Targeted Apoptosis of Melanocortin Type 4 Receptor (MC₄R)-Expressing Cells Within the Preoptic Area of the Hypothalamus Causes Rapid Weight Gain in Adult Mice

The central melanocortin system is a potent controller of energy homeostasis through modulation of food intake and energy expenditure; genetic deletion or interference with the melanocortin 4-receptor (MC 4 R) results in hyperphagia and obesity. MC 4 R is widely expressed throughout the brain, with particularly high expression in regions of the hypothalamus, including the preoptic area (POA). Spatial transcriptomic studies have demonstrated that within the POA, MC 4 R is expressed by only a small number of glutamatergic neurons. Previous studies demonstrated that acute injection of an MC 4 R agonist into the POA stimulates brown fat thermogenesis and metabolism in rats, however, relatively little is known about the long-term functional significance of MC 4 R-expressing cells within the POA for energy balance. We hypothesized that selective ablation of MC 4 R-expressing cells in the POA would disrupt integrative responses to cold stimuli (including food intake, resting metabolic rate, and thermoregulatory behaviors). Adult mice expressing Cre-recombinase via the Mc4r locus ( Mc4r -2a-Cre, Jackson Labs #030759, n=4 male + 4 female) and age-, mass-, and sex-matched Cre-deficient littermate controls (n=4 male + 4 female) underwent targeted bilateral stereotaxic microinjection of an AAV vector encoding Cre-dependent expression of caspase-3 (Addgene 45580-AAV5, pAAV-flex-taCasp3-TEVp). Mice were housed at 22°C with ad libitum access to phytoestrogen-free 2920x chow and drinking water. Beginning within one week after injection, the ablation group exhibited a robust hyperphagic response (5.1±1.0 vs 3.5±0.4 grams chow/day, p=0.001). Feeding efficiency, an inverse metric of total energy expenditure (EE), was significantly increased after ablation (138.4±62.9 vs 0.1±56.3 Δ mg body mass/grams total food consumed, p=0.002), thereby supporting a suppression of EE after ablation. Basal aerobic metabolic rate between two and five weeks after injection, assessed by V· O 2 / V· CO 2 respirometry at 30°C after 5h fast, was not significantly altered by ablation after adjusting for fat-free and fat masses. At 6 weeks after injection, the lesioned group had gained significantly more mass than controls (20.4±7.4 vs 3.4±1.9 grams/6wks, p<0.001), primarily driven by a disproportionate increase in adiposity (16.9±6.1 vs -0.4±3.0 Δ % fat mass/6wks, p<0.001). These data demonstrate a major role for MC 4 R-expressing cells within the POA in long-term energy balance, potentially through the modulation of feeding and EE responses to cold stimuli. Future studies in mice chronically housed at thermoneutral ambient temperatures (30°C) are warranted. HL134850 DK133121 GM080202 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.