Ovarian hormones and high-fat diet duration distinctively modulate hypothalamic chemokine profile.

INTRODUCTION

As a central regulator of energy homeostasis, the hypothalamus integrates peripheral and central cues, including hormonal and nutritional signals, through coordinated activity of anorexigenic and orexigenic neurons.1,2A diet rich in saturated fatty acids (SFAs) rapidly disrupts this hypothalamic neuronal network involved with control of food intake and energy expenditure, contributing to fat mass accumulation.3,4High‐fat diet (HFD)‐induced hypothalamic dysfunction arises from a transient inflammatory response that evolves in distinct temporal phases.5,6In the acute phase, glial cells release high levels of pro‐inflammatory cytokines and undergo morphological changes, a process known as gliosis.7,8Perivascular macrophages contribute to the inflammatory milieu by producing inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF), which promote angiogenesis and increase vascular permeability.9,10,11Following this initial phase, there is an adaptive transition, during which compensatory mechanisms attempt to restore homeostasis.5Chronic phase is marked by the establishment of central resistance to leptin and insulin, further disrupting energy homeostasis and exacerbating metabolic dysregulation.6,12,13,14

Emerging evidence highlights that the chronic stage of HFD‐induced hypothalamic inflammation is accompanied by the directed migration of peripheral immune cells, such as myeloid cells originating from bone marrow progenitors, and Forkhead box P3 (Foxp3+) regulatory T cells (Tregs) derived from the thymus.15,16,17,18Four weeks of HFD exposure triggers the infiltration of bone marrow‐derived cluster of differentiation 169 (CD169+) immune cells into the mediobasal hypothalamus via the fenestrated vasculature of the median eminence (ME).15Upon entry, these cells migrate toward the arcuate nucleus (ARC) and undergo morphological transformation, adopting a microglia‐like phenotype; however, their precise role in HFD‐induced hypothalamic inflammation remains unclear. Recent data indicate that Foxp3+Tregs C–X–C motif chemokine receptor 3 (CXCR3hi), with lymphoid origin, exert a protective effect in this inflammatory context, as their depletion exacerbates HFD‐induced hypothalamic immune activation.16Similarly, our group has recently shown that CXCR3+myeloid cells help mitigate HFD‐induced metabolic impairments.17In this study, we have also found that recruited hypothalamic C–C chemokine receptor type 2 (CCR2+) cells exhibit pronounced sex‐specific messenger RNA (mRNA) expression, including differential expression of genes involved in chemokine signaling and immune response pathways, suggesting that biological sex modulates their contribution to HFD‐induced hypothalamic inflammation.

Sex differences in the architecture and function of the central melanocortin system have been increasingly recognized and may underlie the distinct metabolic impairments observed between males and females in obesity and other metabolic disorders.19However, how biological sex shapes the hypothalamic chemokine profile during the progression of HFD‐induced inflammatory response remains unexplored. We hypothesized that hypothalamic chemokine and chemokine receptor expression is dynamically and differentially regulated throughout the phases of HFD‐induced inflammation in a sex‐dependent manner. To test this, we analyzed the transcripts of the hypothalamus of male and female mice subjected to acute and chronic HFD, as well as ovariectomized females fed a standard diet (SD) with or without estradiol (EST) replacement. We demonstrated that most chemokine pathways show biphasic regulation during HFD exposure, independent of sex, with C–C motif chemokine ligand 2 (also known as MCP‐1) (CCL2)/CCR2 exhibiting particularly pronounced dynamics. In contrast,Cxcr3is sex‐specifically regulated, with higher female expression and sensitivity to dietary fat and ovarian hormones, revealing a critical role for ovarian hormones in hypothalamic immune homeostasis.

MATERIALS AND METHODS

Animal care and diets

Six‐week‐old male and female C57BL/6J mice were obtained from CEMIB (Centro Multidisciplinar para Investigação Biológica—University of Campinas, Brazil). All animal care and experimental procedures were conducted in accordance with the guidelines of the Brazilian College for Animal Experimentation and were approved by the Institutional Animal Care and Use Committee (CEUA 6393‐1/2024 and 6610‐1/2025).

For experiment with a dietary challenge, mice were socially housed (N= 2–3 per cage) in a controlled environment (22 ± 2°C, 12‐h light/dark cycle) with ad libitum access to food and filtered water. After 1 week of acclimatization, mice were randomly assigned to experimental groups receiving a HFD for 1, 3, 14, or 28 days (groups 1d HFD, 3d HFD, 14d HFD, and 28d HFD, respectively). The HFD provided 20.1% of energy from protein, 34.7% from carbohydrate, and 45.2% from fat (4.70 kcal/g; D12451, ResearchDiets, USA). Control group was fed exclusively with a SD containing 12.6% of energy from protein, 77.7% from carbohydrate, and 9.58% from fat (3.76 kcal/g; AIN93M, Pragsoluções, Brazil).

The HFD challenge was conducted during the final days prior to euthanasia, according to the experimental timeline, ensuring that all mice were sacrificed at the same age (Figure1A). Body weight (BW) was measured at baseline and at the end of the dietary intervention, on the day of euthanasia. On that day, mice were fasted from 8 a.m. to 2 p.m., and tissue collection was performed at 2 p.m.

*High‐fat diet induces sex‐specific metabolic alterations. (A) Schematic representation of the experimental protocol of the high‐fat diet (HFD) challenge. (B) Percentage of body weight gain during the experimental protocol. (C) Weight of white adipose tissue (retroperitoneal depot) at the end of the experiment. (D) Six‐hour fasting blood glucose levels. Data are expressed as mean ± standard error of the mean of 10–12 mice per group. Statistical analyses were performed using two‐way ANOVA followed by Sidak's post hoc test. *p< .05, ***p< .0001 compared with the indicated groups. BW, body weight; rWAT, retroperitoneal white adipose tissue; SD, standard diet. dagger:p< .05 when the effect of sex is observed by two‐way ANOVA analysis. double dagger:p< .05 when the effect of interaction of sex and time (HFD duration) is observed by two‐way ANOVA analysis.

Ovariectomy procedure and estradiol replacement

To analyze the role of ovarian hormones in hypothalamic chemokine profile, we conducted a bilateral ovariectomy procedure. The surgery was conducted as described by Mendes et al.17The experimental timeline is shown in Figure5A. Eight‐week‐old C57BL/6J female mice were anesthetized with a ketamine/xylazine solution (100 and 10 mg/kg, respectively). The ventral abdominal area was shaved and sterilized with iodine solution. A small midline incision was made to expose and bilaterally excise the ovaries (OVX group). Half of the ovariectomized mice received EST replacement via subcutaneously implanted 17β‐estradiol pellets (0.05 mg/pellet, 60‐day sustained release; Innovative Research of America, Inc., USA) beneath the dorsal neck region (OVX + EST group). Sham‐operated mice, which underwent the same procedure without ovary removal, were used as controls (Sham group). For analgesia, tramadol hydrochloride (5 mg/kg) was intraperitoneally administered immediately after surgery, and again after 24 and 36 h. Mice were individually housed and monitored during a 7‐day recovery period. From Day 7 to Day 35, we measured BW weekly. On Day 25, mice were fasted for 6 h, and fasting glycemia was measured prior to euthanasia.

Tissue sampling

Prior to tissue collection, mice were deeply anesthetized by intraperitoneal injection of a ketamine/xylazine solution (300 and 30 mg/kg, respectively). The hypothalamus was immediately harvested and frozen in liquid nitrogen. Retroperitoneal white adipose tissue (rWAT) was dissected and weighed. To confirm the efficacy of bilateral ovariectomy and EST replacement, the whole uterus from three mice of each group was collected, weighed, photographed for macroscopic comparison, and processed for histological analysis.

Uterus morphology

Uterine horns from groups Sham, OVX, and OVX + EST (N= 3/group) were fixed in 4% paraformaldehyde, paraffin‐embedded, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (H&E) following standard procedures. Images were acquired using an inverted brightfield microscope (Leica DMI 4000 B) at 10× and 40× magnifications at the National Institute of Photonics Applied to Cell Biology (INFABIC) at the University of Campinas.

Quantitative reverse transcription‐polymerase chain reaction

Hypothalami were homogenized in 500 μL of TRIzol Reagent (Invitrogen) and incubated for 5 min at reverse transcription (RT). After adding 100 μL of chloroform (Merck Millipore) and centrifuging at 12,000 rpm for 15 min at 4°C, the aqueous phase was collected and RNA precipitated with 250 μL of isopropanol. Following a 5‐min incubation at RT, samples were centrifuged (12,000 rpm, 4°C) to form RNA pellets, which were washed sequentially with 75% and 100% ethanol (8400 rpm, 10 min, 4°C), air‐dried, and resuspended in 20 μL RNase/DNase‐free water. For reverse transcription, 2 μg of RNA was converted to complementary DNA (cDNA) using the High‐Capacity cDNA Reverse Transcription Kit (Life Technologies) following the manufacturer's protocol. RNA quantity and purity were measured by absorbance at 260/280 nm.

Real‐time polymerase chain reaction (PCR) was performed using the TaqMan system (Applied Biosystems). cDNA products were diluted 1:10, and a pooled mix was used to generate a standard curve. On a 96‐well plate, each well received 3.5 μL of TaqMan mix (PCR Biosystems, qPCRBIO Probe Mix), 0.25 μL of DNase/RNase‐free water, 0.25 μL of primers, and 4 μL of diluted cDNA. Gene expression was measured using QuantStudio 6 (Applied Biosystems).

Cycle threshold (Ct) values were obtained using QuantStudio software (Applied Biosystems). Relative mRNA expression levels of target genes were normalized to the housekeeping gene Gapdh (Mm99999915_g1) using the ΔCt method. For comparisons between experimental groups, the ΔΔCt method was applied, and fold changes were calculated as 2−ΔΔCt. Gene expression levels are presented as log2fold change relative to the mean of the respective control groups for data visualization and statistical analysis. Primer sequences were obtained from Thermo Fisher Scientific. The list of primers and their assay IDs are shown in TableS1.

Statistical analyses

Statistical analyses were conducted using GraphPad Prism version 8.4.2https://www.graphpad.com/. For the diet challenge experiment, biometric parameters were compared using a two‐way analysis of variance (ANO with HFD duration and sex as factors, followed by Sidak's post hoc tests. Quantitative polymerase chain reaction (qPCR) data transformed to log2fold change values were analyzed using two‐way ANOVA with HFD duration and sex as factors, followed by Sidak's post hoc tests. For the ovariectomy experiment, biometric parameters and qPCR were compared using a one‐way ANOVA, followed by Tukey's post hoc tests. Biometric data are presented as mean ± standard error of the mean, while qPCR data are shown as violin plots highlighting the median and quartiles. Significance is indicated as *p< .05, **p< .01, ***p< .001, and ****p< .0001.

RESULTS

High‐fat diet exposure triggers dynamic, sex‐dependent metabolic alterations and transient hypothalamic inflammation

HFD‐fed males showed greater BW gain and increased rWAT mass than HFD‐fed females, particularly after 28 days of HFD (p< .01; Figure1B,C). Fasting glycemia was elevated in males of HFD 28d group (p< .001; Figure1D). Despite these metabolic differences, caloric intake did not differ between HFD‐fed groups or sexes (data not shown).

Analysis of hypothalamic neuropeptide expression revealed that pro‐melanin concentrating hormone (Pmch) was consistently reduced in females compared to males throughout the HFD challenge, showing significant effects of sex, time, and their interaction (p< .05; Figure2A). Agouti‐related peptide (Agrp) expression was slightly decreased in most HFD‐fed groups, primarily in a time‐dependent manner (p< .01), with no significant sex effects (Figure2B). In females, pro‐opiomelanocortin (Pomc) expression declined only after 28 days of HFD (p< .01; Figure2C), while males exhibited a non‐significant decrease at 14 days followed by a marked rebound at 28 days (p< .0001; Figure2C).

*High‐fat intake induces hypothalamic inflammation and alters the neuropeptide profile in a sex‐ and time‐dependent manner. (A–C) Hypothalamic mRNA levels of neuropeptides. (D–F) Hypothalamic mRNA levels of inflammatory genes. Data are shown as violin plots with median and quartiles for four to six mice per group. qPCR data (log2fold change) were analyzed using Two‐way ANOVA with sex and high‐fat diet (HFD) duration as factors, followed by Sidak's post hoc tests. *p< .05, **p< .01, ***p< .001, ***p< .0001 compared with the indicated groups. SD, standard diet.

The pro‐inflammatory marker tumor necrosis factor‐alpha (Tnfa) was higher in the hypothalamus of males at all time points, with the largest difference at 1 day of HFD (p< .05; Figure2D). Conversely, interleukin‐6 (Il6) increased progressively in females, with significant effects of sex, diet duration, and their interaction forIl6(p< .05; Figure2E). Toll‐like receptor 4 (Tlr4) expression was influenced by HFD duration, displaying a distinct biphasic pattern: reduced in the acute phase and increased in the chronic phase (p< .05; Figure2F). Together, these results show that HFD‐fed male mice exhibit higher hypothalamicTnfaexpression than females at the early stage of diet exposure. These findings indicate a more pronounced early hypothalamic inflammatory response in males. Additionally, the temporal analysis reveals that the hypothalamic inflammatory response to HFD is dynamic and differs between sexes.

HFDexposure dynamically modulates hypothalamic chemokine pathways in a time‐dependent manner

To better understand how hypothalamic chemokine profile changes in the distinct phases of HFD‐induced inflammation, we also evaluated the gene expression of some chemokines and their receptors. For both sexes,Ccr2, a chemokine receptor primarily expressed on key immune cells involved in inflammation and immune surveillance, and its ligandCcl2, showed reduced expression during the acute phase of HFD challenge, followed by recovery in the chronic phase (p< .05; Figures3Aand4A).

*Chemokine receptorsCcr2,Cxcr3, andCx3cr1are modulated by high‐fat diet (HFD) in females. (A–E) Hypothalamic mRNA levels of chemokine receptors. Data are shown as violin plots with median and quartiles for four to six mice per group. qPCR data (log2fold change) were analyzed using two‐way ANOVA with sex and high‐fat diet (HFD) duration as factors, followed by Sidak's post hoc tests. *p< .05, **p< .01, **p< .001 compared with the indicated groups. SD, standard diet.

*High‐fat diet induces sex‐specific modulation of the chemokinesCcl2andCx3cl1. (A–G) Hypothalamic mRNA levels of various chemokines. Data are shown as violin plots with median and quartiles for four to six mice per group. qPCR data (log2fold change) were analyzed using two‐way ANOVA with sex and high‐fat diet (HFD) duration as factors, followed by Sidak's post hoc tests. *p< .05, **p< .01, ***p< .001, ***p< .0001 compared with the indicated groups. SD, standard diet.

Similarly,Cxcr3, a chemokine receptor predominantly expressed on activated T cells, and its ligands (C–X–C motif chemokine ligand 9 [Cxcl9], C–X–C motif chemokine ligand 10 [Cxcl10], and C‐X‐C motif chemokine ligand 11 [Cxcl11]) displayed a transient decrease after 3 days of HFD exposure, followed by later upregulation, with females showing an earlier rise and higher expression ofCxcr3at 14 and 28 days of HFD (p< .05; Figures3Band4B–D).

The C–X–C motif chemokine receptor 4 (Cxcr4), a chemokine receptor expressed not only in infiltrating immune cells, but also in brain resident cells, and its ligand C–X–C motif chemokine ligand 12 [Cxcl12] (also known as stromal cell‐derived factor 1 alpha [SDF1‐α]) mirrored this pattern:Cxcl12decreased after 3 days of HFD in females and increased with chronic HFD (p< .001; Figure4E), whileCxcr4showed time‐dependent changes without post hoc significance (p< .05; Figure3C). The C–X–C motif chemokine receptor 6 (Cxcr6), a chemokine receptor involved in immune cell trafficking, and its ligand C–X–C motif chemokine ligand 16 (Cxcl16) were similarly affected by HFD duration, regardless of sex (p< .05; Figures3Dand4F).

In contrast, the receptorCx3cr1, a classical microglia marker, andCx3cl1(fractalkine), which is its ligand, being primarily produced by neurons, displayed sex‐specific regulation. Females showed a progressive decline inCx3cr1expression over time, whereas males maintained higher levels at 28 days of HFD (p< .001; Figure3E). Corresponding changes in the ligandCx3cl1reflected these dynamics, with early decreases in females and late increases in males (p< .001; Figure4G).

Overall, these findings indicate that HFD induces transient alterations in hypothalamic chemokine signaling, such as in CCL2‐CCR2 and C–X3–C motif chemokine ligand 1‐C–X3–C motif chemokine receptor 1 (CX3CL1‐CX3CR1) pathways, following time‐dependent dynamics in key pathways involved in immune cell recruitment and neuron–microglia communication. Sex effects were not significant, except forCxcr3andCx3cr1, whose expression was, respectively, higher and lower in females during the chronic phase of HFD exposure.

Ovarian hormones absence triggers metabolic impairments and hypothalamic inflammation

Next, to isolate the role of ovarian hormones in hypothalamic chemokine expression, females exclusively fed on SD underwent bilateral ovariectomized (OVX) with or without EST replacement (Figure5A). Both and OVX + EST mice showed increased BW compared with Sham controls (p< .05; Figure5B,C), without significant changes in rWAT depot weight. Fasting glycemia (Figure5D) and total caloric intake (data not shown) remained unchanged.

*Ovariectomy induces metabolic dysfunction independent of dietary composition. (A) Schematic representation of the experimental design. (B) Percentage of body weight gain during 4 weeks after the surgical removal of the ovaries (OVX) surgical procedure. (C) Weight of white adipose tissue (retroperitoneal depot, retroperitoneal white adipose tissue [rWAT]) at the end of the experiment. (D) Six‐hour fasting blood glucose levels. (E) Representative macroscopic images of the uterus collected at euthanasia. (F) Uterine weight measurements. (G) Representative histological images of uterine horns (hematoxylin and eosin staining). Data are expressed as mean ± standard error of the mean of 5–10 mice per group, except for uterine weight (N= 3 per group). Statistical analyses were performed using one‐way ANOVA, followed by Tukey's post hoc test. *p< .05, **p< .001,p< .00001, compared with the Sham group. BW, body weight; EST, estradiol; GE, glandular epithelium; LE, luminal epithelium; S, stroma; UL, uterine lumen.

Macroscopic analysis showed uterus with visible ovaries in intact females (Sham group), whereas OVX uterus was markedly reduced, thin, and atrophic. EST replacement (OVX + EST) restored uterine growth, resulting in enlarged and hypertrophic organs (Figure5E). Consistently, uterine weight was lower in OVX compared to Sham, although without statistical significance, while EST replacement (OVX + EST) significantly increased uterine weight relative to both Sham and OVX groups (Figure5F). Histologically, the uterus from intact females displayed preserved architecture with a well‐developed endometrium, abundant glands, and columnar epithelium. Bilateral ovariectomy resulted in endometrial atrophy with reduced thickness, fewer glands, and lower epithelium. EST treatment reversed these changes, increasing epithelial height and glandular development compared to the OVX group (Figure5G). Altogether, these morphological, histological, and quantitative findings confirm that the ovariectomy and hormone replacement protocol was effective.

The absence of ovarian hormones induced alterations in the hypothalamic transcript levels of the orexigenic neuropeptideAgrp, which was reduced in OVX females (p< .05; Figure6B), whereasPmchandPomc expression remained unchanged (Figure6A–C). These changes were accompanied by signs of inflammatory activation, as indicated by increasedTnfaexpression in OVX females (p< .05; Figure6D). No differences were detected inIl6orTlr4expression levels (Figure6E,F).

*Ovarian hormone deficiency modulates hypothalamic neuropeptides and inflammatory markers. (A–C) Hypothalamic mRNA levels of neuropeptides. (D–F) Hypothalamic mRNA levels of inflammatory markers. Data are shown as violin plots with median and quartiles for five to seven mice per group. qPCR data (log2fold change) were analyzed using one‐way ANOVA, followed by Tukey's post hoc tests. p< .05 compared with the Sham group. EST, estradiol; OVX, ovariectomized.

Hypothalamic chemokine profile is influenced by ovarian hormones

Analysis of hypothalamic chemokine expression in OVX females and their controls revealed a significant increase inCcl2mRNA levels in both OVX and OVX + EST groups (p< .001 andp< .05, respectively; Figure8A), whereasCcr2expression remained unchanged (Figure7A). Consistent with findings from females at 14 and 28 days of HFD,Cxcr3expression was upregulated in the OVX group (p< .05; Figure7B), with no differences detected inCxcl9,Cxcl10, orCxcl11(Figure8B–D). Interestingly,Cxcr4expression increased in OVX + EST females compared with both Sham and OVX groups (p< .05–.01; Figure7C), while other chemokine receptors and their respective ligands remained unaltered (Figures7D,Eand8D,E). Overall, these results indicate that the absence of ovarian hormones alters hypothalamic chemokine and inflammatory profiles in females, fostering a pro‐inflammatory state and metabolic dysregulation that are only modestly mitigated by EST replacement.

*Cxcr3 expression in the hypothalamus is modulated by ovarian hormones. (A–E) Hypothalamic mRNA levels of chemokine receptors. Data are shown as violin plots with median and quartiles for five to seven mice per group. qPCR data (log2fold change) were analyzed using one‐way ANOVA, followed by Tukey's post hoc tests. *p< .05, *p< .01 compared with the indicated groups. EST, estradiol; OVX, ovariectomized.

*Ovariectomy results in increasedCxcr3expression in the hypothalamus. (A–G) Hypothalamic mRNA levels of chemokine receptors. Data are shown as violin plots with median and quartiles for five to seven mice per group. qPCR data (log2fold change) were analyzed using one‐way ANOVA, followed by Tukey's post hoc tests. *p< .05, *p< .01 compared with the Sham group. EST, estradiol; OVX, ovariectomized.

DISCUSSION

This study is the first to characterize how short‐ and long‐term exposure to a HFD modulates the hypothalamic chemokine profile in male and female mice, highlighting the role of ovarian hormones in this process. In line with previous studies showing greater male vulnerability to diet‐induced metabolic dysfunction,20,21,22,23we observed that males gained more BW and adipose mass than females under the same dietary conditions. Several studies have reported that HFD exposure rapidly disrupts the homeostatic balance of hypothalamic anorexigenic and orexigenic neurons.4,24In our study, we observed a sex‐specific effect primarily inPmch, which encodes melanin‐concentrating hormone (MCH), reinforcing the influence of sex on MCH regulation, as recently highlighted by Kuebler et al.25Regarding other neuropeptides, our data align with previous reports demonstrating a transient, time‐dependent compensatory response to HFD in both males and females.20,22

Our results in OVX females further support the critical role of ovarian hormones in metabolic regulation. Consistent with previous studies,26,27,28,29OVX females exhibited increases in BW accompanied by modifications in hypothalamic neuropeptide expression,30,31despite no differences in total caloric intake compared with Sham controls. In OVX females, we observed a reduction inAgrpexpression, with no significant changes inPomclevels. In our previous study, analyses of HFD‐fed OVX females revealed lower hypothalamicAgrpand higherPomcexpression.17These data suggest thatPomcupregulation in females occurs primarily through the interaction between ovarian hormone deficiency and HFD, whereasAgrpregulation appears to depend on both diet and ovarian hormones through distinct mechanisms. These metabolic data indicate that the absence of ovarian hormones renders females susceptible to metabolic alterations and dysregulated neuropeptide expression, similar to changes observed in chronically HFD‐fed mice, highlighting their pivotal role in protecting hypothalamic function and maintaining energy homeostasis.

We also investigated how biological sex and the duration of HFD exposure modulate the inflammatory response in the hypothalamus. After just 1 day of HFD feeding, male mice exhibited higher hypothalamicTnfaexpression compared with females, suggesting an earlier activation of glial cells, as this cytokine is primarily released by microglia in response to excessive saturated fatty acid intake.7This finding is consistent with Cansell et al.24who reported rapid glial activation and increased inflammatory cytokine release in males within only a few hours of HFD exposure. In females, hypothalamic Tnfα expression was increased in OVX mice and restored by estrogen replacement, whereas HFD‐fed intact females showed no changes in this pro‐inflammatory cytokine regardless of HFD duration. Interestingly, in our previous study using OVX mice fed an HFD,17no differences were observed between OVX and Sham groups, suggesting that Tnfα modulation in females is primarily hormone‐dependent rather than directly driven by dietary fat exposure. This apparent discrepancy, together with our previous observations in OVX mice under HFD conditions, suggests that the effects of ovarian hormone deficiency on hypothalamic inflammation are context‐dependent and may be attenuated under chronic metabolic challenge.

BeyondTnfa, we identified additional inflammatory markers showing sex‐specific regulation. Toll‐like receptor 4 (TLR4), a key sensor of dietary saturated fats in the hypothalamus, plays a central role in triggering microglial activation and inflammatory signaling.32,33,34In the present study, HFD‐fed females displayed reduced Tlr4 expression during the early phase of HFD exposure, suggesting a rapid, adaptive protection against inflammatory stimuli. However,Tlr4levels did not differ between Sham and OVX females, indicating that ovarian hormones modulate hypothalamic inflammation through mechanisms beyond TLR4 signaling. Interestingly, Curtis et al.35reported that OVX‐induced changes inTlr4expression are nucleus‐specific, with reductions in the ARC but no alterations in the paraventricular nucleus. Since we measured transcript levels in the whole hypothalamus, this broader approach may have masked such nucleus‐specific effects, representing a limitation of the present study.

Regarding the hypothalamic chemokine profile, HFD‐fed females exhibited a progressive reduction inCx3cl1and its receptorCx3cr1from Day 3 to Day 28, suggesting a sustained disruption of neuron–microglia communication. Interestingly, this pattern contrasts with findings from studies using longer HFD exposure (18 weeks), in which females displayed increased hypothalamic CX3CL1/CX3CR1 signaling, a response proposed to counteract metabolic impairments in this sex.36In our previous work, we sorted hypothalamic CX3CR1+cells by FACS and found no major sex differences in cell number after 4 weeks of HFD.17Thus, the HFD‐induced changes in bothCx3cl1andCx3cr1mRNA observed here likely reflect impaired neuron–microglia communication, potentially contributing to attenuated microglial pro‐inflammatory cytokine release in females.

The anatomic position of the hypothalamus, adjacent to the third ventricle and near the fenestrated capillaries of the ME, facilitates chemokine‐mediated immune cell infiltration under HFD conditions.37In experimental models of diet‐induced obesity, CCL2 (also known as MCP‐1) is a key chemokine in this process, showing raised expression in various tissues.38,39In the present study, we observed that hypothalamic CCL2/CCR2 signaling exhibited a dynamic, biphasic response to HFD exposure. Initially, bothCcl2andCcr2expression were downregulated during the acute phase (Days 1–3), followed by recovery or upregulation during chronic feeding (Days 14–28). This transient pattern likely represents an adaptive mechanism; the early suppression may restrain excessive neuroinflammation, while subsequent reactivation facilitates CCR2+monocyte recruitment that contributes to the chronic inflammatory state. Supporting this interpretation, previous studies have shown that CCR2+cell infiltration into the hypothalamus increases progressively with HFD duration, with substantial parenchymal accumulation evident by 4 weeks.15,17,18

Lainez et al.18quantified cluster of differentiation 11b and high levels of cluster of differentiation 45 (CD11b+CD45high) macrophages in the hypothalamus of 12‐week HFD‐fed mice and found greater accumulation in males than females, correlating with worse metabolic dysfunction in males. Regarding CCR2+cells, we previously found similar frequencies in the hypothalamic parenchyma of males and females after 4 weeks of HFD using flow cytometry.17Consistent with these cellular data, we detected no sex differences inCcr2orCcl2mRNA expression during either acute or chronic HFD exposure. Notably, despite similar CCR2/CCL2 expression between sexes, our previous transcriptomic analysis revealed sex‐specific gene expression profiles within the CCR2+infiltrating cells themselves, particularly in chemotaxis and interferon‐γ pathways.17These findings suggest that functional differences between male and female immune responses extend beyond the transcriptional regulation of chemokine ligands and receptors, potentially involving distinct cellular activation states or effector functions.

An important feature of the infiltrating CCR2+cell population in the central nervous system (CNS) is a subset that co‐expresses CXCR3, a chemokine receptor found on monocytes and activated Tregs. We have previously shown that infiltration of CCR2+CXCR3hicells into the hypothalamus attenuates HFD‐induced metabolic impairments,17suggesting a protective role for this specific immune cell subset. Here, to investigate sex‐specific regulation of CXCR3 during HFD exposure, we have also analyzed its hypothalamic transcript levels. While most chemokines showed similar patterns between sexes,Cxcr3was a notable exception; females consistently exhibited higher expression, particularly during later stages of HFD. Interestingly, OVX females also displayed elevated hypothalamicCxcr3, which was restored by estrogen replacement. The consistent upregulation of CXCR3 in both HFD‐fed and OVX females suggests a controlled recruitment of a subset of immune cells that may aid resolution rather than exacerbate inflammation. This pattern aligns with the milder inflammatory phenotype in females and indicates hormone‐dependent modulation of chemotactic signaling that supports metabolic protection.

In conclusion, our findings show that HFD induces dynamic, sex‐specific changes in hypothalamic chemokine signaling. While the CCL2–CCR2 axis was strongly modulated by HFD in both sexes, females exhibited lowerTnfalevels at HFD onset, a progressive reduction inCx3cl1andCx3cr1transcripts, and increasedCxcr3expression during chronic stages, likely contributing to their early metabolic resilience. Ovarian hormone deficiency abolished this protective profile, shifting females toward a pro‐inflammatory state resembling males. These results highlight ovarian hormones as critical regulators of hypothalamic chemokine networks and metabolic homeostasis, underscoring the importance of sex‐dependent mechanisms in neuroimmune regulation.

Importantly, our quantitative PCR‐based approach captures broad transcriptional dynamics, but future studies using nucleus‐specific analyses and cellular localization will be crucial to resolve region‐specific mechanisms in HFD and OVX models. Additionally, estrous cycle stage was not monitored in the HFD time‐course or Sham cohorts, which may contribute to within‐group variability in females and represents a limitation to be addressed in future studies. It should also be noted that the estrogen dose employed in the OVX replacement model may have exceeded physiological levels, as indicated by the marked increase in uterine mass observed relative to the Sham group, which represents an additional limitation of the present study. Targeted hormone replacement studies could further clarify the roles of estrogen, progesterone, and other ovarian factors in shaping these sex‐specific neuroimmune and metabolic adaptations.

AUTHOR CONTRIBUTIONS

NFM: designed and planned the study, wrote the manuscript, and prepared the figures;AGBAandGAdS: performed most of the experiments;IVSC,VSM,LSB,NPM, andEdAS: contributed to tissue harvesting and/or surgical procedures;LAV: supervisedEdASandLBSduring their doctoral programs and contributed to data discussion. All authors read and approved the final manuscript.

FUNDING INFORMATION

This research was funded by Sao Paulo Research Foundation (FAPESP) (2013/07607‐8 and 2022/06282‐7). Alexia Guimaraes Batista Augusto and Giovanna Ariozi dos Santos received fellowships from FAPESP (2023/11393‐5 and 2024/18774‐7, respectively). Open access publishing facilitation was provided by Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), through its transformative “Read and Publish” agreement with Wiley.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

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Republished from the open web under CC-BY. Authors: Augusto AGB, Dos Santos GA, Cavalheiro IVS, Mantovan VS, Bergantini LS, Marin NP, Dos Santos Alves E, Velloso LA, Mendes NF. Read the original.