0001), energy expenditure (F_{(2, 29.3)}= 68.9; p < 0.0001), and RER (F_{(2, 48.7)}= 187.2; p < 0.0001), compared with the baseline, in both control and LHA^ΔGHR mice (Fig. 4A–F). The absence of GHR in LHA neurons did not affect weight loss (Fig. 4A,B) and energy expenditure (Fig. 4C,D) compared with food-restricted control mice. RER was also not significantly modified in LHA^ΔGHR mice (Fig. 4E,F). In accordance with previous studies (Yamanaka et al., 2003; Pedroso et al., 2020; de Souza et al., 2022a,b), food deprivation robustly increased the ambulatory activity of control mice compared with the baseline (F_{(3, 75.7)}= 10.96; p < 0.0001; Fig. 4G,H). Remarkably, LHA^ΔGHR mice did not exhibit hyperactivity during food restriction, maintaining a significantly lower ambulatory activity during food restriction compared with control mice [effect of GHR ablation (F_(1,25)= 9.9; p = 0.0042); interaction between GHR ablation and food restriction (F_(5,123)= 3.029; p = 0.013); Fig. 4G,H]. To evaluate whether the decreased ambulatory activity of food-deprived LHA^ΔGHR mice was related to changes in feeding behavior, their response to the buried food-seeking test was determined (Fig. 4I). When compared with ad libitum-fed condition, food-deprived control mice exhibited the expected decrease in the latency to find food (t₍₇₎= 4.435; p = 0.003). In contrast, 4 d of food restriction did not reduce the latency to find food in LHA^ΔGHR mice compared with ad libitum-fed condition (Fig. 4I). Consequently, feeding latency was significantly higher in food-deprived LHA^ΔGHR mice compared with control animals (t₍₁₁₎= 3.237; p = 0.0079; Fig. 4I). We recently demonstrated that chronic food restriction changes GH secretion to a tonic/basal pattern instead of the typical pulsative secretion observed in ad libitum-fed mice (de Sousa et al., 2023). Plasma GH levels were analyzed in ad libitum-fed and food-deprived mice used in the current study (Fig. 4J). In both control and LHA^ΔGHR groups, part of the fed mice exhibited high plasma GH levels, indicating that the blood was collected during a GH peak, whereas a subgroup of ad libitum-fed mice showed plasma GH levels near zero, suggesting that blood collection occurred during the interpulse interval (Fig. 4J). On the other hand, practically all mice exhibited intermediate plasma GH levels after 4 d of food restriction, indicating a constitutively high basal GH secretion, as formerly demonstrated (de Sousa et al., 2023). Altogether, these findings indicate that the increased activity in food-deprived mice requires GHR signaling in the LHA and it is associated with increased food-seeking behavior.

GHR ablation in the LHA reduces the percentage of ORX neurons expressing c-Fos in food-deprived mice

LHA^ORX neurons are critical for the regulation of arousal and sleep–wake cycle, including during food deprivation (Chemelli et al., 1999; Eriksson et al., 2001; Yamanaka et al., 2003; Sakurai, 2014; Michael and Elmquist, 2020). To investigate whether the blunted increase in activity showed by food-deprived LHA^ΔGHR mice is associated with ORX neurons, we colocalized c-Fos protein, a marker of neuronal activity (Hoffman and Lyo, 2002), with ORX in food-deprived mice. Notably, a significant decrease in the percentage of ORX neurons expressing c-Fos was observed in the LHA of LHA^ΔGHR mice compared with food-deprived control mice (t₍₁₇₎= 2.137; p = 0.0474; Fig. 5A–C). Additionally, the percentage of double-labeled neurons in relation to the total number of c-Fos + neurons was significantly decreased in LHA^ΔGHR mice compared with control mice (t₍₁₇₎= 3.031; p = 0.0096; Fig. 5A–C). A robust c-Fos expression was also observed in the ARH of food-deprived mice (Fig. 5D,E). However, in this case, no difference in the number of c-Fos-positive neurons was observed in the ARH of control (66.8 ± 9.3) and LHA^ΔGHR mice (77.0 ± 13.4; t₍₁₇₎= 0.6374; p = 0.5324). Since c-Fos expression was mostly restricted to the ventromedial ARH, where AgRP neurons are found (Tritos et al., 1998; Furigo et al., 2019b), a colocalization between c-Fos and AgRP was performed. We observed that most c-Fos-positive neurons in the ARH expressed AgRP (Fig. 5F). These findings indicate that the absence of GHR expression in the LHA reduces the percentage of ORX neurons expressing c-Fos but does not affect the robust activation of AgRP neurons during food restriction.

Figure 5.

GHR ablation in the LHA reduces the percentage of ORX neurons expressing c-Fos in food-deprived mice. A–C, The percentage of ORX neurons expressing c-Fos, the percentage of double-labeled neurons in relation to the total number of c-Fos+ neurons, and representative photomicrographs of control (n = 10) and LHA^ΔGHR (n = 9) mice after 5 d of food restriction. c-Fos protein is observed through black nuclear staining, whereas ORX neurons present cytoplasmic brownish staining. D–E, ARH neurons present intense c-Fos expression in food-deprived control (n = 10) and LHA^ΔGHR (n = 9) mice. F, Representative photomicrograph demonstrating that c-Fos expression in the ARH of food-deprived mice is mainly restricted to AgRP neurons (discreet cytoplasmic brownish staining). Abbreviations: 3V, third ventricle; ARH, arcuate nucleus of the hypothalamus. Scale bar: C, 50 µm; D, 50 µm; F, 25 µm. Differences between groups were analyzed by unpaired two-tailed Student’s t test. *p < 0.05; **p < 0.01 significant difference between groups.

GH injection increases the expression of c-Fos in LHA^ORX neurons

The previous findings suggest that GHR signaling in the LHA can regulate the activity of ORX neurons during chronic food deprivation, which is a situation associated with increased GH secretion (Zhao et al., 2010; Furigo et al., 2019b; de Sousa et al., 2023). In the next experiment, the capacity of an acute GH injection to induce the expression of c-Fos protein in LHA^ORX neurons was evaluated. GH injection significantly increased the percentage of LHA^ORX neurons expressing c-Fos, compared with saline-injected mice (t₍₉₎= 2.521; p = 0.0327; Fig. 6A–E).

Figure 6.

GH injection increases the expression of c-Fos in LHA^ORX neurons. A–E, The percentage of ORX neurons expressing c-Fos and representative photomicrographs of C57BL/6J mice that received an acute injection of saline (A, B; n = 5) or GH (C, D; n = 6). c-Fos protein is observed through black nuclear staining, whereas ORX neurons present cytoplasmic brownish staining. Arrows indicate double-labeled neurons. B and D represent higher-magnification photomicrographs of the selected areas in A and C, respectively. Abbreviation: fx, fornix. Scale bar, 100 µm. Differences between groups were analyzed by unpaired two-tailed Student’s t test. *p < 0.05 significant difference between groups.

GHR expression in non-LepR neurons is necessary to produce hyperactivity during food restriction

GH-induced pSTAT5 is observed in both LepR and ORX neurons of the LHA (Fig. 1). Importantly, ORX- and LepR-expressing cells represent distinct neuronal populations of the LHA (Fig. 7A). To determine the specific role of these neuronal populations in the effects induced by GH, we generated mice carrying ablation of GHR in the entire brain, using Nestin promoters to drive Cre expression, or specifically in LepR-expressing cells (Fig. 7B,C). These mouse models have been extensively characterized and validated in our previous studies (Furigo et al., 2019a,b; Teixeira et al., 2019; Wasinski et al., 2020; Pedroso et al., 2021; Wasinski et al., 2021b,2023). Brain^ΔGHR mice exhibit an ∼90% reduction in the hypothalamic expression of Ghr mRNA (Wasinski et al., 2020,2021b,2023). Furthermore, only a few GH-responsive cells were observed in the LHA- of GH-injected Brain^ΔGHR mice (Fig. 7B), confirming the efficacy of GHR ablation. In contrast, LepR^ΔGHR mice still exhibited numerous GH-induced pSTAT5 cells in the LHA, although LepR neurons were no longer responsive to GH (Fig. 7C). Brain^ΔGHR and LepR^ΔGHR mice were subjected to 5 d of food restriction. As expected, control mice progressively increased ambulatory activity during food restriction [effect of food restriction (F_{(2.8, 56.8)}= 12.18; p < 0.0001); Fig. 7D–E]. Noteworthy, Brain^ΔGHR mice sustained a reduced ambulatory activity throughout the analyzed period, despite the food restriction [effect of GHR ablation (F_(2,21)= 5.865; p = 0.0095); interaction between GHR ablation and food restriction (F_(10,100)= 3.935; p = 0.0002); Fig. 7D–E]. On the other hand, food-deprived LepR^ΔGHR mice increased ambulatory activity similarly to the control group’s animals or showed an even more significant increase on the third day of food restriction (p = 0.0276; Fig. 7D). Food-seeking behavior was also analyzed in control and Brain^ΔGHR mice, and although the time to find food was similar between the ad libitum-fed groups, Brain^ΔGHR mice exhibited increased feeding latency after 4 d of food restriction compared with control mice (t₍₁₂₎= 2.941; p = 0.0123; Fig. 7F). Since GHR expression in non-LepR neurons is necessary to increase activity during food restriction, our findings suggest that the absence of GHR signaling in ORX neurons possibly explains the reduced activity in food-deprived LHA^ΔGHR mice.

Figure 7.

GHR expression in non-LepR neurons is necessary to increase activity and food-seeking during food restriction. A, Epifluorescence photomicrograph showing that ORX neurons (green) and LepR-expressing cells (magenta) represent distinct neuronal populations in the LHA. B, Representative photomicrograph showing that GH-injected Brain^ΔGHR mice exhibit only a few pSTAT5 cells in the LHA. C, Representative photomicrograph showing that GH-injected LepR^ΔGHR mice present numerous pSTAT5 cells in the LHA (green staining), although most LepR neurons (magenta) were no longer responsive to GH. Abbreviations: 3V, third ventricle; fx, fornix; VMH, ventromedial nucleus of the hypothalamus. Scale bar, 100 µm. D–E, Ambulatory activity of control (n = 8), Brain^ΔGHR (n = 5), and LepR^ΔGHR (n = 11) mice in the basal (fed) state and during 5 d of food restriction (40% of basal intake). The arrows indicate when the animals received the food (2 h before lights off). The representative figure is the average of all mice in each group. Gray and white backgrounds represent dark and light cycles, respectively. F, Latency to find food in the buried food-seeking test in ad libitum-fed mice and after 4 d of food restriction (n = 6–8/group). Differences between groups were analyzed by two-way repeated–measure ANOVA. *p < 0.05; **p < 0.01 significant differences between groups.

Discussion

Neurons in the LHA integrate interoceptive and exteroceptive signals to control feeding, arousal, reward, and motivated behaviors (Stuber and Wise, 2016; Arrigoni et al., 2019; Perez-Bonilla et al., 2020; Ramirez-Virella and Leinninger, 2021). Part of these effects are mediated either by LHA^ORX neurons (Chemelli et al., 1999; Eriksson et al., 2001; Yamanaka et al., 2003; Sakurai, 2014; Michael and Elmquist, 2020) or LHA^LepR cells (Leinninger et al., 2009, 2011; Louis et al., 2010). Although LHA^ORX and LHA^LepR cells represent distinct neuronal populations, LHA^LepR neurons project and regulate the activity of neighboring ORX neurons (Leinninger et al., 2009, 2011; Louis et al., 2010). Thus, the primary targets of GH in the LHA essentially represent the same neurocircuit involved in regulating eating-motivated behavior.

To investigate the physiological importance of GHR signaling in the LHA, we used AAV injections to induce genetic ablation of the Ghr gene selectively in LHA neurons. Only mice having injections that targeted the entire LHA bilaterally and without significantly affecting adjacent nuclei were included in the LHA^ΔGHR group. The efficacy of the genetic manipulation was further confirmed by the absence of GH-induced pSTAT5 in the LHA of LHA^ΔGHR mice. An advantage of using stereotaxic injections to manipulate genes is the fact that there is no possibility for compensation during development, a fact that may help explain the lack of phenotype in some models where GHR inactivation occurred early in life (Dos Santos et al., 2021; Chaves et al., 2022).

GHR ablation in the LHA did not affect energy and glucose homeostasis in ad libitum-fed mice. This finding is similar to GHR ablation in numerous neuronal populations, which does not produce physiological consequences unless the mice are subjected to metabolic stress conditions, such as food restriction, hypoglycemia, exercise training, or pregnancy (Furigo et al., 2019a,b; Teixeira et al., 2019; Pedroso et al., 2021). Thus, we hypothesize that central GH action in adult mice is mainly associated with metabolic and behavioral responses to restore homeostasis in metabolic stress situations (Tavares et al., 2023). For example, the absence of GHR signaling in AgRP neurons blunts the energy-saving adaptations and accelerates weight loss in food-deprived mice (Furigo et al., 2019b; Furigo et al., 2020). ORX neurons are also involved in the responses to food restriction. Yamanaka et al. (2003) demonstrated that ORX neurons are necessary to increase activity and arousal in fasted mice. The current study found that the absence of GHR in the LHA did not affect weight loss and energy expenditure during food restriction, which differs from the phenotype of AgRP^ΔGHR mice (Furigo et al., 2019b). In contrast, LHA^ΔGHR mice did not exhibit the well-described hyperactivity demonstrated by starved animals (Yamanaka et al., 2003; Pedroso et al., 2020; de Souza et al., 2022a,b).

Several studies in rodents and nonhuman primates have found that ORX neurons are activated by food deprivation or low glucose levels (Cai et al., 1999,2001; Diano et al., 2003; Linehan and Hirasawa, 2022), whereas increased glucose availability inhibits the activity of ORX neurons (Yamanaka et al., 2003). Hypoglycemia is a powerful stimulus to induce the secretion of GH (Roth et al., 1963; Furigo et al., 2019a). However, it remains unknown whether GH secretion during hypoglycemia contributes to the activation of ORX neurons during low glucose availability (Cai et al., 1999,2001; Yamanaka et al., 2003).

Interestingly, glucose-inhibited LHA^ORX neurons promote food-seeking behavior following caloric restriction (Teegala et al., 2023). Ghrelin is a powerful GH secretagogue (Kojima et al., 1999; Peino et al., 2000). Food deprivation increases circulating levels of ghrelin and GH (Tschop et al., 2000; Zhao et al., 2010; Furigo et al., 2019b; de Sousa et al., 2023). Ghrelin-induced feeding is suppressed in ORX knock-out mice (Toshinai et al., 2003). Furthermore, intra-LHA injections of ghrelin increase food intake and locomotor activity, and pretreatment with an orexin 1 receptor antagonist blocks the orexigenic effect of ghrelin (Barrile et al., 2023). GH-deficient hypophysectomized or dwarf rodents show a blunted orexigenic response to both ghrelin and orexin (Egecioglu et al., 2006; Alvarez-Crespo et al., 2013; Wasinski et al., 2021b). Taken together, these data suggest a possible interrelationship among ORX neurons, ghrelin, and GH.

To gain insight into the neuronal population that mediates the effects of GH in the LHA, we analyzed c-Fos expression in two conditions: in food-deprived mice and after an acute GH stimulus. Inactivation of GHR in the LHA reduced the percentage of ORX neurons expressing c-Fos during food restriction, suggesting that the activation of ORX neurons in this situation requires GHR signaling in the LHA. In accordance with this finding, an acute GH injection increased the percentage of ORX neurons expressing c-Fos, providing additional evidence that GHR signaling stimulates the activity of ORX neurons. However, even though 70% of ORX neurons are responsive to GH, these experiments do not allow us to determine whether the activation of ORX neurons occurs through a direct effect on ORX neurons via GHR signaling or indirectly via neuronal connection with LHA^LepR neurons, as these cells are also responsive to GH, and they regulate the activity of ORX neurons (Leinninger et al., 2009,2011; Louis et al., 2010).

To investigate the possibility that GH action on LHA^LepR neurons is necessary for hyperactivity induced by food deprivation, LepR^ΔGHR mice were generated, as previously described (Furigo et al., 2019a; Furigo et al., 2019b; Teixeira et al., 2019; Pedroso et al., 2021). In contrast to the findings observed in LHA^ΔGHR mice, LepR^ΔGHR mice exhibited a progressive increase in ambulatory activity during food restriction. Actually, the hyperactivity of LepR^ΔGHR mice on the third day of food restriction was even higher than that observed in control mice. Like AgRP^ΔGHR mice, LepR^ΔGHR mice present defects in energy-saving adaptations during food restriction, leading to an earlier depletion of their body energy reserves (Furigo et al., 2019b). Consequently, food-deprived LepR^ΔGHR mice may become hungrier, possibly exacerbating their food-seeking behavior. On the other hand, when GHR was inactivated in the entire brain, hyperactivity induced by food restriction was prevented, reproducing the behavioral alterations exhibited by LHA^ΔGHR mice. By comparing the phenotype of LepR^ΔGHR and Brain^ΔGHR mice, our findings indicate that a non-LepR neuronal population is necessary to produce hyperactivity during food restriction. Thus, given the high responsiveness of ORX neurons to GH, the reduced percentage of ORX neurons expressing c-Fos in food-deprived LHA^ΔGHR mice, and the capacity of GH in inducing c-Fos in ORX neurons, it is very likely that ORX neurons are the primary targets of GH in the LHA to stimulate foraging behaviors in food-deprived animals. Importantly, this effect is independent of metabolic alterations or activation of additional neuronal populations that induce hunger (e.g., ARH^AgRP neurons) during food restriction.

Since we analyzed male mice only, caution must be applied, as it is well described that males and females may exhibit marked sex differences in several metabolic aspects (de Souza et al., 2022a). Furthermore, GH secretion is sexually dimorphic (Steyn et al., 2016), and GHR ablation in specific neuronal populations may have divergent outcomes in different sexes (Furigo et al., 2019b, 2020; Dos Santos et al., 2022,2023). Thus, our findings should not be extrapolated to females, and future studies are needed to analyze the role of GHR signaling in the LHA of female mice. Another aspect that should be acknowledged is that the Nestin-Cre mouse, used here to induce brain-specific GHR ablation, may present mild hypopituitarism due to the insertion of a GH minigene, leading to the expression of human GH (hGH) in the brain (Galichet et al., 2010; Declercq et al., 2015). However, since Brain^ΔGHR mice are intrinsically unresponsive to GH in the central nervous system, the undesired effects of hGH transgenic expression are not observed in Brain^ΔGHR mice (Furigo et al., 2019b; Wasinski et al., 2020,2021b).

In conclusion, the absence of GHR signaling in the LHA disturbs the increased exploratory activity and food-seeking behavior observed in starved animals. Noteworthy, several neurological and behavioral alterations are commonly reported by GH-deficient patients, including modifications in appetite, sleep, mental alertness, motivation, and metabolism (Nyberg, 2000; Nyberg and Hallberg, 2013; Karachaliou et al., 2021). All these functions are closely associated with the described physiological roles of the LHA, particularly ORX neurons (Yamanaka et al., 2003; Sakurai, 2014; Goforth and Myers, 2017; Michael and Elmquist, 2020). Thus, the current study revealed that ORX neurons represent a novel neuronal population affected by GH action to trigger behavioral responses to starvation. This new evidence reinforces our hypothesis that GH secretion during situations of metabolic stress acts in several tissues (e.g., liver, adipose tissue, and muscle) and in different neuronal populations to promote metabolic and behavioral adjustments that help restore homeostasis (Tavares et al., 2023). Regarding the present findings, GH may act as a starvation signal via LHA^ORX neurons to trigger exploratory activity, wakefulness, and foraging behaviors, ultimately increasing the animal’s chances of finding food and surviving.