Primary cilia in the hypothalamic AgRP neurons mediate metabolic effects of butyrate

Domain: neuroscience
Published: 2026-04-09
Editorial outcome: minor_revision

Abstract
The microbiota-derived short-chain fatty acid (SCFA), butyrate influences host metabolism beyond the gut, including central regulation of appetite and energy homeostasis. However, mechanistic insights into central butyrate metabolic actions are undetermined. Here, we demonstrated that primary cilia of agouti-related peptide (AgRP) neurons in the hypothalamic arcuate nucleus are essential for butyrate’s anorexigenic effects and glucose homeostasis. Notably, peripheral or central butyrate administration to the mice significantly enhanced hypothalamic ciliogenesis, possibly through increased histone acetylation and activation of ciliogenic transcription factors, leading to suppressed food intake and improved metabolism. Disrupting cilia in the hypothalamus or specifically in AgRP neurons, but not in ventromedial hypothalamus neurons, abolished these effects. At the neuronal level, butyrate inhibited AgRP neuron activity, and this effect was markedly reduced following primary cilia deletion, indicating that cilia are crucial for butyrate’s inhibitory action. These findings establish primary cilia in AgRP neurons as essential mediators of butyrate’s metabolic effects.

Introduction
Microbiota-derived short-chain fatty acids (SCFAs), produced through the bacterial fermentation of indigestible dietary fiber, are suggested to play an essential role in gut-brain communication1,2. In addition to their local effects in the colon, increasing evidence supports that SCFAs, particularly butyrate, circulate beyond the gut to act on other organs, including the central nervous system (CNS), thereby affecting systemic homeostasis3-10. At physiological levels, butyrate is proposed to modulate brain functions indirectly through immune, endocrine, and vagal pathways1. However, at the higher concentrations used in most in vivo studies, butyrate may cross the blood-brain barrier (BBB), accumulate in the brain, and directly affect CNS functions11-13. Furthermore, SCFAs have been shown to regulate energy metabolism, potentially through central homeostatic mechanisms3,4,7,8,10,14. However, direct evidence that butyrate acts directly on central neurons (CNs) to regulate whole-body metabolism is lacking, and the molecular mechanisms underlying these CNS-mediated effects of butyrate remain to be elucidated.

Butyrate can influence host metabolism either as a cellular energy substrate or by altering host cellular functions and epigenetics. It acts on its receptors, such as G-protein coupled receptors (GPCRs) GPR109A and GPR41/43, or by inhibiting histone deacetylase (HDAC) enzymes2,15-17. HDACs are important factors in neural development and are associated with many brain disorders18. Butyrate's inhibition of HDACs has been implicated in its beneficial effects across a wide range of neurological diseases19. Recent studies suggest that HDACs are involved in the dysfunction of primary cilia, small organelles that act as cellular hubs regulating diverse signaling pathways under various health and disease conditions20-27. Furthermore, many cellular signaling pathways evoked by primary cilia involve different GPCRs, and primary cilia function is required for the localization and trafficking of metabolic receptors, including GPCRs, at the ciliary membrane28-32. Hypothalamic primary cilia have recently been demonstrated to play a critical role in the regulation of whole-body energy homeostasis29,32-39. This body of evidence raises the intriguing question of whether the SCFA butyrate directly modulates hypothalamic primary cilia function to promote its metabolic actions.

In the current study, we examined this hypothesis and demonstrated that primary cilia in hypothalamic AgRP neurons mediate the effects of butyrate on appetite and glucose homeostasis. We found that butyrate treatment, whether administered peripherally or centrally, significantly promoted hypothalamic ciliogenesis and improved whole-body glucose homeostasis, primarily through the suppression of energy intake. These metabolic effects of butyrate were abolished by disrupting primary cilia throughout the hypothalamus or specifically in AgRP neurons, but not by disrupting cilia in ventromedial hypothalamic (VMH) neurons. At the cellular level, we observed that the deletion of primary cilia specifically in AgRP neurons attenuated butyrate's inhibitory effect on these neurons. Collectively, these findings indicate that the SCFA butyrate directly targets primary cilia in hypothalamic AgRP neurons to exert its anorexigenic effect and confer metabolic benefits.

Results
Central butyrate regulates food intake and glucose homeostasis
To determine whether central administration of butyrate is sufficient to elicit systemic metabolic effects, 2.5 µL of butyrate (1.25 µg/µL) was administered into the hypothalamic third ventricle (3V) of mice via intracerebroventricular (ICV) injection. The dose was selected to enhance histone acetylation without causing adverse neurological effects, such as seizures, sluggishness, or impaired feeding behavior40,41. In male mice maintained on a normal chow diet, 3 weeks of ICV administration of butyrate led to a significant reduction in body weight accompanied by decreased food intake (Figs. S1a-d). Prolonged ICV butyrate treatment also protected male mice from high-fat diet (HFD)-induced weight gain and adipose hypertrophy in major fat depots, including epididymal (eWAT) and subcutaneous white adipose tissues (scWAT) (Figs. 1a-e). In female mice fed normal chow, ICV butyrate administration was limited to 1 week, rather than 3 weeks as in males, because their lower body weight (~24 g) made them more vulnerable to repeated long-term injections that could compromise their health. Under this short-term regimen, only trend toward decreased body weight was observed, suggesting that 1 week of butyrate treatment is insufficient to induce significant body weight reduction in female (Figs. S1e–g). After 3 weeks of treatment, HFD-fed male mice receiving butyrate showed improved glucose tolerance and insulin sensitivity, although basal glucose and insulin levels remained unchanged (Figs. 1f-i).

To further explore the metabolic effects of central butyrate, we conducted indirect calorimetry to assess its impact on energy homeostasis. Mice receiving butyrate displayed a significant reduction in daily food intake without any notable changes in energy expenditure or locomotor activity (Figs. 1j-n). Concomitant with the significant reduction in food intake, butyrate treatment resulted in the downregulation of hypothalamic neuropeptide Y (Npy) and Agrp gene expresssion, both of which are crucial orexigenic neuropeptides (Fig. 1o). In addition, central butyrate administration was linked to reduced plasma leptin levels and a targeted enhancement of hypothalamic Signal transducer and activator of transcription 3 (STAT3) activation, collectively suggesting an increase in leptin sensitivity (Figs. 1p, q).

Butyrate directly modulates hypothalamic primary cilia
To seek direct evidence of butyrate actions in the hypothalamus, we examined the expression of its cellular targets in the hypothalamus both in vivo and in vitro. We found that several histone deacetylases (HDACs) including Class I and IIa, are expressed in both the mouse hypothalamus and hypothalamic N38 cells (Fig. S2a). We further examined whether butyrate could also directly induce neuronal HDAC inhibition by assessing histone acetylation under butyrate treatment. We observed that butyrate treatment significantly increased acetylation at several lysine residues on histones 3 (H3) and 4 (H4) in a dose- and time-dependent manner (Figs. 2a and S2b). This effect of butyrate on HDAC inhibition was evident at concentrations as low as 10 μM and became more prominent at concentrations equal to or higher than 1 mM (Fig. 2a). Notably, this HDAC inhibitory effect was specific to butyrate, as the ketone body β-hydroxybutyrate (BHB) did not alter histone acetylation even at high doses (Fig. S2c).

HDAC inhibition is associated with cilia expression, and hypothalamic cilia are known to be critical organelles for whole-body energy homeostasis21-25,38,39,42. Therefore, we next investigated whether butyrate is connected to primary cilia function in hypothalamic neurons. We first treated cells with 1 mM butyrate for 24 hours and examined the expression of several transcriptional regulators involved in ciliogenesis. As shown in Fig. 2b, butyrate treatment significantly induced the expression of regulatory factor X (Rfx) isoforms 2 and 3, both known as cilia-promoting transcription factors43,44. Additionally, it markedly enhanced the transcription of various ciliary component genes involved in intraflagellar transport and the kinesin superfamily, resulting in a significant increase in both the number and length of primary cilia in hypothalamic N38 cells (Figs. 2c, d). Furthermore, direct ICV injection of butyrate into the third ventricle significantly enhanced histone acetylation and induced the expression of Foxj1, Rfx2, and Rfx3, and was notably associated with elongated primary cilia in several hypothalamic nuclei, including the arcuate nucleus (ARC) and the VMH (Figs. 2e-g).

Given that butyrate crosses the blood-brain barrier and potentially influences epigenetic processes in the brain45, we investigated whether the metabolic benefits of peripheral butyrate administration are mediated through HDAC inhibition and associated alterations in primary cilia function within the hypothalamus. To test this, HFD-fed mice were treated with butyrate either orally or via intraperitoneal (i.p.) injection. For oral administration, sodium butyrate was supplied in the drinking water at a concentration of 100 mM, whereas mice in the i.p. group received daily injections of sodium butyrate at a dose of 500 mg/kg body weight. As shown in Figs. 2h-j (oral) and Fig. S2d-h (i.p.), butyrate treatment resulted in reduced body weight, decreased eWAT hypertrophy, and lower blood glucose levels. Notably, these metabolic improvements were accompanied by increased histone acetylation, along with a significant elongation of primary cilia in hypothalamic neurons (Figs. 2k, l and S2g, h). These findings suggest that butyrate may modulate histone acetylation and primary cilia function in the hypothalamus, contributing, at least in part, to its metabolic effects.

Hypothalamic primary cilia are essential for the metabolic effects of butyrate
We next investigated whether hypothalamic primary cilia are required for the metabolic actions of butyrate. To examine this, we stereotaxically injected recombinant adeno-associated virus serotype 2 (AAV2) — either encoding Cre recombinase fused to green fluorescent protein (AAV2-Cre-GFP) or lacking Cre (AAV2-GFP, control) — into the 3V of the hypothalamus in mice carrying floxed alleles of intraflagellar transport protein 88 (IFT88F/F) and a Cre-inducible tdTomato reporter at the ROSA26 locus (ROSA26-tdTomatoF/+) (Fig. 3a). Following vehicle or oral butyrate treatment (100 mM), we observed butyrate-induced primary ciliogenesis in AAV2-GFP–injected mice, whereas AAV2-Cre-GFP–injected mice showed specific deletion of primary cilia in the mediobasal hypothalamus, including the ARC and VMH (Figs. 3b and S3a, b). Different from the control virus-injected IFT88F/F mice, the targeted deletion of primary cilia prevented hypothalamic ciliogenesis in response to butyrate (Fig. 3b).

Interestingly, the metabolic benefits of oral butyrate—such as reductions in body weight, fat mass, and food intake, as well as improvements in glucose and insulin homeostasis—observed in the control virus-injected group (Figs. 3c–h) were largely abolished in mice with disrupted primary cilia in the mediobasal hypothalamus (Figs. 3i–n). Notably, the beneficial effects on food intake and glucose tolerance were lost and became indistinguishable from those in control mice (Figs. 3e, g, k, m). Unexpectedly, butyrate-treated AAV2-Cre-GFP–injected mice exhibited increased body weight (Figs. 3c, d, i, j) and showed impaired insulin tolerance at a specific time point (Figs. 3h, n). These findings strongly suggest that hypothalamic primary cilia are crucial for mediating the metabolic effects of butyrate, particularly in the regulation of body weight and glucose homeostasis.

Primary cilia in the AgRP neuron of the ARC are required for the metabolic actions of butyrate
We next sought to identify the crucial neuronal populations in the hypothalamus whose primary cilia are responsible for mediating the metabolic effects of butyrate35,37,38. Among the hypothalamic regions, ARC and VMH are well-established centers involved in primary cilia-mediated regulation of energy metabolism30,36-39. Steroidogenic factor-1 (SF-1) neurons in the VMH are a well-characterized neuronal population that regulates energy balance through primary cilia38. In the ARC, the orexigenic peptides AgRP and NPY, which are key regulators of feeding behavior, were suppressed by butyrate treatment (Fig. 1o). Therefore, we generated two distinct knockout models to target SF-1 neurons in the VMH and AgRP neurons in the ARC, respectively. This was achieved by crossing IFT88F/F mice with either SF-1-cre (IFT88 KOSF-1) or AgRP-cre (IFT88 KOAgRP) mice (Fig. S4a). The SF-1 neuron- and AgRP neuron-specific IFT88 knockout mice exhibited no detectable cilia in these neurons, confirming the successful and targeted knockout (Figs. S4b, c).

In IFT88 KOSF-1, central butyrate treatment significantly reduced body weight and food intake (Figs. 4a-c). The metabolic effects of central butyrate on leptin and insulin homeostasis (Figs. 4d, e) were also intact in IFT88 KOSF-1 mice. Furthermore, butyrate improved glucose and insulin tolerance (Figs. 4f, g). However, these metabolic effects of butyrate were completely abolished in IFT88 KOAgRP mice, highlighting that the primary cilia function in AgRP neurons is essential for the anorexigenic action of butyrate (Figs. 4h-n).

Butyrate inhibits AgRP neurons and requires primary cilia for its effect
Given that the metabolic effects of butyrate were abolished in IFT88 KOAgRP mice, we next investigated whether primary cilia are required for butyrate’s action on AgRP neurons using whole-cell patch clamp recordings (Fig. 5a). Primary cilia signaling has been reported to modulate neuronal excitability through receptors, ion channels, and secondary messengers, with electrophysiological evidence supporting both synaptic and intrinsic regulatory mechanisms37,46. To address this, we recorded the resting membrane potential (RMP) and spontaneous action potential (sAP) firing frequency in AgRP neurons from wild type (WT, AgRP-cre; tdTomatoF/+) and AgRP neuron-specific primary cilia knockout IFT88 KOAgRP (AgRP-cre; IFT88F/F; tdTomatoF/+) mice (Figs. 5b-d). Baseline measurements revealed no significant differences in either RMP or sAP firing frequency between the two groups (Supplementary Table 1).

To assess primary cilia’s role in butyrate-induced regulation of AgRP neuron activity, we bath applied 1mM butyrate for 3-4 minutes and observed variable response in both WT and IFT88 KOAgRP neurons (Fig. 5e). The change in RMP following butyrate treatment was measured as the difference between the RMP before and after application. Upon butyrate application, 71% of tested WT neurons (n=15/21), exhibited changes in RMP, predominantly hyperpolarization whereas 52% of tested IFT88 KOAgRP neurons (n=12/23) responded, showing either hyperpolarization (43%) or depolarization (9%) (Figs. 5f, h-i). The average hyperpolarization in WT neurons was -10.5 ± 2.13 mV (n=15), while IFT88 KOAgRP neurons showed a slightly reduced average hyperpolarization of -8.07 ± 1.55 mV (n=10) (Fig. 5j). Depolarization observed only in IFT88 KOAgRP neurons (n=2/23) displayed average value of 5.07 ± 1.75 mV.

Additionally, butyrate also modulated sAP frequency. In most cases, the inhibition of sAP frequency was associated with hyperpolarization. However, in some instances, sAP frequency was altered without change in RMP, showing variable effect including increase, decrease, or no change. Based on these observed effects on RMP and sAP activity, neurons were classified as inhibited, excited or unaffected in both WT and IFT88 KOAgRP groups. Butyrate application inhibited neuronal activity (via hyperpolarization and/or decreased sAP frequency) in the majority of WT neurons (81%) compared to IFT88 KOAgRP neurons (48%). Conversely, a higher percentage of IFT88 KOAgRP neurons (22%) were excited (via depolarization and/or increased sAP frequency) relative to WT neurons (14%) (Figs. 5g, k-l). However, no significant difference was observed between the two groups in the inhibition of sAP frequency by butyrate (Fig. 5m).

Primary cilia facilitate butyrate-mediated inhibitory signaling in AgRP neurons
Next, we investigated whether primary cilia in AgRP neurons contribute to synaptic activity by comparing spontaneous inhibitory post-synaptic currents (sIPSCs) and spontaneous excitatory post-synaptic currents (sEPSCs) between the two groups. Notably, IFT88 KOAgRP neurons exhibited significantly reduced sIPSCs frequency and amplitude compared to WT neurons (Figs. 6a-c, and Supplementary Table 1), whereas sEPSCs frequency or amplitude were unchanged (Figs. 6j-l, and Supplementary Table 1). Based on these findings, we speculated that butyrate’s effect on AgRP neuronal excitability may depend upon synaptic transmission. To test this, we examined butyrate’s impact on sIPSCs and sEPSCs in WT and IFT88 KOAgRP neurons. In WT AgRP neurons, bath application of butyrate significantly increased sIPSCs frequency (Figs. 6d, e); however, this effect was not observed in IFT88 KOAgRP neurons (Figs. 6g, h and Supplementary Table 2). Butyrate did not significantly alter sIPSCs amplitude in either group (Figs. 6f, i and Supplementary Table 2). Regarding sEPSCs, butyrate induced no significant changes in frequency or amplitude in either neuron type (Figs. 6m-r and Supplementary Table 2). Taken together, these results indicate that the inhibitory effect of butyrate on AgRP neurons is highly dependent on the presence of primary cilia and may underline its anorexigenic actions.

Methods
Reagents and primary antibodies
Sodium butyrate and β-hydroxybutyrate (BHB) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in Ultra PureTM water (Invitrogen, Waltham, MA, USA) in stock solutions. Stock solutions of butyrate and BHB were filtered through a 0.2 µm membrane and stored at -80 °C. Working solutions were freshly prepared by diluting the stock solutions with UltraPureTM water. Trichostatin A was purchased from Sigma and dissolved in DMSO. All chemicals were obtained from Sigma unless stated otherwise.
Antibodies against Histone 3 (Cat.no.4499), AcH3K9 (Cat.no.9649), AcH3K18 (Cat.no.13998), AcH3K27 (Cat.no.8173), STAT3 (Cat.no.9139), phosphorylated-STAT3 (Cat.no.9145), and a kit of HDACs antibodies (Cat.no.9928) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against Histone 4 (Cat.no.ab177840), AcH4K5 (Cat.no.ab519987) and AcH4K12 (Cat.no.ab177793) were obtained from Abcam (Cambridge, UK). Antibodies against Adenylyl cyclase III (Cat.no.sc-588) and GAPDH (Cat.no.sc-32233) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Acetylated-α-tubulin (Cat.no.T7451) antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture
The hypothalamic N38 cell line was purchased from Cedarlane (Ontario, Canada) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S). For butyrate treatment, cells were incubated with butyrate at 0.01 to 20 mM final concentration for 24 hours period before harvested unless stated otherwise. All of cell culture experiments were performed in biological triplicate unless stated otherwise.

Animal
The Institutional Animal Care and Use Committee (IACUC) at the Avison Biomedical Research Center, Yonsei University, approved all animal protocols utilized in this research (Approval No. 2022-0056). All experiments were performed using inbred C57BL/6J mice. To generate hypothalamic primary cilia knockout mice, mice carrying the Ift88 floxed allele (Jackson Laboratory, Strain no.022409) were crossed with either AgRP-ires-cre (Jackson Laboratory, Strain no.012899) or SF-1-cre (Jackson Laboratory, Strain no.012462) mice. Whenever stated, mice were backcrossed with the Cre-dependent reporter Gt(ROSA)26Sor (Ai9) mouse line (Jackson Laboratory, B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, Strain no.007905) to identify cells with Cre recombinase activity using td-Tomato fluorescence. For comparisons of primary cilia length between hypothalamic AgRP/NPY and POMC neurons, heterozygous transgenic mice expressing humanized Renilla green fluorescent protein (hrGFP) under the control of the Npy (Jackson Laboratory, Strain no.006417) or Pomc1 promoter (Jackson Laboratory, Strain no.006421) were used. Both male and female mice were included in metabolic phenotyping experiments, whereas male mice were used for molecular, histological, and electrophysiological analyses. Mice were housed in individual cages under the specific pathogen-free condition, with a 12:12 h light:dark cycle (light automatically on/off at 08:00-20:00) at 22±1 °C and with 55% humidity. Mice were fed either a high fat diet (HFD, Research Diets, 60% calories from fat, Cat.no.D12492) or control chow diet (PicoLab® Rodent diet 20, LabDiet, 13.2% calories from fat) and provided with distilled water ad libitum.
For experiments with peripheral butyrate administration, 8- to 10-week-old male mice were stabilized on HFD feeding for 7 days before starting butyrate treatment. pH-matched (7.45±0.03) solutions of sodium butyrate (Santa Cruz Biotechnology, Cat.no.sc-202341C) and sodium bicarbonate buffer as the control were prepared at final concentration of 100 mM and provided ad libitum via the drinking water bottles. Butyrate and control solutions in drinking bottles were freshly replaced every 3 days. For experiments with i.p. butyrate administration, sodium butyrate (500 mg/kg of body weight) and sterile control saline were injected i.p. daily after measuring the body weight.
For experiments with ICV injection, mice after full recovery from ICV cannulation were stabilized on HFD feeding for 7 days before starting ICV injection treatment. 2.5 µL of 1.25 µg/µL sodium butyrate solution or control saline were daily infused via inserted ICV cannula into the third ventricle at an infusion rate of 2.5 µL/min using the microinjection syringe pump (LEGATO® 100 syringe pump, KD Scientific, MA, USA). Body weight was monitored daily before injection. At the end of experiments, mice were perfused using 10% neutral-buffered formalin solution or euthanized by decapitation. Blood plasma and tissues samples were snap frozen in dry ice and stored at -80 °C before analyzed. Perfused organs were kept at 4 °C in the 10% neutral-buffered formalin solution.

ICV cannulation and injection procedure
Male and female mice (10-12 weeks old) were anesthetized with an i.p. injection of Zoletil and Rompun (30 and 7.5 mg/kg, respectively) and mounted on a stereotaxic frame (KOPF1900, David Kopf Instruments, CA, USA) under aseptic conditions. Guided by the Paxinos and Franklin mouse brain atlas, a 26-gauge stainless steel guide cannula was stereotaxically implanted into the third ventricle (coordinates from bregma: AP −1.8 mm, DV −5.0 mm) and secured to the skull with dental cement. Post-surgery, the animals received a single i.p. injection of ketoprofen (5 mg/kg) for analgesia and were individually housed for at least a 7-day recovery period. To verify functional cannula placement, mice were centrally infused with angiotensin II (50 ng in 2.5 µL, rate of 2.5 µL/min). Only those exhibiting a robust dipsogenic response within 5 minutes were included in subsequent experiments; non-responders were excluded. Prior to the actual ICV treatments, all mice underwent three days of habituation to handling and restraint to minimize stress. Finally, the anatomical accuracy of the cannula placements was confirmed post-mortem.

Stereotaxic virus injection
Eight-week-old male mice were anesthetized via an intraperitoneal injection of a Zoletil and Rompun cocktail (30 and 7.5 mg/kg, respectively). Under aseptic conditions, the skull was exposed via a minor incision and drilled at the designated coordinates. We stereotaxically injected 500 nL of either AAV2-Cre-GFP (Addgene, Cat. No. 105545) or AAV2-GFP (Addgene, Cat. No. 105530) into the third ventricle (3V) of the hypothalamus (AP: −1.76 mm, ML: 0 mm, DV: −5.25 mm relative to bregma). The viral vectors were delivered at a constant rate of 50 nL/min over 10 minutes, with the injection needle left in situ for another 5 minutes to prevent backflow. Post-operatively, animals recovered on a heating pad to maintain body temperature. A minimum three-week period was provided to ensure complete surgical recovery and adequate Cre expression prior to experimental interventions. Finally, accurate viral targeting in the 3V was confirmed post-mortem using immunohistochemistry (IHC); animals with off-target injections were excluded from the final analysis.

Food intake measurement
Daily food consumption was calculated by subtracting the weight of the remaining chow from the initial amount provided every 24 hours. We monitored this intake for the first 5 days following the onset of treatment and utilized the average daily consumption for our comparative analyses. To ensure data accuracy, measurements from mice exhibiting food-crumbling behavior were excluded from the final analysis.

Glucose and insulin tolerance tests
Following 3 to 4 weeks of butyrate administration, we conducted glucose and insulin tolerance tests (GTT and ITT). Prior to testing, mice were fasted overnight for the GTT or for 2 hours for the ITT, while maintaining free access to water. Mice were i.p. injected with a solution of D-glucose (1.5 g/kg of body weight) or insulin (1.2 U/kg of body weight; HumulinR, Elli&Lilly) prepared in saline for GTTs and ITTs, respectively. Baseline (t0) and post-injection blood glucose levels were recorded at predetermined intervals by sampling tail-vein blood using a ContourTS hand-held glucometer (Bayer, Germany).

Metabolic cage study
To evaluate indirect calorimetry, HFD-fed mice receiving either butyrate or a vehicle control were placed in a 12-channel PhenoMaster Home Cage System (TSE Systems Inc.) featuring triple-beam infrared technology. The animals were single-housed and allowed to acclimatize to the metabolic chambers for 48 hours under standard experimental conditions. Following this habituation, we continuously monitored various metabolic indices—including oxygen consumption (VO2), carbon dioxide production (VCO2), energy expenditure, locomotor activity, and ingestive behaviors (food and water intake)—over a 5-day period. Throughout the evaluation, mice had ad libitum access to chow and water.

Measurement of body composition (Dual-energy X-ray absorptiometry)
For the analysis of body composition, the fat and lean masses of the mice were quantified using a dual-energy X-ray absorptiometry (DEXA) scanner (InAlyzer™, MEDIKORS, Korea) under brief isoflurane anesthesia. Following calibration according to the manufacturer's instructions, the system's lower limit of detection was established at 0.001 g/cm².

Measurements of metabolic hormones
Blood samples obtained via tail-nick were collected into EDTA-treated capillary tubes (Microvette CB300, Sarstedt). Following centrifugation to isolate the plasma, the aliquots were immediately frozen and maintained at −80 °C. The circulating concentrations of insulin and leptin were subsequently quantified using commercial ELISA kits (Morinaga Institute of Biological Science, Yokohama, Japan) in strict accordance with the manufacturer's provided protocols.

Immunoblotting
Cells or tissue homogenates were washed in 1X PBS and subsequently lysed using RIPA buffer (containing 150 mM NaCl, 50 mM Tris pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) enriched with phosphatase and protease inhibitor cocktails (Roche, Basel, Switzerland). Total protein concentration was determined via the Bio-Rad Protein Assay system (Bio-Rad Laboratories, CA, USA). Equivalent quantities of protein lysates were resolved by SDS-PAGE on acrylamide gels and electro transferred to nitrocellulose membranes. For blocking, the membranes were treated with 5% non-fat milk in TBST (Tris-buffered saline containing 0.1% Tween 20) before an overnight incubation at 4 °C with the respective primary antibodies under gentle agitation. After washing the membranes four times in TBST, they were exposed to species-appropriate secondary antibodies for 1 hour at room temperature on a shaker. Following an additional four washes with TBST, the protein bands were developed using an ECL substrate and captured on X-ray film or a chemiluminescence imaging platform.

Quantitative real-time PCR (qPCR)
Total RNA was isolated from cultured cells or tissue homogenates utilizing the Trizol reagent (Life Technologies, CA, USA). Following RNA quantification via a NanoDrop spectrophotometer, 0.5 to 1 µg of the purified RNA was converted into cDNA with the ReverTra Ace-α-™ synthesis kit (Toyobo, Osaka, Japan). The qPCR was subsequently performed on a CFX Connect Real-Time System (Bio-Rad, CA, USA). Each reaction mixture contained 1 µL of the cDNA template, target-specific primers, and iQ SYBR Green Supermix. To ensure accurate quantification, all gene expression data were normalized to 18S ribosomal RNA (18S) as the endogenous control.
Primer sequences used in qPCR were as follows: 18S: F, 5′-AACCCGTTGAACCCCATT-3′, R, 5′-CCATCCAATCGGTAGTAGCG-3′; Agrp: F, 5′-CGGCCACGAACCTCTGTAG-3′, R, 5′-CTCATCCCCTGCCTTTGC-3′; Foxj1: F, 5′-CCCTGACGACGTGGACTATG-3′, R, 5′- GCCGACAGAGTGATCTTGGT-3′; Ift20: F, 5′-AGGCAGGGCTGCATTTTGAT-3′, R, 5′- CAAGCTCAATTAGACCACCAACA-3′; Ift27: F, 5′- GGGACCCATTTCCAGAAGAA-3′, R, 5′- CCGGCTGAGTCAAAAATGAA-3′; Ift54: F, 5′- TGTGTAAGAGTGCGCTTCCC-3′, R, 5′- GCATGCTGCCTGTTCTCACT-3′; Ift88: F, 5′- GCAGTGACAGTGGCCAGAAC-3′, R, 5′- AAGGTTGCATCTGTCCCAGGC-3′; Kif3a: F, 5′- AGCTGCGATAATGTGAAGGTG-3′, R, 5′- GTTCCCCTCATTTCATCCACG-3′; Kif3b: F, 5′- GGACTGTGTGTGGAGCCTTG-3′, R, 5′- CTGACAGAGGCAGGCACCTA-3′; Npy: F, 5′-CTACTCCGCTCTGCGACACT -3′, R, 5′-AGTGTCTCAGGGCTGGATCTC-3′; Pomc: F, 5′-CAGGTCCTGGAGTCCGAC-3′, R, 5′-CATGAAGCCACCGTAACG-3′; Rfx1: F, 5′- GGCAGCCAGAAGCAGTATGT-3′, R, 5′- GCACCTTCCGATACGGTGA-3′; Rfx2: F, 5′- TGTGAGCCGATCCTACAGTG-3′, R, 5′- CCGCAGCACATCTGGGATAAG-3′; Rfx3: F, 5′- AGCTCGGGAGGAACCTATTTG-3′, R, 5′- GACAGACCGTCAGACTTTTGC-3′; and Rfx4: F, 5′- GACAGCTTAACTCAGGTGATCC-3′, R, 5′- GTTTTCCGGGAGATCGTGGAG-3′.

Immunofluorescence (IF) staining and quantification of cilia length
Cells grown on coverslips were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature, followed by an overnight incubation at 4 °C with an anti-acetylated-α-tubulin antibody under gentle agitation. For brain tissue analysis, mice underwent transcardial perfusion with 10% neutral-buffered formalin (Sigma-Aldrich, MO, USA). Extracted whole brains were cryoprotected overnight in a 20% sucrose/PBS solution and subsequently sliced into 25-µm sections utilizing a Leica CM1850 cryostat (Wetzlar, Germany). Fixed cells or brain sections were blocked in 3% goat serum prepared in 1X PBS with 0.25% Triton® X-100 (0.25% PBST) for 1 hour and incubated with the acetylated-α-tubulin antibody (for staining primary cilia in cells) or the adenylyl cyclase III antibody (for staining primary cilia in brain sections) prepared in PBST for 24 hours at 4 °C on a shaker. After washing 4 times with 1X PBS, cells or brain sections were incubated with the Alexa Fluor 488 secondary antibody (Invitrogen, CA, USA) prepared in 0.1% PBST. After washing 4 times (10 min for each) with 1X PBS, cover glasses of stained cells and brain sections were mounted using the Fluoroshield Mounting Medium with DAPI (Abcam, Cat.no.ab188804) and visualized with the Zeiss LSM700 Confocal Laser Scanning Microscope. Captured images generated from the ZEN 3.0 SR software of the microscope system were subjected to the ImageJ CiliaQ Plugins software tool (https://github.com/hansenjn/ CiliaQ) for an automated quantification of ciliary length57 or analyzed by an independent researcher without prior knowledge of experiment identity.

Histological analysis
White adipose tissues collected in 10% neutral-buffered formalin solution were paraffin embedded, sectioned and stained with hematoxylin and eosin (H&E) following a standard protocol. Stained samples were visualized and captured by the Leica ICC50E microscope system (Leica Microsystems, Wetzlar, Germany). Captured images were subjected to the ImageJ Adiposoft Plugin software tool (https://imagej.net/plugins/adiposoft) for an automated quantification of adipocyte and lipid droplet size58.

Brain slice preparation and electrophysiology
Five to seven-weeks-old male WT (AgRP-cre;tdTomatoF/+) or IFT88 KOAgRP (AgRP-cre;IFT88F/F;tdTomatoF/+) mice carrying a Cre-dependent tdTomato reporter allele were used for electrophysiological experiments. For slice preparation, mice were decapitated, and their dissected brains were quickly immersed in ice-cold, oxygenated cutting solution containing (in mM): 208 Sucrose, 2.5 KCl, 0.5 CaCl2, 6 MgCl2, 11 D-glucose, 1.4 NaH2PO4, 25 NaHCO3, and 0.5 sodium ascorbate. Coronal hypothalamic slices (300 µm thick) were prepared using a vibratome (7000SMZ-2, Campden Instruments, Loughborough, England). The slices were then incubated at 30 °C for 1 hour in oxygenated artificial cerebrospinal fluid (ACSF) with composition (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 11 D-glucose, 1.4 NaH2PO4, 25 NaHCO3 and 0.5 sodium ascorbate (osmolarity 310–320 mOsm, pH 7.3-7.4, bubbled with 95% O₂/5% CO₂).
For recordings, slices were transferred to a recording chamber perfused with oxygenated ACSF at a flow rate of approximately 2 mL/min using a gravity-driven perfusion system. AgRP-expressing neurons in the arcuate nucleus (ARC) were identified via brief epifluorescence exposure on a Nikon FN1 upright microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with a 10× water-immersion objective and differential interference contrast optics. Whole-cell signals were amplified using a Multiclamp 700B amplifier (Molecular Devices), filtered at 2 kHz, and digitized at 10 kHz using a Digidata 1440A interface (Molecular Devices). Recording pipettes, fabricated from borosilicate glass using a Flaming-Brown puller (P-1000, Sutter Instruments), had a tip resistance of 3–5 MΩ. The standard pipette solution contained (in mM): 130 K-gluconate, 10 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 4 MgATP, and 10 EGTA, with the pH adjusted to 7.3 using KOH, for current clamp data and spontaneous excitatory post-synaptic currents (sEPSCs) under voltage clamp mode at -70 mV. sEPSCs were recorded in the presence of 100 µM picrotoxin (PTX) in ACSF. For recording spontaneous inhibitory post-synaptic currents (sIPSCs), ACSF containing 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM D-2-amino-5-phosphonopentanoic acid (DL-AP5) was used, along with a high-Cl⁻ intracellular pipette solution (130 mM KCl replacing K-gluconate). Under the high [Cl⁻], the reversal potential for Cl⁻ was 0 mV, resulting in inward GABA-mediated currents at -70 mV. All recordings were performed at room temperature using pCLAMP 10.7 software (Molecular Devices). Resting membrane potential (RMP) and action potential (AP) firing properties were assessed after 5 minutes of whole-cell configuration. Series resistance (Rs) was monitored at the beginning and end of each experiment, and data were discarded if neurons exhibited Rs greater than 20 MΩ or if it changed by more than 20% during the recording. CA neuron was classified as depolarized or hyperpolarized if its RMP shifted by at least 2 mV. Membrane potentials were not corrected for liquid junction potential (-8 mV). AP firing frequency, as well as synaptic current frequency and amplitude, were analyzed using Mini Analysis software (ver. 6.0.7; Synaptosoft Inc., Decatur, GA, USA).

Statistics
Statistical significance was determined using Student’s t-test, chi-square test, and two-way ANOVA, as appropriate, using GraphPad Prism 8.0 software (GraphPad Software, Inc., CA, USA). Detailed statistical test(s) used and corresponding p-value are described in figure legends. For all analyses, p < 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The current manuscript investigates the role of neuronal primary cilia in the central effects of butyrate. The study demonstrates the impact of butyrate on energy metabolism and identifies AgRP cilia as key mediators of its effects. The authors first show that chronic central administration of butyrate protects against diet-induced obesity, which correlates with increased primary cilia length in hypothalamic neurons. Interestingly, by knocking down IFT88 in SF-1 or AgRP neurons, the authors demonstrate that butyrate acts specifically on AgRP neurons to modulate metabolism.\n\nThe present study is clear and well-written; the objectives are well-defined, and the experiments are thoughtfully designed and executed in an elegant manner. The discussion is well-supported by the results.\n\nWhile the overall quality of the manuscript is exceptional, I find a few points that need to be addressed:\n\nMajor Comments:\n-Are the effects of butyrate only observed during high-fat diet (HFD) exposure? All the presented results are from diet-induced obese mice. Does butyrate affect energy metabolism in mice maintained on a chow diet?\n-A considerable amount of work is conducted using the N38 cell line. While this is acceptable, it is evident that this cell line expresses various HDACs at higher levels than those found in the hypothalamus. In this regard, are cilia-related genes regulated in the hypothalamus of mice treated with butyrate?\n-Are female mice also affected by butyrate? Including data or a brief discussion on potential sex differences would strengthen the manuscript.\n-The authors report changes in primary cilia length in ARC neurons. Since they show that primary cilia in AgRP neurons are critical for the metabolic effects of butyrate, would it be possible to quantify the primary cilia length specifically in AgRP neurons? Is there any difference in cilia morphology or length between AgRP and POMC neurons?", "reviewer_2": "Rijal et al.'s work investigates the neuronal and molecular substrates that mediate the behavioral (primarily eating) and metabolic effects of butyrate. The main findings, as stated in the discussion, are that the cilia of Agrp-expressing neurons are required for butyrate-mediated effects and that central administration of butyrate produces effects similar to those reported following peripheral administration. While primarily of interest to a very specialized audience, these results might be relevant to the broader audience interested in the mechanisms controlling body weight regulation.\n\nSome aspects of this work require improving. A general remark is that, although the technical approach is sound and methodologically diverse, some of the experimental procedures requires more details and information. The sex of the mice used in the experiments, which is a critical factor particularly in studies addressing body weight regulation, was not specified in the text. The number of biological replicates is inconsistently reported, as is noted in some figures but absent in others, making it difficult to assess the robustness of the findings. Additionally, the statistical tests used are often not specified, which limits the reader’s ability to evaluate the validity of the comparisons presented. Some procedural details are lacking, at least in the main text, including the concentration and duration of several treatments (e.g., the butyrate concentration is reported in the main text for experiments in Fig. 2a and 5b, but not for most other experiments). Although some of these information is included in the material and methods, I recommend that the authors add these details to the main text, as it would help the reader not only to interpret the findings promptly but also to relate to the dosages reported in the discussion when discussing similar works in the field. I provide a more detailed list of comments below:\n\nLINES 74-76: “The dose was selected to enhance histone acetylation without inducing adverse neurological effects, such as seizures, sluggishness, or eating ability39,40”\nThe information regarding the sex of the mice and the concentration of the drugs is missing. Granted that the authors refer to 2 prior publications, this information should be readily apparent, as it is very important for data interpretation.\n\nLINES 77-78: “Notably, ICV butyrate treatment protected mice from high-fat diet (HFD)-induced weight gain and white adipose tissue (WAT) hypertrophy 78 (Fig. 1a, b).”\nMice have multiple white adipose tissue (WAT) depots, each with distinct anatomical, molecular, and physiological characteristics. It is important to specify which depot(s) were analyzed in this study and to justify the choice. This information should be made readily apparent to the reader, preferably in the main text and figure, to aid in the interpretation of the findings (same applies to Fig. 2h).\n\nabout Figure 1:\n-What is the rationale for showing adipocyte area (e.g., Fig. 1b) in AU rather than in µm2?\n-Fig. 1g-l: I understand that this is a cohort of mice tested in metabolic cages. Why does panel g (FI) have n =6 mice per group, but panels i-l have only 3 mice? It is not ideal to compare food intake measurements (n = 6) to metabolic readouts that depend on food intake (n = 3). Why were the other 3 mice excluded? Three mice in Fig. 1g have caloric intake similar to the average of the controls, whereas another 3 mice have a lower caloric intake. Are these 3 mice with less FI the ones excluded from panel i-g? If so, that could change the results and interpretation. Moreover, I feel that n=3 for metabolic studies is too few replicates to reach firm conclusions, and the number of replicates should be increased. Finally, are these mice males or females?\n\nLINE 108-109: “We first treated butyrate and examined expression of several transcriptional regulators involved in ciliogenesis. “\n-How long did the treatment last and what concentration of butyrate was used?\n\nLINES 115-116: “Furthermore, direct intracerebroventricular (ICV) injection of butyrate into the 3V significantly enhanced histone acetylation and notably elongated primary cilia in h including arcuate (ARC) and VMH (Fig. 2e, f).”\n-The number of mice used is not specified in the main text, figures, or figure legends. Additionally, the type of statistical test used is not reported. In Figure 2f, it appears that the statistical analysis was performed on the number of neurons, rather than on the average per mouse. Without information on the number of cilia analyzed per mouse, a subset of cells from the same mice may drive the observed differences. The authors should present the data as average cilia length per mouse, rather than as measurements from individual cilia, to ensure appropriate statistical interpretation at the animal level.\n\nLINES 121-122: “alterations in primary cilia function within the hypothalamus. To investigate this, mice fed a HFD received butyrate either orally or via intraperitoneal injection”\nThese experiments differ from the ones before because butyrate is administered systemically, rather than directly in the brain. I assume that the route of administration significantly impacts the action of this molecule. The authors should provide the following information, relative to these experiments:\n-Mice received butyrate via two different means, orally or via intraperitoneal injection. Why 2 different means of administration? Are controls and experimental mice treated the same way (i.e. they both received butyrate via the same route)?\n-How long did the treatment last, and what concentration of butyrate was used?\n\nLINE 125: The term “increased ciliogenesis” suggests an increase in the number or proportion of cilia; however, in the figures cited, the authors quantify cilia length. While neurons generally possess a single cilium, the use of this term may be misleading to non-experts, as it could suggest an increase in cilia number rather than in their length, as reported in the manuscript.\n\nLINES 133-136: “To do this, we stereotaxically injected recombinant adeno-associated virus serotype 2 (AAV2) either with or without the Cre recombinase fused to green fluorescent protein (AAV2-GFP as control or AAV2-Cre-GFP) into the hypothalamic 3V of mice carrying floxed alleles for both intraflagellar transport 88 and ROSA26-tdTomato (IFT88F/F::td-TomatoF/+) 37 (Fig. 3a).”\n-The main text does not explicitly mention that these mice were treated with vehicle or butyrate, which should be stated. If the primary goal of these experiments (as I understand them) was to demonstrate that injection of AAV-Cre-GFP removes the cilia, wouldn’t it be sufficient to show this effect in vehicle-treated animals only? Importantly, what is concentration of butyrate, and how is it administered in this context? Systemically, as in the previous figure, or directly to the brain, similarly for data in Figs.. 1 and 3?\n\n-Figure 3b: It is unclear which antibodies were used for staining in the various panels. Please specify the antibodies in each panel to improve clarity.\n\n-Figure 3d: If fat mass increases in one mouse, the proportion (i.e., the % of total mass or body weight) of lean mass will necessarily decrease. To correctly interpret the data, the authors should display the raw values for fat and lean mass, in grams, without normalization.\n\nLINES 141-144 Interestingly, the metabolic benefits of oral butyrate on body weight, body compositions, food intake, and glucose and insulin homeostasis observed in the control virus-injected group (Fig. 3c-h), were completely abolished or even reversed in mice with disrupted hypothalamic primary cilia (Fig. 3i-n).”\n-In general, I feel that the authors could comment more on these findings instead of referring to as “all the effects”. Furthermore, the conclusion that the effects were “completely abolished” is partially contradicted by the data reported in Fig. 3n, showing (graph on the left) the same effect as in Fig. 3h (graph on the left).\n\n-Why are the effects on body weight of 3c, reversed in Fig. 3i? If Butyrate requires the cilia to exert its effects, one could expect that removing the cilia would blunt the action of butyrate, and treated mice would weigh the same as controls. Instead, cilia-less mice grow heavier (Fig. 3i), although they do not eat more (Fig. 3k). Did the authors have data that could explain this phenomenon? For instance, can the authors check whether these mice display a deficit in energy expenditure, which could explain the weight gain despite similar rates of caloric intake?\n\nLINES 161-162: “The SF-1 neuron- and AgRP neuron-specific IFT88 knockout mice exhibited no detectable cilia in these neurons, confirming the successful and targeted knockout (Fig. S3b, c).”\n-These experiments could benefit from more controls. Can the authors show that the cilia in SF1 cells are still present in AGRP models? And vice versa, are the cilia in AGRP-expressing neurons unaffected in SF1 models?\n\nLINES 188-189: “Next, to gain further insights into how primary cilia contribute to butyrate-induced regulation of AgRP neuron activity, we bath applied 1mM butyrate for 3-4 minutes and observed variable response in both WT and IFT88 KOAgRP neurons (Fig. 5b).”\n-The text states that the application period is 3–4 minutes. What accounts for this variability? Could differences in bathing time affect the results or introduce inconsistencies?\n\nLINES 190-193: “Upon butyrate application, 71% of tested WT neurons (n = 15/21), exhibited changes in membrane potential, predominantly hyperpolarization whereas 52% of tested IFT88 KOAgRP neurons (n = 12/23) responded, showing either hyperpolarization or depolarization (Fig. 5c, e-f).”\n-It is not readily apparent whether these measurements were taken during or after the application of butyrate. Additional information regarding these procedures should be provided in the main text to improve clarity.\n\nFig. 5c,d. The authors performed electrophysiological recordings on Agrp-expressing neurons in AgRP-cre; IFT88F/F 179 ; tdTomatoF/F mice (experimental group) and AgRP-cre; tdTomatoF/F mice (control group). One of their findings is that in control mice, application of butyrate inhibits Agrp neurons (Fig. 5d). This could explain the decrease in food intake and body weight gain shown by control mice treated with butyrate in vivo in Fig.1a and in mice with IFT88 floxed in SF-1 neurons (Fig. 4a). On the other hand, bathing slices from experimental mice with butyrate results in a smaller proportion of agrp neurons being inhibited (Fig. 5d). These results suggest that AgRP-cre; IFT88F/F mice treated with Butyrate (in Fig. 4h-n) could eat more and gain more weight compared to their control, where such inhibition should not occur (AgRP-cre; IFT88F/F mice treated with vehicle in Fig. 4h-n). Yet, these mice ate and gained as much weight as their controls (Fig. 4h, j). The electrophysiological recording appears to explain the behavioral and metabolic effects observed in wild-type (WT) mice treated with butyrate, but not the effects displayed by experimental ones in Fig. 4h-n. A better understanding could be provided if the authors were to include the electrophysiological data from Agrp-expressing neurons in wild-type and AgRP-cre; IFT88F/F 179; tdTomatoF/F mice treated with vehicle (not just with Butyrate) to Fig. 5c,d. These controls would help determine whether the effects on neuronal firing shown in Fig. 5c,d are due to the intrinsic deletion of cilia from Agrp neurons or to butyrate treatment, and could aid in interpreting the in vivo data.", "reviewer_3": "In this manuscript, Rijal et al. identify a critical role for primary cilia of hypothalamic AgRP neurons in mediating how butyrate reduces food intake and promotes glucose tolerance.\n\nAfter setting up their mouse model of butyrate’s actions (Fig.1), Rijal et al. show that butyrate promotes ciliogenesis in the mouse hypothalamus and the N38 hypothalamic cell line, which they use to characterize the likely mechanisms of butyrate’s ciliogenic effects: (i) histone deacetylase inhibition, (ii) upregulation of ciliogenic transcription factors Rfx2 and Rfx3, and (iii) upregulation of key ciliary Rfx target genes (Fig.2).\n\nBy injecting AAV2-Cre into the hypothalamus of IFT88 conditional KO mice, they completely eliminate hypothalamic cilia, leading to a complete loss of butyrate’s effects (Fig.3). They then show that butyrate’s effects are also abolished by specifically deleting cilia in the AgRP neurons of the arcuate nucleus, which they achieve using the AgRP-Cre; Ift88 f/f mouse model (Fig.4h-n). By contrast, butyrate’s effects remain intact when the cilia of ventromedial hypothalamus SF1 neurons are deleted instead (Fig.4a-g). The authors conclude their study by showing that cilia are important regulators of AgRP neurons’ electrophysiological behavior (Fig.5).\n\nAltogether, I think the data are of high quality, very convincing, and of great relevance. The manuscript is also well written. Therefore, my opinion is that this manuscript deserves prompt publication. Before that, however, I would recommend addressing the following issues:\n\nMinor Points:\n1. This is the most important of my points: by looking at Figure 5, a non-expert reader could reach the conclusion that cilia do not make much of a difference in AgRP neuron behavior, since there is not a single panel in the figure showing statistically significant differences between WT and IFT88-KO-AgRP. In panels c-d the differences appear large, but there is no statistical analysis to back their significance. Such an analysis should be added. Moreover, Fig.S4e-h in the supplement shows statistically significant differences between WT and KO in the sIPSC measurements: I think it would benefit the paper to move Fig.S4e-h to the main figure.\n2. Line 94. Remove “of butyrate” (it is redundant)\n3. Line 136. The way tdTomato genotype is indicated, it seems as if tdTomato expression should be deleted by Cre, rather than activated as it is. Please clarify this for readers.\n4. Lines 156-7. Revise sentence. Also add reference to Fig.1m.\n5. Line 167. Those > that.\n6. Line 189. Fig.5a is nowhere referenced in the text.\n7. Line 211. sIPSCs > sEPSCs.\n8. Remove “the”.\n9. Lines 227-8. Since correlation alone is no proof of causation, and correlation is what has been shown here, I would not say “our study demonstrates”. A less conclusive statement, like “our study suggests” seems more appropriate.\n10. Line 235. Systematically > systemically.\n11. Line 276. Revise sentence.\n12. Line 318. Write “um” properly.\n13. Line 332. Indicate source of N38 cell line here in methods.\n14. Line 362. It should be “full recovery” or “fully recovering”.\n15. Line 682-3. Write reference 53 properly.\n16. Line 702. Protect > protects from.\n17. Line 703. Improve > improves.\n18. Figure 5 legend. Please spell out the meaning of “WO” and “IEIs”.\n19. Several mistakes in Suppl. Fig.1 legend: (i) remove “the” in first line, (ii) figure shows nothing about GPCRs, (iii) “lysine residues” instead of “lysine residuals”; (iv) HDAC inhibitor used is Trichostatin A, not Triclosan."}, "author_response": "Response Letter (NCOMMS-25-39036)\nWe sincerely appreciate the Editor and Reviewers for taking your valuable time to carefully\nread and consider our work. Here, we would like to address the Reviewer’s comments as\nfollows. We have also provided a marked-up version of the manuscript, in which all revisions\nfrom the original submission are highlighted in yellow.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe current manuscript investigates the role of neuronal primary cilia in the central effects of\nbutyrate. The study demonstrates the impact of butyrate on energy metabolism and identifies\nAgRP cilia as key mediators of its effects. The authors first show that chronic central\nadministration of butyrate protects against diet-induced obesity, which correlates with\nincreased primary cilia length in hypothalamic neurons. Interestingly, by knocking down IFT88\nin SF-1 or AgRP neurons, the authors demonstrate that butyrate acts specifically on AgRP\nneurons to modulate metabolism.\nThe present study is clear and well-written; the objectives are well-defined, and the\nexperiments are thoughtfully designed and executed in an elegant manner. The discussion is\nwell-supported by the results.\nWhile the overall quality of the manuscript is exceptional, I find a few points that need to be\naddressed:\nResponse: We sincerely thank Reviewer #1 for the thoughtful and encouraging feedback on\nour manuscript. Below, we provide detailed responses to each of the reviewer’s comments:\n1. Are the effects of butyrate only observed during high-fat diet (HFD) exposure? All the\npresented results are from diet-induced obese mice. Does butyrate affect energy metabolism in\nmice maintained on a chow diet?\nResponse: We acknowledged the reviewer for the comment. In response to the reviewer’s\ncomment, we conducted additional experiments to evaluate whether the effects of butyrate are\nalso observed under normal chow (NC) conditions. To this end, we established new male mouse\ncohorts and performed intracerebroventricular (ICV) administration of butyrate. We found that\nICV injection of butyrate resulted in a significant reduction in body weight, primarily due to\ndecreased food intake (Supplementary Figs. 1a-d). These findings indicate that central butyrate\nexerts beneficial metabolic effects on body weight regulation not only under high-fat diet (HFD)\nconditions but also in mice maintained on a normal chow diet. Accordingly, we have added a\ndescription of these results to the Results section (lines 67–72) and updated Supplementary Fig.\n1 to include the new data.\n2. A considerable amount of work is conducted using the N38 cell line. While this is acceptable,\nit is evident that this cell line expresses various HDACs at higher levels than those found in the\nhypothalamus. In this regard, are cilia-related genes regulated in the hypothalamus of mice\ntreated with butyrate?\nResponse: This is an important question to determine whether the effects of butyrate on cilia-\nrelated gene regulation are same or differ from cell line and hypothalamus. To address this\nquestion, we run QPCR using hypothalamus after treatment of either saline or butyrate. The\nresults showed that the butyrate treatment significantly increased Foxj1, Rfx2, and Rfx3.\nAccordingly, we have added a description of these results to the Results section (lines 115–118)\nand presented the corresponding data in Fig. 2f.\n3. Are female mice also affected by butyrate? Including data or a brief discussion on potential\nsex differences would strengthen the manuscript.\nResponse: We appreciate the reviewer’s insightful comment regarding potential sex\ndifferences in the response to butyrate. To address this, we conducted an additional experiment\nusing a new cohort of female mice fed a normal chow diet, as described in the Results section\n(lines 72–78). Cannulation surgery was performed at 10 weeks of age, and\nintracerebroventricular (ICV) butyrate administration began at 12 weeks. In contrast to the\nmale mice (Supplementary Fig. 1a), the female mice (body weight ~24 g) showed higher\nsusceptibility to daily injections, which appeared to adversely affect their health; therefore, we\nlimited the treatment period to 1 week, rather than 3 weeks as in males, and monitored body\nweight in females. During this 1-week ICV butyrate regimen in female mice, only a trend\ntoward decreased body weight was observed (Supplementary Figs. 1e–g). Unlike females, male\nmice tolerated the treatment well without noticeable behavioral abnormalities, allowing us to\ncontinue ICV butyrate injections for 3 weeks, as shown in Supplementary Figs. 1a–d. This 3-\nweek injection regimen led to a significant decrease in body weight in male mice. Based on\nthese results, we acknowledge that further studies using older female mice with higher body\nweight or diet-induced obese female mice will be required to determine the precise metabolic\neffects of butyrate in females. Due to time constraints, we were unable to assess the metabolic\neffects of butyrate in older or diet-induced obese female mice in the current study.\n4. The authors report changes in primary cilia length in ARC neurons. Since they show that\nprimary cilia in AgRP neurons are critical for the metabolic effects of butyrate, would it be\npossible to quantify the primary cilia length specifically in AgRP neurons? Is there any\ndifference in cilia morphology or length between AgRP and POMC neurons?\nResponse: Thank you for the insightful comment. In accordance with the reviewer’s\nsuggestion, we performed primary cilia staining in age-matched POMC-hrGFP and NPY-\nhrGFP (AgRP) mice to visualize the primary cilia in each neuronal population. Interestingly,\nwe found that the primary cilia in AgRP neurons are significantly longer than those in POMC\nneurons. We have updated the Discussion section (lines 337-340) to highlight this observation\nand included the corresponding data in Supplementary Figure 5.\nReviewer #2 (Remarks to the Author):\nRijal et al.'s work investigates the neuronal and molecular substrates that mediate the\nbehavioral (primarily eating) and metabolic effects of butyrate. The main findings, as stated in\nthe discussion, are that the cilia of Agrp-expressing neurons are required for butyrate-mediated\neffects and that central administration of butyrate produces effects similar to those reported\nfollowing peripheral administration. While primarily of interest to a very specialized audience,\nthese results might be relevant to the broader audience interested in the mechanisms controlling\nbody weight regulation.\nSome aspects of this work require improving. A general remark is that, although the technical\napproach is sound and methodologically diverse, some of the experimental procedures requires\nmore details and information. The sex of the mice used in the experiments, which is a critical\nfactor particularly in studies addressing body weight regulation, was not specified in the text.\nThe number of biological replicates is inconsistently reported, as is noted in some figures but\nabsent in others, making it difficult to assess the robustness of the findings. Additionally, the\nstatistical tests used are often not specified, which limits the reader’s ability to evaluate the\nvalidity of the comparisons presented. Some procedural details are lacking, at least in the main\ntext, including the concentration and duration of several treatments (e.g., the butyrate\nconcentration is reported in the main text for experiments in Fig. 2a and 5b, but not for most other experiments). Although some of these information is included in the material and\nmethods, I recommend that the authors add these details to the main text, as it would help the\nreader not only to interpret the findings promptly but also to relate to the dosages reported in\nthe discussion when discussing similar works in the field. I provide a more detailed list of\ncomments below:\nResponse: We sincerely thank the reviewer for the thoughtful and constructive comments.\nOur detailed responses are provided below.\n1. LINES 74-76: “The dose was selected to enhance histone acetylation without inducing\nadverse neurological effects, such as seizures, sluggishness, or eating ability39,40” The\ninformation regarding the sex of the mice and the concentration of the drugs is missing. Granted\nthat the authors refer to 2 prior publications, this information should be readily apparent, as it\nis very important for data interpretation.\nResponse: We thank the reviewer for pointing out the lack of experimental detail. This\ninformation has now been included in the revised manuscript (lines 63–67).\nThe original sentence:\nTo determine if central administration of butyrate is sufficient to induce systemic metabolic\neffects, butyrate was directly delivered into the hypothalamic third ventricle (3V) through\nintracerebroventricular (ICV) injection. The dose was selected to enhance histone acetylation\nwithout inducing adverse neurological effects, such as seizures, sluggishness, or eating\nability39,40.\nRevised sentence:\nTo determine whether central administration of butyrate is sufficient to elicit systemic\nmetabolic effects, 2.5 µL of butyrate (1.25 µg/µL) was administered into the hypothalamic third\nventricle (3V) of male mice via intracerebroventricular (ICV) injection. The dose was selected\nto enhance histone acetylation without causing adverse neurological effects, such as seizures,\nsluggishness, or impaired feeding behavior40, 41.\n2. LINES 77-78: “Notably, ICV butyrate treatment protected mice from high-fat diet (HFD)-\ninduced weight gain and white adipose tissue (WAT) hypertrophy 78 (Fig. 1a, b).” Mice have\nmultiple white adipose tissue (WAT) depots, each with distinct anatomical, molecular, and\nphysiological characteristics. It is important to specify which depot(s) were analyzed in this\nstudy and to justify the choice. This information should be made readily apparent to the reader,\npreferably in the main text and figure, to aid in the interpretation of the findings (same applies\nto Fig. 2h).\nResponse: We appreciate the reviewer’s comment. Following the reviewer’s suggestion, we\nhave now specified the white adipose tissue depots analyzed in this study and included\nadditional data. In the revised manuscript, we present results from epididymal white adipose\ntissue (eWAT) and subcutaneous white adipose tissue (scWAT), which are the major depots\ninvolved in systemic metabolic regulation. We thank the reviewer for this helpful suggestion.\nThe sentence has been revised accordingly in the main text (lines 70–72) and the figure legend.\nThe original sentence:\nNotably, ICV butyrate treatment protected mice from high-fat diet (HFD)-induced weight gain\nand white adipose tissue (WAT) hypertrophy (Fig. 1a, b).\nRevised sentence:\nProlonged ICV butyrate treatment also protected male mice from high-fat diet (HFD)-induced\nweight gain and adipose hypertrophy in major fat depots, including epididymal (eWAT) and\nsubcutaneous white adipose tissues (scWAT) (Figs. 1a-e).\n3. about Figure 1:\n3-1). What is the rationale for showing adipocyte area (e.g., Fig. 1b) in AU rather than in µm2?\nResponse: As suggested, we have revised the figure to present the adipocyte diameter in µm\ninstead of arbitrary units (AU). We appreciate the reviewer’s helpful suggestion, which has\nimproved the clarity of data presentation.\n3-2). Fig. 1g-l: I understand that this is a cohort of mice tested in metabolic cages. Why does\npanel g (FI) have n =6 mice per group, but panels i-l have only 3 mice? It is not ideal to compare\nfood intake measurements (n = 6) to metabolic readouts that depend on food intake (n = 3).\nWhy were the other 3 mice excluded? Three mice in Fig. 1g have caloric intake similar to the\naverage of the controls, whereas another 3 mice have a lower caloric intake. Are these 3 mice\nwith less FI the ones excluded from panel i-g? If so, that could change the results and\ninterpretation. Moreover, I feel that n=3 for metabolic studies is too few replicates to reach firm\nconclusions, and the number of replicates should be increased. Finally, are these mice males or\nfemales? (Male)\nResponse: We would like to clarify that the discrepancy in animal numbers is not due to\nexclusion of mice but rather because food intake measurements and metabolic cage analyses\nwere performed in separate cohorts. We apologize for the confusion caused by not clearly\nindicating this in the original version of the manuscript. We fully agree with the reviewer that\nusing only three mice for metabolic cage analyses is insufficient to draw firm conclusions.\nTherefore, we have repeated the experiments with a new cohort, increasing the sample size to\nsix mice per group. The updated results have been incorporated into the revised manuscript\n(Figs. 1k–n). We greatly appreciate the reviewer’s constructive feedback, which has\nsignificantly improved the robustness of our study.\n4. LINE 108-109: “We first treated butyrate and examined expression of several transcriptional\nregulators involved in ciliogenesis.” How long did the treatment last and what concentration\nof butyrate was used?\nResponse: We thank the reviewer for this helpful comment. In response, we clarify that cells\nwere treated with 1 mM butyrate for 24 hours. This information has now been added to the\nmain text (lines 108–109) and the figure legend to improve clarity and reproducibility. We\nappreciate the reviewer’s suggestion, which has strengthened the presentation of our\nexperimental details.\nThe original sentence:\nWe first treated butyrate and examined expression of several transcriptional regulators involved\nin ciliogenesis.\nRevised sentence:\nWe first treated cells with 1 mM butyrate for 24 hours and examined the expression of several\ntranscriptional regulators involved in ciliogenesis.\n5. LINES 115-116: “Furthermore, direct intracerebroventricular (ICV) injection of butyrate\ninto the 3V significantly enhanced histone acetylation and notably elongated primary cilia in\nthe several hypothalamic nuclei including arcuate (ARC) and VMH (Fig. 2e, f).” The number\nof mice used is not specified in the main text, figures, or figure legends. Additionally, the type\nof statistical test used is not reported. In Figure 2f, it appears that the statistical analysis was\nperformed on the number of neurons, rather than on the average per mouse. Without\ninformation on the number of cilia analyzed per mouse, a subset of cells from the same mice\nmay drive the observed differences. The authors should present the data as average cilia length\nper mouse, rather than as measurements from individual cilia, to ensure appropriate statistical\ninterpretation at the animal level.\nResponse:We thank the reviewer for important comment. In response, we have now specified\nthe number of mice used (n = 4) in the main text, figure panels, and figure legend. Additionally,\nthe statistical methods applied have been clearly indicated in the updated figure legend.\nRegarding Figure 2f, we agree with the reviewer that statistical analyses should be performed\nat the animal level to ensure appropriate interpretation. Accordingly, we have revised the\nanalysis to present the average primary cilia length per mouse, rather than individual cilia\nmeasurements. We sincerely appreciate the reviewer’s suggestion, which has helped improve\nthe rigor and clarity of our data presentation.\n6. LINES 121-122: “alterations in primary cilia function within the hypothalamus. To\ninvestigate this, mice fed a HFD received butyrate either orally or via intraperitoneal injection”\nThese experiments differ from the ones before because butyrate is administered systemically,\nrather than directly in the brain. I assume that the route of administration significantly impacts\nthe action of this molecule. The authors should provide the following information, relative to\nthese experiments:\n6-1). Mice received butyrate via two different means, orally or via intraperitoneal injection.\nWhy 2 different means of administration? Are controls and experimental mice treated the same\nway (i.e. they both received butyrate via the same route)?\nResponse: We thank the reviewer for this insightful comment. As noted, these experiments\ndiffer from the previous ones in that butyrate was administered systemically rather than directly\ninto the brain. Given that butyrate crosses the blood–brain barrier and can influence epigenetic\nprocesses in the brain, we sought to determine whether peripherally administered butyrate—\ntogether with its previously observed direct effects in the hypothalamus—is associated with\nHDAC inhibition and the regulation of primary ciliogenesis in this brain region. To assess this,\nwe used two routes of systemic delivery—oral and intraperitoneal (i.p.)—to evaluate the\nrobustness and consistency of butyrate’s central effects. As indicated in the revised manuscript,\nthe data shown in Figs. 2h-l correspond to orally administered butyrate, while Supplementary\nfigures 2d-h show the results from i.p. administration. Importantly, in both cases, control and\nexperimental mice within each cohort were treated using the same route of administration. We\nhave now clarified this information in the main text to avoid any confusion. We appreciate the\nreviewer’s suggestion, which allowed us to improve the clarity and transparency of our\nexperimental design.\n6-2). How long did the treatment last, and what concentration of butyrate was used?\nResponse: We thank the reviewer for this important question. For oral administration, pH-\nmatched (7.45 ± 0.03) sodium butyrate and control sodium bicarbonate solutions were prepared\nat a final concentration of 100 mM and provided ad libitum via drinking water, with solutions\nreplaced every 3 days. For intraperitoneal (i.p.) administration, sodium butyrate was injected\ndaily at a dose of 500 mg/kg body weight, with sterile saline used as the control. These\nexperimental conditions have now been also described both in the main text and in the\nMaterials and Methods section for clarity. We appreciate the reviewer’s suggestion, which\nhelped improve the completeness of our experimental descriptions.\n7. LINE 125: The term “increased ciliogenesis” suggests an increase in the number or\nproportion of cilia; however, in the figures cited, the authors quantify cilia length. While\nneurons generally possess a single cilium, the use of this term may be misleading to non-experts,\nas it could suggest an increase in cilia number rather than in their length, as reported in the\nmanuscript.\nResponse: We appreciate the reviewer’s comment for helping us express our results more\nclearly. According to the reviewer’s suggestion, we revised the wording from “primary\nciliogenesis” to “elongation of primary cilia” (line 129).\n8. LINES 133-136: “To do this, we stereotaxically injected recombinant adeno-associated virus\nserotype 2 (AAV2) either with or without the Cre recombinase fused to green fluorescent\nprotein (AAV2-GFP as control or AAV2-Cre-GFP) into the hypothalamic 3V of mice carrying\nfloxed alleles for both intraflagellar transport 88 and ROSA26-tdTomato (IFT88F/F::td-\nTomatoF/+) 37 (Fig. 3a).”\n8-1). The main text does not explicitly mention that these mice were treated with vehicle or\nbutyrate, which should be stated. If the primary goal of these experiments (as I understand them)\nwas to demonstrate that injection of AAV-Cre-GFP removes the cilia, wouldn’t it be sufficient\nto show this effect in vehicle-treated animals only? Importantly, what is concentration of\nbutyrate, and how is it administered in this context? Systemically, as in the previous figure, or\ndirectly to the brain, similarly for data in Figs.. 1 and 3?\nResponse: We thank the reviewer for these constructive comments and for pointing out the\nneed to clearly state the treatment condition details in the main text. As the primary goal of this\nstudy was to determine whether the metabolic effects of butyrate require primary cilia in the\nmediobasal hypothalamus, we included both vehicle-treated and butyrate-treated groups in\nparallel. Reflecting the reviewer’s comment, we have revised the text as follows (lines 135–\n144).\nWe next investigated whether hypothalamic primary cilia are required for the metabolic actions\nof butyrate. To examine this, we stereotaxically injected recombinant adeno-associated virus\nserotype 2 (AAV2) — either encoding Cre recombinase fused to green fluorescent protein\n(AAV2-Cre-GFP) or lacking Cre (AAV2-GFP, control) — into the third ventricle (3V) of the\nhypothalamus in mice carrying floxed alleles of intraflagellar transport protein 88 (IFT88F/F)\nand a Cre-inducible tdTomato reporter at the ROSA26 locus (ROSA26-tdTomatoF/+) (Fig. 3a).\nFollowing vehicle or oral butyrate treatment (100 mM), we observed butyrate-induced primary\nciliogenesis in AAV2-GFP–injected mice, whereas AAV2-Cre-GFP–injected mice showed\nspecific deletion of primary cilia in the mediobasal hypothalamus, including the ARC and\nVMH (Figs. 3b and S3a, b).\nWe sincerely appreciate the reviewer’s suggestion, which has improved the clarity and\ncompleteness of our description.\n8-2). Figure 3b: It is unclear which antibodies were used for staining in the various panels.\nPlease specify the antibodies in each panel to improve clarity.\nResponse:We thank the reviewer for this helpful comment. In accordance with the reviewer’s\nsuggestion, antibody information has been added to Figure 3b, indicated in the lower right\ncorner with matching color codes, and specified in the figure legend. Detailed antibody\ninformation is also provided in the Materials and Methods section to ensure clarity.\n8-3). Figure 3d: If fat mass increases in one mouse, the proportion (i.e., the % of total mass or\nbody weight) of lean mass will necessarily decrease. To correctly interpret the data, the authors\nshould display the raw values for fat and lean mass, in grams, without normalization.\nResponse: We thank the reviewer for the comment. In accordance with the reviewer’s\nsuggestion, we have revised Figure 3d to present fat and lean mass in grams. We appreciate the\nreviewer’s input.\n9. LINES 141-144 Interestingly, the metabolic benefits of oral butyrate on body weight, body\ncompositions, food intake, and glucose and insulin homeostasis observed in the control virus-\ninjected group (Fig. 3c-h), were completely abolished or even reversed in mice with disrupted\nhypothalamic primary cilia (Fig. 3i-n).”\n9-1). In general, I feel that the authors could comment more on these findings instead of\nreferring to as “all the effects”. Furthermore, the conclusion that the effects were “completely\nabolished” is partially contradicted by the data reported in Fig. 3n, showing (graph on the left)\nthe same effect as in Fig. 3h (graph on the left).\nResponse: We sincerely thank the reviewer for this insightful comment, which allowed us to\nclarify the description of our results. We agree that the original phrasing could have been\nmisleading. In accordance with the reviewer’s suggestion, we have revised the sentence to\nprovide a more precise description of the findings and to avoid overstatement (lines 147–154).\nThe original sentence:\nInterestingly, the metabolic benefits of oral butyrate on body weight, body compositions, food\nintake, and glucose and insulin homeostasis observed in the control virus-injected group (Fig.\n3c-h), were completely abolished or even reversed in mice with disrupted hypothalamic\nprimary cilia (Fig. 3i-n).\nRevised sentence:\nInterestingly, the metabolic benefits of oral butyrate—such as reductions in body weight, fat\nmass, and food intake, as well as improvements in glucose and insulin homeostasis—observed\nin control virus–injected mice (Figs. 3c–h) were largely abolished in mice with disrupted\nprimary cilia in the mediobasal hypothalamus (Fig. 3i–n). Notably, the beneficial effects on\nfood intake and glucose tolerance were lost and became indistinguishable from those in control\nmice (Figs. 3e, g, k, m). Unexpectedly, butyrate-treated AAV2-Cre-GFP–injected mice\nexhibited increased body weight (Figs. 3c, d, i, j) and showed impaired insulin tolerance at a\nspecific time point (Figs. 3h, n).\n9-2). Why are the effects on body weight of 3c, reversed in Fig. 3i? If Butyrate requires the\ncilia to exert its effects, one could expect that removing the cilia would blunt the action of\nbutyrate, and treated mice would weigh the same as controls. Instead, cilia-less mice grow\nheavier (Fig. 3i), although they do not eat more (Fig. 3k). Did the authors have data that could\nexplain this phenomenon? For instance, can the authors check whether these mice display a\ndeficit in energy expenditure, which could explain the weight gain despite similar rates of\ncaloric intake?\nResponse: We thank the reviewer for this insightful comment regarding the reversal of\nbutyrate’s effects on body weight in Fig. 3i. We agree that, if butyrate requires intact\nhypothalamic primary cilia to exert its actions, one would initially expect that removal of cilia\nwould simply blunt the effects of butyrate, resulting in similar body weight between vehicle-\nand butyrate-treated mice. However, in our study, mice with primary cilia ablated throughout\nthe mediobasal hypothalamus and treated with butyrate exhibited greater body weight than\nvehicle-treated controls, despite no increase in food intake (Figs. 3i, k). Unfortunately, we do\nnot currently have direct experimental data to fully explain this phenotype. Therefore, in\nresponse to the reviewer’s comment, we have revised the Discussion section to explain this\nunexpected finding and to describe possible mechanisms, including altered energy expenditure\nand the engagement of energy-conserving or other counter-regulatory pathways (lines 268–\n283).\nThe revised text in the Discussion is as follows:\nInterestingly, however, in mice with primary cilia ablated throughout the mediobasal\nhypothalamus, butyrate-treated animals exhibited greater body weight than vehicle-treated\ncontrols, despite no increase in food intake (Figs. 3i, k). We had initially expected that\ndisruption of primary cilia in the mediobasal hypothalamus would simply abolish the\nanorexigenic and weight-lowering effects of butyrate, resulting in comparable body weight\nbetween vehicle- and butyrate-treated groups. Instead, the greater weight gain observed in\nbutyrate-treated cilia-deficient mice suggests that loss of mediobasal hypothalamic cilia may\nalter energy balance in a more complex manner. One possibility is that deletion of primary cilia\nin this region impairs energy expenditure—such as thermogenesis or locomotor activity—\nthereby overriding the catabolic actions of butyrate. Another, not mutually exclusive,\npossibility is that the absence of primary cilia in the mediobasal hypothalamus engages energy-\nconserving or other counter-regulatory pathways, rather than merely blunting butyrate\nsignaling, thereby predisposing cilia-deficient mice to greater weight gain than their vehicle-\ntreated counterparts. We believe that future studies directly examining this phenotype,\nparticularly in relation to energy expenditure, are warranted.\n10. LINES 161-162: “The SF-1 neuron- and AgRP neuron-specific IFT88 knockout mice\nexhibited no detectable cilia in these neurons, confirming the successful and targeted knockout\n(Fig. S3b, c).” These experiments could benefit from more controls. Can the authors show that\nthe cilia in SF1 cells are still present in AGRP models? And vice versa, are the cilia in AGRP-\nexpressing neurons unaffected in SF1 models?\nResponse: We thank the reviewer for this constructive suggestion, which allowed us to\nstrengthen our results. In response, we have replaced the immunostaining images and now\npresent primary cilia in both the VMH and ARC (Supplementary Fig. 4). The revised images\ndemonstrate that in AgRP neuron-specific IFT88 knockout mice, primary cilia are ablated in\nAgRP neurons but remain intact in SF-1 neurons. Conversely, in SF-1 neuron-specific primary\ncilia knockout mice, primary cilia are ablated in SF-1 neurons but remain intact in AgRP\nneurons in the ARC, thereby confirming the specificity of the knockout in each neuronal\npopulation.\n11. LINES 188-189: “Next, to gain further insights into how primary cilia contribute to\nbutyrate-induced regulation of AgRP neuron activity, we bath applied 1mM butyrate for 3-4\nminutes and observed variable response in both WT and IFT88 KOAgRP neurons (Fig. 5b).”\nThe text states that the application period is 3–4 minutes. What accounts for this variability?\nCould differences in bathing time affect the results or introduce inconsistencies?\nResponse: Thank you for raising this point. In our ex vivo brain slice patch-clamp experiments,\nwe use a simple manual gravity-flow perfusion system, which makes it difficult to maintain a\nperfectly constant flow rate. We typically aim for ~2 ml/min, and the stated application period\nof 3–4 minutes refers to the time during which the brain slice in the recording chamber is\nperfused with butyrate. Because our system is not automated or time-controlled, slight\nvariations of a few seconds may occur when clearing the drug from the chamber. We believe\nsuch minor variability is insufficient to introduce inconsistencies in the observed butyrate\nresponses. Moreover, to further ensure data reliability, we also present results after drug\nwashout (Figs. 5h, i, k, l).\n12. LINES 190-193: “Upon butyrate application, 71% of tested WT neurons (n = 15/21),\nexhibited changes in membrane potential, predominantly hyperpolarization whereas 52% of\ntested IFT88 KOAgRP neurons (n = 12/23) responded, showing either hyperpolarization or\ndepolarization (Fig. 5c, e-f).” It is not readily apparent whether these measurements were taken\nduring or after the application of butyrate. Additional information regarding these procedures\nshould be provided in the main text to improve clarity.\nResponse: We appreciate the reviewer’s suggestion. To clarify this point, we have added the\nfollowing sentence to the main Results section (lines 195–196): “The change in RMP following\nbutyrate treatment was measured as the difference between the RMP before and after\napplication.”\n13. Fig. 5c,d. The authors performed electrophysiological recordings on Agrp-expressing\nneurons in AgRP-cre; IFT88F/F 179 ; tdTomatoF/F mice (experimental group) and AgRP-cre;\ntdTomatoF/F mice (control group). One of their findings is that in control mice, application of\nbutyrate inhibits Agrp neurons (Fig. 5d). This could explain the decrease in food intake and\nbody weight gain shown by control mice treated with butyrate in vivo in Fig.1a and in mice\nwith IFT88 floxed in SF-1 neurons (Fig. 4a). On the other hand, bathing slices from\nexperimental mice with butyrate results in a smaller proportion of agrp neurons being inhibited\n(Fig. 5d). These results suggest that AgRP-cre; IFT88F/F mice treated with Butyrate (in Fig.\n4h-n) could eat more and gain more weight compared to their control, where such inhibition\nshould not occur (AgRP-cre; IFT88F/F mice treated with vehicle in Fig. 4h-n). Yet, these mice\nate and gained as much weight as their controls (Fig. 4h, j). The electrophysiological recording\nappears to explain the behavioral and metabolic effects observed in wild-type (WT) mice\ntreated with butyrate, but not the effects displayed by experimental ones in Fig. 4h-n. A better\nunderstanding could be provided if the authors were to include the electrophysiological data\nfrom Agrp-expressing neurons in wild-type and AgRP-cre; IFT88F/F 179; tdTomatoF/F mice\ntreated with vehicle (not just with Butyrate) to Fig. 5c,d. These controls would help determine\nwhether the effects on neuronal firing shown in Fig. 5c,d are due to the intrinsic deletion of\ncilia from Agrp neurons or to butyrate treatment, and could aid in interpreting the in vivo data.\nResponse: We sincerely appreciate the reviewer’s detailed and insightful comments. To\naddress the reviewer’s request to include electrophysiological data from AgRP-expressing\nneurons in wild-type and AgRP-cre; IFT88F/F; tdTomatoF/F mice treated with vehicle (not only\nwith butyrate), we have now included the requested electrophysiological measurements in Fig.\n5b–d. Specifically, analyses of resting membrane potential and spontaneous action potential\nfiring reveal no differences between groups, indicating that the intrinsic membrane properties\nof AgRP neurons are comparable between WT and IFT88 KOAgRP mice. In addition, the\nhistograms shown in Fig. 5c and d (revised as Fig. 5f and g) represent the proportion of neurons\nresponding to butyrate application, with responses quantified relative to the vehicle condition\n(i.e., baseline activity recorded in ACSF). Together, these data indicate that the altered neuronal\nresponses to butyrate observed in IFT88 KOAgRP neurons are attributable to primary cilia-\ndependent modulation of synaptic inhibition, rather than to baseline differences in intrinsic\nneuronal properties.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Rijal et al. identify a critical role for primary cilia of hypothalamic AgRP\nneurons in mediating how butyrate reduces food intake and promotes glucose tolerance.\nAfter setting up their mouse model of butyrate’s actions (Fig.1), Rijal et al. show that butyrate\npromotes ciliogenesis in the mouse hypothalamus and the N38 hypothalamic cell line, which\nthey use to characterize the likely mechanisms of butyrate’s ciliogenic effects: (i) histone\ndeacetylase inhibition, (ii) upregulation of ciliogenic transcription factors Rfx2 and Rfx3, and\n(iii) upregulation of key ciliary Rfx target genes (Fig.2).\nBy injecting AAV2-Cre into the hypothalamus of IFT88 conditional KO mice, they completely\neliminate hypothalamic cilia, leading to a complete loss of butyrate’s effects (Fig.3). They then\nshow that butyrate’s effects are also abolished by specifically deleting cilia in the AgRP neurons\nof the arcuate nucleus, which they achieve using the AgRP-Cre; Ift88 f/f mouse model (Fig.4h-\nn). By contrast, butyrate’s effects remain intact when the cilia of ventromedial hypothalamus\nSF1 neurons are deleted instead (Fig.4a-g). The authors conclude their study by showing that\ncilia are important regulators of AgRP neurons’ electrophysiological behavior (Fig.5).\nAltogether, I think the data are of high quality, very convincing, and of great relevance. The\nmanuscript is also well written. Therefore, my opinion is that this manuscript deserves prompt\npublication. Before that, however, I would recommend addressing the following issues:\nResponse: We appreciate the reviewer’s careful assessment of our manuscript. As outlined\nbelow, we have addressed each point raised, and these revisions have substantially improved\nthe quality and clarity of the manuscript.\n1. This is the most important of my points: by looking at Figure 5, a non-expert reader could\nreach the conclusion that cilia do not make much of a difference in AgRP neuron behavior,\nsince there is not a single panel in the figure showing statistically significant differences\nbetween WT and IFT88-KO-AgRP. In panels c-d the differences appear large, but there is no\nstatistical analysis to back their significance. Such an analysis should be added. Moreover,\nFig.S4e-h in the supplement shows statistically significant differences between WT and KO in\nthe sIPSC measurements: I think it would benefit the paper to move Fig.S4e-h to the main\nfigure.\nResponse:We thank the reviewer for this constructive suggestion, which helped us strengthen\nour results. We compared the significance levels for figure 5c and d (revised as Fig 5f and g)\nand found that figure 5g showed a statistically significant difference. The significance levels\nhave now been clearly indicated in both the figure panels and the corresponding legends. In\naddition, to further support our findings, the data previously presented in Figure 5 and\nSupplementary figure 4 have been reorganized and now included as the main figure 5 and 6\nand supplementary table 1 and 2 in the revised manuscript. Accordingly, these results are now\nexplained under two separate headings in the revised manuscript (lines 182–214 and 217–233).\n2. Line 94. Remove “of butyrate” (it is redundant)\nResponse:We have removed the word.\n3. Line 136. The way tdTomato genotype is indicated, it seems as if tdTomato expression\nshould be deleted by Cre, rather than activated as it is. Please clarify this for readers.\nResponse: We appreciate the reviewer’s comment and have revised the sentence to clearly\nindicate that tdTomato is Cre-activated (lines 136–141).\nThe original sentence:\nAccordingly, we stereotaxically injected recombinant adeno-associated virus serotype 2\n(AAV2) either with or without the Cre recombinase fused to green fluorescent protein (AAV2-\nGFP as control or AAV2-Cre-GFP) into the hypothalamic 3V of mice carrying floxed alleles\nfor both intraflagellar transport 88 and ROSA26-tdTomato (IFT88F/F::td-TomatoF/+)\nRevised sentence:\nTo examine this, we stereotaxically injected recombinant adeno-associated virus serotype 2\n(AAV2) — either encoding Cre recombinase fused to green fluorescent protein (AAV2-Cre-\nGFP) or lacking Cre (AAV2-GFP, control) — into the third ventricle (3V) of the hypothalamus\nin mice carrying floxed alleles of intraflagellar transport protein 88 (IFT88F/F) and a Cre-\ninducible tdTomato reporter at the ROSA26 locus (ROSA26-tdTomatoF/+).\n4. Lines 156-7. Revise sentence. Also add reference to Fig.1m.\nResponse: We thank the reviewer for the helpful suggestion. The sentence has been revised for\nclarity, and the requested reference to Fig. 1m (now Fig. 1o) has been added (lines 165–167).\nThe original sentence:\nSimilarly, in the ARC, AgRP/NPY are the key regulators of feeding behavior, and butyrate\ntreatment suppressed their expression of the orexigenic peptides AgRP and NPY.\nRevised sentence:\nIn the ARC, the orexigenic peptides AgRP and NPY, which are key regulators of feeding\nbehavior, were suppressed by butyrate treatment (Fig. 1o).\n5. Line 167. Those > that.\nResponse: We thank the reviewer for the comment. The sentence has been revised accordingly.\n6. Line 189. Fig.5a is nowhere referenced in the text.\nResponse:We thank the reviewer for pointing this out. We have corrected the oversight in the\nrevised manuscript.\n7. Line 211. sIPSCs > sEPSCs.\nResponse: We appreciate the reviewer’s comment. We have corrected the wording in the\nrevised manuscript.\n8. Remove “the”.\nResponse: We appreciate the reviewer’s comment. Although we were unable to identify a\nspecific instance in which “the” needed to be removed beyond this point, we noted that in the\nfirst sentence of the Discussion section (line 222 in the original version), “the butyrate” would\nbe more appropriately written as “butyrate.” Accordingly, we have removed “the” from this\nsentence in the revised manuscript.\n9. Lines 227-8. Since correlation alone is no proof of causation, and correlation is what has\nbeen shown here, I would not say “our study demonstrates”. A less conclusive statement, like\n“our study suggests” seems more appropriate.\nResponse: We thank the reviewer for the helpful suggestion and have revised the sentence to\nuse a more appropriate term, changing “demonstrates” to “suggests.”\n10. Line 235. Systematically > systemically.\nResponse:We thank the reviewer for the comment. The word has been revised accordingly.\n11. Line 276. Revise sentence.\nResponse: We sincerely appreciate the reviewer’s suggestion. The sentence has been revised\naccordingly (lines 304–307).\nThe original sentence:\nBased on their observation, oral, but not intravenous (i.v.) administration, butyrate reduced\nchow intake, and that this effect was abolished by total sub-diaphragmatic vagotomy, which\nthey interpreted as evidence supporting a vagal, rather than direct central mechanism.\nRevised sentence:\nThey observed that oral, but not intravenous (i.v.), administration of butyrate reduced chow\nintake, and this effect was abolished by total sub-diaphragmatic vagotomy. Based on these\nfindings, they suggested that butyrate acts through a vagal, rather than a direct central,\nmechanism.\n12. Line 318. Write “um” properly.\nResponse: We have corrected the unit and have properly written “µm” in the revised\nmanuscript.\n13. Line 332. Indicate source of N38 cell line here in methods.\nResponse: We thank the reviewer for pointing this out. We have now indicated the source of\nthe N38 cell line in the Materials and Methods section of the revised manuscript (line 362).\n14. Line 362. It should be “full recovery” or “fully recovering”.\nResponse:We have revised the phrase to “full recovery” in the revised manuscript.\n15. Line 682-3. Write reference 53 properly.\nResponse:We apologize for the oversight and thank the reviewer for the helpful comment. We\nhave properly corrected reference 53 in the revised manuscript. Additionally, we have\nthoroughly reviewed all references to ensure accuracy and consistency.\n16. Line 702. Protect > protects from.\nResponse:We appreciate the reviewer’s comment. We have revised the phrase from “protect”\nto “protects from” in the revised manuscript (line 736).\n17. Line 703. Improve > improves.\nResponse:We appreciate the reviewer’s comment. We have revised the phrase from “improve”\nto “improves” in the revised manuscript (line 737).\n18. Figure 5 legend. Please spell out the meaning of “WO” and “IEIs”.\nResponse: We thank the reviewer for the helpful comment. We have now spelled out the\nmeanings of the abbreviations “WO” (wash out) and “IEIs” (inter-event intervals) at the end of\nthe Figure 5 legend in the revised manuscript.\n19. Several mistakes in Suppl. Fig.1 legend: (i) remove “the” in first line, (ii) figure shows\nnothing about GPCRs, (iii) “lysine residues” instead of “lysine residuals”; (iv) HDAC inhibitor\nused is Trichostatin A, not Triclosan.\nResponse: We appreciate the reviewer’s detailed comment. The legend for Supplementary\nFigure 1 (now Supplementary Figure 2 in the revised manuscript) has been revised to (i)\nremove the article “the” in the first line, (ii) eliminate the incorrect mention of GPCRs, (iii)\nreplace “lysine residuals” with “lysine residues,” and (iv) correct the HDAC inhibitor name\nfrom “Triclosan” to “Trichostatin A.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors provided an updated manuscript that addresses all the concerns raised by this reviewer.\nThe authors have made a good effort to include additional data, which now increases the scientific strength of the study.\nOverall, the current manuscript provides new data that help clarify the role of primary hypothalamic cilia in the regulation of energy homeostasis. For this reviewer, the study has substantially improved in clarity, rigor, and relevance, and is now suitable for publication.", "reviewer_2": "The authors have addressed all my comments and provided explanation for the parts that, in the original manuscript, and in my opinion, deserved clarification. The manuscript is well written, the findings clear and of interest to an interdisciplinary audience. I have no further comments.", "reviewer_3": "The authors have addressed all my points."}, "author_response": ""}]

Decision letter