**Background:** The hypothalamus, particularly the arcuate nucleus (ARH), plays a central role in energy homeostasis. Pro-opiomelanocortin (POMC) neurons in the ARH are anorexigenic, reducing food intake and increasing energy expenditure. Toll-like receptor 4 (TLR4) is a pattern recognition receptor known to mediate hypothalamic inflammation and insulin resistance in obesity. While TLR4 is expressed in microglia and some neurons, its specific role in POMC neurons was unknown. This study investigated the effect of deleting TLR4 specifically in POMC neurons on energy balance, thermogenesis, and lipid metabolism in male and female mice.
**Methods:** POMC-GFP-TLR4^f/f^ mice were generated by crossing POMC-GFP mice with TLR4^f/f^ mice. A Cre recombinase dependent on GFP (CRE-DOG) system was delivered via adeno-associated virus (AAV) into the ARH of 8-week-old mice to delete TLR4 selectively in POMC neurons. Control mice were TLR4^f/f^ littermates injected with the same viral mix. Body weight, body composition, food intake, blood glucose, and energy expenditure (indirect calorimetry) were measured. BAT thermogenesis was assessed by rectal and interscapular temperature measurements, infrared thermal imaging, and cold exposure tests (4°C). Gene and protein expression of thermogenic markers (UCP1, PPARγ, Prdm16, Ppargc1a) and lipolysis markers (ATGL, p-HSL) were analyzed by qPCR and Western blot. Sympathetic innervation was evaluated by tyrosine hydroxylase (TH) staining and norepinephrine (NE) levels. Retrograde tracing with WGA-Cre virus was used to map POMC neuron projections to BAT. RNA-sequencing was performed on iWAT from female mice.
**Key Results:** TLR4 was expressed in approximately 26% of POMC neurons in the ARH. Viral deletion reduced TLR4 mRNA by ~50% in the ARH without affecting other brain regions. In male POMC-TLR4-KO mice, body weight decreased from 10 weeks post-injection, with reduced fat mass but unchanged lean mass. Food intake and blood glucose were normal, but heat production, O2 consumption, and CO2 production were significantly increased. Body temperature was higher during the light phase and remained elevated during cold exposure. BAT showed increased UCP1, PPARγ, Prdm16, and Ppargc1a mRNA and protein levels, along with increased UCP1 staining and smaller adipocytes. TH staining and NE levels in BAT were significantly increased, indicating enhanced sympathetic innervation. In contrast, female POMC-TLR4-KO mice showed increased body weight from 8 weeks post-injection, increased fat mass, decreased lean mass, and decreased heat production, O2 consumption, and CO2 production. BAT thermogenic markers were unchanged, and BAT weight and adipocyte size increased. iWAT and eWAT weights and adipocyte sizes increased in females, with decreased mRNA and protein levels of ATGL and p-HSL, indicating reduced lipolysis. Serum triacylglycerol was increased in iWAT, and NEFA was decreased in BAT and iWAT of females. Retrograde tracing showed that ~45% of POMC neurons projecting to BAT were tdTomato-positive, and POMC neurons coexpressed TH. RNA-Seq of iWAT from female mice identified 1,654 differentially expressed genes (453 upregulated, 1,201 downregulated), with downregulated genes enriched in immune response pathways (T-cell activation, NF-κB signaling, TLR signaling).
**Clinical Implications:** This study reveals a novel, sex-dependent role for TLR4 in POMC neurons in regulating energy balance. In males, TLR4 deletion enhances BAT thermogenesis via increased sympathetic outflow, suggesting that targeting TLR4 in POMC neurons could be a potential strategy for increasing energy expenditure and combating obesity in males. In females, TLR4 deletion impairs lipid mobilization and suppresses immune function, leading to weight gain, indicating that TLR4 modulation may have opposite effects depending on sex. These findings highlight the importance of considering sex as a biological variable in developing therapies targeting hypothalamic inflammatory pathways for obesity treatment. The study also provides evidence for direct POMC neuron projections to BAT, offering a neural circuit basis for central regulation of thermogenesis.