Male Sprague–Dawley rats (6–8 weeks, 170–210 g) were housed in independent cages in a temperature-controlled room with a daily light and dark cycle. Food and water were provided ad libitum except during the fasting–refeeding test. Surgical procedures, drug administration, and outcome assessments (body weight, food intake, histology, and molecular assays) were performed by investigators blinded to group allocation. All procedures followed our institutional guidelines and were approved by the ethics committee of Nanfang Hospital, Southern Medical University.

Animals were randomized into groups using a computer-generated randomization sequence, prepared and assigned by a technician not involved in subsequent experiments. For each experiment, animals were assigned to: sham surgery (n = 12), lesion surgery (n = 15), refeeding lesion rats (), short-term setmelanotide treatment following lesion (n = 14), short-term saline treatment (n = 13), long-term saline (n = 13), long-term setmelanotide (n = 14). Sample sizes were determined based on prior literature and power calculation to ensure sufficient statistical power (Song et al., 2022).

Bilateral ARC/VMH lesions were induced under isoflurane anesthesia (5% induction, 2% maintenance; Reward small animal gas device) and stereotaxic guidance. Using coordinates relative to bregma (2.6 mm posterior, 0.6 mm lateral to midline, 9.6 mm ventral), a 1-mm-thick insulated stainless-steel electrode (0.45-mm tip) was lowered bilaterally into the hypothalamus (Song et al., 2022). A cathodic current of 1.2 mA was delivered for 25 s using a constant power supply (53500, UGO Basile, Italy). Sham-operated controls underwent the same procedure without current application.

Regions and extent of hypothalamic damage were confirmed in all animals postmortem via immunohistochemical staining. Only animals with bilateral, well-confined ARC/VMH lesions, and no damage to adjacent hypothalamic nuclei, were included. Exclusion criteria comprised off-target or unilateral lesions, overt perioperative complications, significant tissue loss, or failure to recover baseline feeding within 72 h post-surgery.

All animals were housed in independent cages, and body weight and food intake were recorded weekly. The linear distance between the tip of the rat’s nose and the anus was recorded at the end of 4^th week as the body length of the rat, and the evaluation of obesity was indicated by the Lee index (Lee index = ∛wt (g)/length (cm) × 1,000) and the perirenal adipose tissue (PRAT) weight (Jasim, 2021; Baumann et al., 2025).

In the short term of fasting-refeeding test (Igarashi et al., 2023), rats were fasting for 24 h with water available, and then the food consumption was recorded every half hour during the total 2 h refeeding process.

Setmelanotide (MCE, CAS No. HY-19870) was freshly dissolved in sterile saline before use and kept at 4 °C, shielded from light, for no longer than 24 h. In the short term, after fasting for 24 h with water available, rats of the setmelanotide (SET) group were administered with setmelanotide (0.05 mg/kg body weight) by intraperitoneal injection (I.P.) according the previous studies (Pressley et al., 2022; Lazare et al., 2024). The dose for each injection was calculated based on the animal’s body weight measured immediately prior to injection. Saline rats were injected with saline. 0.5 h later the refed process began, and the food consumption was recorded every half hour during the total 2 h refeeding process.

In the long-term treatment paradigm, starting on postoperative day 21, rats were singly housed with ad libitum access to chow and water and received once-daily I.P. of either vehicle (saline) or setmelanotide (0.05 mg/kg). The injection volume and dose were recalculated and adjusted for each rat’s current body weight prior to every injection throughout the experiment. All injections were performed at the same time each day. Food intake was recorded at 1 h and 24 h after each injection.

Rats were deeply anesthetized with 1% sodium pentobarbital, frozen with normal saline, and then instilled with 4% paraformaldehyde. Then, the brain tissues were immersed in 4% paraformaldehyde for 48 h and transferred to 70%–100% ethanol. Individual tissues for brain biopsy material were placed in processing cassettes, dehydrated through a serial alcohol gradient, and embedded in paraffin wax blocks. Before immunostaining, 3-µm-thick brain tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. In addition, the samples were then stained with hematoxylin and eosin. After staining, the sections were dehydrated with increasing concentrations of ethanol and xylene, and sealed with resin. The images were captured with a microscope (Olympus, BX63). Only animals with confirmed bilateral, well-confined ARC/VMH lesions and no adjacent tissue damage were included.

For immunofluorescence staining of coronal frozen brain sections, the sections were rinsed with PBS, followed by blocking with 5% nonspecific antigen goat serum for 1 h at 37 °C. Then, the sections were incubated overnight at 4 °C with specific primary antibodies. The next day, after rinsing three times with PBS containing 0.5% Triton X-100, the sections were incubated for 1 h at 37 °C with the corresponding secondary antibodies conjugated with Alexa 488 or Alexa 594 (Thermo Fisher Scientific, United States). The primary and secondary antibodies were diluted in PBS containing 5% normal goat serum and 0.2% Triton X-100. After incubation with secondary antibodies, the sections were mounted on glass slides, and cover glasses were mounted in mounting medium. Fluorescence images were captured with a confocal microscope (LSM880, Zeiss, Germany). For cell number counted by ImageJ, the data are presented as the number of cells/mm². For double staining analysis, the data are presented as the ratio of the number of double-positive cells to the number of single-positive cells.

Hypothalamic PVN tissues were harvested under a microscope, and washed with PBS three times and lysed with RIPA buffer (50 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA pH 8.0, 5 mmol/L DTT, 2% SDS) containing protease inhibitor and phosphoric acid protease inhibitor at 4 °C for 30 min. The protein concentration was determined using a BCA assay kit (Beyotime Inc., China), after which the protein was loaded onto a 10% SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, United States). The membranes were incubated with primary antibodies against p-AMPK (1:1,000; CST), AMPK (1:1000; CST), p-CaMKK2 (1:1000; CST), CaMKK2 (1:1000; PTG) and GAPDH (1:5000, PTG) at 4 °C overnight. Subsequently, the membranes were incubated with secondary antibodies (1:1000, ABclonal). The immunoreactive signals were visualized with enhanced chemiluminescence reagents, and images were captured with a digital camera (Pierce, Rockford, IL, United States).

The following primary antibodies were used: rabbit anti-POMC (ab94446; Abcam); mouse anti-POMC (66358-1-Ig; Proteintech Group); rabbit anti-MC4R (GTX31382; Genetex); mouse anti-cFos antibody (GTX60996; Genetex); rabbit anti-p-AMPK (2535; CST), mouse anti-AMPK (66536-1-Ig, Proteintech Group), rabbit anti-p-CaMKK2 (12716; CST), rabbit anti-CaMKK2 (13730-1-AP; Proteintech Group); and rabbit anti-GAPDH (10494-1-AP, Proteintech Group). The following secondary antibodies were used: goat anti-rabbit 488 fluorescent antibody (A-11008; Thermo Fisher Scientific); goat anti-mouse 594 fluorescent antibody (A-11005; Thermo Fisher Scientific); and anti-rabbit IgG-HRP antibody (1:1000, AS014, ABclonal).

All statistical analyses were performed using GraphPad Prism 10.0 software. Data were represented as mean ± SEM and analyzed with a two-tailed Student’s t-test between two groups, and one-way ANOVA followed by LSD correction between four groups. Exact sample sizes for each analysis are reported in figure captions. P < 0.05 was considered to indicate significance.

Hypothalamic lesions often damage the ARC/VMH area and result in HO (Song et al., 2022; King, 2006; Joly-Amado et al., 2014). To investigate the effects of hypothalamic injury on feeding behavior and metabolic function, we induced HO by targeted electrolytic injury of ARC/VMH in SD rats, with representative coronal sections confirming lesion localization (Figure 1A). A total of 7.35% of animals were excluded due to off-target lesions, perioperative complications, or failure to recover baseline feeding; only animals with bilateral, well-confined ARC/VMH lesions were included in the analyses.

Body weight analysis showed no difference in the beginning between the two groups; however, 4 weeks after injury, the body weight of the lesion rats was 27% higher than the sham group, which were considered to be obesity in animal research (Figures 1B,C). On the first week, the food intake of lesioned rats increased significantly and this hyperphagic response persisted, and by the 4^th week, the food intake of lesion rats was reached to 1.96 times than that of the sham group (Figure 1D). In addition, the nasal anal length of lesioned rats was significantly shortened, and the Lee index and PRAT weight were significantly increased, further confirming the occurrence of HO after lesion (Figures 1E–G).

These findings demonstrate that ARC/VMH lesions effectively model HO, characterized by increased appetite and body weight. This surgery-induced obesity model provides a platform for evaluating potential pharmacological interventions.

In the PVN, POMC and MC4R neurons play a key role in inhibiting food intake and maintaining metabolic balance (Guo et al., 2024; Talbi et al., 2025). To explore the underlying mechanism, we first used immunofluorescence to detect POMC expression in the PVN of HO rats. The immunofluorescence showed that POMC fiber density was significantly decreased compared to the sham group (Figures 2A–C). Western blot analysis further confirmed the decreased POMC protein expression in HO rats (Figures 2D,E).

Given that MC4R neurons are the primary recipients of POMC within the PVN (Griffin et al., 2024), we further performed double immunofluorescence staining, which revealed the distribution and contact of POMC fiber density with MC4R neurons (Figure 2F). Double immunofluorescence staining of cFos, a classical marker for neuron activation, confirmed that activation of MC4R neurons is significantly reduced in HO rats (Figures 2G–J). Reduced activation of MC4R is associated with impaired appetite suppression and the development of HO in rats.

Refeeding, the process of reintroducing food after fasting, plays a critical role in restoring metabolic balance and regulating appetite mechanisms in obesity research (Imoto et al., 2021). We then examined the role of MC4R neurons in appetite suppression during the fasting-refeeding treatment. Rats were fasted for 24 h at 4 weeks post-surgery and then refeed for 2 h (Figure 3A). Although there was no difference in food intake between two groups in the initial 0.5 h, HO rats showed impaired appetite inhibition in the following 0.5h–2 h, and resulting in increased food consumption compared with sham rats in total refeeding time (Figures 3B,C).

During the fasting period, there was no difference in MC4R activation between the two groups. Interestingly, after refeeding, the activation of MC4R neurons in HO rats was significantly reduced compared with sham rats (Figures 3D–F).

These findings indicate that reduced MC4R activation in HO rats, especially during refeeding, weakens the ability to suppress appetite, leading to short-term hyperphagia.

MC4R neurons were reported to be associated with the regulation of the AMP-activated protein kinase (AMPK) pathway, which plays a critical role in various metabolic processes (Zhang et al., 2017). Recent studies have highlighted the involvement of calmodulin-dependent protein kinase kinase 2 (CaMKK2) in enhancing the phosphorylation and activation of AMPK, thereby influencing metabolic regulation (Chen et al., 2023).

Western blot analysis demonstrated a significant decrease in the activation of the AMPK and CaMKK2 signaling pathway in lesion rats compared to sham rats (Figures 4A–C). Furthermore, to evaluate these signaling pathways within the appetite inhibition in HO rats, we examined the CaMKK2/AMPK expression in fasting-refeeding treated rats. The results showed that refeeding significantly reduced pAMPK in controls, whereas pCaMKK2 did not differ between states. In lesioned rats, pCaMKK2 was lower than controls under both fasting and refeeding and remained non-responsive to refeeding; the refeeding-induced decrease in pAMPK was blunted relative to controls (Figures 4D–F), which indicated a major role of AMPK signaling pathway in the appetite regulation post-lesion surgery.

In summary, the above data underscore that AMPK pathway and MC4R exhibit consistent effects in modulating appetite inhibition in HO rats, highlighting their interconnected roles in this regulatory mechanism and offering potential mechanisms for managing HO.

Previous findings suggest that activation of MC4R contributes to decreased appetite inhibition and HO in rats through the CaMKK2/AMPK pathways. To treat obesity in this rodent model, the agonist of MC4R, setmelanotide was used in HO rats.

To verify the role of setmelanotide in suppressing appetite, rats were treated with setmelanotide (Figure 5A). After refeeding, the activation ratio of MC4R in lesion-SET rats was increased, significantly higher than the lesion-saline rats (Figures 5B–D).

During the refeeding time, the food consumption was measured every 0.5 h, and results showed that lesion-SET rats were completely capable of controlling food consumption in the refeeding time, and there was no difference in total amount of food intake compared to the sham-SET rats. During the entire refeeding period, the lesion-SET group rats and sham-SET group rats showed similar food intake (Figures 6A,B), indicating that setmelanotide normalized feeding behavior to levels statistically indistinguishable from sham controls in the short term.

To delve into the mechanism of setmelanotide, we assessed the expression of CaMKK2/AMPK pathways. Immunoblotting confirmed that setmelanotide enhanced the activation of these 2 pathways after refeeding (Figures 6C–E). Furthermore, higher expression of p-CaMKK2/p-AMPK validated the effect of setmelanotide on restoring the metabolic homeostasis in HO rats.

These results highlight that in the short term, setmelanotide could normalize feeding behavior in HO rats by activating MC4R via the CaMKK2/AMPK pathways, underscoring its potential as a therapeutic agent for inhibiting hyperphagia in HO.

To assess the long-term weight management of setmelanotide in HO, we treated rats with setmelanotide for 1 week (Figure 7A). Immunofluorescence analysis revealed that setmelanotide significantly strengthened the activation of MC4R. In the lesion-SET rats, the proportion of activated MC4R increased compared to the lesion-saline rats (Figures 7B–E).

Furthermore, we assessed the impact of setmelanotide in the long term. Body weight analysis showed no difference at the baseline and on the first day after administration between the lesion-saline and lesion-SET groups. Subsequently, the body weight of the lesion-SET group decreased significantly. After 1 week, body weight in the lesion-SET group was restored to levels comparable to the sham-SET group (Figures 8A,B), and food intake was significantly less than lesion-saline controls. While metabolic indices such as the Lee index and PRAT weight were significantly improved, shortened body length did not recover, indicating full reversal of some but not all parameters.

Postoperative monitoring revealed that food intake was significantly lower in lesion-SET rats compared to lesion-saline rats (Figure 8C). Additionally, while the shortened body length of lesioned rats did not recover, both the Lee index and PRAT (perirenal adipose tissue) weight were significantly reduced in lesion-SET rats versus lesion-saline rats, further confirming the anti-obesity effect of setmelanotide in HO rats (Figures 8D–F).

In summary, our findings indicate that setmelanotide treatment effectively activates PVN MC4R by upregulating the CaMKK2/AMPK pathways, thereby inhibit hyperphagia and reverse obesity in HO rats. These results underscore the potential of setmelanotide as a therapeutic strategy for managing obesity after hypothalamic injury.