Albornoz et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03154-z(2024) 21:191

Journal of Neuroinflammation

Open Access

RESEARCH

Targeting the immunoproteasome
in hypothalamic neurons as a novel
therapeutic strategy for high-fat diet-induced
obesity and metabolic dysregulation
Nicolás Albornoz1, Javiera Álvarez-Indo1, Adely de la Peña1, Eloisa Arias-Muñoz1, Alanis Coca2,
Fabián Segovia-Miranda2, Bredford Kerr1, Mauricio Budini3, Alfredo Criollo4,5, María A. García-Robles2,
Eugenia Morselli6, Andrea Soza1,7* and Patricia V. Burgos1,7*
Abstract
Objective Obesity represents a significant global health challenge characterized by chronic low-grade inflammationand metabolic dysregulation. The hypothalamus, a key regulator of energy homeostasis, is particularly susceptible toobesity’s deleterious effects. This study investigated the role of the immunoproteasome, a specialized proteasomal
complex implicated in inflammation and cellular homeostasis, during metabolic diseases.
Methods The levels of the immunoproteasome β5i subunit were analyzed by immunostaining, western blotting, andproteasome activity assay in mice fed with either a high-fat diet (HFD) or a regular diet (CHOW). We also characterized
the impact of autophagy inhibition on the levels of the immunoproteasome β5i subunit and the activation of
the AKT pathway. Finally, through confocal microscopy, we analyzed the contribution of β5i subunit inhibition on
mitochondrial function by flow cytometry and mitophagy assay.
Results Using an HFD-fed obese mouse model, we found increased immunoproteasome levels in hypothalamic
POMC neurons. Furthermore, we observed that palmitic acid (PA), a major component of saturated fats found in
HFD, increased the levels of the β5i subunit of the immunoproteasome in hypothalamic neuronal cells. Notably,
the increase in immunoproteasome expression was associated with decreased autophagy, a critical cellular process
in maintaining homeostasis and suppressing inflammation. Functionally, PA disrupted the insulin-glucose axis,
leading to reduced AKT phosphorylation and increased intracellular glucose levels in response to insulin due to theupregulation of the immunoproteasome. Mechanistically, we identified that the protein PTEN, a key regulator of
insulin signaling, was reduced in an immunoproteasome-dependent manner. To further investigate the potential
therapeutic implications of these findings, we used ONX-0914, a specific immunoproteasome inhibitor. We
demonstrated that this inhibitor prevents PA-induced insulin-glucose axis imbalance. Given the interplay between*Correspondence:
Andrea Soza
andrea.soza@uss.cl
Patricia V. Burgos
patricia.burgos@uss.cl
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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in this article, unless otherwise stated in a credit line to the data.

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

Page 2 of 20

mitochondrial dysfunction and metabolic disturbances, we explored the impact of ONX-0914 on mitochondrial
function. Notably, ONX-0914 preserved mitochondrial membrane potential and attenuated mitochondrial ROS
production in the presence of PA. Moreover, we found that ONX-0914 reduced mitophagy in the presence of PA.
Conclusions Our findings strongly support the pathogenic involvement of the immunoproteasome in hypothalamic
neurons in the context of HFD-induced obesity and metabolic disturbances. Targeting the immunoproteasome
highlights a promising therapeutic strategy to mitigate the detrimental effects of obesity on the insulin-glucose axis
and cellular homeostasis. This study provides valuable insights into the mechanisms driving obesity-related metabolic
diseases and offers potential avenues for developing novel therapeutic interventions.
Keywords Obesity, Immunoproteasome, Hypothalamus, Neurons, Insulin-glucose axis, Autophagy, Mitochondrial
function, Metabolic disturbances, Redox biology

Introduction
Obesity, acknowledged as a global epidemic, is significantly promoted by unbalanced diets high in saturated
fats such as palmitic acid (PA) [1]. Chronic consumptionof such HFD has been associated with increased metabolic diseases, including an imbalance in the insulinglucose signaling axis, leading to insulin resistance [2, 3].
In mice, chronic consumption of these diets leads to theaccumulation of PA in the brain, specifically the hypothalamus, the critical region responsible for maintaining
energy homeostasis and body metabolism [4].
Macroautophagy, hereafter autophagy, a crucial cellular
homeostatic mechanism, is inhibited in the hypothalamus by this type of diet [5, 6]. Indeed, in mice exposed
to a 16-week HFD, the protein levels of Beclin1 and LC3II decreased, while the protein content of p62/SQSTM1
increased when compared to lean mice fed with a regular diet [6]. The autophagy flux directly quantifies theamount of autophagy-dependent degradation of cellularcomponents such as Beclin1, p62/SQSTM1, LC3 ratio,
among others. In hypothalamic neurons, a decrease in
autophagy inhibits insulin signaling and insulin-dependent glucose uptake [7]. Consistently, inhibition of
autophagy in the hypothalamus increases blood glucose
and insulin levels and drives chronic low-grade inflammation via IKKβ/NF-κB activation [5].
One of the major functions of autophagy is to counteract the inflammatory responses [8]. Persistent inflammation, broadly identified in obesity, disrupts the
hypothalamic function in energy balance, leading toaccelerated development of obesity and its comorbidities [9]. The energy balance is finely regulated to maintainan equilibrium between energy intake (food we eat) andenergy expenditure (calories we burn). It acts as a control center, constantly monitoring and integrating signals
from various body parts to regulate hunger, satiety, andmetabolism.
The molecular components activated in response
to a decrease in autophagic flux that could lead to an
imbalance in the insulin-glucose axis remain largely
unknown, a question of significant interest in the fieldof biomedicine. One possible candidate is the immunoproteasome, prevalent in inflammatory scenarios [10,11]. The immunoproteasome is a proteolytic protein
complex ubiquitously expressed during inflammation
where catalytic subunits of the standard proteasome, β1,
β2, and β5, are replaced by the inducible subunits; β1i/
LMP2, β2i/MECL-1, and β5i/LMP7, respectively, forming an alternative isoform [12, 13]. Immunoproteasomes
are capped at one (26 S) or both ends (30 S) by the 19 S
regulatory particle that captures and prepares appropriate substrates for breakdown [13]. Since the ratio of
immunoproteasome, 26 S immunoproteasome or 30 S
immunoproteasome varies across different cellular conditions, a dynamic equilibrium between the two species
may be part of an adaptive response to cellular needs [13].
Deleting the immunoproteasome’s β5i subunit in mice
reduces the obesogenic phenotype and insulin resistancein response to an HFD [14]. Yet, whether HFD leads toan increase in the immunoproteasome in the hypothalamus or if this complex could be linked to an imbalancein the insulin-glucose axis is unknown. Furthermore, thepotential contribution of the immunoproteasome to cellular metabolism, particularly in hypothalamic neurons,remains entirely unexplored.

Materials and methods
Chemical reagents

Products were purchased from several suppliers. From
Sigma-Aldrich (St. Louis, MO, USA), we procured Amersham™ Protran® Nitrocellulose (cat#GE1060002), Palmitic
Acid (PA, cat#P0500), Stearic Acid (SA, cat#S4751), TypeA Porcine Skin Gelatin (cat#G-2500), 2-b-Mercaptoethanol (cat#M3148), and protease inhibitor “cocktail”(cat#P8340). Bovine Serum Albumin (BSA, cat#BSA-05)
for antibody dilution was obtained from Rockland
Immunochemicals (Pottstown, PA, USA), while fatty
acid-free BSA (cat#152,401), used as a treatment, camefrom MP Biomedicals (Santa Ana, CA, USA). ONX-0914(cat#HY-13,207) was procured from MedChemExpress(Monmouth Junction, NJ, USA), and insulin (humulin R
100UI/ml F-A) was acquired from Cruz Verde (Santiago,

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

Chile). Merck Millipore (Burlington, MA, USA) supplied us with Methanol (cat#1,060,092,500), Acetic Acid
(cat#100064.1000), and HEPES-free acid (cat#391,340).
ThermoFisher Scientific (Waltham, MA, USA) provided
numerous items including the BCA Kit (cat#23,225),
DMEM (cat#D5030-10 L), and many other biologicalreagents. From Winkler (Santiago, Chile), we purchased
Acrylamide: Bisacrylamide 29:1 30% (cat#BM0100), Bromophenol Blue (cat#A2-0395), and several other chemicals. SDS (cat#C15081006) and Triton X-100 (cat#T8655)
were purchased from USBiological Life Science (Salem,
Massachusetts, USA). Our lab also used FluoromountG (cat#17984-25) and 16% Paraformaldehyde molecular
grade solution (PFA cat#15,710) from Electron Microscopy Science (Hatfield, PA 19,440, USA), and Glycine(cat#FER00G500G) and Tris Base (cat#FER00B500) from
Fermelo Biotec (Santiago, Chile). The AccuRuler RGB
Plus Ladder Protein (cat#02102 − 250) was purchasedfrom MaestroGen (Hsinchu City, Taiwan, China). Other
critical reagents include Ammonium persulfate (APS,
cat#0486 − 256) from Avantor-VWR (Radnor, PA, USA),
Fetal bovine serum (FBS, cat#04-127-1 A) from BI Biological Industries/Sartorius (Aubagne, France), Phosphatebuffered saline (PBS, cat#46-013-CM) from Corning Inc.
(Glendale, Arizona, USA), Tween-20 (cat#SC29113B)
from ChemCruz™ Biochemicals-Santa Cruz Biotechnology (Dallas, TX, USA), and the fluorogenic peptide AcANW-AMC specific for β5i (Cat#26,640) from Cayman
Chemical.
Antibodies

Monoclonal antibodies used in this study were sourced
from three companies. From Abnova, we used Mouse
p62/SQSTM1 (Clone 2C11, cat#H00008878-M01, Dilution 1:5000 in 3% BSA and 1:300 in 0.2% Gelatin). All
primary and secondary antibodies were diluted in 3%
BSA. From Santa Cruz Biotechnology, we utilized Mouse
BECN1 (cat#sc-11,427, Dilution 1:1000), Mouse PTEN
(cat#sc-7974, Dilution 1:1000), Mouse β5 (cat#sc-393,931,
Dilution 1:1000), Mouse GLUT4 (cat#sc-53,566, Dilution 1:1000), and Mouse β-Actin (cat#sc-47,778, Dilution
1:2000). Lastly, from Abcam, we used Mouse Hexokinase-II (Clone 3D3, cat#ab104836, Dilution 1:1000). For
polyclonal antibodies, we used four suppliers. From Cell
Signaling Technology, we used Rabbit p-AKT (Ser473)(cat#9271, Dilution 1:2000), Rabbit AKT (cat#9272,
Dilution 1:2000), and Rabbit TOM20 (Clone D8T4N,
cat#42,406, Dilution 1:200 in 0.2% Gelatin). We also utilized Rabbit FIP200 from ProteinTech (cat#172,501-AP,
Dilution 1:1000), Rabbit β5i from Abcam (cat#Ab3329,
Dilution 1:1000), Rabbit Glucose 6 Phosphate Dehydrogenase (Clone EPR6292, cat#ab133525, Dilution 1:1000),
and Rabbit Lactate Dehydrogenase (Clone EPR1564,
cat#ab101562, Dilution 1:1000). Lastly, Rat LAMP1 wasPage 3 of 20

sourced from BDPharmingen (Clone 1D4B, cat#553,792,
Dilution 1:300 in 0.2% Gelatin).
Use of mice and HFD diet

Four-week-old male C57BL/6 mice were obtained fromthe animal facility of Fundación Ciencia & Vida. Then,
mice were housed for 8 weeks in the mouse room of the
CEBICEM-USS. Housing conditions included an averagetemperature of 21 °C, daily 12-h light-dark cycles, and ad
libitum access to water. Four-week-old male mice were
fed either a CHOW diet (cat # 0001495, Lab Diet Inc.) or
HFD (cat# D12492, Research Diets, Inc.) for 8 weeks.
Cell culture and treatments with fatty acids, drugs, insulin,or siRNAs

Human HeLa WT cells (ATCC # CCL-2), HeLa
ATG9aKO cells (kindly donated by Dr. Juan Bonifacino,
NIH and referred to in [15]), human HeLa ATG8KO cells(kindly donated by Dr. Maho Hamasaki and referred to in[16]), mouse embryonic hypothalamic N43/5 cells (Cellutions Biosystems, code #CLU127), and adult mouse
hypothalamic CLU177 cells (Cellutions Biosystems,
code #mHypoA-2/12- CLU177) were used. All cell lines
were cultured and grown in DMEM culture medium(cat#11965092, ThermoFisher Inc), supplemented with
10% v/v fetal bovine serum (FBS), 100 U/mL penicillin,and streptomycin at 37°C with 5% CO2. Cells were cultured on glass coverslips at 15,000 cells per well. In 6-wellplates, 200,000 cells were seeded per well. Treatments
were performed no earlier than the third day after seeding. Treatment with fatty acids: N43/5 and CLU177 cells
were pretreated with DMEM (1 g/L glucose; #A1443001,
ThermoFisher Inc) supplemented with 2% FBS for 24 h.
Then, they were incubated with 100 µM PA, or SA conjugated to BSA 100 µM for 6 and 24 h. BSA 100 µM
alone at the same concentration was used as a control.
Drug treatment: CLU177 cells were assessed with thecommercial inhibitor ONX-0914 (10 nM prepared in
DMSO) to study the effect of immunoproteasome inhibition in response to fatty acids. DMSO was used as avehicle control. Insulin treatment: CLU177 cells werepretreated with DMEM 1 g/L glucose supplemented with
2% FBS for 24 h. Subsequently, the cells were incubatedwith commercial insulin (10 nM) for 5, 15, or 30 min,diluted in PBS 1X. siRNA treatment: N43/5 or CLU177
cells were cultured in 6-well plates and transfected withsiRNAs when the cells reached 20% confluence. siRNAs
for murine Beclin1 (5´-cugagaaugaaugucgaa-3´, SigmaAldrich), for FIP200 (5’- ​G​G​A​G​A​U​U​U​G​G​U​A​C​U​C​A​U​C​
A​U​C​A-3’, DNA Technologies IDT), or for β5i (5´-AGG​
A​A​A​G​G​A​A​U​G​U​U​C​A​A​A​U​U​G-3´, Horizon Discovery Ltd) were used. Each of the siRNAs was prepared at
a concentration of 100 µM and used at a concentrationof 10 nM. According to the manufacturer’s instructions,Albornoz et al. Journal of Neuroinflammation(2024) 21:191

the transfection was performed using Lipofectamine
RNAiMAX® reagent (Invitrogen, Carlsbad, CA, USA). Asa negative control, cells were incubated with siControl(5’- ​U​U​C​U​C​C​G​A​A​C​G​U​G​U​C​A​C​G​U-3’, DNA Technologies IDT). After 72 h, the cells were processed for protein
extraction using RIPA buffer (50 mM Tris-HCl, 150 mM
NaCl, 5 mM EDTA, 1% NP40, 1% sodium deoxycholate,
0.1% SDS, pH 7.4).
Tissue immunofluorescence

C57BL/6J 16-week-old-Mice or POMC-eGFP mice wereanesthetized intraperitoneally with Ketamine/Xylazine(90 mg/kg/10 mg/kg) and vascular perfused first with
50 mL of PBS and second with 50 mL of 4% PFA-0.1%
Tween. Subsequently, brain tissue embedded in agarose was cut at 100 μm using a vibratome (Leica, model
VT1000 S), following the protocol previously published(Morales-Navarrete et al., 2019). In addition, 50 μm sections were alternatively made using the cryostat (Leica,model 1900-1-1). Then, the sections were processedusing an immunofluorescence protocol with the floating section technique (Potts et al., 2020). The agarose
was removed with a 0.5% Triton X-100 solution in PBS
for 1 h for those sections embedded in agarose. The sections obtained by the cryostat were blocked with 3%
BSA in PBS for 1 h. Subsequently, the immunofluorescence protocol proceeded without changes. Primary
antibodies were diluted in buffer TX (0.2% gelatin, 300
mM NaCl, 0.3% Triton X-100 in PBS 1X), and a volumeof 1:150 µL was incubated with the section for 2 days at
4 °C in a humid chamber. The primary antibodies usedwere β5i (Abcam, Ab3329), chicken anti-GFP (Synaptic system, # 132,006), and GFAP (Santa Cruz Biotechnology, sc-33,673). Then, 5 washes of 15 min each were
performed with PBS-Tween 0.3%. A 200 µL of secondary antibodies diluted 1:1000 in buffer TX coupled toAlexa Fluor® (Life Technologies) was incubated with thesections for 2 days at 4 °C in a humid chamber. Nuclei
were co-stained with DAPI (1:10,000) and added to thesecondary antibody dilution. Then, the samples were
washed 6 times for 5 min in Tris-phosphate buffer (pH7.8) and mounted with a fluorescence mounting medium(Dako Cytomation, Campintene, CA, USA). Images of
the hypothalamus slices were obtained by confocal spectral microscopy (Carl Zeiss, LSM780, Jena, Germany)at ×40 magnification (bidirectional X, speed 600 Hz,
pinhole 1.00 AU) and processed using ImageJ software(National Institutes of Health, Bethesda, MD, USA).
Images Fig. 1-B and D are two overlapping and correlative images. The images of the tissue samples obtainedwith the Zeiss LSM780 confocal microscope were separated into three channels. One to observe POMC-GFP
neurons, astrocytes (GFAP) coupled with secondary
antibody Donkey anti-Mouse IgG H + L, Alexa-488

Page 4 of 20(#A-21,202, ThermoFisher Inc), immunoproteasome(β5i) coupled with secondary antibody Donkey anti-Rabbit IgG H + L, Alexa-647 (#A-31,573, ThermoFisher Inc)or alternatively nucleus (DAPI). Using ImageJ/Fiji, theAutoNeuriteJ plugin (https://github.com/Grenoble-Institute-Neurosciences/AutoNeuriteJ) was used to generate
superimposed images (neurons with nuclei or astrocyteswith nuclei). Subsequently, a third overlay is performed
with the immunoproteasome (β5i) channel, quantifying
the fluorescence intensity, as detailed in [17].
Indirect immunofluorescence in cell lines

Cells grown on coverslips were washed with 1X PBS and
fixed in 4% (v/v) paraformaldehyde for 20 min at room
temperature. After fixation, cells were washed in 1X PBS
and permeabilized with 0.2% (v/v) Triton X-100 in 1X
PBS for 10 min at room temperature. Subsequently, 0.2%
pig gelatin in 1X PBS was added for 15 min. Next, cells
were incubated with the primary antibodies TOM20,
LAMP1, and p62/SQSTM1 for 1 h at 37 °C. Then, coverslips were washed in 1X PBS and incubated with thecorresponding secondary antibodies conjugated with
Donkey anti-Mouse IgG H + L Alexa-488 (cat#A-21,202,
ThermoFisher Inc), Donkey anti-Rat IgG H + L Alexa594 (cat#A482711, ThermoFisher Inc), and Donkey
anti-Rat IgG H + L Alexa-647 (#A32795, ThermoFisherInc), respectively, at a concentration of 1:1000 in 0.2%
pig gelatin in 1X PBS for 30 min at 37ºC. Finally, cells
were washed with 1X PBS and incubated for 10 min atroom temperature with DAPI (1:10,000). After the finalwash, coverslips were mounted on glass slides using Fluoromount-G (Electron Microscopy Science, USA). For
quantification of fluorescent signals, 16-bit images wereacquired under identical settings avoiding signal saturation and corrected for background signal on each image.
The corrected fluorescent signal in each cell of each
image was used in Image J (version 1.44o; Wayne Rasband, NIH, http://imagej.nih.gov) to determine the total
integrated pixel intensity per cell area. Colocalization
analyses were performed with sets of immunofluorescence images of the same cells for each marker. Quantification was performed with the software Image J software
version 1.44o(Wayne Rasband, NIH, http://imagej.nih.
gov) and the plugin “colocalization threshold” to determine the Manders’ coefficients for each channel. Scores
are calculated for pixels above an automatically determined threshold for both channels, according to the algorithm described by [18].
Live cell imaging in cell lines

HEK293T cells were seeded into 6-well plated at 1 × 105
cells per well in DMEM supplemented with 10% FBS for
lentiviral production. A transfection mix contained 1 µgof the plasmid mt-Keima (kindly donated by Dr. Thomas

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

Mund), 0.5 µg of plasmids viral capsid (VSV-G) andretroviral gag/pol (OPG), and 0.1 mg/ml of Polyethyleneimine (PEI) was incubated for 20 min at room temperature before adding to the cells. The virus-containing
supernatants were collected after 48 h of incubation at
37 °C and centrifugated at 8000 x g for 3 min at room
temperature. Viral supernatants were titrated in 500 µl of
DMEM containing 10% FBS and 80 µg/ml of polybrene(hexadimethrine bromide) and added to Clu177 cells
that were prior seeded at 5 × 104 cells per well in a 6-wellplate. 48 h after transduction, the cells were analyzed
using a confocal microscope to test the expression of
the mt-Keima. Clu177-mt-Keima were seeded at 1 × 105
cells on a 35 mm cover glass in cells were pretreated withDMEM supplemented with 2% FBS and penicillin-streptomycin for 24 h, and incubated with BSA (vehicle), PA,
BSA + ONX-0914 or PA + ONX-0914 for 24 h. Before theexperiment, the cell medium was replaced with DMEM
Hepes 25 mM. Cells were imaged using a Leica SP8 confocal microscope and a 63X oil immersion 1.4 ma lens in
an environmental chamber maintained at 37 °C. Fluorescence of mt-Keima was imaged in two channels via twosequential excitations (440 nm, cyan; 586 nm, red) andusing a 620 nm emission range. The images were analyzed using ImageJ software experiments [19]. The number of acid puncta per cell, representing the number ofmitochondria undergoing degradation (mitophagy), wasquantified in 80 cells from 3 independent.
Preparation of protein extracts and Western blot analysis

Cells were washed with PBS and lysed with RIPA buffer supplemented with a cocktail of protease and phosphatase inhibitors (1 mM NaF, 0.3 mM Na2P2O7, and 1
mM Na3VO4; Sigma-Aldrich). Cells were collected andlysed for 30 min at 4 °C. The extracts were centrifuged
for 20 min at 12,000 × g at 4 °C. The supernatants werecollected, and protein concentration was quantified using
the BCA assay (ThermoFisher Scientific). The extracted
proteins were denatured at 70 °C for 5 min. For the gels,
30–50 µg of denatured proteins from each sample wereresolved on 7.5%, 10%, or 12% SDS-PAGE gels. Gels weretransferred to nitrocellulose membranes and incubatedwith 5% BSA (BM-0150, Winkler, RM, Chile) in trisbuffered saline (TBS) to block nonspecific binding, withagitation for 1 h. The membranes were incubated withprimary antibodies p62/SQSTM1, p-AKT (Ser473), AKT,BECN1, FIP200, PTEN, β5, β5i, β-actin, prepared in 1X
TBS − 5% BSA overnight. Then, the membranes were
washed 3 times for 10 min in TBS-0.25% Tween-20 andrevealed with the appropriate horseradish peroxidaselabeled secondary antibodies (Goat Anti-Mouse IgG(H + L)-HRP Conjugate, #1,706,516; Goat Anti-Rabbit
IgG (H + L)-HRP Conjugate, #1,706,515; Bio-Rad, CA,
USA) and the chemiluminescent substrate.

Page 5 of 20

Native Western blot

Hypothalamus from mice fed a CHOW or HFD diet for
8 weeks were frozen and stored at -80 °C, then resuspended in 100 µL of TSDG lysis buffer (Tris/HCl 10 mM,
MgCl2 1.1 mM, NaCl 10 mM, EDTA 0.1 mM, NaN3 1
mM, DTT 1 mM, ATP 2 mM, Glycerol 10% (v/v), H2O).
The tissue was ground with a plastic douncer, and subsequently, it was subjected to 5 cycles of freezing/thawing
in liquid nitrogen, followed by centrifugation at 20,000 x
g for 20 min at 4 °C. The supernatant was taken, and after
quantification, the native buffer was added. Samples were
loaded into pre-made gels of NativePAGE™ 4 to 16%, BisTris, 1.0 mm (Cat# BN1002BOX, Thermo Fisher Scientific), and a running buffer containing TBE buffer 1x (Tris
Base 712 mM, boric acid 712 mM, EDTA 10 mM, H2O),
ATP 413 µM, MgCl2 2 mM, DTT 0.5 mM, and H2O. The
electrophoresis chamber was placed on ice, and the electrophoresis was run at 150 V for 4.5 h. Afterward, thegel was incubated in a buffer containing the peptide that
detects chymotrypsin activity (Tris pH 7.5 0.5 µM, ATP/MgCl2, DTT 1 mM, AC-ANW-AMC 50 µM, H2O) for
30 min at 37 °C, protected from light, and then visualized under UV light. To remove the residual activity buffer, it was washed with a buffer containing SDS 2% w/v,
Na2CO3 66 mM, β-mercaptoethanol 1.5% v/v, and H2Ofor 15 min at room temperature. For transfer, a bufferof 10% tris-glycine and 10% methanol in H2O was used,
and the PVDF membrane was activated in methanol for
5 min. It was then transferred on ice in the cold room for
1.5 h at 400 mA. Afterward, it was blocked in 1X PBS 5%
milk for 1 h and incubated overnight with the primary
antibody β5i or RPN11. Subsequently, washes were performed with PBS Tween, incubated with the secondary
antibody for 1 h, and washed with PBS 0.25% Tween-20
until visualization on the iBright™ CL1500 Imaging System (Cat#A44114, ThermoFisher Scientific Inc).
Intracellular glucose assay

The Amplex RED kit (Cat#A22189, Thermofisher) wasused to measure glucose levels in CLU177 cells treatedwith BSA or PA concentration for 24 h in the presenceor absence of the ONX-0914 inhibitor. The cells weretrypsinized, centrifuged at 800 x g for 5 min, and replated to keep them suspended. After being trypsinizedfor 30 min, insulin at 10 nM was added for 5–15 min, or
1X PBS as a control. The samples were then centrifuged
at 800 x g for 5 min, and the supernatant was removed
and subsequently washed with 1X PBS. The samples were
centrifuged again, the supernatant was removed and thenresuspended in 100 µL of 1X PBS. The samples were thensonicated for 3 s at 30 mA in the sonicator. Subsequently,
50 µL of the sample collected in 1X PBS was added to
50 µL of the Amplex RED kit in black 96-well plates. It
was allowed to react for 30 min at room temperature

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

and measured on a fluorimeter (Biotek Synergy HTX) at
EX530/EM590. The values were normalized based on theprotein amount quantified with the BCA kit.
Flow Cytometry measurements

Cells were treated with BSA or PA at the indicated concentration for 24 h in the presence or absence of theinhibitor ONX-0914. After this time, cells were incubatedwith 1 µM of TMRE probe (Cat# T669, ThermoFisher
Inc) or 5 µM of Mitosox-Red probe (Cat# M36008, ThermoFisher Inc) in combination with 0.5 µM of Mitotracker
Deep Red probe (Cat# M46753, ThermoFisher Inc), alldiluted in HBSS medium for 30 min. After two washes
with PBS 1X, the cells were trypsinized, washed again in
PBS 1X, and finally resuspended in 200 µL of HBSS for
analysis in the cytometer (BD FACSCanto II).
Statistical analysis

Densitometric quantification of the Western blot signal was estimated using Fiji version 2.1.0 (http://imagej.
net/software/Fiji/). Experiments were performed at least
three times for each assay. Data analysis was conductedusing Microsoft Excel 2021 (Microsoft Corporation) andPrism 9.0 (GraphPad Software, San Diego, CA, USA).
The graphs are presented as points ± median (U-MannWhitney) or mean ± standard error of the mean (SEM, for
ANOVA-ONE-WAY), and comparisons were made using
the non-parametric U Mann-Whitney t-test or ANOVAONE-WAY. Values of *p < 0.05, **p < 0.01, ***p < 0.001, and****p < 0.0001 were considered statistically significant.

Results
HFD increases the levels and activity of theimmunoproteasome in the hypothalamus

Mice lacking the β5i subunit of the immunoproteasome
and fed with HFD for 2 months accumulate less fat andimprove glucose intolerance and insulin sensitivity thanwild-type mice fed with the same diet [14]. Importantly,
this improvement in the metabolic phenotype occurs
despite no difference in food intake, energy expenditure, or locomotor activity. Furthermore, these mice
have decreased macrophage infiltration and chemokine
expression, reducing inflammation in the adipose tissuecompared to HFD-fed WT mice [14].
In this study, we examined how HFD impacts immunoproteasome levels in the hypothalamus of male micecompared to age-matched mice fed a regular chow diet.
We focus on the β5i subunit because it is the one thatwould be mainly altered in obesity [14, 20]. Furthermore, this subunit is involved in the differentiation ofpreadipocytes to adipocytes [21] and it has been also
related to lipodystrophy diseases [22]. Our findings indicate that the HFD feeding significantly increases the levels of the β5i subunit of the immunoproteasome in the

Page 6 of 20

hypothalamus, compared to mice fed a chow diet (Fig. 1A
and B). No changes were observed in the constitutive
proteasome β5 subunit (Fig. 1A and C). These results
indicate that HFD feeding specifically triggers the immunoproteasome, which may contribute to the low-grade
chronic inflammation associated with obesity.
We analyzed the immunoproteasome complex levels in
the hypothalamus of mice fed with an HFD or chow using
protein extracts resolved on native gels. We used immunodetection to analyze the 19 S and 20 S complexes,
focusing on the RPN11 (19 S) and β5i (20 S) subunits. By
detecting the overlapping signal of both antibodies coupled to a secondary antibody, we identified the 26 S and
30 S proteasome complexes. These complexes are formedby the interaction of a single regulatory particle 19 S withthe 20 S proteasome to create the 26 S proteasome or bybinding two regulatory particles 19 S with the 20 S proteasome to form the 30 S proteasome. We observed that
HFD feeding significantly increases the 20 S complex in
the hypothalamus of mice fed with an HFD compared
to mice fed with the chow diet (Fig. 1D-E). However, we
did not observe significant differences in the amount of
the 26 S and 30 S complexes (Fig. 1D, F, D and G). The
activity of the β5i subunit of the immunoproteasome wasalso measured in native gels using the fluorescent substrate Ac-ANW-AMC. This assay allowed us to visualize
the 26 S and 30 S complexes (Fig. 1H). An increase in theactivity of the 30 S complex of the immunoproteasome
was detected (Fig. 1J) without changes in the activity of
the 26 S complex (Fig. 1I). These experiments showedthat consumption of an HFD increases the levels of the
β5i subunit and 20 S complex in the hypothalamus andboosts the activity of the 30 S complex. These results
suggest that the immunoproteasome may have a role in
HFD-induced hypothalamic inflammation.
HFD increases immunoproteasome levels in astrocytes and
POMC neurons in the hypothalamus

To analyze the distribution and levels of the immunoproteasome, we performed immunofluorescence staining of the β5i subunit of the immunoproteasome on the
ARC (arcuate nucleus), obtained from mice fed with an
HFD or chow diet. We observed an increase in the total
fluorescence intensity of the immunoproteasome in
the ARC, close to the 3 V, and in the median eminence(Fig. 2B-C). According to the location, shape, and immunopositivity of GFAP, some correspond to astrocytes(Fig. 2D-E). However, the increase in total fluorescenceof β5i is probably attributable to other parenchymal
cells like neurons or microglia. Therefore, we explore thelocalization of immunoproteasome in POMC-GFP mice
exposed to either diet. We observed a significant increasein the number of POMC neurons positive to β5i in mice
fed with a HFD compared to CHOW (Fig. 2F-G). On

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

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Fig. 1 Consumption of an HFD increases the levels of immunoproteasome in hypothalamic astrocytes and POMC neurons (A) Protein extracts fromhypothalamic lysates of male mice fed with chow or HFD for 16 weeks were analyzed by western blot to evaluate the total levels of β5i and β5 subunits.
β-actin was used as internal control. Quantification of β5i (B) and β5 (C) levels normalized to β-actin. (D) Representative native western blot showing thesubunits RPN-11 and β5i levels incorporated into the 20 S, 26 S, and 30 S of the immunoproteasome. The RPN11 subunit of the 19 S regulatory particle(green) and β5i subunit of the 20 S complex (red) were analyzed from hypothalamic lysates of male mice fed either with chow or HFD for 16 weeks.
Quantification of the β5i levels incorporated to the 20 S (E), 26 S (F), and 30 S (G) complexes is shown. (H) Activity of the β5i subunit incorporated into
the 26–30 S complex was measured in native gels using the fluorescent substrate Ac-ANW-AMC in hypothalamic lysates from male mice fed either withchow or HFD diet for 16 weeks. Quantification of the activity of the 26 S and 30 S complexes is shown in (I) and (J), respectively. Data are represented asmean ± SEM. Analysis: Mann-Whitney test. *p < 0.05, n = 4

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

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Fig. 2 HFD feeding increases the levels and activity of the immunoproteasome in the hypothalamus (A) Schematic representation of a coronal
section of the brain showing the location of the arcuate nucleus, where
the confocal images of mice fed with chow or HFD were obtained. (B)Immunofluorescence using anti-β5i antibodies (red) and DAPI as a nuclei
marker (blue). (C) Quantification of total fluorescence of the β5i subunit
in all parenchyma of the hypothalamus. (D) Co-immunostaining using
anti-β5i antibodies (red, left panel), anti-GFAP (green, middle panel), andmerge image (right panel), including nuclear staining with DAPI (blue).
(E) Quantification of total fluorescence of the β5i subunit in segmented
GFAP-positive astrocytes. (F) Immunostaining using anti-β5i (red, left
panel), POMC-GFP (green, middle panel), and composite image (rightpanel), including nuclear staining with DAPI (blue) Scale bar: 50 μm. (G)Quantification of percentage of POMC neurons segmented to B5i neurons. (A, C, E) shows a 4X magnification of the area indicated in the whitedashed-line box projected in the lower-right inset. Scale bar: 15 μm. Data
are represented as mean fluorescence intensity/cell ± SEM. Analysis: UMann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 4

the other hand, we observed that the HFD feeding also
increases the immunoproteasome fluorescence intensity in different brain areas, such as the motor cortex M1
and M2, where suggestive labeling of pyramidal neurons
is observed (Fig. S1 A-B). This suggests that the effectsof HFD feeding on brain might affect global cerebral
homeostasis, impacting extrahypothalamic regions.
The inhibition of the immunoproteasome with ONX-0914
restores the imbalance of the insulin-glucose axis induced
by PA in the presence of insulin

It has been observed that the process of autophagy is not
functioning properly in the hypothalamus of male mice
who have been fed an HFD for a prolonged period of time[5, 6, 23]. Additionally, an increase in saturated fatty acids
such as PA has been found in the brains of mice fed with
an HFD for 16 weeks [4, 24, 25]. Consistently, it has also
been observed that the PA reduces the autophagic flux
in N43/5 cells, a model of embryonic hypothalamic neurons [7]. We observed that in CLU177 cells, a model ofadult hypothalamic neurons [26], treatment with PA for
24 h increases the β5i subunit of the immunoproteasome
but not the β5 subunit of the proteasome (Fig. 3A-C).
This result confirms that saturated fatty acids, enrichedin the HFD diet, upregulate the immunoproteasome. This
increase in the immunoproteasome could be partly due
to a loss of autophagy since the silencing of the essential autophagy-related genes Beclin1 and FIP200, both
involved in the formation of the autophagosome [27],augment immunoproteasome levels (Fig. 3D-E, Fig. S2).
Given that autophagy inhibition blunts insulin signaling in hypothalamic neurons [7] and that consumption of HFDs causes hypothalamic insulin resistancein rodents [17], we studied whether the increased in
immunoproteasome upon PA treatment (Fig. 3D-E)
could play a role in the insulin pathway. To this aim, weused ONX-0914, a specific inhibitor of the immunoproteasome [11]. First standardized the concentration of theAlbornoz et al. Journal of Neuroinflammation(2024) 21:191

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Fig. 3 Inhibition of autophagy increases the β5i subunit of the immunoproteasome in CLU177 hypothalamic neuronal cells (A) CLU177 cells wereincubated with vehicle (BSA) or PA (100 µM) for 24 h. Protein extracts were analyzed by western blot to assess the levels of the β5i subunit of the immunoproteasome, β5 subunit of the proteasome, and p62/SQSTM1. (B) Quantification of β5i subunit levels normalized to the internal control β-actin. (C)Quantification of β5 subunit levels normalized to the internal control β-actin. (D) CLU177 cells were transfected for 72 h with liposomes containing specific siRNA sequences targeting Beclin1 and FIP200, including a siControl sequence. Extracts were analyzed by western blot to assess the levels of the β5i
subunit of the immunoproteasome and the silenced proteins FIP200 and Beclin1. (E) Quantification of β5i subunit levels in CLU177 relative to the internal
control β-actin with the indicated treatments. Data are represented as mean ± SEM. Analysis: U-Mann-Whitney test. *p < 0.05. B and D (n = 5) and C (n = 3)

immunoproteasome inhibitor ONX-0914 to evaluate its
effect on the insulin pathway (Fig. S3).
CLU177 cells were treated with PA for 24 h, in theabsence and presence of ONX-0914. As expected, insulin
treatment increased the phosphorylation of AKT(S-473)
in the BSA condition, while the addition of PA along withinsulin decreased its phosphorylation, a phenomenon
previously documented [7]. Interestingly, the ONX-0914
inhibitor rescued the AKT(S-473) phosphorylation levelsin the presence of PA (Fig. 4A-B). To further understandthe relationship between insulin signaling, the immunoproteasome, and glucose, we analyzed intracellularglucose levels in CLU177 cells after insulin treatment.
To this purpose, we evaluated intracellular glucose levels using a kit with glucose oxidase enzyme. We studied
glucose levels after 5 and 15 min of exposure to insulin.
Insulin stimulation was performed in cells previously

incubated with vehicle (BSA) or PA (100 µM) for 24 h,both in the absence and presence of the ONX-0914
inhibitor. In the conditions with the BSA vehicle and theabsence of ONX-0914, we observed that insulin caused
a significant time-dependent increase in intracellularglucose levels. Specifically, we observed an increase inglucose levels after 5 min of insulin, which was not evident after 15 min of insulin (Fig. 4C). A similar result
was obtained in the presence of the BSA vehicle andthe ONX-0914 inhibitor (Fig. 4C). However, unexpectedly, we observed a different pattern when the cells wereincubated with PA. In this case, intracellular glucose levels continued to increase at 15 min of insulin incubation(Fig. 4C), indicating a dysregulation due to the effect of
PA. Interestingly, the increase in glucose at 15 min by PA
was reduced in the presence of ONX-0914, approaching
the control condition with BSA (Fig. 4C).

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

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Fig. 4 ONX-0914 attenuates the PA-induced reduction in PTEN levels and insulin sensitivity in CLU177 (A) CLU177 cells were incubated with vehicle(BSA) or PA (100 µM) for 24 h in the presence or absence of 100 nM ONX-0914. Protein extracts were analyzed by western blot to evaluate the levels of
p-AKT(Ser-473) and total AKT. (B) Quantification of the P-AKT/AKT(S-473) ratio normalized to the internal control β-actin. (C) Intracellular glucose levels
measured by AmpliRed Glucose/Glucose Oxidase assay in CLU177 cells treated with vehicle (BSA) or PA (100 µM) for 24 h in the presence or absence of
ONX-0914, followed by stimulation with 10 nM insulin or PBS 1X (vehicle) for 5 or 15-min. (D) Protein extracts were analyzed by western blot to assess thelevels of the β5i subunit of the immunoproteasome, PTEN, and p62/SQSTM1. (E) Quantification of PTEN levels normalized to the internal control β-actin.
Data are represented as mean ± SEM. Analysis: U-Mann-Whitney test. *p < 0.05. (B and E) n = 5 and (C) n = 3.

It has been shown that PTEN, a negative regulator of
the insulin pathway, is a substrate of the immunoproteasome [28]. We observed that PTEN levels decreased after
24 h of treatment with PA, accompanied by an increasein the b5 subunit of the immunoproteasome and p62/
SQSTM1, an autophagy marker (Fig. 4D-E). We observedthat the reduction of PTEN, induced by PA, was attenuated in the presence of ONX-0914 (Fig. 4D-E), suggestingthat the decrease in PTEN by PA could be partly dependent on the immunoproteasome, as previously describedin UO2S cells [29]. Interestingly, the ONX-0914 inhibitor did not cause significant changes in the levels of the
β5i subunit of the immunoproteasome under any of thetested conditions (Fig. 4A, C). Additionally, although theincrease in p62/SQSTM1 levels, a characteristic phenomenon of PA treatment due to autophagic flux inhibition, did not show significant changes in the presenceof ONX-0914, there was a subtle inclination towards adecrease. These results suggested that the inhibition of
the immunoproteasome and the inflammation caused by
PA could have a preventive effect on the imbalance of theinsulin-glucose axis. This effect could be partly related to
the restoration of PTEN levels.
ONX-0914 restores mitochondrial membrane potential,
decreasing ROS and mitophagy enhanced by PA treatment

We considered the implications of the Randle cycle inline with the idea that PA might influence cellular metabolism and consequently alter how cells handle the useof glucose following its entry [30]. This cycle proposesa metabolic competition between glucose and free fatty
acids, considering that there should always be a balance
between glycolysis and mitochondrial β-oxidation [30].
Under high concentrations of fatty acids and mitochondrial β-oxidation conditions, glycolysis should be inhibited, a mechanism that would occur due to high citrate
production in the mitochondria. This metabolite has
been shown to inhibit key enzymes of glycolysis resulting in intracellular glucose accumulation [30, 31]. Mitochondrial membrane potential is crucial for maintaining
the physiological function of the respiratory chain andgenerating ATP, while reactive oxygen species (ROS)Albornoz et al. Journal of Neuroinflammation(2024) 21:191

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Fig. 5 ONX-0914 reverses the decrease in mitochondrial membrane potential and increase in ROS induced by PA (A) Representative image of mitochondrial membrane potential labeled with TMRE probe (red) and mitochondria labeled with Mitotracker probe (green) in CLU177 cells treated with BSA or
PA (100 µM) in the presence or absence of ONX-0914 for 24 h. (B) Flow cytometry to detect changes in Mitosox-Red for ROS measurement together with
Mitotracker-Deep Red for mitochondrial mass measurement. (C) Quantification of TMRE fluorescence intensity normalized to the amount of Mitotracker
via flow cytometry. (D) Quantification of Mitosox fluorescence intensity (ROS) via flow cytometry. Data are represented as mean ± SEM. Scale bar = 10 μm.
Analysis: U-Mann-Whitney test. *p < 0.05. n = 4

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

are signaling molecules that lead to oxidative and cellular stress [32]. Therefore, we assessed the integrity
and activity of mitochondria in the presence of PA in
CLU177 cells. Initially, we evaluated mitochondrial function through ROS and membrane potential marked with
Tetramethylrhodamine ethyl ester perchlorate (TMRE),
aiming to assess whether mitochondria in the PA + ONX0914 condition could show functional improvements.
We observed that PA causes a decrease in mitochondrial membrane potential, manifested by a reduction in
TMRE fluorescence intensity (Fig. 5A, C). Additionally,
PA increases Mitosox fluorescence intensity, which measures ROS levels (Fig. 5B, D). However, both the decreasein mitochondrial membrane potential [33, 34] and theincrease in ROS [35, 36] have been documented in othercell types.
Surprisingly, we observed that the ONX-0914 inhibitor reduces the effects of PA on mitochondrial function,
revealing an unexpected role of the immunoproteasomein this regulation.
Next, we proceeded to analyze whether PA causedchanges at the level of the mitochondrial network in
CLU177 cells compared to the BSA condition. For thispurpose, we analyzed mitochondria using the protein
TOM20, located in the outer membrane of mitochondria(Fig. S4). These results indicated that PA generates a lesscomplex mitochondrial network with less branched andinterconnected mitochondria, suggesting a more fragmented mitochondrial network (Fig. 4S). This confirms
previous studies with PA in other cell types [34, 37, 38].
Unexpectedly, when we tested the combined effect of PA
with ONX-0914, we observed even greater mitochondrial network fragmentation. Since ONX-0914 enhances
mitochondrial function in combination with PA, theincreased mitochondrial fragmentation could positively
impact mitochondrial activity (Fig. S4).
Mitochondrial dynamics play a crucial role in responding to mitochondrial dysfunction by promoting the elimination of irreversibly damaged mitochondria throughmitochondrial fission [39]. In this regard, mitophagy
is a protective mechanism that removes dysfunctional
mitochondria while preserving the population of healthy
mitochondria. It has been observed that PA plays a dualrole in endothelial cells [40]. Depending on the stresslevel, adding PA triggers mitophagy activation to remove
damaged mitochondria. However, under severe stress,
this mechanism can be inhibited, leading to the accumulation of damaged mitochondria [40].
Based on these findings, we aimed to evaluate mitophagy in our model. For this purpose, we studied the colocalization of LAMP1, an endolysosomal marker, andTOM20, an outer mitochondrial membrane marker.
This would allow us to assess whether PA, by blocking autophagic flux, could prevent the degradation ofPage 12 of 20

damaged mitochondria, potentially resulting in co-localization between both markers. Thus, we observed that
PA caused a significant increase in the co-localization of
TOM20 and LAMP1 compared to BSA. This suggested
that PA, by blocking autophagic flux in the presence ofdysfunctional mitochondria, could inhibit the final stageof mitophagy (Fig. 6A-C). Interestingly, we observedthat ONX-0914, in the presence of PA, where the mitochondria are even smaller, reduced the co-localization
between TOM20 and LAMP1 (Fig. 6A-C).
Since we observed a more significant co-localization of
LAMP1 with TOM20 in the PA condition compared tothe control, we conducted experiments to confirm if thiswas related to mitophagy.
To differentiate free mitochondria from mitochondria
within lysosomes (mito-lysosomes), we used the pHsensitive mt-Keima reporter. This tool has dual excitation
peaks in its protonated and deprotonated states, making
it a ratiometric and reversible pH measure.
In the presence of BSA, we observed a significant emission at 440 nm (Fig. 7A-B). Conversely, when PA wasused, an emission at 586 nm was detected (Fig. 7A-B).
These findings confirm PA caused accumulation of damaged mitochondria in lysosomes through mitophagy.
Further, we found ONX-0914 caused a decrease in theemission at 586 nm compared to PA alone (Fig. 7.A-B),
consistently with the idea that this inhibitor prevents
mitochondrial damage, and therefore degradation, by
PA. Together, these results confirm the pathogenic roleof the immunoproteasome in response to PA in hypothalamic neurons, an effect related to the unexpected regulatory role of the immunoproteasome on mitochondrial
function.

Discussion
Our results demonstrated that the β5i subunit of theimmunoproteasome is increased in proteins and activity
levels in the hypothalamus of mice consuming HFD incomparison to those on a chow diet. Furthermore, using
native western blotting, we observed an increase in theassembly of the 20 S proteasome complex and a rise in
the 30 S proteasome complex (19–20 S-19 S), coupledwith a significant increase in its activity. Although theactivity of the 30 S complex is higher in the hypothalamus of this mouse model of diet-induced obesity, theimplications of these findings on the hypothalamic neuroendocrine integrative function, as well as the effecton other body systems that contribute to the functionof this important area of the brain remain to be characterized [41]. We suggest that this increase may correlatewith a tendency towards higher protein levels of the 30 Scomplex in the hypothalamus of HFD-induced obese
mice, suggesting that the immunoproteasome is elevatedand active in the hypothalamus of obese mice. Several

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

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Fig. 6 ONX-0914 reduces the co-localization of TOM20 with LAMP1 increased by PA treatment (A) Representative confocal image of CLU177 cells treatedwith BSA or PA (100 µM) in the presence or absence of ONX-0914 for 24 h and co-stained with anti-TOM20 antibodies (Red, left panel), anti-LAMP1 antibodies (Green, middle panel), and composite image with DAPI (Blue, right panel). (B) % co-localization between TOM20 and LAMP1 according to Manders’coefficient analysis. (C) % co-localization between LAMP1 and TOM20 according to Manders’ coefficient analysis. Scale bar = 10 μm. Data are represented
as mean ± SEM. Analysis: ONE-WAY ANOVA test. *p < 0.05, ***p < 0.001, ****p < 0.0001. n = 3

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

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Fig. 7 ONX-0914 Reverses Mitophagy Induced by PA treatment (A) Representative confocal images of CLU177 cells treated with BSA or PA in the presence or absence of ONX-0914 for 24 h and transfected with mt-Keima were excited by the 440 and 586 nm laser. (B) Quantification of the number of acidpuncta per cell (C) Schematic depiction of the ratiometric mitophagy reporter mt-Keima. Scale bar = 10 μm. Data are represented as mean ± SEM. Analysis:
ONE-WAY ANOVA test. *p < 0.05, ***p < 0.001, ****p < 0.0001. n = 3

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

reports have shown that proinflammatory cytokines orpathological conditions can increase the immunoproteasome in neurons, astrocytes, and microglia [42–45]. Our
results suggest this could also be the case in the context
of obesity induced by HFD. Additionally, we describe forthe first time that the immunoproteasome is also present
in POMC neurons, where it is increased following HFD
consumption, suggesting a potential role in neuroinflammation. However, immunoproteasome expression on
the function of these and other hypothalamic neurons
remains to be elucidated.
On the other hand, previous studies have shown that
PA reduces AKT phosphorylation levels [7], which havebeen associated with insulin resistance in different models [46–48]. Consistently, we observed a decrease in AKT
phosphorylation after 24 h of PA treatment, which wasrestored when the immunoproteasome was chemicallyinhibited by treatment with ONX-0914. We observedthat insulin stimulation for 5 min increases intracellular glucose levels in both control conditions and in thepresence of PA, with or without the ONX-0914 inhibitor. However, after 15 min of insulin stimulation, we
observed that in control conditions, intracellular glucose
levels decrease, suggesting that, after glucose uptake, thisfuel is rapidly utilized. In contrast, with PA, we observeda sustained increase in intracellular glucose, a phenotype that is rescued in the presence of the ONX-0914
inhibitor. There is evidence indicating that PA treatment
decreases glucose consumption [7, 49, 50], while otherstudies report the opposite [51, 52]. This suggests thatthe effects of PA on glucose levels vary depending on thetime, concentration, and cell type. A potential explanation for this phenomenon is the metabolic pathway glucose undergoes upon entering the cells. For example, it
has been shown that PA affects the activity of hexokinase,the first enzyme in glycolysis [53–55]. It is possible that
PA could affect glycolysis, upregulating fatty acid oxidation to increase energy production or other pathways,
such as the acetylation of STAT3, which enhance phospholipid biosynthesis, thereby increasing mitochondrial
membrane lipid content to resist apoptosis. as previously
reported [56]. Therefore, studying various metabolic
pathways and different cell types is essential to gain adeeper understanding of the effects of PA on cell metabolism. To investigate this, it is necessary to thoroughly
examine the various metabolites that are enhanced in
response to PA overload using metabolomics and otheromics techniques. This approach could provide a broader
view of metabolism under the overload of saturated fatsor upon the overexpression of the immunoproteasome,an aspect not addressed in this study.
Another possibility is that PA itself could exert aneffect on GLUT4, GLUT1, or the main glucose transporter localized in neurons GLUT3 on the cell surface ofPage 15 of 20

hypothalamic neurons, which might explain the increase
in intracellular glucose, a hypothesis that requires future
investigation. Nonetheless, it is still possible that a reduction in PTEN levels by PA could at least partially mediate
these effects.
It has been described that the immunoproteasome
degrades PTEN, a lipid and protein phosphatase sensitive
to reduction-oxidation, playing an essential role in regulating the PI3K-AKT pathway [28]. PTEN often exhibits
genetic alterations in cancer, such as point mutations,chromosomal deletions, or epigenetic mechanisms. Furthermore, it is also dysregulated in PIP3 metabolism, aneffect observed in diabetes [57]. It is important to notethat PTEN activity is regulated by post-translational
modifications, such as phosphorylation or oxidation,
which affect its activity and interaction with other proteins. The relationship between obesity and PTEN is stilla subject of discussion and controversy in the scientificcommunity. However, it has been consistently observedthat PTEN loss is associated with improved insulin sensitivity in obesity models [58–60]. However, a decrease
in PTEN levels may also promote fat storage due to theincreased efficiency of the PI3K-AKT pathway, limiting
the organism’s ability to burn excess calories. It is essential to consider that decreasing PTEN levels can also
increase the proliferation of cancer cells in an obesitycontext [61].
In our study, we observed that PTEN levels were significantly decreased following 24 h-PA exposure. Similar findings were recently published in U2OS cells [29].
The decrease in PTEN suggests a disruption in the PI3KAKT pathway. Although PTEN activity can be measured,
we did not assess it in this study. Instead, we used thedecrease in its levels as an indicator of its dysfunction.
This limitation highlights the need for future studies todirectly measure PTEN activity to confirm the impact of
PA in the axis PI3K-AKT/PTEN pathway. We observedthat the reduction of PTEN levels, caused by 24 h of PA
treatment, was reversed when using the immunoproteasome inhibitor ONX-0914. This finding suggests that theimmunoproteasome plays a major role in metabolism,
controlling the entry of nutrients such as glucose and PA,
and regulating the capacity of hypothalamic neurons toorchestrate systemic metabolism to either burn or store
excess nutrients. In this regard, the immunoproteasome can emerge as a promising therapeutic target withpotential impact in the treatment of metabolic diseases.
Although our results strongly support this hypothesis,
further in vivo studies with the ONX-0914 inhibitor are
needed to confirm it.
Additionally, we used the protein p62/SQSTM1 todemonstrate that PA inhibits the autophagic flux; nonetheless immunoproteasome inhibition is insufficient torestore autophagy basal levels in the cells used in thisAlbornoz et al. Journal of Neuroinflammation(2024) 21:191

study. However, we do not rule out that immunoproteasome inhibition could account for a distinct function of
p62/SQSTM1. p62/SQSTM1 is modified and regulatedby phosphorylation, ubiquitination, acetylation, proteolytic processing, and the formation of disulfide bridges,
where these modifications fine-tune p62/SQSTM1
activities in autophagy [62, 63]. On the other hand, it isessential to note that we would expect that a decrease in
PTEN levels would increase the PI3K-AKT pathway and,
therefore, enhance AKT phosphorylation. Nonetheless, it
has been described that a reduction in PTEN levels canactivate the ERK pathway, and ERK activation can inhibit
AKT phosphorylation, which could explain our findings[64, 65]. Therefore, we attribute the decrease in AKT
phosphorylation to the recovery of PTEN levels due toimmunoproteasome inhibition.
Dysfunction in glucose metabolism could also indicate alterations in fatty acid metabolism and essential
organelles that control these pathways. The interaction
between glucose and fatty acid metabolism, known as
the Randle cycle or glucose-fatty acid cycle, is critical tounderstanding processes that may be linked to the insulin-glucose imbalance. This raised the possibility that
excess of PA and the immunoproteasome could affectglucose metabolism. In this regard, the functional relationship between the immunoproteasome and mitochondria is related to the fact that PA promotes mitochondrial
fragmentation. This phenomenon has been described inmany cell types and observed in CLU177, a cellular modelof adult hypothalamic neurons. Our results showed that
ONX-0914, in combination with PA, promotes evengreater mitochondrial fragmentation than PA alone. In
this regard, it is known that mitochondrial fission is often
responsible for mitochondrial fragmentation, favoring
the metabolism of exogenous fatty acids and the transfer
of fatty acids between mitochondria and lipid droplets[37]. Therefore, a possibility suggested by our results isthat the inhibition of the immunoproteasome by ONX0914 could favor mitochondrial fission to reduce theexcess of PA and its lipotoxicity. However, mitochondrial
fragmentation can also promote the elimination of dysfunctional mitochondria through mitophagy. This option
is supported by (i) the fact that while PA leads to a lossof mitochondrial membrane potential and an increasein ROS, these alterations are not observed in the presence of ONX-0914, (ii) the colocalization of TOM20 with
LAMP-1 in the presence of PA is lost with ONX-0914 and(iii) mt-Keima shows mitochondria in acid compartmentin the presence of PA, an effect that is reversed by ONX0914 exposure. This strongly suggests that inhibiting theimmunoproteasome prevents mitochondrial damage inthe presence of PA, allowing mitochondria to be more
active in carrying out PA β-oxidation. How could theimmunoproteasome affect mitochondrial integrity? We

Page 16 of 20

observed that ONX-0914 reduces mitochondrial damage
in the presence of PA, as demonstrated by a reduction in
ROS, an increase in membrane potential, and the absenceof mitophagy. It is well-known that the proteasome plays
a crucial role in mitochondrial protein quality control[66]. Mitochondria-associated degradation (MAD) actson two levels: firstly, the proteasome removes mature,functionally compromised, or mis-localized proteinsfrom the mitochondrial surface; secondly, the proteasome clears import intermediates that are stalled duringtranslocation from the mitochondrial import pore [54].
However, little is known about how the immunoproteasome regulates mitochondrial function. In this regard,
it has been observed that mitochondrial damage or dysfunction leads to increased immunoproteasome levels,
which has been linked to contributing to autoimmunity[67]. On the other hand, it has been observed that theinduction of the subunit β1i of the immunoproteasome isrequired to regulate cell proteostasis upon mitochondrial
dysfunction [68] or subunit β1i deficiency causes abnormal metabolism, oxidative stress, and neuroinflammation[69]. In contrast, the β2i subunit of the immunoproteasome ameliorates myocardial ischemia/reperfusion
injury by regulating Parkin-Mfn1/2-mediated mitochondrial fusion [70]. As for the effect of the β5i subunit of theimmunoproteasome on mitochondria, it is unknown. A
report using the inhibitor ONX-0912, which inhibits the
β5i subunit, showed that it triggered apoptosis throughthe intrinsic mitochondrial pathway and induced mitophagy by activating the Parkin/Pink pathway in liver cancer[71]. In contrast, another report indicates that inhibitionof chymotrypsin-like activity of the proteasome by Ixazomib prevents mitochondrial dysfunction during myocardial ischemia by blocking the release of cytochrome c andsignificantly preserving mitofusin-2 integrity [72].
This disparity in these results could indicate that it
depends on the cell type and the cellular context. Basedon our results, the effect of the immunoproteasome on
mitochondrial function could be related to PTEN. PTEN
has two isoforms: PTENα (PTEN-L) and PTENβ; PTENαpromotes the recruitment of Parkin, an E3 ubiquitin
ligase, to damaged mitochondria, while PTENβ can prevent Parkin from recognizing and tagging damaged mitochondria for degradation [73]. This dual effect allows themaintenance of mitochondrial integrity. In our case, we
observed a decrease in total PTEN levels. However, it islikely that the PTENα isoform is the one that decreases
the most and accounts for this effect, so future research
could delve into this mechanism.
Therefore, another possibility is that the immunoproteasome inhibits the autophagic flux. In this regard,
it has been recently reported that the immunoproteasome degrades the autophagy-related proteins ATG5 and
ATG12 in cardiomyocyte and retinopathy models [74,Albornoz et al. Journal of Neuroinflammation(2024) 21:191

75], something that could be occurring in the context
of hypothalamic neurons. Our results suggest that PA
elevates the immunoproteasome due to autophagy inhibition. This protein complex could further enhance theinhibition of autophagy by degrading proteins involvedin this pathway. This would explain the rescue effectof ONX-0914 on mitochondrial function by favoring
mitophagy and improvements in LAMP-1 positive compartments in the presence of PA. Additionally, the immunoproteasome would impact the insulin-glucose axis by
reducing PTEN levels, resulting in increased intracellularglucose. This phenotype could also be a consequence ofexcessive β-oxidation, supported by mitochondrial fragmentation in the presence of PA, an effect that could bepartially compensated by efficient mitophagy.
Our results show an increase in the immunoproteasome in POMC neurons. These neurons induce satiety
and respond to adipogenic signals such as insulin. Thus,
we propose that the immunoproteasome is a molecularpathogenic factor in hypothalamic neurons within thecontext of HFD-induced obesity and that its pharmacological inhibition may offer a promising therapeuticstrategy to prevent the onset of metabolic alterations
associated with the development of obesity-related metabolic diseases, such as insulin resistance. Additionally, weobserved an increase in the immunoproteasome in astrocytes. Other authors have already documented that theimmunoproteasome increases in astrocytes and microglia, a product of inflammation [42, 76]. Therefore, it was
expected to observe this increase. However, it remains
to be determined if this increase can be harmful, as in
the case of neurons. Our findings support the hypothesis that the immunoproteasome may play a crucial rolein the hypothalamic neuroinflammation caused by HFD
and obesity. We also underscore the role of the immunoproteasome in controlling mitochondrial homeostasis,
revealing a new and unexpected mechanism involved
in regulating the insulin/glucose balance in hypothalamic neurons. However, it is still necessary to investigate how this mitochondrial dysfunction induced by the
immunoprpteosome impacts the function of these neurons and the impact on the neuroendocrine integrationcommanded by these neurons to maintain body energy
homeostasis.
Altogether, our findings reveal the unexpected role ofthe immunoproteasome in cell metabolism and obesity, highlighting the pathogenic role of hypothalamic
low-grade inflammation and the necessity of maintaining intracellular integrity to prevent energy imbalances
caused by key actors in proteostasis and inflammation.
While further studies are needed to validate this hypothesis and understand the impact of immunoproteasome
inhibition on metabolic imbalances related to hypothalamic function and obesity, it is crucial to address the

Page 17 of 20

limitations associated with immune system inhibition by
ONX-0914, which affects antigen presentation requiredfor virus and tumor cell elimination [11]. Additionally,a thorough characterization is necessary to determine
whether ONX-0914 can cross the blood-brain barrier
and be delivered to the brain, particularly in the context
of obesity [77]. Our findings underscore the significant
role of hypothalamic organelle integrity and the potential of immunoproteasome inhibition as a therapeutic
approach for preventing metabolic and inflammatory
diseases.
Abbreviations
APS	Ammonium persulfate
ARC	arcuate nucleus
CHOW	Regular diet
FBS	Fetal bovine serum
HFD	High Fat Diet
MAD	Mitochondria-associated degradation
OPG	retroviral gag/polPA	Palmitic Acid
PEI	Polyethyleneimine
PFA	Paraformaldehyde
ROS	Reactive oxygen species
SA	Stearic Acid
TMRE	Tetramethylrhodamine ethyl ester perchlorate
VSV-G	Plasmids viral capsid

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03154-z.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Acknowledgements
We thank Felix Randow and Thomas Mund of the MRC Laboratory of
Molecular Biology, Cambridge, CB2 0QH, United Kingdom, for their generous
provision of the mt-Keima vectors.
Author contributions
N.A conceived, designed, performed and analysed data from all experiments,
and wrote the paper. J A-I fed the mice and mounted the sections tobe observed under confocal microscope. EA-M cultured and performed
treatments on CLU177 cells for the mt-Keima experiment. AdP transfected
and analyzed CLU177 cells with mt-Keima and subsequently acquired
images by confocal microscopy and performed analysis. Alanis C fed themice and mounted the sections to be observed under confocal microscope,acquired, and analyzed images. FS-M: review & editing, Funding acquisition,
contributed to the acquisition of confocal microscopy images. BK: review &editing, Funding acquisition, contributed with animals and animals feeding.
MB: review & editing, Funding acquisition. AC: review & editing, Funding
acquisition, contributed with essential reagents MdAG: review & editing,Funding acquisition, contributed with animals and animals feeding andacquisition of confocal microscopy images. EM: Writing – review & editing,Visualization, Funding acquisition, contributed with essential reagents.
AS Supervised Project, Writing – review & editing, Visualization, Funding
acquisition, contributed with essential reagents. PVB: Conceptualization,
conceived, designed and supervised project, Writing – review & editing,Visualization, Funding acquisition.

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

Funding
This work was funded by grants from Fondo Nacional de Desarrollo Científico
y Tecnológico, FONDECYT 1211261 to PB, FONDECYT 1211829 to AS,
FONDECYT 1240623 to EM, FONDECYT 1230905 to BK, FONDECYT 1211329
to AC, FONDECYT 1200965 to FS-M; FONDECYT 1221508 to MdAG, VRIDFAPPE22/05 to PB; Anillo ACT172066 to EM, MB, AC and PVB; Anillo ACT210039
to BK; ANID/BASAL (https://anid.cl/centros-e-investigacion-asociativa/
centros-basales/ (accessed on 17 December 2023) grant number FB210008
to PB; ANID Doctoral Fellowship 21212138 to N.A and 21221618 to AdP. This
work was supported by Vicerrectoría de Investigación y Doctorados de la
Universidad San Sebastián – Fondo USS-FIN-24-APCS-13.
Data availability
Data is provided within the manuscript or supplementary information files.

Page 18 of 20

7.

8.
9.

10.
11.

Declarations
Ethics approval
All procedures with experimental mice were approved and conducted inaccordance with the regulations of the Committee for the Care and Use ofExperimental Animals-Universidad San Sebastián and in accordance with
the ANID Biosafety Standards Manual. We have ethical approval from the
Universidad San Sebastián scientific ethics committee, code # 22-2021-20.
Competing interests
The authors declare no competing interests.
Author details
1
Centro de Biología Celular y Biomedicina, Facultad de Medicina y
Ciencia, Universidad San Sebastián, Santiago, Chile2
Departamento de Biología Celular, Facultad de Ciencias Biológicas,
Universidad de Concepción, Concepción, Chile
3
Laboratory of Molecular and Cellular Pathology, Institute in Dentistry
Sciences, Dentistry Faculty, University of Chile, Santiago, Chile
4
Cell and Molecular Biology Laboratory, Institute in Dentistry Sciences,
Dentistry Faculty, Universidad de Chile, Santiago, Chile
5
Advanced Center for Chronic Diseases (ACCDiS), Faculty of Chemicaland Pharmaceutical Sciences, Faculty of Medicine, Universidad de Chile,
Santiago, Chile
6
Department of Basic Sciences, Faculty of Medicine and Sciences,
Universidad San Sebastián, Santiago, Chile
7
Centro Basal Ciencia & Vida, Universidad San Sebastián, Santiago, Chile
Received: 22 April 2024 / Accepted: 14 June 2024

12.
13.
14.
15.
16.

17.
18.

19.
20.

21.
References
1. Qiu T, et al. Obesity-induced elevated palmitic acid promotes inflammationand glucose metabolism disorders through GPRs/NF-κB/KLF7 pathway. Nutr
Diabetes. Apr. 2022;12(1):23. https://doi.org/10.1038/s41387-022-00202-6.
2. von Frankenberg AD, Marina A, Song X, Callahan HS, Kratz M, Utzschneider KM. A high-fat, high-saturated fat diet decreases insulin sensitivity
without changing intra-abdominal fat in weight-stable overweight andobese adults. Eur J Nutr. Feb. 2017;56(1):431–43. https://doi.org/10.1007/
s00394-015-1108-6.
3. Gupte AA et al. Aug., High-fat feeding-induced hyperinsulinemia increases
cardiac glucose uptake and mitochondrial function despite peripheral insulin
resistance. Endocrinology. 2013;54(8):2650–2662, , https://doi.org/10.1210/
en.2012-2272.
4. Rodriguez-Navas C, Morselli E, Clegg DJ. Sexually dimorphic brain fatty
acid composition in low and high fat diet-fed mice. Mol. Metab. Aug
2016;5(8):680–689. https://doi.org/10.1016/j.molmet.2016.06.014.
5. Meng Q, Cai D. Defective hypothalamic autophagy directs the central pathogenesis of obesity via the IkappaB kinase beta (IKKbeta)/NF-kappaB pathway.
J Biol Chem. Sep, 2011;286(7):32324–32332. https://doi.org/10.1074/jbc.
M111.254417.
6. Portovedo M et al. Mar., Saturated fatty acids modulate autophagy’s
proteins in the hypothalamus. PLoS ONE. 2015;10(3):e0119850 https://doi.
org/10.1371/journal.pone.0119850.

22.
23.
24.
25.

26.
27.
28.

Hernández-Cáceres MP, et al. Palmitic acid reduces the autophagic fluxand insulin sensitivity through the activation of the free fatty acid receptor
1 (FFAR1) in the hypothalamic neuronal cell line N43/5. Front Endocrinol(Lausanne). Mar. 2019;10:176. https://doi.org/10.3389/fendo.2019.00176.
Gan T, Qu S, Zhang H, Zhou X-J. Modulation of the immunity and inflammation by autophagy. MedComm. Aug. 2023;4(4):e311. https://doi.org/10.1002/
mco2.311.
Le Thuc O, Stobbe K, Cansell C, Nahon J-L, Blondeau N, Rovère C. Hypothalamic inflammation and energy balance disruptions: spotlight on chemokines. Front Endocrinol (Lausanne). Aug. 2017;8:197. https://doi.org/10.3389/
fendo.2017.00197.
Arimochi H, Sasaki Y, Kitamura A, Yasutomo K. Dysfunctional immunoproteasomes in autoinflammatory diseases. Inflamm Regen. May 2016;36:13.
https://doi.org/10.1186/s41232-016-0011-8.
Çetin G, et al. Immunoproteasomes control activation of innate immune signaling and microglial function. Front Immunol. Oct 2022;13:982786. https://doi.org/10.3389/fimmu.2022.982786.
Murata S, Yashiroda H, Tanaka K. Molecular mechanisms of proteasome
assembly. Nat Rev Mol Cell Biol. Feb 2009;10(2):104–115. https://doi.
org/10.1038/nrm2630.
Albornoz N, Bustamante H, Soza A, Burgos P. Cellular responses to proteasome inhibition: molecular mechanisms and beyond. Int J Mol Sci. Jul
2019;20(14). https://doi.org/10.3390/ijms20143379.
Kimura H, et al. Immunoproteasome subunit LMP7 Deficiency improves
obesity and metabolic disorders. Sci Rep. Oct. 2015;5:15883. https://doi.
org/10.1038/srep15883.
Guardia CM, et al. Structure of human ATG9A, the only transmembrane protein of the core autophagy machinery. Cell Rep. Jun. 2020;31:107837. https://doi.org/10.1016/j.celrep.2020.107837.
Nguyen TN, Padman BS, Usher J, Oorschot V, Ramm G, Lazarou M. Atg8
family LC3/GABARAP proteins are crucial for autophagosome-lysosome
fusion but not autophagosome formation during PINK1/Parkin mitophagy
and starvation. J Cell Biol. Dec 2016;215(6):857–874. https://doi.org/10.1083/
jcb.201607039.
Boulan B et al. Jul., AutoNeuriteJ: An ImageJ plugin for measurement andclassification of neuritic extensions. PLoS ONE. 2020;15(7):e0234529. https://doi.org/10.1371/journal.pone.0234529.
Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S. Automatic and quantitative measurement of protein-protein colocalizationin live cells. Biophys J. 2004;86(6):3993–4003, , https://doi.org/10.1529/
biophysj.103.038422.
Liu K, et al. Evaluating mitophagy in embryonic stem cells by using
fluorescence-based imaging. Front Cell Dev Biol. Sep 2022;10:910464. https://doi.org/10.3389/fcell.2022.910464.
Fletcher E, Wiggs M, Greathouse KL, Morgan G, Gordon PM. Impaired proteostasis in obese skeletal muscle relates to altered immunoproteasome activity.
Appl Physiol Nutr Metab. May 2022;47(5):555–64. https://doi.org/10.1139/
apnm-2021-0764.
Arimochi H, Sasaki Y, Kitamura A, Yasutomo K. Differentiation of preadipocytes and mature adipocytes requires PSMB8. Sci Rep. May 2016;6:26791.
https://doi.org/10.1038/srep26791.
Kitamura A et al. Oct., A mutation in the immunoproteasome subunit
PSMB8 causes autoinflammation and lipodystrophy in humans. J Clin Invest.
2011;121(10):4150–4160. https://doi.org/10.1172/JCI58414.
Morselli E, Criollo A, Rodriguez-Navas C, Clegg DJ. Chronic high fat dietconsumption impairs metabolic health of male mice. Inflamm Cell Signal.
2014;1(6):e561. https://doi.org/10.14800/ics.561.
Morselli E, et al. Hypothalamic PGC-1α protects against high-fat diet exposure
by regulating ERα. Cell Rep. Oct. 2014;9(2):633–45. https://doi.org/10.1016/j.
celrep.2014.09.025.
Melo HM, et al. Palmitate is increased in the Cerebrospinal Fluid of humans
with obesity and induces memory impairment in mice via pro-inflammatory
TNF-α. Cell Rep. Feb 2020;30:2180–e21948. https://doi.org/10.1016/j.
celrep.2020.01.072.
Yi C-X, et al. TNFα drives mitochondrial stress in POMC neurons in obesity. Nat
Commun. May 2017;8:15143. https://doi.org/10.1038/ncomms15143.
Pietrocola F, Bravo-San JM, Pedro. Targeting autophagy to counteract obesityAssociated oxidative stress. Antioxid (Basel). Jan 2021;10(1). https://doi.
org/10.3390/antiox10010102.
Xie X, et al. Selective inhibition of the immunoproteasome β5i prevents
PTEN degradation and attenuates cardiac hypertrophy. Front Pharmacol. Jun
2020;11:885. https://doi.org/10.3389/fphar.2020.00885.

Albornoz et al. Journal of Neuroinflammation(2024) 21:191

29. Bai D, et al. Palmitic acid negatively regulates tumor suppressor PTEN
through T366 phosphorylation and protein degradation. Cancer Lett. Jan
2021;496:127–33. https://doi.org/10.1016/j.canlet.2020.10.007.
30. Randle PJ. Regulatory interactions between lipids and carbohydrates: the glucose fatty acid cycle after 35 years. Diabetes
Metab Rev. Dec 1998;14(4):263–83. https://doi.org/10.1002/(sici)1099-0895(199812)14:4%3C263::aid-dmr233%3E3.0.co;2-c.
31. Newsholme EA, Randle PJ, Manchester KL. Inhibition of the phosphofructokinase reaction in perfused rat heart by respiration of ketone bodies, fatty acids
and pyruvate. Nature. Jan 1962;193:270–1. https://doi.org/10.1038/193270a0.
32. Suski JM, Lebiedzinska M, Bonora M, Pinton P, Duszynski J, Wieckowski MR. Relation between mitochondrial membrane potential and
ROS formation. Methods Mol Biol. 2012;810:183–205. https://doi.
org/10.1007/978-1-61779-382-0_12.
33. González-Giraldo Y, Forero DA, Echeverria V, Garcia-Segura LM, Barreto GE.
Tibolone attenuates inflammatory response by palmitic acid and preserves
mitochondrial membrane potential in astrocytic cells through estrogen
receptor beta. Mol Cell Endocrinol. Apr 2019;486:65–78. https://doi.
org/10.1016/j.mce.2019.02.017.
34. Sergi D, Luscombe-Marsh N, Naumovski N, Abeywardena M, O’Callaghan
N. Palmitic acid, but not lauric acid, induces metabolic inflammation, mitochondrial fragmentation, and a drop in mitochondrial membrane potential
in human primary myotubes. Front Nutr. May 2021;8:663838. https://doi.
org/10.3389/fnut.2021.663838.
35. Chen P et al. Apr., Palmitic acid-induced autophagy increases reactive oxygen
species via the Ca2+/PKCα/NOX4 pathway and impairs endothelial function
in human umbilical vein endothelial cells. Exp Ther Med. 2019;17(4):2425–
2432. https://doi.org/10.3892/etm.2019.7269.
36. He Y, Zhou L, Fan Z, Liu S, Fang W. Palmitic acid, but not high-glucose,
induced myocardial apoptosis is alleviated by Nacetylcysteine due toattenuated mitochondrial-derived ROS accumulation-induced endoplasmic
reticulum stress. Cell Death Dis. May 2018;9(5):568. https://doi.org/10.1038/
s41419-018-0593-y.
37. Song J-E, et al. Mitochondrial fission governed by drp1 regulates exogenous
fatty acid usage and storage in hela cells. Metabolites. May 2021;11(5).
https://doi.org/10.3390/metabo11050322.
38. Zezina E, et al. Mitochondrial fragmentation in human macrophages
attenuates palmitate-induced inflammatory responses. Biochim Biophys
Acta Mol Cell Biol Lipids. Apr 2018;1863(4):433–46. https://doi.org/10.1016/j.
bbalip.2018.01.009.
39. Twig G, et al. Fission and selective fusion govern mitochondrial segregationand elimination by autophagy. EMBO J. Jan. 2008;27(2):433–46. https://doi.
org/10.1038/sj.emboj.7601963.
40. Wu W, et al. PINK1-Parkin-mediated Mitophagy protects mitochondrial Integrity and prevents metabolic stress-Induced Endothelial Injury. PLoS ONE. Jul.
2015;10(7):e0132499. https://doi.org/10.1371/journal.pone.0132499.
41. Sahu I, Glickman MH. Proteasome in action: substrate degradation by the
26S proteasome. Biochem Soc Trans. Apr. 2021;49(2):629–644. https://doi.
org/10.1042/BST20200382.
42. Orre M, et al. Reactive glia show increased immunoproteasome activity in
Alzheimer’s disease. Brain. May 2013;136:1415–31. https://doi.org/10.1093/
brain/awt083.
43. Moritz KE, et al. The role of the immunoproteasome in interferon-γ-mediatedmicroglial activation. Sci Rep. Aug. 2017;7(1):9365. https://doi.org/10.1038/
s41598-017-09715-y.
44. Shanley KL, Hu C-L, Bizzozero OA. Proteasome Composition in CytokineTreated Neurons and Astrocytes is Determined Mainly by Subunit Displacement. Neurochem Res. Apr. 2020;45(4):860–871.https://doi.org/10.1007/
s11064-020-02958-8.
45. Zhang Y, et al. Expression of immunoproteasome subunits in the brains of
Toxoplasma Gondii-infected mice. Exp Mol Pathol. Dec. 2021;123:104684.
https://doi.org/10.1016/j.yexmp.2021.104684.
46. Feng X-T et al. Jul., Palmitate contributes to insulin resistance through downregulation of the Src-mediated phosphorylation of Akt in C2C12 myotubes.
Biosci Biotechnol Biochem. 2012;76(7): 1356–1361. https://doi.org/10.1271/
bbb.120107.
47. Calvo-Ochoa E, Sánchez-Alegría K, Gómez-Inclán C, Ferrera P, Arias C. Palmitic
acid stimulates energy metabolism and inhibits insulin/PI3K/AKT signaling indifferentiated human neuroblastoma cells: The role of mTOR activation andmitochondrial ROS production. Neurochem Int. Nov. 2017;110:75–83. https://doi.org/10.1016/j.neuint.2017.09.008.

Page 19 of 20

48. Aggarwal R, et al. Chronic exposure to Palmitic Acid Down-regulates AKT in
Beta-Cells through activation of mTOR. Am J Pathol. Jan. 2022;192(1):130–45.
https://doi.org/10.1016/j.ajpath.2021.09.008.
49. Huang F, Chen J, Wang J, Zhu P, Lin W. Palmitic Acid Induces MicroRNA-221
Expression to Decrease Glucose Uptake in HepG2 Cells via the PI3K/AKT/
GLUT4 Pathway. Biomed Res In. Nov. 2019;2019:8171989. https://doi.
org/10.1155/2019/8171989.
50. Xu L, et al. Palmitic acid causes insulin resistance in granulosa cells via
activation of JNK. J Mol Endocrinol. May 2019;62(4):197–206. https://doi.
org/10.1530/JME-18-0214.
51. Pu J, Peng G, Li L, Na H, Liu Y, Liu P. Palmitic acid acutely stimulates glucose
uptake via activation of Akt and ERK1/2 in skeletal muscle cells. J Lipid Res.
Jul. 2011;52(7):1319–1327. https://doi.org/10.1194/jlr.M011254.
52. Kakimoto PA, Serna JDC, de Miranda Ramos V, Zorzano A, Kowaltowski
AJ. Increased glycolysis is an early consequence of palmitate lipotoxicity
mediated by redox signaling. Redox Biol. Sep. 2021;45:p102026. https://doi.
org/10.1016/j.redox.2021.102026.
53. Alnahdi A, John A, Raza H. Augmentation of glucotoxicity, oxidative stress,
apoptosis and mitochondrial dysfunction in hepg2 cells by palmitic acid.
Nutrients. Aug. 2019;11(9). https://doi.org/10.3390/nu11091979.
54. Sohlang MN, Majaw S. Altered VDAC-HK association and apoptosis in mouse
peripheral blood lymphocytes exposed to diabetic condition: an in vitro andin vivo study. Arch Physiol Biochem. Jan. 2021;1–11. https://doi.org/10.1080/1
3813455.2020.1867187.
55. Sun J, et al. aFGF alleviates diabetic endothelial dysfunction by decreasingoxidative stress via Wnt/β-catenin-mediated upregulation of HXK2. Redox
Biol. Feb. 2021;39:101811. https://doi.org/10.1016/j.redox.2020.101811.
56. Li Y-J, et al. Fatty acid oxidation protects cancer cells from apoptosis byincreasing mitochondrial membrane lipids. Cell Rep. May 2022;39:110870.
https://doi.org/10.1016/j.celrep.2022.110870.
57. Molinari F, Frattini M. Functions and regulation of the PTEN gene in colorectal
cancer. Front Oncol. 2013;3:326. https://doi.org/10.3389/fonc.2013.00326.
58. Kurlawalla-Martinez C, Stiles B, Wang Y, Devaskar SU, Kahn BB, Wu H. Insulin
hypersensitivity and resistance to streptozotocin-induced diabetes in micelacking PTEN in adipose tissue. Mol Cell Biol. Mar. 2005;25(6):2498–2510.
https://doi.org/10.1128/MCB.25.6.2498-2510.2005.
59. Wijesekara N, et al. Muscle-specific pten deletion protects against insulin
resistance and diabetes. Mol Cell Biol. Feb. 2005;25(3):1135–45. https://doi.
org/10.1128/MCB.25.3.1135-1145.2005.
60. Stiles BL et al. Apr., Selective deletion of Pten in pancreatic beta cells leads
to increased islet mass and resistance to STZ-induced diabetes. Mol Cell Biol.
2006;26(7):2772–2781. https://doi.org/10.1128/MCB.26.7.2772-2781.2006.
61. Venniyoor A. PTEN: a thrifty gene that causes disease in times of plenty? Front
Nutr. Jun. 2020;7:81. https://doi.org/10.3389/fnut.2020.00081.
62. Wani WY, Boyer-Guittaut M, Dodson M, Chatham J, Darley-Usmar V, Zhang
J. Regulation of autophagy by protein post-translational modification. Lab
Invest. Jan. 2015;95(1):14–25. https://doi.org/10.1038/labinvest.2014.131.
63. Hennig P, Fenini G, Di Filippo M, Karakaya T, Beer H-D. The pathways underlying the multiple roles of p62 in inflammation and cancer. Biomedicines. Jun.
2021;9(7). https://doi.org/10.3390/biomedicines9070707.
64. Tan B-J, Chiu GNC. Role of oxidative stress, endoplasmic reticulum stressand ERK activation in triptolide-induced apoptosis. Int J Oncol. May
2013;42(5):1605–12. https://doi.org/10.3892/ijo.2013.1843.
65. Shin JW, Kim S-H, Yoon JY. PTEN downregulation induces apoptosis
and cell cycle arrest in uterine cervical cancer cells. Exp Ther Med. Oct.
2021;22(4):1100. https://doi.org/10.3892/etm.2021.10534.
66. Rödl S, Herrmann JM. The role of the proteasome in mitochondrial
protein quality control. IUBMB Life. Oct. 2023;75(10):868–879. https://doi.
org/10.1002/iub.2734.
67. Wang X et al. Apr., DNA sensing via the cGAS/STING pathway activates the immunoproteasome and adaptive T-cell immunity. EMBO J.
2023;42(8):e110597. https://doi.org/10.15252/embj.2022110597.
68. Kim M, et al. Immunoproteasome-specific subunit PSMB9 induction
is required to regulate cellular proteostasis upon mitochondrial dysfunction. Nat Commun. Jul. 2023;14(1):4092. https://doi.org/10.1038/
s41467-023-39642-8.
69. Chen X, et al. LMP2 deficiency causes abnormal metabolism, oxidative stress,
neuroinflammation, myelin loss and neurobehavioral dysfunctions. J Transl
Med. Mar. 2023;21(1):226. https://doi.org/10.1186/s12967-023-04071-0.
70. Su H-X, Li P-B, Shi K-N, Gao J, Zhang H-J, Li H-H. The immunoproteasome
subunit β2i ameliorates myocardial ischemia/reperfusion injury by regulating

Albornoz et al. Journal of Neuroinflammation

71.
72.

73.
74.
75.

(2024) 21:191

Parkin-Mfn1/2-mediated mitochondrial fusion. Cell Mol Life Sci. Jul.
2023;80(8):231. https://doi.org/10.1007/s00018-023-04867-9.
Wu M, et al. ONX0912, a selective oral proteasome inhibitor, triggering
mitochondrial apoptosis and mitophagy in liver cancer. Biochem Biophys Res
Commun. Apr. 2021;547:102–10. https://doi.org/10.1016/j.bbrc.2021.02.037.
Sánchez G, et al. Inhibition of chymotrypsin-like activity of the proteasomeby ixazomib prevents mitochondrial dysfunction during myocardial ischemia.
PLoS ONE. May 2020;15(5):e0233591. https://doi.org/10.1371/journal.
pone.0233591.
Wang L, Lu G, Shen H-M. The long and the short of PTEN in the regulationof mitophagy. Front Cell Dev Biol. May 2020;8:299. https://doi.org/10.3389/
fcell.2020.00299.
Xie X, et al. The immunoproteasome catalytic β5i subunit regulates cardiac
hypertrophy by targeting the autophagy protein ATG5 for degradation. Sci
Adv. May 2019;5(5):eaau0495. https://doi.org/10.1126/sciadv.aau0495.
Ji L, et al. Immunosubunit β5i knockout suppresses neovascularization
and restores autophagy in retinal neovascularization by targeting ATG5 forPage 20 of 20

degradation. Invest Ophthalmol Vis Sci. Dec. 2020;61(14):30. https://doi.
org/10.1167/iovs.61.14.30.
76. Smith BC, Sinyuk M, Jenkins JE, Psenicka MW, Williams JL. The impact ofregional astrocyte interferon-γ signaling during chronic autoimmunity:
a novel role for the immunoproteasome. J Neuroinflammation. Jun.
2020;17(1):184. https://doi.org/10.1186/s12974-020-01861-x.
77. Rhea EM, Salameh TS, Logsdon AF, Hanson AJ, Erickson MA, Banks WA.
Blood-Brain Barriers in Obesity, AAPS J. Jul. 2017;19(4):921–930. https://doi.
org/10.1208/s12248-017-0079-3.

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