Ketone Body β-Hydroxybutyrate Enhances Hypothalamic Leptin and Insulin Responsiveness

Abstract

Background/Objectives: Obesity is characterized by dysregulated hypothalamic energy homeostasis and reduced central responsiveness to the anorexigenic hormones leptin and insulin. β-Hydroxybutyrate (β-HB), a major ketone body, has recently garnered attention as a signaling metabolite. However, its effects on hypothalamic leptin and insulin responsiveness remain unclear. This study aimed to investigate the effects of β-HB on hypothalamic hormone responsiveness and the associated molecular mechanisms, primarily using a high-fat diet (HFD)-induced obese mouse model. Methods: Male mice were fed an HFD to induce obesity and treated with β-HB via oral or intracerebroventricular (ICV) administration. Feeding behavior following leptin and insulin administration was evaluated, and activation of hypothalamic leptin-induced STAT3 signaling and insulin-induced Akt signaling was analyzed. In addition, mRNA expression of inflammation-related and appetite-regulating genes was assessed by quantitative PCR. Normal mice also received chronic ICV administration of β-HB from the onset of HFD feeding, and changes in body weight and cumulative food intake were measured. Results: Both oral and ICV administration of β-HB significantly enhanced the anorexigenic responses to leptin and insulin in HFD-induced obese mice. At the molecular level, leptin-induced STAT3 phosphorylation and insulin-induced Akt phosphorylation were enhanced in the hypothalamus. Gene expression analysis revealed reduced SOCS3 and TNFα expression and increased POMC expression. Furthermore, chronic ICV administration of β-HB from the onset of HFD feeding significantly suppressed body weight gain and the increase in cumulative food intake. Conclusions: This study demonstrates that β-HB improves hypothalamic leptin and insulin responsiveness in obese mice and modulates the associated molecular environment. These findings suggest that β-HB acts as a metabolically responsive signaling molecule regulating hypothalamic function, providing a basis for novel metabolic intervention strategies against obesity.

1. Introduction

Obesity has been increasing rapidly worldwide and represents a major public health challenge as a primary risk factor for metabolic disorders, including type 2 diabetes, hypertension, dyslipidemia, and cardiovascular disease [1,2,3]. The central nervous system, particularly the hypothalamus, plays a pivotal role in the regulation of energy homeostasis by integrating peripheral hormonal and nutritional signals to control food intake and metabolic functions [4,5,6]. Leptin and insulin are representative anorexigenic hormones that typically suppress food intake and promote energy expenditure. However, in obesity, hypothalamic signaling of these hormones is markedly attenuated, leading to impaired leptin and insulin responsiveness [7,8,9,10]. This central hormonal resistance promotes persistent excess energy intake and progressive weight gain. Therefore, identifying factors that restore or enhance hypothalamic leptin and insulin responsiveness is critical for understanding the pathophysiology of obesity and for developing future therapeutic strategies.

β-Hydroxybutyrate (β-HB), the predominant circulating ketone body, has long been recognized as an alternative energy substrate for the brain under conditions of energy insufficiency, such as fasting or carbohydrate restriction. More recently, β-HB has been reported to function as an endogenous signaling molecule that modulates diverse cellular processes, including neuronal activity and inflammatory responses [11,12,13]. These findings suggest that elevations in β-HB induced by dietary or metabolic alterations may influence central nervous system function, particularly within the hypothalamus, the key regulatory center of energy homeostasis. However, the extent to which β-HB directly affects hypothalamic responsiveness to leptin and insulin remains poorly understood.

In this context, the present study aimed to elucidate the effects of β-HB on hypothalamic responsiveness to anorexigenic hormones. We hypothesized that β-HB acts on the hypothalamus to enhance leptin and insulin responsiveness. To test this hypothesis, we primarily employed high-fat diet (HFD)-induced obese mice and examined the effects of both oral and intracerebroventricular (ICV) administration of β-HB. Specifically, we evaluated feeding responses following leptin and insulin administration, assessed activation of downstream hypothalamic signaling pathways (leptin-induced STAT3 and insulin-induced Akt), and analyzed changes in the expression of inflammation-related genes and appetite-regulating neuropeptides in the hypothalamus.

2. Materials and Methods

2.1. Animals and Diets

Male C57BL/6 mice (3.5 or 7 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and housed under constant conditions (22–24 °C, 50–60% relative humidity, 12 h light/dark cycle, lights on 7:00 a.m.–7:00 p.m.) with ad libitum access to food and water. Male mice were used to minimize potential variability related to the estrous cycle. To induce obesity, 3.5-week-old mice were fed an HFD (60 kcal% fat; Research Diet, D12492, New Brunswick, NJ, USA) for 3 months. In the chronic β-HB administration test, 7-week-old mice were fed a normal diet (MF, Oriental Yeast Co., Ltd., Tokyo, Japan) until the start of administration and then switched to an HFD at the beginning of administration. Animals were monitored at least once daily for general health and signs of pain or distress, with increased monitoring after surgery. Humane endpoints were predefined according to institutional guidelines. At the end of the study, animals were euthanized by cervical dislocation under isoflurane anesthesia. No unexpected adverse events occurred. All experimental procedures were approved by the Animal Committee of Meiji University (approval number: MUIACUC2022-05, approved on 6 May 2022). The experimental unit was an individual mouse for all in vivo experiments; for organotypic slice culture experiments, the experimental unit was an individual slice. Sample sizes were chosen based on our previous experience with these assays and similar published studies; no a priori sample size calculation was performed. Animals were allocated to groups after matching for age and body weight; a formal randomisation sequence was not used. Investigators were not blinded to group allocation during the experiments. A priori exclusion criteria included incorrect cannula placement, postoperative complications, or technical failure preventing outcome assessment. The final numbers for each analysis are reported in the corresponding figure legends. Assumptions for parametric analyses (normality and homogeneity of variance) were assessed in Prism 10. When assumptions were not met, appropriate non-parametric tests were used. The study protocol was not preregistered.

2.2. Cannula Implantation

Mice were anesthetized with isoflurane and placed in a stereotaxic brain fixation device. A 26-gauge single stainless steel guide cannula (C315GS-5-SPC, Plastics One, Roanoke, VA, USA) was implanted into the lateral ventricle (−0.45 mm from the bregma, ±0.9 mm lateral, and −2.5 mm from the skull). The cannula was secured with screws and dental cement. Mice were then housed individually and allowed a one-week recovery period. Cannula placement was histologically confirmed at the end of the experiment.

2.3. Central Leptin Sensitivity Test

In the oral experiment, β-HB (300 mg/kg/mouse, kindly provided by Osaka Gas Co., Ltd., Osaka, Japan) or saline was administered orally once daily at 5:00 PM for three consecutive days to age- and weight-matched mice. After the day 3 oral administration, all mice received an ICV injection of saline, followed by a 24 h measurement of food intake and body weight. On day 4, the same mice received an ICV injection of leptin (0.5 µg/mouse; PeproTech, Cranbury, NJ, USA), and measurements were repeated for the next 24 h. Data were analyzed as four conditions based on oral (saline or β-HB) and ICV (saline or leptin) treatments. In the ICV experiment, age- and body-weight–matched mice received daily ICV injections of either β-HB (10 µg/mouse) or saline at 5:00 PM for three consecutive days. From day 4 to day 6, leptin (0.5 µg/mouse, PeproTech) was additionally administered once daily via ICV injection. Food intake and body weight were measured daily. Saline-treated mice served as the control group in all in vivo experiments.

2.4. Central Insulin Sensitivity Test

In the oral experiment, mice were administered β-HB (300 mg/kg/mouse) or saline orally once daily for two consecutive days. After an overnight fast, β-HB or saline was administered orally again at 10:00 a.m. Two hours later, insulin (1.5 mU, Humulin-R, 100 U/mL, Eli Lilly, Indianapolis, IN, USA) was administered via ICV injection at the onset of refeeding. Cumulative food intake was measured at 30, 60, 120, and 240 min after insulin administration. For the ICV experiments, the fasting schedule was the same as above, but β-HB (10 µg/mouse) or saline was administered only once on the experimental day (without the 2-day pretreatment).

2.5. Immunohistochemical Staining

Male mice were placed under deep anesthesia and underwent cardiac perfusion with physiological saline and 4% paraformaldehyde. After perfusion, the brains were removed and fixed overnight in 4% paraformaldehyde. The brains were then immersed in 20% sucrose and sectioned at 30 μm. The slices were washed with PBS and incubated in 1% hydrogen peroxide and 1% sodium hydroxide for 20 min to quench endogenous peroxidase activity. After PBS washing, sections were treated with 0.3% glycine and 0.03% SDS for 10 min each, then incubated at 4 °C for 48 h in PBT (PBS containing 0.25% Triton X-100) solution and 3% normal donkey serum (NDS; Jackson ImmunoResearch Labs, West Grove, PA, USA) containing 0.1% sodium azide, using a phospho-STAT3 antibody (1:3000, Cell Signaling Technology, Danvers, MA, USA, 9131) at 4 °C for 48 h. Sections were then incubated at room temperature for 1 h with a biotinylated secondary antibody (1:1000, Jackson ImmunoResearch Labs, 711-065-152), followed by incubation for 1 h with an avidin-biotin-peroxidase complex (ABC) kit (1:1000, Vector Laboratories, Newark, CA, USA, PK-6100). Finally, sections were stained with 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO, USA, 7304). Sections were mounted, dehydrated in graded ethanol, washed with xylene, and coverslipped. Images were captured using a Nikon ECLIPSE Ci-L plus microscope (Nikon Corporation, Tokyo, Japan), background-corrected in Adobe Photoshop, and DAB intensity was quantified using ImageJ software (NIH, Bethesda, MD, USA; version 1.54g, Java 1.8.0_345). For relative quantification, intensities were normalized to the Saline+Saline group and expressed as fold change.

2.6. Western Blot Analysis

The hypothalamus was prepared using a brain matrix (1 mm thickness), immediately flash-frozen in liquid nitrogen, and stored at −80 °C. Total protein was extracted using a lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA, 87788) supplemented with a protease and phosphatase inhibitor cocktail (1:100, Thermo Fisher Scientific, 78442), and quantified using the BCA protein assay (Thermo Fisher Scientific, 23227). Equal amounts of protein were separated by SDS-PAGE and transferred to a Nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA, 1620115). Primary antibodies used were anti-phospho-Akt antibody (1:1000, Cell Signaling Technology, 4060), anti-Akt antibody (1:1000, Cell Signaling Technology, 4691), and anti-β-actin antibody (1:1000, Cell Signaling Technology, 4970), incubated overnight at 4 °C. After incubation for one h with HRP-conjugated anti-rabbit IgG secondary antibody (1:5000, Cell Signaling Technology, 7074), immunoreactive bands were visualized using a high-sensitivity chemiluminescence detection kit (Nacalai Tesque, Kyoto, Japan, 02230) and captured with a WSE-6100 LuminoGraph I imaging system (ATTO Corporation, Tokyo, Japan). Relative protein levels were determined by normalizing band intensities to the Saline + Saline group using ImageJ software and are expressed as fold change.

2.7. RNA Extraction and Quantitative Real-Time PCR

Hypothalamic samples were collected from C57BL/6 mice that received central injections of β-HB (10 μg/mouse) or saline for three consecutive days. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized using the PrimeScript^® RT Master Mix (Takara, Osaka, Japan). Quantitative real-time PCR was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and THUNDERBIRD^® qPCR Mix (Toyobo, Osaka, Japan) with gene-specific primer sets for SOCS3, TNFα, IL6, POMC, AgRP, NPY, XBP1s, BIP, ATF4, and CHOP. β-Actin was used as the reference gene. PCR amplification consisted of 40 cycles of denaturation at 95 °C for 15 s, followed by annealing and extension at 60 °C for 60 s. mRNA expression levels were normalized by dividing the efficiency-corrected expression value of each target gene by that of the reference gene. The resulting values were expressed as fold changes relative to the mean level of the control group. Primer sequences used were as follows: SOCS3 (F-CACCTGGACTCCTATGAGAAAGTG, R-GAGCATCATACTGATCCAGGAACT), TNFα (F-CCCTCACACTCAGATCATCTTCT, R-GCTACGACGTGGGCTACAG), IL-6 (F-TAGTCCTTCCTACCCCAATTTCC, R-TTGGTCCTTAGCCACTCCTTC), POMC (F-GAGGCCACTGAACATCTTTGTC, R-GCAGAGGCAAACAAGATTGG), AgRP (F-CGGCCACGAACCTCTGTAG, R-CTCATCCCCTGCCTTTGC), NPY (F-TCCGCTCTGCGACACTAC, R-GGGACAGGCAGACTGGTT), XBP1s (F-CTGAGTCCGAATCAGGTGCAG, R-GTCCATGGGAAGATGTTCTGG), BIP (F-TTCAGCCAATTATCAGCAAACTCT, R-TTTTCTGATGTATCCTCTTCACCAGT), ATF4 (F-GGGTTCTGTCTTCCACTCCA, R-AAGCAGCAGAGTCAGGCTTTC), CHOP (F-CCACCACACCTGAAAGCAGAA, R-AGGTGAAAGGCAGGGACTCA), and β-actin (F-CTGCGCAAGTTAGGTTTTGTCA, R-TGCTTCTAGGCGGACTGTTACTG).

2.8. Organotypic Hypothalamus Slice Culture

Hypothalamic slices were prepared as previously described [14]. C57BL/6 mouse pups (postnatal day 8–11) were decapitated, and the brains were rapidly removed. Hypothalamic tissue was sectioned at a thickness of 250 µm in ice-cold Gey’s balanced salt solution supplemented with glucose (0.5%) and KCl (30 mM) using a vibratome (Leica VT1200S, Wetzlar, Germany). Coronal slices containing the arcuate nucleus (ARC) were then placed on Millicell-CM filters (Millipore, Burlington, MA, USA; pore size, 0.4 µm; diameter, 30 mm) and maintained at the air–medium interface in minimum essential medium supplemented with heat-inactivated horse serum (25%), glucose (32 mM), and GlutaMAX (2 mM). Slices were typically cultured for 8–10 days in standard medium, with medium changes performed 2–3 times per week. After 10 days of culture, healthy slices were selected and randomly assigned to experimental and control groups.

2.9. Statistical Analysis

All data are presented as mean ± standard error of the mean (SEM). One-way or two-way analysis of variance (ANOVA) was performed, followed by Tukey’s or Sidak’s post hoc tests for multiple comparisons. Two-group comparisons were performed using two-tailed unpaired Student’s t-tests. All statistical analyses were conducted using Prism 10 (GraphPad Software, San Diego, CA, USA; version 10.6.1 (892), Windows 64-bit), and p < 0.05 was considered statistically significant. Sample sizes (n) are provided in each figure legend and denote the number of mice per group unless otherwise specified. Where possible, exact p-values are reported; otherwise, significance thresholds are indicated.

3. Results

3.1. Oral Administration of β-HB Enhances Hypothalamic Leptin and Insulin Responsiveness in HFD-Fed Obese Mice

To examine the effects of oral β-HB administration on hypothalamic anorexigenic hormone responsiveness in HFD-induced obese mice, we evaluated the anorexigenic effects of leptin and insulin following ICV administration in mice treated with β-HB or saline. In saline-treated mice, neither leptin nor insulin induced a statistically significant reduction in food intake (Figure 1A,C,D), and leptin did not significantly reduce body weight (Figure 1B). In contrast, oral β-HB pretreatment exhibited a marked reduction in 24 h cumulative food intake in response to leptin (Figure 1A) and a significant decrease in body weight 24 h after leptin administration (Figure 1B). In the insulin-induced refeeding test, oral β-HB pretreatment significantly enhanced insulin’s anorexigenic effect, leading to reduced refeeding at 240 min and a lower cumulative food intake compared with saline-treated controls under insulin-stimulated conditions (Figure 1C,D). These results suggest that oral administration of β-HB enhances hypothalamic responsiveness to both leptin and insulin in HFD-induced obese mice.

3.2. ICV Administration of β-HB Enhances Hypothalamic Leptin Responsiveness in HFD-Fed Obese Mice by Promoting Hypothalamic STAT3 Phosphorylation

Because oral β-HB administration enhanced hypothalamic leptin and insulin responsiveness in HFD-induced obese mice, we next examined whether β-HB directly acts within the hypothalamus to enhance anorexigenic hormone signaling. For this purpose, β-HB was administered by ICV injection to HFD-induced obese mice. Leptin-induced body weight loss and the reduction in cumulative food intake were both significantly greater in the β-HB-treated group than in the saline-treated group (Figure 2A,B). Consistently, immunohistochemical analysis of the arcuate nucleus of the hypothalamus revealed that leptin-stimulated pSTAT3 levels were significantly higher in the β-HB-treated group under the same leptin-stimulated conditions (Figure 2C,D). Taken together, these results indicate that β-HB acts within the hypothalamus of HFD-induced obese mice and enhances leptin responsiveness, at least in part by potentiating leptin signaling.

3.3. ICV Administration of β-HB Enhances Hypothalamic Insulin Responsiveness in HFD-Fed Obese Mice by Promoting Akt Phosphorylation

Because Figure 2 demonstrated that β-HB directly acts on the hypothalamus to enhance leptin signaling, we next examined whether similar effects are observed in the insulin signaling pathway. To this end, we evaluated the effects of ICV administration of β-HB on insulin-induced anorexigenic responses and downstream signaling activation in HFD-fed obese mice. In the refeeding test, insulin did not significantly suppress food intake in saline-treated mice at any time point, whereas mice treated with β-HB showed a significant suppression of food intake at 240 min after refeeding, resulting in lower cumulative food intake compared with saline-treated controls under insulin-stimulated conditions (Figure 3A,B). To further assess activation of insulin downstream signaling in the hypothalamus, Western blot analysis of phosphorylated Akt (p-Akt) and total Akt was performed. Consistent with the behavioral results, insulin administration did not significantly increase the p-Akt/Akt ratio in saline-treated mice, whereas a significant increase in the p-Akt/Akt ratio was observed in β-HB-treated mice following insulin administration (Figure 3D). However, the p-Akt/Akt ratio did not differ significantly between the saline- and β-HB-treated groups under insulin-stimulated conditions. Taken together, these results suggest that β-HB enhances hypothalamic insulin responsiveness in obesity, at least in part by facilitating activation of the hypothalamic insulin signaling pathway.

3.4. Long-Term ICV Administration of β-HB Suppresses HFD-Induced Body Weight Gain and Hyperphagia in Normal Mice

Based on the results shown in Figure 1, Figure 2 and Figure 3, short-term β-HB administration enhanced hypothalamic leptin and insulin responsiveness in HFD-fed obese mice. To investigate the long-term anti-obesity potential of central β-HB, we next conducted a chronic ICV administration study in normal mice initiated at the onset of HFD feeding. Specifically, normal mice received daily ICV administration of β-HB or saline for 28 consecutive days, beginning at HFD initiation, and changes in body weight and cumulative food intake were monitored. In the saline-treated group, body weight progressively increased. However, β-HB-treatment significantly suppressed the body weight gain from day 14 onward (Figure 4A). Similarly, cumulative food intake was significantly lower in the β-HB-treated group, with the difference becoming statistically significant from day 21 (Figure 4B). These results demonstrate that chronic ICV administration of β-HB suppresses both food intake and body weight gain in mice under HFD feeding conditions. The sustained anti-hyperphagic effect suggests that the hypothalamic enhancement of anorexigenic hormone responsiveness by β-HB may contribute to its long-term anti-obesity action.

3.5. Effects of ICV Administration of β-HB on Hypothalamic Gene Expression in HFD-Fed Obese Mice

Based on the findings in Figure 1, Figure 2, Figure 3 and Figure 4, β-HB enhanced hypothalamic leptin and insulin actions and exerted short- and long-term suppressive effects on food intake and body weight gain. To determine whether these effects are accompanied by changes in hypothalamic gene expression related to inflammation and appetite regulation, we next analyzed hypothalamic mRNA expression in HFD-fed obese mice after 3 days of ICV β-HB administration. Expression levels of SOCS3 and TNFα were significantly reduced in the β-HB-treated group compared with those in the saline-treated group (Figure 5A,B). IL6 showed a decreasing trend, but no significant difference was observed (Figure 5C). Expression of the anorexigenic neuropeptide POMC was significantly increased in the β-HB-treated group compared with the saline-treated group (Figure 5D). In contrast, no significant differences were detected in the expression of the orexigenic genes AgRP or NPY between the two groups (Figure 5E,F). These results demonstrate that short-term ICV administration of β-HB modifies hypothalamic gene expression in HFD-fed obese mice, characterized by the reduced expression of inflammation-related genes and the increased expression of the anorexigenic neuropeptide gene POMC.

4. Discussion

Obesity is known to disrupt hypothalamic regulation of energy homeostasis and to reduce central responsiveness to the anorexigenic hormones leptin and insulin [7,8,9,10]. Therefore, enhancing responsiveness to these anorexigenic hormones is considered an important strategy for anti-obesity interventions. Although ketone bodies, particularly β-HB, have traditionally been viewed as alternative energy substrates during starvation, they have recently attracted attention as signaling metabolites with diverse biological functions [15,16]. In this study, we primarily used an HFD-induced obese mouse model to evaluate the effects of both ICV and oral administration of β-HB on hypothalamic anorexigenic hormone responsiveness, to explore its potential as a hypothalamus-targeted intervention for obesity.

First, β-HB tended to restore the behavioral responses to leptin and insulin in HFD-fed obese mice. Oral administration of β-HB to HFD-fed mice significantly enhanced the anorexigenic effects of ICV leptin and insulin administration (Figure 1), suggesting its capability to act on the brain after gastrointestinal absorption. Furthermore, in assessing direct central effects via ICV administration, β-HB significantly promoted leptin-induced body weight reduction and leptin- and insulin-induced appetite suppression (Figure 2 and Figure 3). These results indicate that β-HB partially restores the anorexigenic actions of leptin and insulin that are attenuated by obesity. Consistent with the reduction in food intake, immunohistochemical analyses revealed that β-HB markedly enhanced leptin-induced STAT3 phosphorylation (Figure 2). STAT3 is a key downstream mediator of leptin receptor signaling, and its phosphorylation is reduced under obese conditions [17,18,19,20]. Notably, the enhancing effect of β-HB on STAT3 phosphorylation was also reproduced in ex vivo experiments using hypothalamic organotypic slice cultures (Figure S1). In contrast, Western blot analyses showed that insulin stimulation did not induce a robust increase in the p-Akt/Akt ratio in the saline-treated group. In contrast, β-HB treatment significantly promoted insulin-induced Akt phosphorylation (Figure 3). Activation of the PI3K/Akt pathway is essential for insulin-mediated suppression of food intake and is reported to be impaired in obesity [21,22,23]. Thus, these results suggest that β-HB enhances hypothalamic leptin and insulin signaling, thereby improving leptin and insulin responsiveness, which are diminished in the obese state.

To further investigate how β-HB affects the hypothalamic molecular environment in obese mice, we next performed gene expression analyses. β-HB administration significantly reduced the mRNA expression levels of SOCS3 and TNFα (Figure 5A,B). SOCS3 is a negative regulator of leptin receptor signaling that suppresses STAT3 phosphorylation and contributes to hypothalamic leptin resistance [24,25,26,27]. The observed reduction in SOCS3 expression, together with increased STAT3 phosphorylation, is consistent with previous reports and suggests improved leptin responsiveness through attenuation of inhibitory signaling. However, because we did not directly manipulate SOCS3 and did not perform time-course or cell-type–resolved analyses, these findings should be interpreted as associative rather than causal. Future studies using SOCS3-targeted approaches (e.g., knockdown/overexpression) and cell-type–specific analyses will be required to establish causality. In addition, inflammatory factors, such as TNFα, have been implicated in obesity-associated impairment of hypothalamic leptin and insulin signaling [28]. Therefore, the reduction in the expression of these genes by β-HB may represent one of the molecular bases supporting the improved downstream signaling observed in this study. These findings are also consistent with previous reports describing the anti-inflammatory effects of β-HB [29,30].

Furthermore, analysis of appetite-regulating genes revealed that β-HB administration significantly increased POMC expression (Figure 5D). POMC neurons are located in the hypothalamus and play a central role in appetite suppression, as major targets of leptin and insulin [31,32]. In contrast, expression levels of AgRP and NPY were not significantly altered (Figure 5E,F), suggesting that the effect of β-HB may not be uniform across all appetite-related genes but selectively modifies pathways closely associated with hormone responsiveness. In addition, we analyzed the expression of endoplasmic reticulum (ER) stress-related genes, but no significant changes were observed with β-HB administration (Figure S2).

Regarding the diverse biological functions of β-HB, it has been reported that these functions are mediated by GPR109A signaling, HDAC inhibition, or modulation of hypothalamic microglial activity [12,16,33,34]. However, the present study did not directly verify these molecular pathways or cell-type-specific mechanisms, and further studies are required to clarify how β-HB alters the hypothalamic molecular environment.

Moreover, chronic ICV administration of β-HB significantly suppressed body weight gain and cumulative food intake under HFD feeding conditions (Figure 4). These results suggest that intervention from the early stages of obesity development may mitigate excessive energy intake and body weight accumulation.

Overall, this study demonstrates that β-HB enhances hypothalamic responsiveness to leptin and insulin in an HFD-induced obese mouse model. Nevertheless, several limitations should be acknowledged. First, we did not directly quantify plasma, cerebrospinal fluid, or brain β-HB concentrations following oral administration in the present experiments. For context, nutritional ketosis is commonly defined as circulating ketone levels > 0.5 mM and can reach ~3–5 mM with ketogenic diets; oral exogenous d-β-hydroxybutyrate has been reported to elevate blood ketones to millimolar levels in humans [35]. Future studies measuring plasma/CSF/brain β-HB time courses in our model will be important to define exposure–response relationships. Second, although ICV administration is useful for evaluating direct hypothalamic actions of β-HB, it does not fully reflect physiological brain β-HB dynamics. In particular, ICV delivery bypasses peripheral metabolism and transport across the blood–brain barrier and therefore represents a pharmacological route. Also, in the case of oral administration, the quantitative relationship between blood ketone body concentration and brain uptake has not been sufficiently examined. Third, the causal links between β-HB–induced reductions in SOCS3 expression and activation of STAT3/Akt signaling, as well as the specific target cell types involved, remain to be elucidated in future studies. Fourth, immunohistochemical and Western blot analyses were performed with relatively small sample sizes; these findings should therefore be interpreted with appropriate caution. Finally, we focused on male mice in this study, and potential sex-specific effects should be addressed in future work.

5. Conclusions

In conclusion, this study demonstrates that β-HB enhances anorexigenic responses to leptin and insulin in the hypothalamus of HFD-induced obese mice. β-HB administration potentiated activation of leptin-induced STAT3 signaling and insulin-induced Akt signaling, accompanied by reduced expression of inflammation-related genes, such as SOCS3 and TNFα, and increased expression of the anorexigenic neuropeptide POMC. In addition, chronic ICV administration of β-HB from the onset of HFD feeding suppressed body weight gain and cumulative food intake, suggesting that central modulation of the metabolic environment by β-HB may contribute to the regulation of energy intake and body weight gain during obesity development. Collectively, these findings indicate that β-HB functions not only as an energy substrate but also as a metabolically responsive signaling metabolite that modulates hypothalamic function, providing a basis for novel nutritional and metabolic intervention strategies against obesity.