De Paula et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03168-7(2024) 21:173

Journal of Neuroinflammation

Open Access

RESEARCH

Extracellular vesicles released from microglia
after palmitate exposure impact brain
function
Gabriela C. De Paula1,2, Blanca I. Aldana3, Roberta Battistella1,2, Rosalía Fernández-Calle1, Andreas Bjure1,
Iben Lundgaard1,2, Tomas Deierborg1,2 and João M. N. Duarte1,2*Abstract
Dietary patterns that include an excess of foods rich in saturated fat are associated with brain dysfunction.
Although microgliosis has been proposed to play a key role in the development of brain dysfunction in dietinduced obesity (DIO), neuroinflammation with cytokine over-expression is not always observed. Thus, mechanisms
by which microglia contribute to brain impairment in DIO are uncertain. Using the BV2 cell model, we investigated
the gliosis profile of microglia exposed to palmitate (200 µmol/L), a saturated fatty acid abundant in high-fat dietand in the brain of obese individuals. We observed that microglia respond to a 24-hour palmitate exposure withincreased proliferation, and with a metabolic network rearrangement that favors energy production from glycolysis
rather than oxidative metabolism, despite stimulated mitochondria biogenesis. In addition, while palmitate did
not induce increased cytokine expression, it modified the protein cargo of released extracellular vesicles (EVs).
When administered intra-cerebroventricularly to mice, EVs secreted from palmitate-exposed microglia in vitro led
to memory impairment, depression-like behavior, and glucose intolerance, when compared to mice receiving EVs
from vehicle-treated microglia. We conclude that microglia exposed to palmitate can mediate brain dysfunction
through the cargo of shed EVs.
Keywords Obesity, Neuroinflammation, Energy metabolism, Glycolysis, LPS*Correspondence:João M. N. Duarte
joao.duarte@med.lu.se
1
Department of Experimental Medical Science (EMV), Faculty of Medicine,
Lund University, Sölvegatan 19, BMC C11, Lund 221 84, Sweden
2
Wallenberg Centre for Molecular Medicine, Lund University, Lund,
Sweden3
Department of Drug Design and Pharmacology, Faculty of Health andMedical Sciences, University of Copenhagen, Copenhagen, Denmark

Introduction
Excessive consumption of diets rich in saturated fat leads
to obesity that, together with its metabolic complications
and comorbidities, can impact the brain [1–3]. In animalmodels, metabolic syndrome upon diet-induced obesity(DIO) has been reported to trigger hippocampal metabolic alterations, synaptic dysfunction, and impairments
in learning and memory processes [4]. Interestingly, justas in animal studies, a relatively short exposure to a highfat and high-sugar diet (four days) is sufficient to deteriorate hippocampal-dependent learning and memory inhealthy humans [5].
Obesity is associated with a state of low-grade inflammation and, in the brain, DIO triggers neuroinflammation© 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
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Paula De et al. Journal of Neuroinflammation(2024) 21:173

and microgliosis [6–11]. However, a controversy on neuroinflammatory processes induced by DIO can be foundin the literature, such as a limited extension of gliosis andlack of cytokine overexpression in certain brain areas,such as hippocampus and cortex [12–16]. Moreover, cellular communication mechanisms other than cytokine
release are likely to play a role in neuroinflammationin DIO, including the ability of microglia to shed eicosanoids and pro-resolving mediators [17], and extracellular vesicles (EVs) [18].
EVs are cell-derived membrane-surrounded vesicles(exosomes, ectosomes, microvesicles, apoptotic bodies,among others) that carry bioactive molecules (metabolites, nucleic acids and proteins) and, depending on theirsize, even cellular organelles. Thus, EVs participate in an
orchestrated strategy of intercellular communication,
including mediating inflammatory cues from microglia toother brain cells [19, 20] EVs are also transmitted acrossmultiple bodily organs [21], although transfer across the
blood-brain-barrier (BBB) might be restricted to particles of smaller size [22].
Palmitate, an abundant saturated fatty acid in diet, wasfound to increase in the cerebrospinal fluid of overweight
and obese individuals relative to lean controls, and cerebrospinal palmitate concentration is correlated with bodymass index and abdominal circumference, but not withobesity associated comorbidities, such as diabetes, dyslipidemia, or hypertension [23]. Melo et al. [23] further
reported that intra-cerebroventricular (i.c.v.) injectionof palmitate impairs synaptic plasticity and memory in
mice, and increased astrocyte and microglia reactivity.
Using the BV2 microglia cell line, we investigated thegliosis profile and energy metabolism alterations inducedby palmitate exposure, and tested the hypothesis that palmitate-exposed microglia utilize EVs as means of transmitting inflammatory messages to other cells.

Materials and methods
BV2 cells and treatment

Murine BV2 microglial cells (#CRL-2469, ATCC, Manassas, VA-USA; RRID: CVCL_0182) were cultured in T75
polystyrene flasks (#83.3911, Sarstedt, Nümbrecht, Germany) at 37 °C with an atmosphere of 5% CO2, using
Dulbecco’s modified Eagle’s medium (DMEM), containing 5 mmol/L glucose, 1 mmol/L pyruvic acid, and 4
mmol/L glutamine (#11,885, Gibco, ThermoScientific,
Göteborg, Sweden) supplemented with 10% fetal bovine
serum (FBS, Gibco #10,500,064), 100 U/mL penicillin/
streptomycin (Gibco #15,140,122). Cells were split every
2 days. For that, media was removed, cells were washedin Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS, Gibco #14,190,250), and 1
mL of 0.05%(w/v) trypsin-EDTA with phenol red (Gibco#25,300,054) was added to detach the cells. After 5 min,Page 2 of 16

9 mL of fresh culture medium was added for re-seeding.
For cell counting, suspended cells (10 µL) were mixed 1:1
with 0.4%(w/v) Trypan Blue (Sigma-Aldrich #T8154), themixture was loaded in a hemocytometer, and live cells(unstained) were counted under a microscope.
For experiments, cells at passage 14–24 were seededin polystyrene plates (unless otherwise stated) to a ~ 40%
confluence as described below for each method. After
6 h, medium was replaced by DMEM without FBS for
12–16 h before treatment with either with vehicle, or
palmitate (PA, 200 µmol/L) in 0.25%(w/v) bovine serum
albumin (BSA) for 24 h (palmitate solution preparation as in supplementary methods). This palmitate concentration was selected from results of a dose-response
pilot experiment, in which palmitate at 500 µmol/L for
24 h induced a cell detachment from the culture support
above 50%. As a positive control of microglia reactivity,
cells were incubated for 3 h with 1 µg/mL lipopolysaccharides (LPS) from Escherichia coli O111:B4 (SigmaAldrich #L2630; Lot #000110081) in 0.25%(w/v) BSA.
Cell proliferation, viability and apoptosis

Cellular proliferation rates were determined at given
time points by counting cells as described above. The
CyQuant MTT Cell Viability kit (#V13154, Invitrogen,
ThermoScientific) and the Caspase-Glo 3/7 assay kit(#G8090, Promega, Nacka, Sweden) were used in 96-wellplates (Sarstedt #82.1581.001) to determine cell viability
and apoptosis, respectively, following the manufacturer’s
instructions. Experiments were performed in quadruplicates with seeding at a density of 104 cells/well.
Oxygen consumption and proton efflux rates

Cellular oxygen consumption rate (OCR) and proton
efflux measured as extracellular acidification rate (ECAR)
were analyzed in the Seahorse XF96 (Agilent, Santa
Clara, CA-USA) following the manufacturer’s instructions (detailed in supplementary methods). Briefly, 104
cells/well were seeded on the Seahorse cell culture plates
using a total volume of 200 µL of culture medium. After
24 h, cells were incubated with the treatment solutions(treatments are described in the figure legends). OCR
and ECAR values were normalized to total protein content, as determined with the bicinchoninic acid assay (kitfrom Pierce, #23,227, ThermoScientific).
Quantitative real-time polymerase chain reaction (qPCR)

Cells were seeded onto 24-well plates (Sarstedt#83.3922.005) at a density of 5 × 104 cells/well. After
treatments, cells were washed with ice-cold phosphatebuffered saline (PBS, Gibco #18,912,014), treated with
100 µL trypsin as above, and re-suspended in 400 µL of
culture medium. Total RNA was extracted with TRIzol(Invitrogen #15,596,026) following the manufacturer’sPaula De et al. Journal of Neuroinflammation(2024) 21:173

instructions, and RNA samples (500 ng) were reverse
transcribed using the qScript cDNA Synthesis Kit(#95,047, QuantaBio, England). The resulting cDNA wasused for qPCR with the reaction cocktail PerfeCTa SYBRGreen FastMix (QuantaBio #95,072), and the primer
pairs in Table 1. Reactions were run in duplicates, andrelative gene expression levels were calculated with the
ΔΔCT method using L14 as internal control gene.
TNF-α ELISAMedia were centrifuged at 2,000 x g and 4 °C during 10 min to remove debris, and the supernatant was
saved at -20ºC until use. Concentration of TNF-α wasdetermined with a Mouse TNF-α ELISA kit (Abcam,#ab208348).
Immunoblotting

Cells were seeded onto 6-well plates (Sarstedt#83.3920.005) at a density of 3 × 105 cells/well, and treatedas described above. Cells were washed with PBS, andproteins were extracted with a lysis buffer [in mmol/L:
150 NaCl, 1 ethylenediaminetetraacetic acid (EDTA), 50
tris(hydroxymethyl)aminomethane (Tris)-HCl, 1% (v/v)
Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.5%(w/v) sodium dodecylsulfate (SDS), pH 8.0] containing
Table 1 Nucleotide sequence of primers used for real-time PCR.
These primers were previously validated by Greene et al. [69] andSkoug et al. [24]
Gene
Il1b

Accession
number
NM_008361.4

IL6

NM_031168.2

Slc2a1

NM_011400.4

Rpl14

NM_025974.3

Tnfa

NM_013693.3

Ppargc1aNM_008904.3

Opa1

NM_001199177.2

Mff

NM_029409.3

Mfn1

NM_024200.5

Mfn2

NM_133201.3

Dnm1l

NM_001025947.3

Fis1

NM_001163243.1

Forward Primer(5’→3’)​T​G​G​A​C​C ​T ​T​C​C​A​G​
G​A​T​G​A​G​G​A​C​A​T​A​C​C​A​C ​T ​T​C​A​C​A​A​
G​T​C​G​G​A​G​G​C​C ​T ​T​C​A​T ​T​G​T​G​G​G​C​
A​T​G​T​G​C ​T ​T​C
​G​G​C ​T ​T ​T​A​G​T​G​G​A​T​
G​G​A​C​C​C ​T​G​G​T​G​C​C ​T​A​T​G​T​C ​T​C​A​G​C​C ​T​C ​T ​T

Reverse Primer(5’→3’)​G​T ​T​C​A​T​C ​T​C​G​G​A​G​C​C ​T​G​T​A​G​T​G​C ​T​G​C​A​A​G​T​G​C​A​
T​C​A​T​C​G​T ​T​G​T ​T​C
​A​G​G​T ​T​C​G​G​C​C ​T​
T​T​G​G​T​C ​T​C​A​G​A​T ​T​G​A​T​A​T​C​C​G​C​C​T ​T​C ​T​C​C​C​G​C​C​A​T​A​G​A​A​C​
T​G​A​T​G​A​G​A​G​G​
G​A​G​T​G​A​T​G​T​G​A​A​T​G​​G​C ​T​C​A​T ​T​G​T ​T​G​A​C ​T ​T​G​G​A​T​A​C​A​
T​A​C ​T​G​G​T ​T​G​G​A​
G​A​C​A
T​A​T​G​T​C ​T​G​A​G​G​C​C​C ​T ​T​C​ ​T​C ​T​G​A​C​A​C​C ​T ​T​C​
T​C ​T ​T​G​T ​T
C​T​G​T​A​A​T​G​C ​T​T​C​G​G​G​T​C ​T​G​T​C​C ​T​ ​C​A​A​C​A​C​A​G​G​T​C​C​C​C​C​A​T​A
T​G​C​G​G​T ​T ​T ​T​C​A​T ​T​G​C​C​A​C​A​A​G​C ​T​ ​T​C ​T​A​G​G​G​A​C​C ​T​
G​T​G​T ​T​C​G​G
G​A​A​A​G​A​T​G​G​G​C​A​G​A​G​G​C​A​G​T ​T ​T​G​ ​A​T​G​A​T​G​A​G​A​C​G​
A​G​G​A​G​T​G​C
A​A​C​G​G​C​C ​T​C
T​ ​C​A​C​C​C​G​G​A​G​A​C​ ​T​G​C ​T ​T​C​A​A​C ​T​C​C​C​T​C ​T​C​A​T ​T
A​T​T​T​T​C​T​T​C​T​C​C​ ​C​G​A​A​G​C ​T​G​C​A​A​ ​A​A​C​C​A​G​G​C​A​C​C​A
G​G​A​A​T ​T ​T ​T​G​A
A​G​G​C​A​T​A​T ​T

Page 3 of 16

cOmplete protease inhibitors (Roche, Switzerland). After
protein determination, we separated 30 µg of protein by
SDS-PAGE and transferred onto nitrocellulose membranes as described elsewhere [24]. Membranes were
blocked for 1 h at room temperature with 5%(w/v) BSA
in Tris-buffered saline (in mmol/l: 20 Tris, 150 NaCl, andpH 7.6) containing 1% Tween-20 (TBS-T), incubatedovernight at 4 °C with the OxPhos Complexes antibody
cocktail (Invitrogen #45-8099), diluted 1:1250 in blocking solution. Membranes were then washed three times
in TBS-T for 15 min and incubated for 2 h at room temperature with horseradish peroxidase-conjugated antimouse IgG secondary antibody (dilution 1:5000, Abcam#ab6789). After washing again, immunoblots were developed on the ChemiDoc (Bio-Rad, Sundbyberg, Sweden)
using the SuperSignal West Pico PLUS Chemiluminescent substrate (ThermoScientific #34,580). For each independent experiment, the 3 experimental groups were run
in parallel in the same gel, and signal from each band was
normalized to the average of that in the 3 groups.
Mitotracker staining and immunofluorescence microscopy

Cells (5 × 104) were seeded onto a 35-mm glass-bottom
dish coated with poly-D-lysine (#P35GC-1.5-14-C, MatTek, Ashland, MA-USA), and treated as described above.
Then, cells were incubated for 30 min at 37ºC with 100
nmol/L of MitoTracker (Invitrogen #M7512; 1 mmol/L
stock solution in DMSO). Stained cells were rinsed in
PBS, and fixed with buffered 4% formaldehyde (Histolab,
Askim, Sweden) for 15 min at room temperature. Fixedcells were rinsed 3 times, permeabilized during 10 min
with 0.2%(v/v) Triton X-100 in PBS, blocked for 30 min
in 5%(v/v) goat serum and 0.3%(v/v) Triton X-100 in PBS,
and incubated overnight at 4°C with blocking solution
containing the primary antibodies rabbit anti-allograft
inflammatory factor 1 (Iba1, dilution 1:200; #019-19741,
Fujifilm Wako, Japan) and mouse anti-β-actin (dilution
1:400, Sigma-Aldrich #A3854). Cells were then incubatedfor 1 h with the secondary antibodies AF488-conjugatedanti-rabbit IgG (dilution 1:500, #AB150077, Abcam,
Cambridge, UK) and AF647-conjugated anti-mouse IgG(dilution 1:500, Invitrogen #A21235), washed in PBS,
mounted with 4,6-diamidino-2-phenylindole (DAPI)containing Fluoroshield (Sigma-Aldrich #F6057), andimaged in a Nikon A1RHD confocal microscope with a
CFI Apochromat TIRF 100 × Oil, NA 1.49 (Nikon Instruments, Tokyo, Japan). Images were acquired with NISelements (Laboratory Imaging, Nikon), and analyzed in
ImageJ (NIH, Bethesda, MD-USA).
13

C tracing experiments

Cells (2.2 × 106) were seeded onto 10-cm dishes (#89089612, ThermoScientific) and treated as above, using
glucose-free DMEM (#11,966,025, ThermoScientific)

Paula De et al. Journal of Neuroinflammation(2024) 21:173

supplemented with 5 mmol/L [1-13 C]glucose (99% C13
atom, Cortecnet, Voisins le Bretonmeux, France). [1-13 C]
glucose was present during the 24 h of treatment withpalmitate or vehicle, or for 21 h ahead and during the 3 hof LPS treatments. Cells were washed with ice-cold DPBS
and frozen in N2 (l), and metabolites were extracted with
1 mL 80%(v/v) methanol. Then, samples were sonicatedfor 30 min at 4 °C, and centrifuged at 13,000 x g, 4℃, for
30 min. Supernatants were dried using a Savant SpeedVac(ThermoScientific) operating at room temperature andwere saved for nuclear magnetic resonance spectroscopy(supplementary methods). Spectra were analyzed by line
fitting using NUTS (Acorn NMR, Fremont, USA), as inprevious studies [25]. Multiplet fractions from aliphatic
carbons of glutamate were group-averaged and usedin tcaCALC running on MATLAB 2019a (MathWorks,
Natick, MA-USA) to determine rates (relative to citratesynthase) of pyruvate dehydrogenase (PDH), anaplerosis
through pyruvate carboxylase (YPC), anaplerosis throughother pathways such as propionate or glutamine (YS), andpyruvate kinase (PK) [26].
EVs isolation

Cells were seeded at a density of 2 × 107 in 75 cm2flasks with 20 mL of medium. After treatment, EVs wereobtained from culture medium as previously described[18]. Briefly, cell media were centrifuged at 400 x g for
5 min at room temperature to remove any cells. The
obtained supernatant was centrifuged at 2,000 x g at 4 °C
for 10 min to remove cell debris, and then at 10,000 x g
at 4 °C for 30 min to remove large particles such as apoptotic cell bodies. The supernatant was again ultracentrifuged at 100,000 x g at 4 °C for 70 min to pellet the EVs.
The resulting pellets were gently re-suspended in DPBS,
and the ultracentrifugation step was repeated. Pelleted
EVs were re-suspended in 20 µL of DPBS, and either kept
at 4 °C overnight (for NTA and injection into the mousebrain) or stored at -20 °C (for proteomics).
Nanoparticle tracking analysis (NTA)

EVs were analyzed using the NanoSight LM10 (Malvern
Panalytical, Malvern, UK). Samples were diluted in PBS
to 106-109 particles/mL and injected at 50 µL/min and atroom temperature (21–22 °C). Particles were tracked 5times during 60 s and analyzed with NanoSight NTA 3.4(Malvern Panalytical) to determine particle diameter.
Proteome analysis

Mass spectrometry (MS) was conducted as reported insupplementary methods. We adopted an all versus all
contrast approach for the analysis of the protein signal
intensities. Only proteins that were present in at least 2/3
independent experiments of each group were used forfurther analysis. A quantile-based regression was used

Page 4 of 16

to replace missing values with random draws. R version
3.6.2 (RRID: SCR_001905) was used for principal component analysis (PCA), and for differential enrichment
testing using the package DEP: Differential Enrichment
analysis of Proteomics data (https://rdrr.io/bioc/DEP).
Significance thresholds were set to adjusted α = 0.05 andlog2 of the fold change = 1. Significant findings were used
for gene ontology analysis in using ShinyGO 0.77 (http://bioinformatics.sdstate.edu/go) and the Reactome pathway database.
Animals

Sixteen 8-week-old male Swiss mice were obtained from
Janvier Labs (Le Genest-Saint-Isle, France) and housedin groups of 4 under controlled conditions of humidity(55–60%) and temperature (21–23 ◦C) with a 12-hour
light: dark cycle (lights on at 7:00). Chow and water were
provided ab libitum. Mice were randomly selected (bycoin tossing) to receive EVs either palmitate- or vehicletreated BV2 cells in the lateral ventricle (supplementary
methods), so that each cage housed 2 mice from either
experimental group (total n = 8/group). Power calculations were not performed, but this sample size is sufficient to reveal DIO-induced memory impairment [11,27]. Body weight was evaluated at the start and end of thestudy.
Glucose tolerance test (GTT)

Food was removed for 5 h and, thereafter, blood glucose
was measured from tail tip blood with the Accu-Chek
Aviva glucometer (Roche, Manheim, Germany). Then,
mice were given 2 g/kg glucose i.p. from a 30% (w/v)
solution in saline, and blood glucose was measured fromthe tail tip at 15, 30, 60, 90 and 120 min after injection.
Behavior

Mice were tested between 8:00 and 17:00, in a cubic
open-field arena with side length of 50 cm, with room
light adjusted to an illuminance of 15 lx. All experiments
were recorded by an infrared camera into AnyMaze 6.0.1(Stoelting, Dublin, Ireland). Mice were habituated to theexperimental setup by exploring the empty arena during
5 min in 3 consecutive days. In the last habituation session, arena exploration was analyzed for total numberof crossings between quadrants, time spent in center orperimeter (delineated at 8 cm from the walls), numberof rearing events, and the clockwise and anti-clockwise
rotations.
In the following day, memory performance wasassessed with novel object recognition (NOR) and novel
location recognition (NLR) tasks, as detailed previously[27] Briefly, mice were allowed to explore the arena for
5 min with two identical objects (familiarization phase).
The mice returned to their home cage for 1 h (retention

Paula De et al. Journal of Neuroinflammation(2024) 21:173

Page 5 of 16

phase), and after were reintroduced for 5 min with one ofthe objects either replaced by a novel object or relocatedin space (retention phase). The time spent exploring eachobject was analyzed in both familiarization and recognition phases.
The sucrose-plash test was used to evaluate depression-related behavior. Mice were sprayed with ~ 1 mL of
a 10% sucrose solution on their dorsal coat and analyzed
for latency between spraying and first grooming event,and total duration of grooming for 5 min [11].

distribution are represented in boxplots extending fromthe 25th to 75th percentiles, line at median, and whiskers to the minimum and maximum values. Two-groupcomparisons were made with 2-tailed Student’s t-tests orMann Whitney tests. Multiple groups were analyzed witheither a Kruskal-Wallis test followed by Dunn’s multiple
comparisons or with ANOVA followed by post hoc comparisons using the Holm-Šídák method. Significance was
accepted for P < 0.05. All ANOVA results are available in
Supplementary Table S1.

Immunofluorescence microscopy in brain slices

Results

Animals under isoflurane anesthesia were transcardially perfused with ice-cold PBS, followed by 4% formaldehyde. Fixed brains were removed, embedded in
4% formaldehyde for 24 h, and cryoprotected in a 30%
sucrose solution in PBS at 4 °C. Free-floating coronalSect. (30 μm) were blocked for 2 h at room temperature with 5%(v/v) goat serum solution in PBS containing
0.3%(v/v) Triton X-100, and then incubated overnight at
4 °C with primary antibodies anti-Iba1 (1:200), and with
anti-glial fibrillary acidic protein (GFAP) pre-tagged with
AF488 (1:500; ThermoScientific #53-9892-82), or with
anti-CD68 (1:500, Biorad #MCA1957). After washingwith PBS, sections were incubated with the AF568-conjugated goat anti-rabbit IgG antibody (1:500, ThermoScientific #A21069) or AF647-conjugated donkey anti-rat
IgG antibody (1:500, ThermoScientific #A78947) for 2 h
at room temperature, washed again, and incubated with
4,6-diamidino-2-phenylindole (DAPI; 1 µg/mL in PBS;
ThermoScientific #62,247) for 10 min. Slices werethen mounted onto slides with ProLong Glass antifade
medium (Invitrogen #P36980) and imaged in a A1RHD
confocal microscope interfaced with NIS-elements(Nikon). Z-stack-projected images were processed andanalyzed in ImageJ for area of Iba1, CD68 and GFAP
immunoreactivity as previously described [16] Morphology of Iba1+ cells (3 cells/region/mouse) was analyzed
with the AnalyzeSkeleton plugin to extract the numberof cell processes and their length, and the number ofbranching points [28] One brain per group were excluded
due to damage during processing.

Palmitate induces gliosis without cytokine overexpression

Statistical analysis

Researchers were blind to experimental group during
analysis of behaviour videos and microscopy images. Alldata were analyzed using Prism 10.2.0 (GraphPad, San
Diego, CA-US). Unless otherwise stated, data are presented as mean ± SD of n independent experiments. Normality was assessed with the Kolmogorov-Smirnov test,or the Shapiro-Wilk test for small sample sizes. Data notshowing a Gaussian distribution were either analyzed
with non-parametric tests, or log-transformed before
ANOVA. Behaviour results deviating from a normal

BV2 cells responded to 200 µmol/L palmitate exposure
during 24 h with increased proliferation, but at a slower
rate than with LPS treatment (Fig. 1A), which was confirmed by an increased cell viability in the MTT assay(+ 66% for palmitate; +60% for LPS; Fig. 1B). There wasalso increased caspase activity, indicative of cell death or
microglial activation [29], in palmitate- and LPS-treatedcells relative to vehicle (+ 19% for palmitate; +26% for
LPS; Fig. 1C).
Transcriptional levels of the cytokines TNF-α, IL-1β
and IL-6, and levels of TNF-α released into the medium
were unaffected by palmitate treatment, while significantly increasing upon LPS exposure, relative to vehicletreated cells (Fig. 1D-E). We tested whether expression ofcytokines was transiently modified at shorter palmitate
exposure periods. Palmitate failed to increase cytokine
expression at any of the time points assessed up to 9 hof exposure (Fig. 1F). Then, we set to determine whether
palmitate expanded the area of the cells and their mitochondria, since palmitate can stimulate mitochondria
metabolism and oxygen utilization in other cell models[30]. When compared to vehicle, palmitate-treated cells
were of similar size, while LPS-treated cells increased thecell area as assessed by the immunoreactivity to either
β-actin or the microglia marker Iba1 (Fig. 1G-H). The
area of mitotracker staining was not substantially affectedby either palmitate (P = 0.054) or LPS (P = 0.061) relative
to vehicle (Fig. 1G-H). However, palmitate but not LPS
exposure resulted in increased fraction of mitotracker
fluorescence area relative to total cell area depicted byeither β-acting or Iba1 (respectively, + 78% and + 73%;
Fig. 1I).
Altogether, these findings suggest that BV2 microglia
exposed to palmitate respond with increased proliferation and expanded mitochondrial density, but not with atypical neuroinflammatory response that involves cytokine overexpression.

Paula De et al. Journal of Neuroinflammation(2024) 21:173

Page 6 of 16

Fig. 1 Palmitate exposure induces gliosis without exacerbated cytokine production and increases mitochondria content. BV2 cells were incubated withvehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. (A) Cell counts before, and after 6, 12 and 24 h of treatment, or after 1.5 and 3 hfor LPS. (B) Relative cell viability measured by MTT reduction. (C) Relative activity of caspase 3/7. (D) Relative expression of TNF-α, IL-6 and IL-1β, and (E)concentration of TNF-α in the medium after treatment (3 h LPS, 14 h vehicle or palmitate). (F) Relative expression of cytokines during the initial 9 h ofpalmitate exposure. (G) Representative immunofluorescence micrographs for mitotracker, β-actin and Iba1 (scale bar is 10 μm). (H) Mean area occupied
by mitotracker, β-actin and Iba1 signal per cell, and (I) area ratios of mitotracker to β-actin and to Iba1. Cells were analyzed within 3–4 fields of view from
3 independent experiments. Data is shown as mean ± SD of 3–8 independent experiments, represented by the individual symbols. *P < 0.05, **P < 0.01,***P < 0.001 depict differences in comparisons following significant effects in ANOVADistinct metabolism alterations after palmitate and LPS
exposure

Quiescent microglia mainly rely on oxidative phosphorylation for ATP production [31, 32]. Upon activation, microglia stimulate glucose uptake and glycolysis
to meet enhanced energy demands [33, 34]. Thus, we
set to measure palmitate-induced alterations of mitochondrial respiration. When compared to vehicle, cells
exposed to either palmitate or LPS showed significantly
lower baseline respiration, ATP-associated respiration,
maximal respiration, and spare capacity (Fig. 2A-C). Proton leakage and non-mitochondrial oxygen consumption
were similar across all groups (Fig. 2C). Since palmitate lowered mitochondrial respiration capacity, despite
increased mitochondrial density, we then measured thedensity of mitochondrial respiration complexes and ATP
synthase. Relative to vehicle-treated cells, palmitate but
not LPS significantly increased the density of complexes
I, II and IV by 57%, 63% and 73%, respectively (Fig. 2D-E).

We further determined the expression of genes involved
in mitochondrial dynamics and observed that both palmitate and LPS induced a significant increase in theexpression of PGC-1α (Fig. 2F), which is a key regulatorof mitochondria biogenesis. In turn, the expression ofgenes involved mitochondrial quality control by fusion(Opa1, Mfn1/2) and fission (Mff, Drp1, Fis1) was similar
between the three groups (Fig. 2F).
Then, we analyzed ECAR as a surrogate of glycolysis (Fig. 2G-I). When compared to vehicle, both palmitate and LPS treatments increased basal glycolysis andthe total glycolytic capacity, but only LPS increased theglycolytic reserve significantly (Fig. 2I). Non-glycolytic
medium acidification was similar across treatments.
We further measured the expression of Slc2a1, the geneencoding the glucose carrier GLUT1, which tended to
be increased by palmitate (adjusted P = 0.065) and LPS(adjusted P = 0.097) treatments compared to vehicle(ANOVA F(2,21) = 3.54, P = 0.048; Fig. 2J).

Paula De et al. Journal of Neuroinflammation(2024) 21:173

Page 7 of 16

Fig. 2 (See legend on next page.)

Finally, we performed 13C metabolic tracing to infer on
the rearrangement of fluxes through anaplerotic pathways. After treatment, cells were allowed to metabolize[1-13 C]glucose for 24 h, and metabolite extracts wereanalyzed by 13C NMR spectroscopy to determine labelingof glutamate carbons (Fig. 2K). Glutamate (Glu) multiplet fractions, were mainly affected by LPS when compared to vehicle, namely Glu C3 (Fig. 2L). However, there
were negligible modifications in the labeling of individual
multiplets for palmitate versus vehicle. A metabolic flux

Paula De et al. Journal of Neuroinflammation(2024) 21:173

Page 8 of 16(See figure on previous page.)
Fig. 2 Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or
1 µg/mL LPS for 3 h. (A) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration(leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). (B-C) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state (B), and calculated respiration parameters (C).
(D) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. (E) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal
within each band was normalized to the average of that in the 3 experimental groups. (F) Expression of genes involved in mitochondria biogenesis, fusionand fission. (G) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium
acidicitation (NGA). (H-I) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic
parameters. (J) Relative expression of Slc2a1 gene (GLUT1). (K) Representation of 13C incorporation into glutamate omitting, for simplicity, generationof isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in 13C NMR spectra measured in extracts after
metabolizing [1-13 C]glucose for 24 h. (L) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). (M)Model used in the TCAcalc analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS,
citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (YPC) or succinyl-CoA (YS). Data is shown asmean ± SD of 3–12 independent experiments, represented by the individual symbols. *P < 0.05, **P < 0.01, ***P < 0.001 depict differences in comparisonsfollowing significant effects in ANOVA

analysis using TCAcalc estimated that, relative to thecitrate synthase flux, pyruvate carboxylation was higher
in palmitate-treated than vehicle and blunted in LPS
treated cells (Fig. 2M). LPS-treated cells had, instead,faster rate of anaplerosis feeding succinyl-CoA than theremaining groups. Lactate labeling was a fitted parameter in the model, and the result correlated well with thatmeasured experimentally (Pearson r = 0.9998, P = 0.014).
In sum, the increased proliferation induced by palmitate is accompanied by a rearrangement of energy
metabolism fluxes, namely increased glycolysis, reduced
mitochondrial respiration, and increased pyruvate carboxylation that can support de novo oxaloacetate synthesis for replenishment Krebs cycle intermediates used inbiosynthetic pathways.
Microglia communication via EVs

Palmitate-treated cells did not exacerbate cytokine production, we thus tested whether palmitate could modulate EVs as means of communication to other cells. Cells
treated with either palmitate or LPS produced EVs of size
like vehicle-treated cells, as determined by NTA (Fig. 3AB). The estimated amount of EVs released into the 20
mL of media ranged between 3.3 × 1010 – 8.9 × 1011 forvehicle, 5.2 × 109 – 6.3 × 1011 for palmitate, and 1.0 × 1011
– 1.1 × 1012 for LPS. This suggests that despite higher proliferation, there is no substantial increase in EV releaseduring palmitate exposure.
We then investigated the proteome of microgliaderived EVs in response to each treatment. The abundance of EV markers was larger than that of typical
proteins from intracellular compartments (Supplementary Figure S1), in line with the high enrichment of EVs
in the preparation. A false discovery rate (FDR) of < 0.01
was set as a threshold protein identification, resulting ina total of 3739 proteins present across a total of 9 experiments (3 per treatment group). From these, only 1636

proteins were present in all analyzed samples. For subsequent analysis, only proteins that were present in at least
2 out of the 3 experiments of each group were kept.
Data was not found missing at random. In fact, EVsfrom palmitate-exposed cells seemed to lack of proteinsthat are present in the other groups. Moreover, missing
proteins had lower average abundance in samples that
did have a measurable signal, suggesting that these aremost likely proteins that have very low abundance or arecompletely absent (true zeroes).
In a PCA using the 1000 most abundant proteins, PC1
allowed to separate palmitate from both LPS and vehicle
treatments (Fig. 3C), suggesting that 24 h of exposure
to palmitate triggers a unique proteomic signature of
released EVs. A differential enrichment analysis revealed
significant differences (adjusted P < 0.05) in the abundance of 102 proteins in-between either of the 3 comparisons performed (Fig. 3D; supplementary figure S2;data can be explored in Supplementary Table S2). Notably, palmitate-induced alterations on the EV proteome
included a reduction in the abundance of several ribosomal proteins and proteins involved in protein metabolism. Indeed, a gene ontology analysis to the EV proteinsthat were differentially enriched between palmitate andvehicle treatments revealed pathways related to RNA
processing, ribosome assembly, and translation processes(Fig. 3E).
EVs from palmitate-treated cells impact brain function

EVs were obtained from BV2 cells exposed to either
palmitate or vehicle, and injected into the lateral ventricle mice, which allows spreading the infusate throughthe whole brain (confirmed in pilot experiments withtrypan blue). A week after injection, we assessed
exploratory behavior, memory performance, and depressive-like behavior (Fig. 4A). The i.c.v. injection of EVs
had no impact on body weight (Fig. 4B), suggesting good

Paula De et al. Journal of Neuroinflammation(2024) 21:173

Page 9 of 16

Fig. 3 Palmitate alters the proteome of BV2-retreased EVs, including a reduction of proteins involved in RNA processing and protein synthesis. (A) Histograms of EV size distribution evaluated by Nanoparticle tracking analysis (NTA) of 6 independent EV isolations, and (B) estimated mean particle size forvehicle (Veh), palmitate (PA) and LPS. Data shown as mean ± SD of n = 6. (C) Score plots of a PCA of the 1000 most abundant proteins in EVs (n = 3/group).
Each symbol shape represents an independent experiment. (D) Heatmap of significant EV proteome differences between either of the 3 experimentalgroups (fold-change comparisons in supplementary figure S1), and (E) significant findings at FDR < 0.01 from the gene ontology analysis of differentiallyexpressed proteins between EVs from palmitate- and vehicle-treated BV2 cells. (1)Full pathway name: “Activation of the mRNA upon binding of the capbinding complex and eIFs and subsequent binding to 43S”

recovery from surgery. In NOR and NOL, neither treatment group showed signs of object bias in the familiarization session (Fig. 4C-D). In the recognition session,
mice injected with EVs from vehicle-treated BV2 cells

showed preserved memory performance as assessed in
the NOR (+ 15%, P = 0.029 versus random exploration)
and NLR (+ 18%, P = 0.010 versus random exploration).
In turn, mice receiving EVs from BV2 cells exposed toPaula De et al. Journal of Neuroinflammation(2024) 21:173

Page 10 of 16

Fig. 4 Intracerebroventricular (i.c.v) injection of microglia-derived EVs following palmitate exposure affects cognition and depressive-like behavior, andalters glucose metabolism in mice. (A) Mice were injected in the lateral ventricle with EVs (500 ng of protein) collected from BV2 cells after exposure toeither palmitate (PA, 200 µmol/L) or vehicle (Veh). (B) Body weight of mice before and 8 days after surgery for EV administration. Impaired memory wasobserved novel object recognition (NOR) test (C), and a similar trend was observed in the novel location recognition (NLR) test (D). (E) Depression-related
behavior was assessed by grooming behavior in the sucrose splash test. (F) locomotor activity and exploratory behavior assessed in the last habituation
day to the open-field arena. (G) Glucose clearance in the GTT in the 8th day following EV injection. The inset is the area under the curve (AUC) of the
glucose excursion for each mouse. Symbols representing each mouse (n = 8) are overlaid on bar graphs showing mean ± SD or box plots showing interquartile ranges. *P < 0.05 from either Student’s t-test or with Mann Whitney test

palmitate did not show increased exploration of either
the novel object (-2%, P = 0.710 versus random exploration; Fig. 4C) or novel location (+ 7%, P = 0.117 versus
random exploration; Fig. 4C). When comparing memory
performance between the treatment groups, significantly
lower memory performance between palmitate and vehicle was observed in the NOR task (Fig. 4C).
The sucrose splash test is used to infer on self-carebehavior. Mice injected i.c.v. with EVs from palmitateexposed microglia showed significantly larger latency
to start grooming, when compared to vehicle (P = 0.037;
Fig. 4E). Such reduced self-care behavior is characteristicof depression, although there was no significant changeon the total duration of grooming between treatments.
Several parameters of locomotion and exploratory
behavior were measured in the empty open field arena,
showing no significant differences between the treatments (Fig. 4F). Notably, the ratio of counterclockwiseto-clockwise rotations was also similar between groups

and did not show signs of lateralized motor impairment(Fig. 4F).
EVs impact glucose homeostasis

EVs injected i.c.v. may impact the hypothalamus, or evenreach the blood stream and act on peripheral organs,[22] thus affecting metabolism and glucose homeostasis.
Therefore, we performed a GTT after behavior assessments. While baseline glucose was similar between
treatment groups (P = 0.083), mice receiving EVs frompalmitate-exposed BV2 cells showed a larger glucose
excursion than the vehicle group, as typified by increased
GTT area-under the curve (+ 24%, P = 0.018; Fig. 4G).
EVs can impact microglia in the mouse brain

EVs are likely to afford exchange of cellular material inbetween microglia, and from microglia to other cells.
We then tested whether the injected EVs induced alterations in the morphology of microglia and astrocytes in
the mouse hypothalamus (arcuate nucleus, ARC), andPaula De et al. Journal of Neuroinflammation(2024) 21:173

in the hippocampal cornu Ammonis (CA1 and CA3) anddentate gyrus (DG) (Fig. 5A). We determined the area
covered by immunoreactivity against Iba1 and GFAP,
which are microglia and astrocyte markers, respectively.
Compared to vehicle, mice treated with EVs from palmitate-exposed BV2 cells showed significantly larger Iba1
coverage across all regions analyzed (ANOVA: region x
treatment F(3,36) = 0.064, P = 0.979; region F(3,36) = 1.081,

Page 11 of 16

P = 0.369; treatment F(1,12) = 14.250, P = 0.003; Fig. 5B).
In contrast, GFAP area was similar in the 2 groups, suggesting that despite microglia expansion, the EVs frompalmitate-treated BV2 microglia did not cause overt
astrogliosis in vivo (Fig. 5C). Next, we set to explore themorphology of Iba1+ cells, that is microglia (Supplementary Figure S3). Microglia from the brain of mice inpalmitate and vehicle groups showed similar number ofFig. 5 EVs from palmitate-challenged microglia in vitro activate microglia in the hippocampus and hypothalamus of mice in vivo. (A) Representative
micrographs showing DAPI signal from nuclei, GFAP immunoreactivity in red, and Iba1 immunoreactivity in red in the dentate gyrus (DG) and cornu ammonis (CA1/CA3) areas of the hippocampus, and arcuate nucleus (ARC) of the hypothalamus at 8 days after intraventricular EVs injection. (B-C) Fractionof area occupied by iba1 and GFAP immunoreactivity in DG, CA1, CA3 and ARC. (D) Mean number of processes, (E) number of branching points, (F) total
cell process length, and (G) maximum process length of microglia, as determined from skeleton analysis of 3–4 iba1+ cells per mouse. (H) Representative
micrographs showing CD68 staining at 8 days after EV injection. (I) Fraction of are occupied by CD68 immunoreactivity, and (J) number of CD68-positive
cells. Data is shown as mean ± SD of 7 independent experiments, represented by the individual symbols. *P < 0.05 depicts differences in comparisonsfollowing significant effects in ANOVA. Scale bars over the micrographs indicate 50 μm; 3 V = third ventricle

Paula De et al. Journal of Neuroinflammation(2024) 21:173

processes sprouting from the cell soma (Fig. 5D). In turn,
compared to vehicle, mice injected i.c.v. with EVs frompalmitate-exposed cells showed more branching points
in the microglia processes (ANOVA: region x treatment F(3,36) = 0.142, P = 0.934; region F(3,36) = 0.546,
P = 0.654; treatment F(1,12) = 5.141, P = 0.043; Fig. 5E).
Total length and maximum length of the cell processes
were similar between groups (Fig. 5F-G). CD68 immunoreactivity was also increased in the brain of mice receiving EVs from palmitate-treated cells, relative to vehicle(ANOVA: region x treatment F(3,36) = 0.476, P = 0.701;
regionF(2.41,28.96) = 5.397,
P = 0.007;
treatment
F(1,12) = 12.910, P = 0.004; Fig. 5H-I), suggesting larger
microglia activation. However, the number of CD68-positive cells was similar between the groups (Fig. 5J).
Our findings indicate that EVs shed by palmitateexposed microglia induce microglia activation andbranching in vivo, despite no overt gliosis after one weekof direct administration to the brain.

Discussion
Our results suggest that microglia respond to palmitate exposure with increased proliferation, and with ametabolic network rearrangement that favors energy
production from glycolysis rather than oxidative metabolism, despite increased mitochondria density. In addition, while palmitate did not induce increased cytokine
expression, it modified the protein cargo of released EVs
which, alone, can contribute to the development of memory impairment, depression-like behavior and glucose
intolerance.
Rearrangement of energy metabolism after palmitate
exposure

Microglia are metabolically flexible cells that can utilize a variety of substrates, and their activation upon
injury or infection is an energetically costly process [35,36] When activated by inflammatory stimuli, microglial
cells exacerbate glucose uptake and glycolysis [33, 34]. A
similar metabolic shift occurs in activated macrophages[37]. Indeed, we have observed this switch from oxidative metabolism to anaerobic glycolysis in BV2 microglia
activated by either palmitate or LPS. In contrast, Chausse
et al. [38] have reported that BV2 activation by palmitateis sustained by oxidative metabolism, while LPS activation is mostly dependent on glycolysis. It should be
noted, however, that Chausse et al. have cultured theircells in the presence of 25 mmol/L of glucose, which isbelieved to represent hyperglycemic conditions (e.g.,
see Duarte et al. [39] for brain glucose levels observedin the brain of humans and rodents). Primary microglia
and BV2 microglial cells express several glucose carriers,
with GLUT1 being at least one order of magnitude moreabundant than the others, and the one that is upregulated

Page 12 of 16

upon microglia activation [40] Our results also support GLUT1 upregulation in both LPS and palmitate
exposure.
Activation of BV2 cells and primary microglia by
LPS is known to induce mitochondrial fragmentation[41, 42]. Enhanced expression of genes coding for proteins involved in mitochondria fragmentation was notobserved in our short (3 h) LPS exposure or upon palmitate treatment. Namely, mRNA levels of mitochondrial fission markers dynamin-related protein 1(Drp1),
fission protein 1 (Fis1), and mitochondrial fission factor(Mff ) were similar across experimental groups. Instead,
we observed a palmitate-induced expression of PGC-1α,which is a key regulator of mitochondrial biogenesis.
Indeed, palmitate also induced an increase in mitochondria content, and in the density of proteins belonging to
the mitochondrial complexes of the electron transport
chain. Together, our findings suggest that although palmitate potentially increases mitochondria biogenesis,
it reduces the efficiency of respiration and exacerbates
glycolysis. Using 13C tracing experiments, we found thatpalmitate further increases the rate of pyruvate carboxylation, which is needed for de novo oxaloacetate synthesis[43], and supports the drainage of Krebs cycle intermediates used in biosynthetic pathways during increased cellular proliferation.
Lack of palmitate-induced cytokine overexpression

Obesity and saturated fat exposure is known to stimulate NF-κB transcriptional activity and the secretion of
pro-inflammatory mediators in peripheral organs, suchas liver, white adipose tissue, and leukocytes [44], In thebrain, the neuroinflammatory process in DIO remains
unclear, and somehow controversial. That might bebecause microglia reactivity is modulated by specificneurochemical signals within the cell’s microenvironment, and due to transient release of inflammatory mediators. Indeed, there have been reports of both differential
microgliosis profiles across distinct brain areas, [45, 46]
and biphasic gliosis with transient cytokine release inchronic noxious stimuli, such as that in DIO [7, 11].
We have conducted this study under the assumptionthat palmitate is the saturated fatty acid in the context ofobesity driving neuroinflammation [23]. In fact, palmitate induces much stronger expression of inflammatory
factors in primary astrocytes than saturated fatty acids
such as laurate or stearate [47]. In addition, 200 µmol/Lof palmitate but not stearate impairs mitochondrial function in the C6 astrocyte cell line. [48] In neuronal cell
models, both palmitate and stearate can cause cell death.
[49] In our hands, while the exposure of BV2 cells to LPS
resulted in an enormous expression of pro-inflammatory
cytokines, palmitate was devoid of such effect. Accordingly, Chausse et al. [38] also failed to find increased

Paula De et al. Journal of Neuroinflammation(2024) 21:173

inflammatory mediators in palmitate- or oleate-exposed
BV2 cells. Others have suggested that palmitate can evenactivate anti-inflammatory pathways and reduce TNF-α
expression in BV2 microglia [50, 51].
EVs as neuroinflammatory mediators

DIO leads to depression-like behavior and hippocampaldependent memory impairment in rodents, although
not all diet intervention studies have found pronounced
increases in neuroinflammatory markers [6, 11, 14–16,
27, 52, 53]. Microglial inhibition protocols have supported the notion that microgliosis is an important contributor to neuronal dysfunction in the arcuate nucleusof the hypothalamus upon DIO, [8] and can both prevent
peripheral inflammation and reduce the extent of metabolic syndrome development [54].
However, the exacerbated cytokine production appears
to be transient in both the hippocampus and hypothalamus during DIO [7, 11] In later stages of obesity
development, in the absence of cytokine overproduction, EVs could contribute with both inflammatory andnon-inflammatory molecular messages from microglia to other cells. EVs isolated from palmitate- or LPStreated cells had unaltered size but displayed significant
proteome changes. Surprisingly, our pattern recognition analysis demonstrated larger proteome differences
between palmitate and vehicle than between LPS andvehicle groups.
Most significant differentially expressed proteins inpalmitate were related to mRNA processing and translation into protein, including the downregulation of several
ribosome subunits (Rps5, Rps7, Rps13, Rps14, Rps15a,
Rps29, Rpl11, Rpl35a). Ribosome subunits in neurons
exhibit a dynamic assembly that is locally regulated indendrites and synapses [55, 56] Moreover, RNA stressgranules and smaller dendritic trees were previously
observed when ribosomal proteins were depleted fromneurons with already established dendrites [57]. Thus,
it is likely that microglia EVs deliver components of theribosomal machinery to support protein synthesis inneuronal processes, and we speculate that the reduction
in such components within microglial EVs after palmitate exposure can result in alterations of synaptic plasticity and neuronal connectivity. Decreased expressionof ribosomal proteins has been reported in metabolically healthy obese individuals, [58] including Rps29 and
Rpl35a, also found to be downregulated in the present
study. Tardif et al. further reported that palmitate exposure induced endoplasmic reticulum stress and suppress
protein synthesis while upregulating proteolytic systems
in skeletal muscle [59]. Since modulation of ribosomal
activity and translation is a key determinant of innate
immune gene translation in microglia, [60] we speculatethat reduced levels of ribosome-associated components

Page 13 of 16

in EVs after palmitate exposure might also contributeto refrain protein translation in target microglia. Suchmechanism of control of innate immune response in resident microglia would explain why limited microgliosis isfound in diet-induced obesity models [12–16].
Previous reports demonstrated that EVs from microglia activated by ATP, IFN-γ or LPS can impact neurons
and astrocytes [61, 62]. Namely, exosomes from IFN-γ/
LPS-activated microglia trigger dopaminergic neurodegeneration, [61] and EVs secreted from ATP-stimulated
microglia induce stronger astrocyte activation withupregulation of IL-10 and IL-6 expression, compared toconstitutive EVs. [62] In our experimental setup, when
EVs were given i.c.v., mice receiving EVs from palmitatetreated microglia developed memory impairment, asassessed by the lower capacity to recognize the novelty
in the NOR test. An increase in latency to grooming inthe sucrose splash test further indicated a depression-likebehavior. Moreover, mice injected with EVs derived frompalmitate-exposed microglia developed glucose intolerance, when compared to the vehicle group, suggestingalterations in central regulation of metabolism.
In DIO, impaired glucose homeostasis occurs before
insulin resistance is installed, [15, 63] which is in linewith direct actions of EVs on hypothalamic glucosesensing neurons. However, one must keep in mind that
smaller brain-born EVs, such as exosomes, reach the systemic circulation, [64] and could act on peripheral organs
as well.
Finally, we investigated whether EVs alone could be
mediators of neuroinflammation. Thus, we evaluated gliosis in the mouse brain following EV administration, withfocus on the hippocampus that controls learning andmemory, and the hypothalamus that is the main central
glucose sensing area. Notably, we have observed somedegree of microgliosis but not astrogliosis when injecting
i.c.v. the EVs from palmitate-exposed microglia, relative
to EVs from vehicle experiments. The absence of astrogliosis is somehow surprising since microglia-derived EVs
after ATP stimulation were found to modulate astrocytes[62].
Limitations

We have characterized energy metabolism of BV2 cells
but have not performed in-depth investigations into themechanisms that drive mitochondrial alterations, which
could include intracellular accumulation of lipids, andthe impairment in insulin signaling. Moreover, although
this study puts forward novel ideas for further testing,it is important to note that we have studied the BV2
microglial cell line and not primary microglia, although
we know they tend to behave similarly [65, 66]. The cellline allowed us to have access to larger amounts of material (namely EVs) without collecting tissue from large

Paula De et al. Journal of Neuroinflammation(2024) 21:173

numbers of animals, but our findings might not fully represent microglia responses in vivo. In addition, we havestudied protein content of EVs, but not other types ofcargo, such as small metabolites, lipids, and nucleic acids.
In fact, it is thought that EVs carry microRNAs that control the expression of a variety of proteins, namely proteins involved in synaptic pruning, energy metabolism,
immune response, and translation/transcription [18, 67,68]. Whether these microRNAs are present and modified
after palmitate exposure was not tested, but further studies on this topic are warranted.
The concentration of palmitate used in our study is in
the range of that used in many other studies on cell cultures, which is at least 10-fold larger than that found in
the cerebrospinal fluid (CSF) of humans (5–20 µmol/L).
[70, 71]. However, stearate a more abundant saturated
fatty acid in the human CSF, ranging from 17 to 120µmol/L [70, 71] .Together, these two main brain free fatty
acids appear at concentrations in the same order of magnitude of our study. Melo et al. reported that obese individuals show a 2-fold increase in CSF palmitate but have
not reported changes in levels of stearate [23].
We have tested the injection of EVs in young mice,although metabolic disease is expected to be a risk factor
mainly in elderly individuals. [1–3] Published researchshows that a very short exposure to a high-fat diet isalready sufficient to impact the brain of mice at 2–3
months of age. Thus, we decided to test whether microglia-derived EVs exposed to palmitate mimic such effects
at this age. Notably, microglia responses have been shownto vary with age, including metabolism, cytokine release,
the important phagocytic capability and the expression ofvarious pattern recognition receptors to regulate migration [72–74]. Therefore, one should indeed expect differences in activation and responses of resident microglia at
young and old age.

Conclusion
In this study, using BV2 microglia, we demonstrate thatpalmitate exposure does not drive expression of cytokines that is typical of microglia activation. Instead, palmitate led to the release of EVs with modified protein
cargo that, alone, is sufficient to induce some degree of
microgliosis in the mouse, and to impact brain function.
The EV-mediated palmitate dysfunction is thus a plausible mechanism by which microglia participate in brain
dysfunction during DIO.
Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03168-7.
Supplementary Material 1
Supplementary Material 2

Page 14 of 16

Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Acknowledgements
The authors thank Charlotte Welinder for the mass spectrometry analysis,
Daria Rago for help with the proteomic analysis, and Patricia F. Nunes(The Francis Crick Institute) for discussions on palmitate preparation andcell treatments. We acknowledge the Strategic Research Area MultiPark(Multidisciplinary Research on Parkinson’s Disease) for access to mouse
behavior labs, the Lund University Bioimaging Centre for access to microscopy
resources, and the BioMS for access to proteomics facility.
Author contributions
JMND designed the study. GCdP, RB, RF-C, AB, BIA and JMND carriedout experiments. IL, TD and JMND provided resources and supervised
experimental work. GCdP and JMND wrote the manuscript. All authors revised
the manuscript.
Funding
Open access funding provided by Lund University. This work was supportedby the Swedish foundation for International Cooperation in Research andHigher education (#BR2019-8508), Swedish Research Council (#2019 − 01130),
Diabetesfonden (#Dia2019-440, #Dia2021-637), Direktör Albert Påhlssons
Foundation, and Royal Physiographic Society of Lund. JMND acknowledges
support from The Knut and Alice Wallenberg foundation, infrastructure
funding of Lund University (Dnr STYR 2019/318) and Lund University Facultyof Medicine (Dnr STYR 2021/2984), and Lund University Diabetes Centre,
which is funded by the Swedish Research Council (Strategic Research Area
EXODIAB; grant no.: 2009 − 1039) and the Swedish Foundation for Strategic
Research (grant no.: IRC15-0067). RB and IL are funded by the Parkinson
Research Foundation.
Open access funding provided by Lund University.
Data availability
Data is provided within the manuscript or supplementary information files.

Declarations
Ethical approval
Experiments on mice adhered to the EU Directive 2010/63/EU, were approvedby the Malmö/Lund Committee for Animal Experiment Ethics (permit#5123/2021) and are reported following the ARRIVE guidelines (Animal
Research: Reporting In Vivo Experiments, NC3Rs initiative, UK).
Competing interests
The authors declare no competing interests.
Received: 29 April 2024 / Accepted: 9 July 2024

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