Sex-specific vulnerabilities in human astrocytes underpin the differential impact of palmitic acid

Obesity and neurometabolic diseases have been linked to neurodegenerative diseases. Our hypothesis is that the endogenous estrogenic component of human astrocytes plays a critical role in cell response during lipotoxic damage, given that obesity can disrupt hormonal homeostasis and cause brain inflammation. Our findings showed that high concentrations of palmitic acid (PA) significantly reduced cell viability more in male astrocytes, indicating sex-specific vulnerabilities. PA induced a greater increase in cytosolic reactive oxygen species (ROS) production in males, while female astrocytes exhibited higher superoxide ion levels in mitochondria. In addition, female astrocytes treated with PA showed increased expression of antioxidant proteins, including catalase, Gpx-1 and Nrf2 suggesting a stronger cellular defence mechanism. Interestingly, there was a difference in the expression of estrogenic components, such as estrogen, androgens, and progesterone receptors, as well as aromatase and 5 α -reductase enzymes, between males and females. PA induced their expression mainly in females, indicating a potential protective mechanism mediated by endogenous hormones. In summary, our findings highlight the impact of sex on the response of human astrocytes to lipotoxicity. Male astrocytes appear to be more susceptible to cellular damage when exposed to high concentrations of fatty acids.


Introduction
Obesity is a risk factor for the development of neurodegenerative diseases (ND) (Mazon et al., 2017a).According to the World Health Organization, 16% of the population over 18 years old lived with obesity in 2022.The global prevalence of obesity has more than doubled in both men and women between 1990 and 2022 (Okunogbe et al., 2022).Previous studies have found that this medical condition is more prevalent among women, regardless of age, geographic region, or socioeconomic status (Chooi et al., 2019).
A significant correlation between high-fat diet (HFD) and cognitive decline in humans has been observed, in particular those diagnosed with obesity and type 2 diabetes (T2D) (Martin-Jiménez et al., 2017;Mazon et al., 2017b).A 50% likelihood of developing conditions such as dementia and Alzheimer's disease (AD) has been identified as a result of inflammatory processes along with their close association with obesity (Cunnane et al., 2011).Furthermore, an excess of fatty acids can result in metabolic, structural, and functional deficits in mitochondria, all of which are common hallmarks of neurodegenerative diseases (Sandu et al., 2015).
Exposure to HFD induces brain inflammation (Pugazhenthi et al., 2017) and activates astrocytes (Wu and Yin, 2022).Astrocytes are brain cells that use fatty acids as energy substrates (Wong et al., 2014a) to cope with high neuronal energy expenditure (Cardoso et al., 2016;McGrattan et al., 2019).Nevertheless, an excess of fatty acids directly impacts their functionality and integrity (Mendell and MacLusky, 2018).Fatty acids predominantly affect mitochondria, where increased mitochondrial β-oxidation (Audano et al., 2018) and cytochrome P450 induction produce oxidative stress and nitrogen free radicals.These changes alter brain cell dynamics and contribute to the development of ND (Fraser et al., 2010;Gupta et al., 2012).
Palmitic acid (C 16 H 32 O 2 ) constitutes 21-30% of adipose tissue (Fernández-Quintela et al., 2007) and is the primary saturated fatty acid in the human diet (El Akoum et al., 2011).As a significant energy source, fatty acids (FAs) produce more than twice the energy of carbohydrates or proteins through β-oxidation in mitochondria (Miyoshi et al., 2007).Compromised fatty acid oxidation leads to mitochondrial dysfunction, causing oxidative stress (Schönfeld and Reiser, 2013) and mitochondrial uncoupling.Moreover, failure to oxidise FAs results in mitochondrial damage and the activation of programmed cell death, ultimately leading to organelle breakdown (Schönfeld and Reiser, 2017).
Early hormonal exposure, endogenous hormones, and chromosomal organization influence sex differences in the brain (Brown et al., 2005;Khan et al., 2019), particularly in astrocytes.To ensure a more precise interpretation of outcomes, it is crucial to investigate the response of estrogen (ERα and ERβ), androgen (AR) and progesterone (PGR) receptors, as well as the modulation of enzymes responsible for estrogen and androgen production such as aromatase or 5α-reductase under damaging conditions.As part of the hormonal component, aromatase is an enzyme that is activated under pathological conditions, leading to an increase in the endogenous synthesis of estradiol in the tissue or cell.This is a defence mechanism against oxidative damage and inflammation (Saldanha, 2021;Wang et al., 2021) in neurons and astrocytes mitochondria (Saldanha, 2021;Ventura-Clapier et al., 2017).Therefore, it is essential to study the sex-dependent responses, specifically regarding the impact of fatty acids and metabolic stress on this organelle.The aim of our study was to investigate the responses of male and female human astrocytes to lipotoxic damage caused by palmitic acid and to examine the participation of endogenous hormonal components in this mechanism.

Human astrocytes culture
For this study, we used human astrocytes isolated from the male and female cortex (Catalog No. 1800, USA).These cells were cultured and maintained in astrocyte medium (AM, Catalog No. 1801) supplemented with astrocyte growth supplement (AGS, Catalog No. 1852) and 2% fetal bovine serum (FBS,Catalog No.0010).All cells and cell culture reagents were purchased from ScienCell Research Laboratories.The cells were kept at 37 • C with 5% CO 2 and seeded to approximately 80% confluence in 96-well plates for viability assays, 24-well plates for flow cytometry, 12-well plates for RNA extraction, and 6-well plates for protein extraction.

Palmitic acid treatment
Palmitic acid (P5585, Sigma, St. Louis, MO, USA) was utilized to create an in vitro model of cellular stress induced by fatty acids.Metabolic stress stimulation with fatty acids involved preparing a 5 mM palmitic acid stock solution, which contained palmitic acid in 100% ethanol and 5% bovine serum albumin (BSA) (A2153, Sigma, St. Louis, MO, USA) in 1× PBS.This solution was filtered through a 0.22 μm filter and stored at − 20 • C. For the working solution, various concentrations of palmitic acid (250-1000 μM) were prepared in culture medium without supplements.L-carnitine (C0283, Sigma, St. Louis, MO, USA) was added to a final concentration of 2 mM and incubated for the specified time in the experimental protocol (24 h).A control vehicle containing 1% BSA was used for each assay.

MTT test
Cell viability was assessed by conducting the MTT assay, which involves the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) through succinate dehydrogenase, a mitochondrial enzyme.The resulting product generates violet crystals (Formazan).After the treatments, the cells were incubated with the MTT reagent at 0.5 mg/mL concentration for 3 h.Following incubation, the crystals were diluted in DMSO and their colorimetric reading was taken using a spectrophotometer at a length of 595 nm.The obtained data were normalized to the control and expressed as a percentage, assuming 100% viability of the control.

Viability assay Propidium iodide (PI) and Annexin V
In the initial assay, cell death was assessed using fluorometry.After the treatment period with palmitic acid, cells were washed with a 1× PBS solution and stained with propidium iodide (P1304MP, Thermo Fisher Scientific) at a final concentration of 10 μg/mL for 20 min.Subsequently, the fluorescence reading was conducted using the Fluoromax OMEGA fluorometer.
To differentiate whether the treatment induced cell death through apoptosis or necrosis, flow cytometry was employed using Annexin V (A23204, Invitrogen) and Propidium Iodide (PI) staining.Annexin V, a recombinant protein, specifically binds to phosphatidylserine residues exposed at the cell membrane during the early stages of apoptosis.It was combined with the DNA marker propidium iodide, which is impermeable to membranes unless they are compromised (indicating later stages of apoptosis), allowing the distinction between apoptotic and necrotic cells.After treatment, cells were harvested and suspended in 1× annexin buffer (containing 100 mM Hepes, 140 mM NaCl, and 2.5 mM CaCl2) and then incubated with Annexin V-FITC and propidium iodide for 15 min at room temperature.The samples were immediately analyzed using the Millipore® Guava EasyCyte cytometer, and the data were processed using FlowJo software (Tree Star Inc).Doxorubicin at a concentration of 100 nM served as a positive control.

Production of oxidative stress
The production of reactive oxygen species (ROS) was determined using the following fluorescent probes: 1. Superoxide radical (O 2 − ) by dihydroethidine (DHE) (D7008, Sigma) at a concentration of 10 μM.After treatments, the cells were incubated with the respective probe in the dark for 30 min at 37 • C in 1× PBS.Rotenone was used as a positive control for DHE and MitoSOX, and H2O2 was used as a positive control for DA-DCFA.Assays were quantified by a Fluoromax OMEGA fluorometer, and the results were normalized against BSA.

Determination and quantification of proteins by Western blot
For protein determination, cells were lysed and solubilized in RIPA buffer (89,900, Invitrogen, IL, USA) containing 1 mM sodium fluoride, 10 mM sodium pyrophosphate, 200 mM methyl-phenyl-sulfonyl PMSF, 10 mM sodium orthovanadate and 1× Halt Thermo protease inhibitor cocktail (78,425,Invitrogen).The lysates were centrifuged at 10,000 rpm for 10 min at 4 • C and finally denatured by heating for 5 min at 95 • C. A sample of the proteins was used for quantification by the bicinchoninic acid method (23,227, BCA Protein Assay Kit, Thermo Scientific).The proteins were separated on a 12-15% SDS-PAGE electrophoresis gel at 100 V for 90 min at 25 • C.They were then transferred electrophoretically to PVDF membranes (88,518, Thermo Scientific) for 90 min at 350 mA and 4 • C. The PVDF membranes were then blocked for 1 h at 37 • C with 5% BSA o Milk free flat 5% in 0.1% Tween-20 in Trissaline buffer pH 7.4.The membranes are then incubated with the primary antibody overnight at 4 • C. The antibodies used are listed in Table 1.Immunoreactivity is obtained by incubating the membrane with a specific IRDye® 800CW Goat anti-Rabbit IgG (H + L) and IRDye® 680RD Goat anti-Mouse IgG (H + L) secondary antibody (1:10000) (LI-COR Biosciences) for 1 h in Tris-saline buffer pH 7.4.Immunoreactivity is obtained by incubating the membrane with a specific IRDye® 800CW Goat anti-Rabbit IgG (H + L) and IRDye® 680RD Goat anti-Mouse IgG (H + L) secondary antibody (1:10000) for 1 h in Tris-saline buffer pH 7.4.Images were obtained using Odyssey CLx (LI-COR Biosciences).Images were analyzed using Image Studio Lite version 4 software (LI-COR Biosciences).All proteins were normalized with β-actin as loading control.

Reverse transcription
After exposing cells to different experimental paradigms for 24 h, RNA was isolated from the cultures using Trizol (Invitrogen; 15,596,026) according to the manufacturer's instructions and as previously reported (29).RNA was resuspended in 50 μl Rnase and Dnase-free H2O and stored at − 80 • C. A Dnase I (Rnase-free) treatment was then performed to eliminate traces of DNA present in the extraction assay according to the manufacturer's instructions (Biolabs, M0303S).RNA concentration was determined using a NanoDrop (TM) 1'00UV/VIS spectrophotometer (Thermo Fisher).OD ratios of 260/280 nm close to 2.0 were obtained for all samples, indicating high purity.To obtain complementary DNA (cDNA), RNA was first at 400-500 ng/μL with Rnase and Dnase-free H2O.To 11 μL of RNA, 1 μL of Oligo d(T)18 mRNA primer (Invitrogen™; SO131) and 1 μL of dNTPs mix (2.5 nM) (Bioline, BIO-39029) were added.This mixture was pre-incubated for 5 min at 65

Real-time PCR
Levels of the genes evaluated were analyzed using the QuantStudio ™ 3 Real-Time PCR System (Applied Biosystems).Forward and reverse primers for specific amplification of genes were selected using the Primer Bank database and a basic local alignment search tool (BLAST, NCBI) to confirm the target gene.The sequences used are listed in Table 2. Subsequently, 1 μL of cDNA from controls and treated cells obtained after reverse transcription was amplified to a final volume of 10 μL with Power SYBR® Green PCR Master Mix (1×) (Applied Biosystems;4,367,659), forward and reverse for each gene of interest (400 nM) and the remainder with Rnase and Dnase-free H2O.The protocol for the QuantStudio™ 3 Real-Time PCR System consists of a step of denaturation at 95 • C for 2 min, then 40 cycles of 10 s at 95 • C and 10 s at the optimal temperature for each gene, 20 s at 72 • C and 10 s at 95 • C.Then, to obtain melting curves for the resulting PCR products, temperature increase cycles from 70 • C to 95 • C at 0.15 • C/s were added.Relative quantification of the PCR products was performed using the comparative method Ct (Scientific, 2015) and as a ratio between the control gene (RPL27) and the signal of the gene of interest.All tests were performed in duplicate to ensure data quality.Experiments were repeated at least times.

17β-estradiol quantification
Quantification of 17β-estradiol was performed using the Estradiol Human ELISA Kit (KAQ0621, Thermo Scientific) according to the manufacturer's instructions.Briefly, at the end of each treatment, an aliquot of the supernatants was taken and assayed using the kit with the highly specific anti-estradiol antibody.A standard curve was generated between 0 and 935 pg/mL.Estradiol-HRP conjugated capture and detection antibodies were used to achieve maximum sensitivity and linearity for each assay.Optical density was read at 450 nm using a Fluoromax OMEGA fluorometer.

Statistical analysis
GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA) was used for data analysis.A two-way ANOVA (factors: sex and treatment) was performed.Also, data were then examined by one-way analysis of variance, followed by Dunnet's post hoc test for comparisons between controls and treatments and Tukey's post hoc test for multiple comparisons between treatment means and time points.All data are presented as mean ± SEM of 3 independent experiments.Results were considered statistically significant at p < 0.05.The p-values in the figures represent the results of ANOVA, Student's t-test after separation of factors.In the graphs, p-values are represented in * for differences between treatments and numerically for differences between sexes.

Palmitic acid has a greater impact on the cell viability of male human astrocytes
The MTT assay was initially used to evaluate cell viability in human male and female astrocytes.The results indicated that cell viability was influenced by both sex (F (1, 40) = 227.7,p < 0.0001) and treatment (F (4, 40) = 1832, p < 0.0001).Concentrations of 0.5 mM (p < 0.001) and mM (p < 0.001) demonstrated significant differences in cell viability after 24 h.The cytotoxic effect of PA on male astrocytes exceeded that on female astrocytes by 20%, with an interaction between these factors (F (4, 40) = 36.44,p < 0.0001) (Fig. 1A).We stained DNA damage using the propidium iodide (PI) assay to confirm this outcome, which helps identify living and dead cells.In a similar way, sex (F (1, 55) = 123.0,p < 0.0001) and treatment (F (3, 55) = 328.2,p < 0.0001) using PA  significantly influenced cell death.At a concentration of 1 mM (p < 0.001), male astrocytes displayed a 20% increase in PI fluorescence in comparison to their female counterparts (Fig. 1B).
Next, we investigated whether PA exposure results in apoptotic cell death in astrocytes.Fig. 1C shows that most male and female cells were Annexin V-/PI+, indicating that necrosis accounted for most of the death.Only 2% or less of cells were Annexin V+.Accordingly, we conducted further analysis on the subset of cells testing positive for both Annexin V and PI, as shown in Fig. 1D.At a concentration of 1 mM (p = 0.0134), a 20% higher rate was observed in male astrocytes compared to female cells, with sex identified as a significant factor in the outcome (F (1,12) = 27.26,p = 0.0002).These findings suggest that sex is a critical variable, revealing a differential response in the presence of PA, with male astrocytes exhibiting more pronounced detrimental effects than female cells.

Characterization of the endogenous hormonal component of human female and male astrocytes
Given the sex-specific findings in cell viability, we postulated whether differences could be explained by endogenous estrogen regulation in these cells.To investigate this hypothesis, we employed RT-qPCR to measure the expression of estrogen, androgen and progesterone receptors.While the expression of ERα (ERS1) is twice as high in Fig. 1.The cytotoxic effects of palmitic acid (PA) on human astrocytes.Both female and male astrocytes were exposed to various PA concentration levels for 24 h. A. The assessment of cytotoxicity was conducted through the MTT method, with optical density at 540 nm being directly proportional to cell viability.Bovine serum albumin (BSA) was used to normalize the results, with the obtained values set as 100% viability.B. The examination of viability was performed by propidium iodide using fluorometry.C. The research displays contour plots of Annexin V/IP labelled cells which were exposed to PA for 24 h.DOXO at 100 nM was utilized as a positive control (data not presented).Calculations were conducted on BSA vehicle.D. Statistical analysis documented the percentage of PI-Anexin-positive cells.Statistical findings are presented as the mean ± SEM for three independent experiments.male astrocytes (p = 0.0388) (Fig. 3A), ERβ expression is nearly three times higher in female cells (p = 0.0005) (Fig. 3B).The androgen receptor (AR) expression in males is 90% higher (p < 0.0001) (Fig. 3C).Female astrocytes have a 10-fold higher expression of the progesterone receptor (PGR) (p = 0.0068) (Fig. 3D).Next, we evaluated the enzymes responsible for the production of estradiol (E2) and dihydrotestosterone (DHT) -aromatase and 5α-reductase, respectively.Basal expression of aromatase (CYP19A1) was twice as high (p < 0.0001) (Fig. 3E), whilst 5α-reductase (Srd5a) was three times higher (p < 0.0001) (Fig. 3F) in female astrocytes.To assess the potential relationship between the increased aromatase expression and endogenous hormone levels in these cells, we performed an ELISA to quantify the levels of 17β-estradiol in the culture medium.Our findings indicated a 1.6-fold rise in estradiol production in female cells (119 ± 13 pg/mL), in comparison to male cells (72 ± 6 pg/mL) (p = 0.0128) (Fig. 3G).

PA modifies the endogenous estrogenic component and estradiol synthesis in human astrocytes
To determine whether PA affects sex-specific endogenous hormone regulation, male and female astrocytes were stimulated with either 0.5 mM or 1 mM PA.Interestingly, two-way ANOVA analysis revealed that both PA (F (2, 42) = 33.78,p < 0.0001) and sex (F (1, 42) = 6.366, p = 0.0155) influenced aromatase expression (Fig. 4A).At a concentration of 0.5 mM, PA elicited an 11% higher increase in aromatase expression in males compared to females (p = 0.0212).In contrast, a notable rise was observed in relation to BSA, while no significant differences were observed between the sexes at the concentration of 1 mM PA (Fig. 4A).
For 5α-reductase, the expression of the enzyme was significantly impacted by sex (F (1, 29) = 53.36,p < 0.0001) (Fig. 4B).PA increased the enzyme's expression by approximately 15% in females, while reducing it by about 16% in males, indicating substantial sex differences at 0.5 mM (p = 0.0002) and 1 mM (p < 0.0001) (Fig. 4B).Because cells can increase endogenous hormone production by upregulating aromatase after inflammatory stimulation, we next measured the endogenous estradiol production in astrocytes treated with PA.Although there was a greater increase in aromatase RNA levels in males exposed to 0.5 mM PA (Fig. 4A), a significant 2.5-fold elevation was observed only in females (p = 0.0084) for E2 levels, with no significant differences seen in males, irrespective of PA concentrations (Fig. 4C).Finally, in Fig. 4D, a correlation analysis was conducted between cell viability and aromatase RNA expression.A dependency linked to both sex and palmitic acid concentration was found.In female astrocytes, a negative correlation was observed at 0.5 mM (r = − 0.3001), while a positive correlation was observed at 1 mM (r = 0.7718) between these factors.In contrast, the analysis of males revealed a slightly positive correlation at 0.5 mM (r = 0.3492) and a negative correlation at 1 mM (r = − 0.9412).

PA induces cytosolic ROS in males and mitochondrial ROS in females
Mitochondrial fatty acids oxidation is crucial for cellular homeostasis.However, an excessive accumulation of free fatty acids can saturate this mechanism, leading to increased production of ROS (Van Houten, 2019) ROS have been linked to mitochondrial dysfunction, protein damage and cell death.To investigate the effect of PA on cellular antioxidant capacity, we analyzed ROS production using two probes -DCFA-DA and DHE -in the cytosol, as well as MitoSOX, which specifically targets the mitochondrial superoxide ion.
To evaluate O 2 − production in mitochondria, we utilized the

Sex-dependent expression of antioxidant proteins in human astrocytes stimulated with PA
As PA may induce sex-dependent cell death, possibly through the increased ROS production, we next hypothesized whether this fatty acid reduces the expression of antioxidant enzymes in male and female astrocytes.We assessed the protein levels of superoxide dismutase (SOD2), catalase and glutathione peroxidase 1 (Gpx-1) (Fig. 6A).No differences in basal expression of catalase were observed between female and male astrocytes.However, catalase expression was significantly influenced by both sex (F (1, 8) = 160.7,p < 0.0001) and PA (F (3, 8) = 65.55,p < 0.0001), and two-way ANOVA analysis showed a strong interaction between these two factors (F (3, 8) = 39.84,p < 0.0001).At 0.5 and 1 mM, PA significantly increased catalase expression in females by more than double compared to the control (BSA) (p < 0.0001), while no change was observed in males (Fig. 6B).For SOD2, there were no significant differences in basal expression between the two sexes.However, in male astrocytes, palmitic acid increased expression by 50% in a dosedependent manner, although not significantly.In contrast, female astrocytes showed no change in expression levels (Fig. 6C).
The third enzyme evaluated was Gpx-1.Interestingly, we found that its expression was affected by both sex (F (1, 16) = 98.14, p < 0.0001) and palmitic acid (F (3, 16) = 4.483, p = 0.0182).Despite slightly lower basal expression in male astrocytes, sex differences were observed for 0.5 mM (p < 0.0162) and 1 mM (p < 0.0001).Similarly, treatment with 1 mM PA significantly reduced its expression by 50% in males compared to their baseline.In contrast, in females, no significant differences between treatments were observed (Fig. 6D).Overall, these results demonstrate a distinct effect of PA on the antioxidant system, revealing notable differences between females and males in the catalase and Gpx-1 enzymes.This finding aligns with the lower H 2 O 2 production observed in females.Regarding SOD2, while a significant difference exists, there appears to be a tendency for an increase in males.This observation could partially account for the absence of superoxide detection in these cells.
We also evaluated the expression of the transcription factor Nrf2, which regulates the inducible expression of a large number of detoxifying enzymes and antioxidants, including those analyzed above.The results revealed that, at basal levels, male astrocytes exhibited 1.7-fold higher expression than female astrocytes (p = 0.0165) (Fig. 6E).Twoway ANOVA analysis indicated a significant interaction between sex and PA treatment on Nrf2 expression (F (2,30) = 5.402, p = 0.0099).PA treatment (F (2, 30) = 11.05,p = 0.0003) significantly influenced Nrf2 expression.We observed that PA increased Nrf2 expression in female cells by 20-30% at both 0.5 mM and 1 mM compared to the control condition, an effect that was not observed in males.Similarly, sex (F (1,30) = 21.79,p < 0.0001) was a key factor; significant differences between males and females were apparent at 0.5 mM (p = 0.0067) and 1 mM (p = 0.0027) (Fig. 6F).

Discussion
The incidence of CNS pathologies affecting the brain has substantially increased, highlighting the importance of their study.Metabolic disorders associated with medical conditions such as diabetes and obesity can affect brain function and contribute to the development of neurological disorders.In recent years, there has been a growing interest in examining sex as a variable, which has long not been traditionally considered in many studies previously.It has become evident that males and females exhibit different susceptibilities to developing certain CNS disorders and respond differently to treatments.However, treatment outcomes are not consistently uniform.The findings of this study demonstrate the diverse mechanisms at play in male and female astrocytes when exposed to metabolic damage with palmitic acid, shedding light on and how the regulation of the endogenous estrogenic component can influence cellular responses.
Our findings reveal that there are sex-specific differences in cell viability, with higher concentrations of PA causing more damage to male astrocytes than to females.This is consistent with previous observations in cortical astrocytes, where concentrations of 250 μM and 500 μM of palmitic acid administered for 24 h resulted in significantly more damage to male astrocytes (Ortiz-Rodriguez et al., 2019).Metabolic imbalances in the transport of fatty acids to the mitochondria and the activation of distinct signaling pathways (Frago et al., 2017) may have contributed to decreased viability.Viability was assessed through two aspects: 1) mitochondrial metabolism using the MTT assay, and 2) changes in cell membrane and nucleus integrity using the PI assay (Fig. 1A-B).Previous studies have shown that astrocyte cell death induced by PA was not associated with endoplasmic reticulum stress (ER stress) or changes in cytosolic Ca 2+ signaling (Wong et al., 2014b).Instead, it was caused by the production of ROS followed by deterioration of the mitochondrial membrane potential (Guo et al., 2013).Our findings suggest that 24-h PA stimulation mainly leads to necrotic cell death, as indicated by the Annexin/PI assay.This form of cell death is associated with environmental perturbations and uncontrolled release of inflammatory cell contents, such as cytokines.It is more consistent with secondary or apoptotic necrosis, as evidenced by morphological changes and membrane damage (Fink and Cookson, 2005).To assess this hypothesis, we examined the apoptosis-related proteins Bax, Bcl-2 and Bak, with the Bax/Bcl-2 expression ratio regulating the mitochondria-related apoptotic pathway.The results showed no significant changes in this ratio (Fig. 2B-C) between both male and female astrocytes.This balance of proteins could partially explain the delayed onset of apoptotic processes.However, male astrocytes exhibited a significant increase in the pro-apoptotic protein Bak.The increase in this protein, along with Bax, may form protein complexes that promote pore formation in the outer mitochondrial membrane (Peña-Blanco and García-Sáez, 2018).This can result in augmented apoptosis due to mitochondrial dysregulation, cytochrome c release, and oxidative stress (Huo et al., 2019).To understand sex differences, it is crucial to consider the role of estrogenic components such as steroid receptors and enzymes that regulate endogenous estrogen and androgen action and production.Our study found that male astrocytes have higher basal expression of ERα compared to females, while ERβ showed higher expression in females.Although ERα is expressed more abundantly in the brain, ERβ is more effective in modulating the effects of estradiol in this organ (Itoh et al., 2023), particularly within astrocyte mitochondria, suggesting enhanced estrogenic protection in female mitochondria (Lejri et al., 2018).As we reported, males have a higher expression of the androgen receptor, possibly due to higher endogenous levels of androgens.Similarly, female brains present higher levels of progesterone.This could explain our finding of higher expression of the progesterone receptor in female astrocytes.Furthermore, there was a correlation between increased production of 17β-estradiol and higher expression of aromatase and 5αreductase in female astrocytes (Figs.3E-G), which is consistent with previous studies on neonatal female cortical astrocytes (Liu et al., 2007).
These enzymes play a crucial role in the production of both estrogen (Azcoitia et al., 2021) and androgen (Steckelbroeck et al., 2001), as well as exerting protective functions on the brain.Their activity at the cellular level is regulated through alterations in mRNA expression (McCullough et al., 2003), which are influenced by hormones, transcription factors, and signaling molecules (Liu et al., 2007).The observed sex differences in astrocytes suggest that female cells may have better survival and resistance when exposed to inflammatory stimuli caused by palmitic acid.
A crucial link to cell protection and survival in astrocytes is aromatase.Previous studies have reported the increased expression of this enzyme in astrocytes following cellular stress (Brocca and Garcia-Segura, 2019).In our model, subjecting human astrocytes to stress with high concentrations (0.5 mM-1 mM) of palmitic acid resulted in a significant elevation in aromatase in both female and male astrocytes.When it comes to 5α-reductase, some studies have reported that certain fatty acids exhibit inhibitory activity against 5α-reductase by blocking the conversion of testosterone to dihydrotestosterone (DHT) (Azzouni et al., 2012;Liang and Liao, 1992).However, our results showed that palmitic acid induced an increase in the expression of 5α-reductase only in female astrocytes, suggesting an elevated production of dihydrotestosterone (DHT) and dihydroprogesterone (DHP) (Cermenati et al., 2017;Yang et al., 2020).This could also potentially enhance protective functions in women.Additionally, female astrocytes exhibited a slight increase in 17β-estradiol levels (Fig. 4C).Astrocytes exposed to glucose and oxygen deprivation, lipopolysaccharide (LPS), interleukin-1β (IL-1β), and tissue necrosis factor (TNF-α) showed similar results (Han et al., 2015).Female cultures exposed to palmitic acid exhibited a greater increase in aromatase, suggesting a greater resistance to lipotoxicity, possibly due to endogenous estradiol as a defence mechanism (Giatti et al., 2019).However, further studies are required to comprehensively address the protective mechanisms of aromatase.
As previously suggested, the impact of palmitic acid on cell viability appears to be associated with mitochondrial changes, given their pivotal role in fatty acid oxidation and cellular energy regulation.To establish the involvement of mitochondria in mediating oxidative processes, we conducted a thorough analysis of mitochondrial dysfunction induced by Fig. 6.Impact of palmitic acid (PA) on the modulation of the antioxidant system in astrocytes.The astrocytes were exposed to various concentrations of PA for 24 h, and then a protein extraction was carried out. A. Western blot analysis showed representative images of antioxidant protein expression, B-C-D.Densitometric analysis was conducted on the relative expression of Catalase, SOD2, and Gpx-1 proteins in relation to the β-actin loading control.E. Basal expression of Nrf2 mRNA was measured using the reference gene RPL27.F. Relative expression of Nrf2 mRNA was also measured in human astrocytes, with expression normalized against BSA control.The basal group are cells with astrocyte medium only.Statistical results present the mean ± SEM for 3 independent experiments.palmitic acid in our model.Our findings revealed that exposure to PA notably increased the production of reactive oxygen species, with significantly higher levels observed in male astrocytes for both superoxide and hydrogen peroxide radicals (Fig. 5A-B).Oxidative stress, a product of an excess of free radical production that surpasses the antioxidant capacity of the central nervous system (Abdul-Muneer et al., 2015;Chen et al., 2020), is implicated in the observed effects.Particularly noteworthy was the significantly higher level of mitochondrial superoxide in females compared to males, suggesting an enhanced inability to regulate this radical in female mitochondria.Analysis of the antioxidant enzyme SOD2, responsible for eliminating mitochondrial superoxide, revealed an elevation in its expression within astrocytes in male cells following palmitic acid (Fig. 6C).This may explain the absence of O 2 − production in males, but its presence in females, where variations in SOD2 expression were observed.Moreover, PA induced an increase in the expression of catalase and glutathione peroxidase (GPx-1), two antioxidant enzymes that help reduce H 2 O 2 levels.H 2 O 2 was found at higher levels in males, possibly due to reduced Gpx-1 expression caused by palmitic acid (Fig. 6B-D).Excessive free radicals in the CNS microenvironment may contribute to reactive astrogliosis, exacerbating inflammation and glial scarring, ultimately imposing a burden on the CNS (Chen et al., 2020).The astrocyte's antioxidant system is primarily governed by nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor crucial for maintaining redox homeostasis and regulating the expression of multiple antioxidant enzymes (Zhang et al., 2013).Nrf2 can respond to oxidative stress and become activated (Ahmed et al., 2000).Our findings showed that, despite higher basal levels of Nrf2 expression in human astrocytes, PA induces greater Nrf2 expression in females (Fig. 6F), indicating better protection in female cells.Previous studies have reported that 17β-estradiol induces Nrf2 expression and reduces oxidative stress by upregulating the expression of vital antioxidant enzymes, SOD1/2 and catalase (Lin et al., 2021).The estradiol levels reported in this study suggest a connection between increased antioxidant protection in female astrocytes when stimulated with palmitic acid.

Conclusions
In conclusion, our findings offer new insights into the metabolic alterations experienced by human astrocytes under palmitic acid-induced stress.Firstly, we observed increased resilience in female astrocytes to high concentrations of palmitic acid through the mechanisms involved in cell survival.
By considering sex as a determining variable in the results, we demonstrated differences in the basal expression of receptors and enzymes associated with the endogenous estrogenic regulation.We observed higher levels of aromatase and 5α-reductase enzymes in females.Interestingly, we found that despite palmitic acid induced higher expression of aromatase in male astrocytes, at high concentrations of this fatty acid there is a positive correlation between enzyme expression and an increase in the viability of female astrocytes.Similarly, our results showed a differential mitochondrial oxidative response in astrocytes based on sex, revealing a more significant effect in males, where mitochondria appear considerably more compromised in terms of ROS and a reduction in antioxidant proteins.Taken together, the results suggest that female astrocytes are better equipped and protected against exposure to fatty acids, possibly due to differences in the expression of the endogenous estrogenic component and antioxidant defence.
• C and immediately placed on ice.The reaction was then completed with 4 μL of 5× First-Strand Buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl], 2 μL of DTT (0.1 M) and 1 μL of M-MLV reverse transcriptase (200 U/μL) (Invitrogen™, Cat.No. 28025013) to a final volume of 20 μL.Reaction mixtures were incubated at 37 • C for 50 min and then at 70 • C for 15 min to inactivate the enzyme in a MasterCycler gradient thermal cycler.

Fig. 2 .
Fig. 2. Palmitic acid induces alterations in the pro-and anti-apoptotic protein expression in astrocytes.Astrocytes were treated with palmitic acid at varying concentrations for 24 h. A. Western blot analysis presented as representative images showing apoptotic protein expression; B-C-D.Densitometric analysis of relative expression of Bax-Bcl2 and Bak proteins compared to β-actin loading control.The basal group are cells with astrocyte medium only.The statistical outcome is indicated as the mean ± SEM for three independent experiments.

Fig. 3 .
Fig. 3. Basal Expression of Endogenous Estrogenic Component in Human Astrocytes.A-B-C-D: Basal evaluation of the relative mRNA expression levels of ERS1, ERS2, AR, and PGR receptors in human astrocytes using RT-qPCR, relative to the RPL27 control.E-F: Determination of basal expression levels of CYP19A1 and Srda5 enzymes in human astrocytes using RT-qPCR.G: Quantification of the release of 17β-estradiol into the culture medium by human astrocytes.The basal group are cells with astrocyte medium only.The statistical results are expressed as the mean ± SEM from 3 independent experiments.

Fig. 4 .
Fig. 4. The impact of palmitic acid on the estrogenic component in human astrocytes.Results are displayed in sections A and B. Human astrocytes (HA) were exposed to palmitic acid (PA) at varying concentrations for 24 h.Relative mRNA expression of the CYP19A1 and Srda5 enzymes in human astrocytes was measured in relation to the reference gene RPL27.The expression was normalized with respect to the BSA control.C. The 17β-Estradiol in medium of astrocytes stimulated with PA was quantified, with MA referring to medium without supplementation.D. Correlation analysis between cell viability and the expression of the aromatase enzyme in astrocytes stimulated with 0.5 and 1 mM of palmitic acid.Statistical results are presented as the mean ± SEM for three independent experiments.

Fig. 5 .
Fig. 5. Production of reactive oxygen species in astrocytes stimulated with palmitic acid (PA).The mean fluorescence intensity (MFI) of astrocytes was recorded over 24 h using different concentrations of PA.The findings indicate that A. Superoxide ion production was evident through DHE staining, rotenone serving as a positive control; B. hydrogen peroxide ion production was identified by DCFA-DA staining; C. Cells labelled with Mitosox (200 nM) were analyzed, revealing representative histograms; D. Mitochondrial superoxide ion quantification was confirmed by Mitosox staining.The basal group are cells with astrocyte medium only.The mean ± SEM for 3 independent experiments is presented as statistical results.

Table 1
Proteins assessed in the study.

Table 2
Primers used in the study.