(2024) 21:192
Meng et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03183-8

Journal of Neuroinflammation

Open Access

RESEARCH

Inhibition of ANGPTL8 protects
against diabetes‑associated cognitive
dysfunction by reducing synaptic loss
via the PirB signaling pathway
Xiaoyu Meng1,2†, Danpei Li1,2†, Ranran Kan1,2, Yuxi Xiang1,2, Limeng Pan1,2, Yaming Guo1,2, Peng Yu1,2,
Peiqiong Luo1,2, Huajie Zou1,2, Li Huang1,2, Yurong Zhu1,2, Beibei Mao1,2, Yi He1,2, Lei Xie1,2, Jialu Xu1,2,
Xiaoyan Liu3, Wenjun Li4, Yong Chen1,2, Suiqiang Zhu3, Yan Yang1,2* and Xuefeng Yu1,2*

Abstract
Background Type 2 diabetes mellitus (T2D) is associated with an increased risk of cognitive dysfunction. Angiopoietin-like protein 8 (ANGPTL8) is an important regulator in T2D, but the role of ANGPTL8 in diabetes-associated cognitive
dysfunction remains unknown. Here, we explored the role of ANGPTL8 in diabetes-associated cognitive dysfunction
through its interaction with paired immunoglobulin-like receptor B (PirB) in the central nervous system.
Methods The levels of ANGPTL8 in type 2 diabetic patients with cognitive dysfunction and control individuals were measured. Mouse models of diabetes-associated cognitive dysfunction were constructed to investigate
the role of ANGPTL8 in cognitive function. The cognitive function of the mice was assessed by the Barnes Maze test
and the novel object recognition test, and levels of ANGPTL8, synaptic and axonal markers, and pro-inflammatory
cytokines were measured. Primary neurons and microglia were treated with recombinant ANGPTL8 protein (rA8),
and subsequent changes were examined. In addition, the changes induced by ANGPTL8 were validated after blocking
PirB and its downstream pathways. Finally, mice with central nervous system-specific knockout of Angptl8 and PirB−/−
mice were generated, and relevant in vivo experiments were performed.
Results Here, we demonstrated that in the diabetic brain, ANGPTL8 was secreted by neurons into the hippocampus,
resulting in neuroinflammation and impairment of synaptic plasticity. Moreover, neuron-specific Angptl8 knockout
prevented diabetes-associated cognitive dysfunction and neuroinflammation. Mechanistically, ANGPTL8 acted
in parallel to neurons and microglia via its receptor PirB, manifesting as downregulation of synaptic and axonal markers in neurons and upregulation of proinflammatory cytokine expression in microglia. In vivo, PirB−/− mice exhibited
resistance to ANGPTL8-induced neuroinflammation and synaptic damage.

†

Xiaoyu Meng and Danpei Li have contributed equally to this study.

*Correspondence:
Yan Yang
yangyan6910@163.com
Xuefeng Yu
xfyu188@163.com
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-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Meng et al. Journal of Neuroinflammation(2024) 21:192

Page 2 of 21

Conclusion Taken together, our findings reveal the role of ANGPTL8 in the pathogenesis of diabetes-associated cognitive dysfunction and identify the ANGPTL8-PirB signaling pathway as a potential target for the management of thiscondition.
Keywords Diabetes-associated cognitive dysfunction, Angiopoietin-like protein 8, Paired immunoglobulin-likereceptor B, Neurons, Microglia

Introduction
Substantial epidemiological evidence supports the association of diabetes mellitus with decrements in cognitive
function and changes in brain structure [1]. Increasingly,
cognitive dysfunction is recognized as an important
comorbidity of type 2 diabetes mellitus (T2D), referred
to as ‘diabetes-associated cognitive dysfunction’ [2].
Changes in hippocampal neurons have been reported inpatients and animals with diabetes, including damage tosynaptic structures, axonal degeneration and loss of neurons [3–6]. Moreover, the expression of proinflammatory
cytokines in the brain is increased and plays a vital role incognitive impairment through toxic effects on neurons,participation in dendrite clearance, or limiting synaptic
connections [7, 8]. In light of the effect of cognitive dysfunction on affected individuals and society, a preventive
treatment for cognitive dysfunction in diabetes is needed,
particularly for retardation of progress to the more severe
stages. However, our understanding of the potential
mechanisms and therapeutic targets underlying cognitive
dysfunction in diabetes is incomplete.
Angiopoietin-like protein 8 (ANGPTL8), also
described as betatrophin, lipasin, and refeeding inducedin fat and liver (RIFL), is a new nutritionally regulated
liver-enriched factor with a role in lipid metabolism [9].
ANGPTL8 was also shown to be associated with diabetes
and insulin resistance [10]. Circulating ANGPTL8 levels
were significantly increased in patients with T2D [11, 12].
Previous studies have shown that ANGPTL8 is found in
the brain [13, 14]. However, the source of ANGPTL8 inthe brain remains to be elucidated in the context of diabetes-associated cognitive dysfunction.
Our previous study suggested that ANGPTL8 interacts
with its receptor paired immunoglobulin-like receptor B(PirB) and regulates downstream signaling pathways [15].
Notably, PirB and its human ortholog receptor (leukocyte
immunoglobulin-like receptor B, LILRB) are distributedin various hematopoietic cells and extensively in different
regions of the injured central nervous system (CNS) [16].
Emerging evidence suggests that PirB contributes to theneuropathology of human Alzheimer’s disease [16, 17](including synaptic loss and neurotransmitter release),hippocampal function decline, and aging-induced cognitive dysfunction [18]. Although an association between
PirB and cognitive dysfunction has been reported, themechanisms by which PirB signals and which ligands are
involved have yet to be determined.
Considering this, we speculate that ANGPTL8 isinvolved in the mechanisms of diabetes-associated cognitive dysfunction through interaction with PirB in
the CNS. In this study, we demonstrated the role of
ANGPTL8 in regulating synaptic plasticity and microglial inflammatory activity and identified the ANGPTL8PirB signaling pathway as a potential target for the
management or treatment of diabetes-associated cognitive dysfunction.

Materials and methods
Participants

The present study consisted of 30 individuals from the
Tongji Hospital of Wuhan, including 20 patients with
T2D and 10 individuals without T2D as controls. The
inclusion and exclusion criteria are listed in the Supplementary Methods. The global cognitive function of all
subjects was measured by the Mini-Mental State Examination (MMSE) [19] and Montreal Cognitive Assessment(MoCA, available at www.​mocat​est.​org) [20]. Higher
MMSE and MoCA scores indicated better cognition; the
MMSE cutoff score for dementia was 23/24 [21], and theMoCA cutoff score for mild cognitive impairment (MCI)
was 25/26 [22]. Cognitive dysfunction was assessed ona case-by-case basis by the study neuropsychologist following stratification of scores according to education.
The serum and cerebrospinal fluid (CSF) of subjects
were collected. The study protocols (approval number:
TJ-IRB20230452) were approved by the Committee on
Human Research at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.
All participants provided written informed consent.
Animals

All animal protocols were approved by the Institutional
Animal Care and Use Committee of the Institute ofModel Animals of Tongji Hospital, Huazhong Universityof Science and Technology. The animals received humane
care according to the criteria outlined in the Guide for
the Care and Use of Laboratory Animals prepared by
the National Academy of Sciences and published by the
National Institutes of Health [23].

Meng et al. Journal of Neuroinflammation(2024) 21:192

The mice were given free access to food and waterand housed in a temperature-controlled environment(23 ± 2 °C) with a 12-h/12-h light/dark cycle. Several
animal models were used in this study, including wildtype (WT) mice, db/m, db/db, CNS-specific Angptl8
depletion mice and PirB knockout mice. Six-monthold male db/m and db/db mice (BKS-Leprem2Cd479/
Gpt, strain number: T002407) were purchased from
GemPharmatech Co., Ltd. (Jiangsu, China). Male
CNS-specific Angptl8-depletion (Angptl8Nestin CKO)
mice on a C57BL/6 genetic background were generated by Cyagen Biosciences Inc. (Guangzhou, China)
using CRISPR/Cas9-mediated genome engineering.
The mouse Angptl8 gene (GenBank accession number: NM_001080940.1) is located on mouse chromosome 9, and 4 exons have been identified, with the
ATG start codon in exon 1 and the TGA stop codon
in exon 4 (transcript: ENSMUST00000058777). Exons
1–2 were selected as the conditional knockout (cKO)region. Deletion of this region should result in the lossof function of the mouse Angptl8 gene. For the genetically targeted model, Cas9 and guide RNA (gRNA)
were coinjected into fertilized mouse eggs to generatetargeted knockout offspring. Angptl8Nestin CKO mice
were generated by intercrossing these heterozygoustransgenic founder mice with homozygous Nestin-Cre
mice. Genomic DNA extracted from tail biopsies wasused for genotyping with a PCR primer set (P1: 5ʹ-ATG​
GCA​G CC​TAT​G GA​AAT​AAA​AGG​-3ʹ, P2: 5ʹ-TCT​
TCA​TAT​TGA​G CT​G GC​TCT​G GA​-3ʹ) that amplified a
168-bp band in samples from mice with the wild-typeallele and a 202-bp band in samples from mice with
the Angptl8 floxed allele. Moreover, genomic DNA
extracted from tail biopsies was used for genotyping with a PCR primer set (P1: 5ʹ-CCT​TCC​TGA​AGC​
AGT​AGA​G CA-3ʹ, P2: 5ʹ-GCC​T TA​T TG​TGG​AAG​GAC​
TG-3ʹ; P3: 5ʹ-TTG​C TA​AAG​CGC​TAC​ATA​G GA-3ʹ, P2:
5ʹ-GCC​T TA​T TG​TGG​AAG​GAC​TG-3ʹ) that amplified a
246-bp band in samples from mice with the wild-typeallele and a 150-bp band in samples from mice with the
Nestin-Cre allele. PirB knockout mice (PirB−/−) werepurchased from Cyagen Biosciences Inc. Models of diabetes-associated cognitive dysfunction were induced byfeeding 8-week-old male C57BL/6J mice a high-fat diet
(HFD, D12492, Research Diets, 60 kcal% fat, 20 kcal%
carbohydrate, and 20 kcal% protein, Jiangsu Xietong,Inc., Nanjing, China) for 9 months. Control mice
were maintained on a normal diet (ND, 10 kcal% fat,
70 kcal% carbohydrate, and 20 kcal% protein, Jiangsu
Xietong, Inc., Nanjing, China, Cat# SWS9102).
According to previous studies, neuroinflammation andmemory impairment occurred in mice fed a HFD for
4–6 months [24, 25] and db/db mice fed a ND for overPage 3 of 21

6 months [26–28]. Therefore, cognitive dysfunction andneuropathology could occur in the diabetic mice in thisstudy.
Stereotaxic injections of recombinant ANGPTL8 (rA8)

For injection of rA8 into the CA1 region of the hippocampus, 8-week-old mice were anesthetized using a
mixture of ketamine (90 mg/kg) and xylazine (2.7 mg/kg) and placed in a stereotaxic device (Kopf Instruments). Then, 0.5 μl rA8 (2 μg/μl) or scrambled protein
was injected into the CA1 region of the bilateral hippocampus with a 10 μl microsyringe (Hamilton, Fisher
Labosi, France) (0.1 μl/min, single dose). Hippocampal
CA1 injections were made at the following coordinates
relative to the bregma: − 2 mm at the anterior/posterioraxis, ± 1.8 mm at the lateral/medial axis and -1.5 mm at
the dorsal/ventral axis. Before returning to the original
coordinates and administering the injection, the syringe
was inserted to an extra depth of 0.5 mm [29, 30]. The
syringe was left in place for an additional 5 min (min)to allow rA8 to spread into the brain. One month after
injection, behavioral tests were performed in mice before
tissue collection [30].
Stereotaxic injections of AAV9‑Nestin‑Cre

For injection of AAV-Nestin-Cre into the hippocampal
CA1 region, the mice were anesthetized using a mixture of ketamine (90 mg/kg) and xylazine (2.7 mg/kg)
and placed in a stereotaxic device (Kopf Instruments).
Then, 0.5 μl AAV9-Nestin-Cre virus (7.77 × ­1012 v.g./ml,
Obio Technology, Shanghai, China) or control virus was
injected into the bilateral hippocampal CA1 region witha 10 μl microsyringe (Hamilton, Fisher Labosi, France)(0.1 μl/min, single dose).
Glucose tolerance test (GTT) and insulin tolerance test (ITT)

To perform in vivo glucose tolerance tests (GTTs) andinsulin tolerance tests (ITTs), mice were intraperitoneally injected with 1.5 g of glucose (Sigma‒Aldrich Co.
St. Louis, MO, USA) or 1 U insulin (Novolin R, Novo
Nordisk Co., Bagsvaerd, Denmark) per kg body weight,
respectively. Corresponding blood glucose concentrations were examined at the indicated time points [15, 31].
Behavioral assessment

The behavioral tests used in this study were the open field
test, novel object recognition test and Barnes maze test.
The open field test was conducted on the first day, andthe novel object recognition test was performed with 2
trials on the second day. The Barnes maze test was conducted over the following 5 days. These behavioral tests
were adapted according to previous studies [27, 28, 32].

Meng et al. Journal of Neuroinflammation(2024) 21:192

Open field test

The open field test was used to assess general activity inrodents [27]. Each mouse was placed in the center of asquare open field box (45 × 45 × 45 cm), which was placedin a quiet and well-lit room, and allowed to roam freely
for 10 min with an overhead video camera recording
the movements. The total distance moved as well as thevelocity of each mouse during that period was recorded
and analyzed by a computerized EthoVision detection
system (Noldus, Wageningen, The Netherlands). The area
between each test was cleaned with 70% ethanol.
Novel object recognition test

Based on the spontaneous tendency of rodents to spend
more time exploring a novel object than a familiar object,
the novel object recognition test was used to evaluate
hippocampal- and cortex-dependent contextual learningand recognition memory in rodents. The test was conducted in the same area as the open field test describedabove. In the first trial, mice were individually allowed
to investigate two identical objects for 5 min and thenreturned to their home cage for an interval of 30 min.
During the second trial, mice were allowed to explore
the objects with one familiar and one novel object for
5 min. All trials were recorded and analyzed by automated EthoVision tracking software (Noldus). The recognition index was then calculated as the difference intime spent exploring the novel and familiar objects or
the frequency of explorations of each object using theformulas: [(frequency exploring novel object−frequency
exploring familiar object)/(total frequency exploring
both objects)*100] or [(time exploring novel object−time
exploring familiar object)/(total time exploring bothobjects)*100], respectively [33]. The recognition index
was used as a measure of memory.
Barnes maze test

The Barnes maze was used to test spatial learning andmemory [28, 32]. Spatial learning and memory ability
were evaluated using a Barnes maze, which was a darkgray circular platform (100 cm in diameter, raised 90 cm
above the floor) that contained 20 holes (5 cm in diameter). One of the holes led to an escape box. There were
spatial cues, which were visible to the mice, located in
fixed positions around the platform in the testing room.
Bright light served as a mildly aversive environmental
stimulus from which mice tried to escape. The simplified Barnes maze test included spatial acquisition andprobe trials. The mice were each given 5 min to adapt tothe maze and the existence of the escape box on the firstday. The first acquisition session was performed for 5 minafter an intertrial interval of 30 min. The mice were gently

Page 4 of 21

guided to the escape box if they did not find it and stayed
there for 2 min. Then, from the second to third day, twotraining trials (5 min per trial) with an intertrial intervalof 30 min were performed in the acquisition test. On the
fourth day, the mice were allowed to rest for 24 h. On thefifth day, the probe test with the escape box removed was
conducted for 5 min. The errors each mouse made before
locating the escape hole were recorded by a video camera
and analyzed using EthoVision as an assessment of spatial
memory retention.
Lipopolysaccharide (LPS) and flag‑targeted recombinant
ANGPTL8 protein (rA8‑flag) injection

For the LPS injection experiment, 8-week-old mice wererandomly divided into two groups and intraperitoneally(i.p.) injected with a dose of LPS (1 mg/kg) (Sigma) orsaline (control) for 36 h before tissue collection.
rA8-flag, an exogenously labeled protein tagged witha short hydrophilic amino acid sequence, DYKDDDDK,
can be detected by flow cytometry analysis, Western
blotting, and immunofluorescence staining and has anegligible influence on the biological function of therecombinant protein [34]. In the rA8-flag injection
experiment, two-month-old mice were randomly divided
into two groups and injected i.p. with a dose of rA8-flag(1 mg/kg) or saline (control) for 8 h.
Plasma, CSF and tissue collection

After the completion of all tests, mice were anesthetizedwith isoflurane, and plasma and CSF were collected followed by cervical dislocation. Then, the brains wereextracted and quickly dissected on ice. The right hemispheres of the brains were fixed in 4% paraformaldehyde
for immunofluorescence analysis, and the hippocampus
was separated from the left hemisphere and frozen at– 80 ℃ for Western blotting and quantitative polymerase
chain reaction (qPCR) analysis.
Enzyme‑linked immunosorbent assay (ELISA)

The serum ANGPTL8 levels of participants were quantified using commercially available ELISA kits (Eiaab
Science, Wuhan, China; Catalog No. E11644h). Commercially available mouse ELISA kits (Eiaab Science Inc.,
Wuhan, China, Cat# E11644 m) were used to measure
ANGPTL8 levels in the CSF of mice.
Hippocampal triglyceride (TG) level measurement

Hippocampal TG levels were measured using a TG assay
kit (Nanjing Jiancheng Bioengineering Institute, Nanjing,
China, Cat#A110-1-1). All assays and data analyses were
performed according to the manufacturer’s protocol.

Meng et al. Journal of Neuroinflammation(2024) 21:192

Brain immunofluorescence

The paraffin-embedded sections were deparaffinized andstained with the following primary antibodies (pAbs)
overnight at 4 ℃: an anti-ANGPTL8 rabbit pAb (Thermo
Fisher Scientific, USA, Cat# PA5-38043, 1:100 dilution),an anti-PirB rat monoclonal antibody (mAb) (Thermo
Fisher Scientific, USA, Cat# MA5-24049, 1:50 dilution),a PE-conjugated anti-Flag mAb (BioLegend, USA, Cat#637310, 1:200 dilution), an anti-NeuN antibody (Servicebio, Wuhan, China, Cat#GB11138-100, 1:200 dilution),an anti-CD16/32 antibody (Abcam, Cambridge, UK, Cat#ab223200, 1:200 dilution), an anti-CD206 antibody (AbD
Serotec, Oxford, UK, Cat# MCA2235T, 1:200 dilution),an anti-IBA-1 antibody (Servicebio, Wuhan, China, Cat#GB11105, 1:200 dilution) and an anti-CD11b antibody(Servicebio, Wuhan, China, Cat# GB11058, 1:200 dilution). After washing in phosphate-buffered saline (PBS),
sections were visualized by Alexa Fluor-conjugatedsecondary antibody (Servicebio, Wuhan, China, Cat#
GB25303, 1:200 dilution) or Cy3-conjugated secondary antibody (Servicebio, Wuhan, China, Cat# GB21303,
1:200 dilution). Nuclear counterstaining was performed
using 4ʹ,6-diamidino-2-phenylindole (DAPI), and thesections were viewed with a fluorescence microscope.
Similar and comparable histological areas were selected.
Transmission electron microscopy of synapses

The hippocampus was carefully separated, rapidly cut
into small pieces (1 ­mm3) and then fixed in 2.5% glutaraldehyde in PBS and postfixed in 1% OsO4 at 4 ℃. After
fixation, the samples were dehydrated in a graded seriesof alcohol and embedded in Poly/Bed 812 resin, followed
by ultrathin (80-nm) sectioning with an ultramicrotome.
Images were generated with a Hitachi H-7650 transmission electron microscope (Hitachi, Japan) at 80 kV. Threevisual fields of each tissue section were imaged (12,000 ×)
to produce a low-resolution map for quantitative analysis, and then high-resolution images (40,000 ×) were
captured. Synapses were identified by the presence ofsynaptic vesicles and postsynaptic densities. The number of synapses was measured using ImageJ software. All
electron microscopy quantification was performed byat least two independent observers who were blinded togenotype.
Nissl staining

After the mice were perfused with 0.1 mol/l PBS followed
by 4% paraformaldehyde (PFA), the hippocampal tissues
were immersed in 4% PFA for 24 h and transferred to
30% sucrose solution until they sank. Subsequently, thehippocampal tissues were cut into 20-um-thick sections
using a freezing microtome (Thermo, USA). After thesections were incubated with 0.1% cresyl violet for 5 min

Page 5 of 21

at room temperature, they were rinsed in double distilledwater followed by 95% ethanol, dehydrated in 100% ethanol and cleared in xylene, and covered with neutral resin.
Images were acquired with a microscope (Nikon, Tokyo,
Japan) and the hippocampal neurons were counted with
Image J software (Media Cybernetics, Bethesda, MD,
USA). Quantitative analysis of histological staining andfluorescence signals was performed by ImageJ [35].
Primary neuron and microglia culture

Hippocampi were dissected from C57BL/6J mouse pups
less than 24 h old, dissociated using trypsin (2 mg/ml)
and Dnase I (0.6 mg/ml), and plated at a density of 1 × ­106
cells per well in 6-well plates coated with poly-l-lysine(PLL, 0.01 mg/ml, Sigma). Neurons were maintained inneurobasal medium (Gibco) supplemented with 2% B27(Invitrogen), 1% penicillin‒streptomycin (Sigma) and
0.5 mM l-glutamine at 37 ℃ in a humidified atmosphere
containing 5% ­CO2.
Primary microglial cultures were prepared from thecerebral cortices of postnatal day 1–2 C57BL/6J mice.
Briefly, the meninges were removed from the brains, andthe cortex was enzymatically dissociated (0.25% trypsin–
EDTA, Sigma, St Louis, MO, USA). Then, the cells weresuspended in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12, Gibco, Carlsbad, CA,
USA) supplemented with 10% fetal bovine serum (FBS,
Gibco) and seeded into PLL precoated T75 tissue culture
flasks. After 3, 7, and 10 days, the culture medium wasrenewed. After achieving confluency at approximately
14 days in vitro, microglia were isolated from mixed glia
cultures with mild trypsinization. Incubation of mixed
glial cultures with a trypsin solution (0.25% trypsin–
EDTA diluted 1:4 in DMEM/F12) for 15–25 min resulted
in the detachment of an intact layer of cells in one piece.
Microglia remained attached to the bottom of the flask.
Then, microglia were collected and plated at a density of
1 × ­106 cells per well in 6-well plates coated with PLL.
Recombinant ANGPTL8 protein treatment

Before rA8 treatment, the culture medium of neurons
and microglia was renewed. The neurons were stimulatedwith 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml
rA8 (Cusabio, Wuhan, China, Cat# CSB-MP844436MO)
or scrambled ANGPTL8 after being cultured for 4–6 h
for an additional 5-day period. The microglia were stimulated with 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/
ml rA8 or scrambled ANGPTL8 after plating in culture
plates for 1 day for an additional 24-h period.
LPS or TNF‑α treatment

Before LPS treatment, the culture medium of the neurons
and microglia was renewed. The neurons were stimulated

Meng et al. Journal of Neuroinflammation(2024) 21:192

with 1000 ng/ml LPS (Sigma) or PBS after culturing for
5 days for an additional 24-h period. The microglia were
stimulated with 1000 ng/ml LPS or PBS for an additional
24-h period after being plated in culture plates for 1 day.
Before TNF-α treatment, the culture medium of neurons was renewed. The neurons were stimulated with
10 ng/ml, 100 ng/ml, and 300 ng/ml TNF-α (Sigma) or
scrambled TNF-α for an additional 24-h period after
being cultured for 5 days.
Antibody blockade assay

Neurons and microglia were pretreated with control IgG(10 µg/ml) and anti-PirB ectodomain antibody (10 µg/ml,
Thermo Fisher Scientific, MA, USA, Cat# MA5-24049)
for 30 min before incubation with rA8 (1000 ng/ml).
Signaling pathway inhibitors

Neurons were pretreated with inhibitors of ERK1/2(U0126; 10 nM, MedChem Express, NJ, USA, Cat#
HY-12031) and AKT (MK-2206; 10 nM, MedChem
Express, NJ, USA, Cat# HY-10358) for 30 min before
incubation with rA8 (1000 ng/ml).
Microglia were pretreated with inhibitors of ERK1/2(U0126; 10 nM, MedChem Express, NJ, USA, Cat#
HY-12031), AKT (MK-2206; 10 nM, MedChem Express,
NJ, USA, Cat# HY-10358), P38 (SB203580; 10 nM,
MedChem Express, NJ, USA, Cat# HY-10256), or P65(Bay11-7082; 10 nM, MedChem Express, NJ, USA,
Cat# HY-13453) for 30 min before incubation with rA8(1000 ng/ml).
RNA‑sequencing analysis

Neuronal mRNA was sequenced by Shanghai Biotechnology Corporation. Differential gene expression analysis was performed using the limma R package (version
3.44.3). The fold change (FC) in expression of each gene
was log2 transformed and further analyzed using RStudioversion 1.1.442 (RStudio, inc., USA).
Flow cytometry

Microglia were plated in 6-well plates at 1 × ­106 cells perwell and collected using 0.25% trypsin–EDTA at 37 ℃
after treatment with rA8 or scrambled ANGPTL8 for
24 h. Cell suspensions were washed with flow cytometry buffer (PBS with 2% FCS, 2 mM EDTA and 25 mM
HEPES, pH 7.4) and then incubated with blocking
buffer (5% BSA in flow cytometry buffer) for 15 min at
4 ℃. Cells were then stained with the desired antibodies for 30 min on ice. The following controls were used:unstained cells, single-stained cells, and dead cells. The
cells were gated using forward and side scatter, as well aslive-dead staining using DAPI (Thermo Fisher Scientific).
Cells were analyzed with FlowJo software (FlowJo). The

Page 6 of 21

antibodies included FITC-CD11b (BioLegend, CA, USA,#101206; 1 μg per ­106 cells), APC-CD206 (BioLegend,
CA, USA, #141707; 2 μg per ­106 cells), and PE-Iba-1 (Cell
Signaling Technology, USA, #10513; 1 μg per ­106 cells).
All antibodies were diluted according to the manufacturer’s instructions. Dead cells and doublets were removed
by dead-cell dye staining (Zombie Aqua Fixable Viability
Kit, BioLegend, CA, USA, #B297827).
Cellular immunofluorescence

Cells were seeded in a 24-well plate with coverslips. Neurons and microglia were treated with exogenous rA8-flag(1000 ng/ml) or scrambled ANGPTL8 for 5 days and
24 h, respectively. After treatment, the cells on the coverslips were fixed with 4% paraformaldehyde at room
temperature for 10 min and then permeabilized with
0.1% Triton X-100 for 15 min. A blocking step was performed with 5% bovine serum albumin (BSA) for 1 h.
Subsequently, the cells were incubated with the primary
antibodies overnight at 4 °C. After washing, the cells
were incubated with appropriate fluorescent secondary antibodies for 1 h. After three washes, the cells on
the coverslips were incubated with DAPI (5 μg/ml) at
room temperature for 5 min and observed under a fluorescence microscope. The antibodies used in this study
included PE-conjugated anti-Flag mAb (BioLegend, USA,
Cat# 637310, 1:100 dilution), anti-PirB rat mAb (Thermo
Fisher Scientific, USA, Cat# MA5-24049, 1:100 dilution),anti-βIII-tubulin rabbit mAb (Cell Signaling Technology,
USA, Cat#5568, 1:200 dilution), Alexa Fluor-conjugatedsecondary antibody (Servicebio, Wuhan, China, Cat#
GB25303, 1:500 dilution), and Cy3-conjugated secondary antibody (Servicebio, Wuhan, China, Cat# GB21303,
1:500 dilution).
RNA isolation and real‑time quantitative PCR (RT‒qPCR)

Total RNA was extracted using TRIzol (Thermo Fisher
Scientific, MA, USA, Cat# 15596018) following the
manufacturer’s instructions and quantified using a Nanodrop spectrophotometer. To prepare RNA for PCR
analysis, 1 μg of total RNA was converted to cDNA using­Hifair® II 1st Strand cDNA Synthesis SuperMix (Yeasen,
Shanghai, China, Cat#11120ES60). RT–qPCR was performed using the qPCR Hieff ­UNICON® qPCR SYBRGreen Master Mix (Low Rox) (Yeasen, Shanghai, China,
Cat#11199ES08) under the following conditions: initialdenaturation at 95 °C for 30 s, 40 cycles at 95 °C for 10 s,
and 60 °C for 30 s. Each sample was analyzed in triplicate.
Relative mRNA levels were calculated using the ­2−ΔΔCt
method and normalized to GAPDH mRNA levels. The
gene-specific primer sequences are listed in Supplementary Table 1.

Meng et al. Journal of Neuroinflammation(2024) 21:192

Western blotting

Cells and frozen hippocampal samples were lysed
in RIPA extract buffer (Beyotime, Shanghai, China,
Cat# P0013B) supplemented with protease inhibitor
cocktail (MedChem Express, USA, HY-K0010), phosphatase inhibitor cocktail I (MedChem Express, USA,
HY-K0021) and phosphatase inhibitor cocktail II (MedChem Express, USA, HY-K0022) according to the manufacturer’s protocol. Then, the lysis buffer containing
protein was centrifuged at 14,000×g for 15 min at 4 °C.
Equal amounts of protein (20 μg) were loaded into eachlane, separated by 10% SDS polyacrylamide gel electrophoresis, and electro transferred to polyvinylidene
fluoride (PVDF) membranes. The PVDF membranes
were blocked in blocking buffer for 2 h at RT, followed
by incubation with the following antibodies overnightat 4 °C: mouse anti-β-actin (Proteintech, Wuhan,
China, Cat# 66009-1-Ig, 1:1000 dilution), rabbit antiANGPTL8 (Thermo Fisher Scientific, MA, USA, Cat#
PA5-38043, 1:1000 dilution), anti-ERK1/2 (Cell Signaling Technology, MA, USA, Cat# 4695T, 1:1000 dilution), anti-phospho-ERK1/2 (Thr202/Tyr204; Cell
Signaling Technology, MA, USA, Cat# 4370T, 1:1000
dilution), anti-AKT (Cell Signaling Technology, MA,
USA, Cat# 4691S, 1:1000 dilution), anti-phospho-AKT(Ser473; Cell Signaling Technology, MA, USA, Cat#
4060T, 1:1000 dilution), anti-ROCK (Abclonal, China,
Cat# A2359, 1:1000 dilution), anti-Cofilin-1 (Abclonal,
China, Cat# A1704, 1:1000 dilution), and anti-phospho-Cofilin-1 (Abclonal, China, Cat# AP0178, 1:1000
dilution). The blots were subsequently rinsed with
Tris-buffered saline containing Tween-20 (TBST) threetimes and incubated with anti-rabbit peroxide-conjugated secondary antibodies for 90 min. The protein
bands were visualized and detected using an enhanced
chemiluminescence system.
Statistical analyses

For animal studies, mice were randomized by bodyweight prior to dietary challenge, and no blinding was
performed for subsequent analyses. Statistical analyses
were performed with Prism 7 (GraphPad Software, Inc.,
USA). For data with a normal distribution and homogeneity of variance, one-way or two-way ANOVA was performed for comparisons among more than two groups.
Two-tailed Student’s t tests were performed to evaluate
significant differences between two groups. The statistical tests used for each experiment are indicated in the
figure legends. All data are expressed as the mean ± s.e.m.
unless otherwise indicated. P < 0.05 was considered statistically significant. All in vitro experiments were performed in triplicate. Animal feeding, treatments andPage 7 of 21

histological analyses were performed in a single-blindedfashion. No blinding was used for the remaining analyses.

Results
ANGPTL8 levels were increased in the serum and CSF
of patients and mice with diabetes‑associated cognitive
dysfunction

After completing the MMSE and MoCA scales, clinicalparticipants were divided into three groups matched forsex, age, and body mass index (BMI): healthy control,
diabetes without cognitive dysfunction, and diabetes withcognitive dysfunction (Supplementary Table 2). There
was no significant difference in circulating ANGPTL8levels regardless of whether the cognition of diabetic
patients declined, although the levels were higher in diabetes patients (Fig. 1A). However, ANGPTL8 levels in the
CSF of diabetes patients with cognitive dysfunction werehigher than those in the CSF of patients without cognitive dysfunction (Fig. 1B).
Nine-month-HFD-fed mice and 6-month-ND-fed db/
db mice, both diabetic mice marked by significantly elevated blood glucose levels and insulin resistance (sFig. 1C
and D), were used to mimic the pathogenesis of diabetes-associated cognitive dysfunction. Significantly lessdistance traveled and velocity were observed in bothdiabetic mouse models than in controls in the open fieldtest (sFig. 1E). The impaired cognitive function of bothdiabetic mouse models was confirmed by a decreased
recognition index (Fig. 1C–F), but not general activity (sFig. 1F and G), in the novel object recognition test
and increased errors in the Barnes maze compared withthe control group (Fig. 1G–I). Similar to patients withdiabetes‑associated cognitive dysfunction, diabetic mice
exhibited higher circulating and CSF ANGPTL8 levels
relative to the control group (Fig. 1J and K). Moreover,
ANGPTL8 levels were increased in the serum and CSF of
STZ-induced diabetes model mice with diabetes-associated cognitive dysfunction (sFig. 2A–N).
Furthermore, the body weight of diabetic mice, HFDfed mice and db/db mice, was higher than that of micein the control groups (sFig. 1A), but this was not foundin STZ-induced diabetes model mice (sFig. 2I). Moreover, diabetic mice exhibited obvious insulin resistance(sFig. 1C and D; sFig. 2K and L). No difference in lipids
levels was observed between diabetic mice with diabetesassociated cognitive dysfunction and the control group(sFig. 1B; sFig. 2J).
ANGPTL8 expression was increased in the hippocampus
of diabetic brains

Consistently, the mRNA expression of Angptl8 wasincreased in the hippocampal tissue of diabetic mice withcognitive impairment (Fig. 1L; sFig. 2G), and intracellularMeng et al. Journal of Neuroinflammation(2024) 21:192

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Fig. 1 ANGPTL8 levels were increased in the serum and CSF of patients and mice with diabetes-associated cognitive dysfunction. A Serum
ANGPTL8 levels in the indicated groups (n = 10 per group). B ANGPTL8 levels in the CSF of the indicated groups (n = 10 per group). C Schematic
depicting the novel object recognition task (the green square represents the familiar object, and the purple square represents the novel object).
F, familiar object; N, novel object. D Representative traces of paths by the indicated mice to explore novel objects. E, F Recognition index
of the indicated mice, calculated as the difference in the amount of time spent exploring the novel and familiar objects (left) and in the frequency
of novel object explorations and familiar object explorations (right) (n = 4–7 mice per group). G Representative traces of paths to the target hole(red) by the indicated mice on the day of testing in the Barnes maze. H, I Barnes maze acquisition and probe trials for the indicated mice, shownby average errors. J Serum ANGPTL8 levels in the indicated groups. K CSF ANGPTL8 levels in the indicated groups. L Angptl8 mRNA expression
in the hippocampus in the indicated groups. M Immunofluorescence analysis of ANGPTL8 protein expression in the hippocampal CA1 regionof the indicated mice. N Immunofluorescence staining of IBA-1 (red) and CD11b (green) in the hippocampal CA1 region of the indicated mice.
O mRNA expression of proinflammatory cytokines (Il-1β, Il-6, and Tnf-α) and axonal growth and synaptic plasticity markers (Gap-43, Snap-25,
Nfl, and Syp) in the hippocampus of the indicated groups. The data are shown as the mean ± s.e.m. and were statistically analyzed by two-way
ANOVA with Bonferroni’s test (A, B) and two-tailed Student’s t test (C–O). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS: nonsignificant. CSF:
cerebrospinal fluid; HC: healthy control; T2DM: type 2 diabetes mellitus

ANGPTL8 protein expression in diabetic brain tissue
was visualized by immunofluorescence of tissue sections (Fig. 1M). Further analysis revealed that diabetic

mice with impaired cognitive function exhibited more
microglia (marked by IBA-1) infiltrating the hippocampal CA1 region (Fig. 1N) as well as higher inflammatory

Meng et al. Journal of Neuroinflammation(2024) 21:192

markers (CD11b and CD16/32) and fewer anti-inflammatory markers (CD206) (Fig. 1N; sFig. 1H). Moreover,the mRNA expression of proinflammatory cytokines (Il1β, Il-6, and Tnf-α) (Fig. 1O; sFig. 2H) was increased in
the hippocampal tissue of diabetic mice with cognitive
impairment, while the mRNA expression of the axonal
growth and synaptic plasticity markers growth-associated protein-43 (Gap-43), synaptosomal-associatedprotein 25 (Snap-25), neurofilament light chain (Nfl)and synaptophysin (Syp), which play vital roles in manyneuronal functions, including axonal growth and theexpression of long-term potentiation (LTP), in the hippocampus [36–44] was decreased (Fig. 1O; sFig. 2H). The
results indicated that diabetic mice exhibited obvious
neuroinflammation, decreased growth of neuronal axons,and damaged synapses in the hippocampus.
ANGPTL8 derived from neurons in the diabetic brain

It was unclear whether the increased ANGPTL8 levels
in CSF were due to more circulating ANGPTL8 crossing
the blood–brain barrier or increased production frombrain tissue. Purified flag-targeted ANGPTL8 protein(rA8-flag) was injected peripherally and was not detectedin brain tissues (sFig. 3A). Furthermore, LPS was injected
intraperitoneally, and Angptl8 expression was upregulated in the hippocampus (sFig. 3B and C). Moreover,an in vitro study showed that the mRNA expression of
Angptl8 was also increased in hippocampal neurons
but not microglia when treated with LPS or TNF-α(sFig. 3D and E), indicating that increased ANGPTL8 inbrain tissue mainly originated from neural cells rather
than microglia. Furthermore, the mRNA expression of
Angptl8 was also increased in hippocampal neurons
but not microglia after exposure to high glucose levels(sFig. 2O). Based on the above observation, the increased
levels of ANGPTL8 protein in diabetic CSF probably
originated directly from brain tissue rather than fromcirculation.
Depletion of Angptl8 in the CNS protectedagainst diabetes‑associated cognitive dysfunction
and alleviated neuroinflammation

Given the association between neuroinflammation andcognitive impairment, we reasoned that a reduction inneuronal ANGPTL8 levels and the consequent alleviation of inflammation and synaptic injury might protect
cognitive function in diabetic mice. Considering that
ANGPTL8 in the CNS was mainly expressed on neurons independent of circulating ANGPTL8, we crossed
Nestin-Cre mice (broadly used for selective gene deletion in neurons of the CNS [45]) with floxed Angptl8transgenic mice to generate neuronal Angptl8 cKO mice(referred to as Angptl8Nestin CKO) to study the function ofPage 9 of 21

neuronal ANGPTL8 in vivo (Fig. 2A). Angptl8 CKO wasconfirmed by the fact that neuronal ANGPTL8 expression was significantly eliminated in the hippocampus(Fig. 2C and D) and that ANGPTL8 was undetectable in
the CSF (Fig. 2B). We next examined whether the conditional deletion of Angptl8 in neurons affected hippocampal development and cognitive function. As shown in
Fig. 2E, F and Fig. 3A, there was no significant difference
in body weight (Fig. 3A), neuronal number or neuronal
distribution in the hippocampus (Fig. 2E and F) between
Loxp/loxp and Angptl8Nestin CKO mice. Moreover, wefound that conditional deletion of Angptl8 in neurons
did not affect the cognitive function of normal diet-fed
mice based on behavior tests including the open fieldtest (sFig. 4A), novel object recognition test and Barnes
maze test (Fig. 2G–J). Taken together, these results indicated that conditional deletion of Angptl8 in neurons
did not affect the development of neurons or cognitive
function. Furthermore, depletion of Angptl8 in the CNS
did not affect body weight (Fig. 3A) and levels of glucose(Fig. 3B–E) or lipids (Fig. 3F) in mice fed an ND or HFD.
Similar to the results shown in Fig. 1, mice fed an HFD
for 9 months exhibited higher CSF ANGPTL8 levels thanND-fed mice in Loxp/loxp group but not Angptl8Nestin
CKO group mice (Fig. 2B). Blood glucose levels and insulin resistance were also significantly elevated in mice fed
an HFD compared to ND-fed mice, but there was no difference between Loxp/loxp and Angptl8Nestin CKO mice
regardless of whether they were fed an HFD (Fig. 3B–E).
Significantly less distance traveled and velocity wereobserved in 9-month-HFD-fed mice than in controls in
the open field test (sFig. 4A). Hippocampus-dependent
spatial memory is particularly vulnerable to diabetesassociated cognitive dysfunction, so the performance ofmice in the new object recognition test and the Barnes
maze task was tested. The cognitive function of Loxp/
loxp mice fed an HFD for 9 months was impaired, as
compared with control mice, they exhibited decreases inrecognition index (Fig. 2G and H), but not general activity (sFig. 4B), in the novel object recognition test; anincreased number of errors in the Barnes maze (Fig. 2I
and J); more microglia (marked by IBA-1) infiltrating
the hippocampal CA1 region (Fig. 3G); higher levels of
inflammatory markers (CD11b and CD16/32) (Fig. 3G
and H); fewer synapses (Fig. 3I and J); and more swollen
postsynaptic membranes, blurred synaptic sites or missing synaptic sites (Fig. 3I); upregulation of inflammatory
cytokine mRNA expression (Il-1β, Il-6, and Tnf-α); anddecreased expression of axonal growth and synaptic plasticity markers (Gap-43, Snap-25, Nfl, and Syp) (Fig. 3K).
Notably, we observed that compared with ND-fed
Angptl8Nestin CKO mice, HFD-fed Angptl8Nestin CKO mice
exhibited no obvious changes. In both tasks, Angptl8Nestin

Meng et al. Journal of Neuroinflammation(2024) 21:192

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Fig. 2 Depletion of Angptl8 in the central nervous system (CNS) protected against diabetes-associated cognitive dysfunction. A Strategy used
to generate Angptl8Nestin CKO mice. B ANGPTL8 levels in CSF in the indicated groups (n = 5 mice per group). C Western blotting analysis of ANGPTL8
in the brain tissue of the indicated mice. D Immunofluorescence staining showing Angptl8 knockout efficiency in the hippocampus of HFD-fed
Angptl8 conditional knockout (CKO) mice. E Nissl staining images showing Nissl bodies in the hippocampus of male Loxp/loxp and Angptl8Nestin
CKO mice (left). Quantitative analysis of the number of neurons (right) (n = 5 per group). F Double immunostaining of NeuN (green) and IBA-1(red) in the hippocampus of male Loxp/loxp and Angptl8Nestin CKO mice (left). Quantitative analysis of the number of NeuN + and IBA-1 + cells(right) (n = 5 per group). G Representative traces of paths by the indicated mice to explore a novel object (the green square represents the familiar
object, and the purple square represents the novel object). F, familiar object; N, novel object. H Recognition index of the indicated mice (n = 5 mice
per group), calculated as the difference in the amount of time spent exploring the novel and familiar objects (left) and in the frequency of novel
object explorations and familiar object explorations (right). I Traces of paths to the target hole (red) in Barnes maze probe trials for the indicatedmice. J Barnes maze acquisition and probe trials for the indicated groups (n = 5 mice per group). *P < 0.05, **P < 0.01, ****P < 0.0001; + P < 0.05: Loxp/
loxp + HFD group vs. Angptl8Nestin CKO + HFD group; ##P < 0.01: Loxp/loxp + ND group vs. Loxp/loxp + HFD group; ΔP < 0.05: Angptl8Nestin CKO + ND
group vs. Angptl8Nestin CKO + HFD group. NS, nonsignificant. The data are shown as the mean ± s.e.m. and were statistically analyzed by two-way
ANOVA with Bonferroni’s test. CSF: cerebrospinal fluid

CKO mice fed an HFD for 9 months exhibited better cognitive function than their floxed littermates (as controls),
as evidenced by a higher recognition index for the novel
objects (Fig. 2G and H), not general activity (sFig. 4B),
and fewer errors in the Barnes maze test (Fig. 2I and J).
Thus, a reduction in neuronal ANGPTL8 expression indiabetic mice protects hippocampus-mediated memory.
Moreover, the 9-month-HFD-fed Angptl8Nestin CKO
mice exhibited fewer microglia (marked by IBA-1) infiltrating the hippocampal CA1 region (Fig. 3G), fewer
inflammatory markers (CD16/32 and CD11b) and moreanti-inflammatory markers (CD206) (Fig. 3H). Next, we

assessed the synaptic integrity of hippocampal neurons
by electron microscopy analysis (Fig. 3I). Comparedwith 9-month-HFD-fed floxed mice, 9-month-HFD-fed
Angptl8Nestin CKO mice showed increases in synapse
number (Fig. 3I and J). In higher magnification images ofbrain slices of Angptl8Nestin CKO mice, we also observedthat the presynaptic and postsynaptic membranes wereclear and had complete outlines, and the synaptic cleft
was evident (Fig. 3I). Similarly, decreased neuroinflammation and increased synaptic connectivity wereobserved in the hippocampal tissue of Angptl8Nestin CKO
mice, as shown by decreased expression of inflammatory

Meng et al. Journal of Neuroinflammation(2024) 21:192

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Fig. 3 Depletion of Angptl8 in the CNS protected against synaptic damage and alleviated neuroinflammation. A Body weight of the indicatedmice. B, C GTT and area-under-the-curve (AUC) values of the indicated mice. D, E ITT and AUC values of the indicated mice. F Hippocampal
TG levels of the indicated mice. G Immunofluorescence staining of IBA-1 (red) and CD11b (green) in the CA1 region of the hippocampus
in the indicated mice. H Immunofluorescence staining of CD16/32 (red) and CD206 (green) in the hippocampal CA1 region in the indicated mice.
I Representative images of synaptic ultrastructure (red arrow) in the hippocampus. J Quantification of the number of synapses in the indicatedmice. K mRNA expression of proinflammatory cytokines (Il-1β, Il-6, and Tnf-α) and axon growth and synaptic plasticity markers (Gap-43, Snap-25,
Nfl, and Syp) in the hippocampus of the indicated groups (n = 5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001,####P < 0.0001: Loxp/loxp + ND group vs Loxp/loxp + HFD group; ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001, ΔΔΔΔP < 0.0001: Angptl8Nestin CKO + ND groupvs Angptl8Nestin CKO + HFD group. NS: nonsignificant. The data are shown as the mean ± s.e.m. and were statistically analyzed by two-way ANOVA
with Bonferroni’s test

Meng et al. Journal of Neuroinflammation(2024) 21:192

cytokines (Il-1β, Il-6, and Tnf-α) and increased expression of axonal growth and synaptic plasticity markers(Gap-43, Snap-25, Nfl, and Syp) (Fig. 3K).
Moreover, to exclude the off-target effects of Nesting-Cre, AAV-Nestin-Cre was directly injected into
the hippocampus of diabetic floxed mice fed an HFD
for 6 months (sFig. 5A and B). After HFD feeding foran additional 3 months, AAV-Cre + HFD mice exhibited improved cognitive function (sFig. 5C-F), improved
synaptic structure (sFig. 5H-J), and alleviated neuroinflammation (sFig. 5G and J; sFig. 6G) in comparison
to AAV-Control + HFD group mice, with no discernible effects on body weight (sFig. 6A), glucose (sFig. 6C
and D) or lipid (sFig. 6B) metabolism, or general activity(sFig. 6E and F).
ANGPTL8 induced synaptic injury and neuroinflammation

In our previous study, GST pulldown assays confirmed
ANGPTL8-PirB binding [15]. In light of the immunofluorescence colocalization of ANGPTL8 and PirB in the
hippocampal CA1 region (Fig. 4A), we isolated and cultured primary hippocampal cells to identify the potential
cellular location of ANGPTL8-PirB in the hippocampus. We observed that rA8-flag colocalized with PirB
on neurons and microglia cells (Fig. 4B) but not astrocytes or endothelial cells (data not shown). Given that
ANGPTL8 is a secreted hormone and is unable to penetrate the cell membrane, we speculated that PirB is thereceptor for ANGPTL8 on neurons and microglia cells.
To identify the potential functions of ANGPTL8 in neurons, we further analyzed ANGPTL8-inducced changes
in the transcriptional profile of neurons through mRNA
sequencing. Pathway enrichment analysis revealed thatthe upregulated genes in neurons after ANGPTL8 treatment were enriched in pathways associated with axonal
injury (Fig. 4C). Markers of axonal growth and synaptic
plasticity were downregulated after ANGPTL8 treatment (Fig. 4D). Based on the above analysis, the effects of
ANGPTL8 in neurons were further analyzed. We foundthat ANGPTL8 significantly inhibited axonal growth,
reduced synaptic connectivity (Fig. 4E), and downregulated the mRNA expression of axonal growth and synaptic plasticity markers (Gap-43, Snap-25, Nfl, and Syp)
in a dose-dependent manner (Fig. 4F). In addition, we
analyzed transcriptional changes in microglia and foundthat M1 (inflammatory phenotype) marker genes werehighly upregulated after ANGPTL8 treatment (Fig. 4G).
Consequently, we referred to CD11b as an M1 marker
and CD206 as an M2 (anti-inflammatory phenotype)marker with IBA-1 costaining to identify the polarizationof microglia by flow cytometry. After ANGPTL8 stimulation, the CD11b + population was increased, whereas
the CD206 + population was decreased (Fig. 4H). Taken

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together, these results suggest that ANGPTL8 is a proinflammatory protein resulting in aggravated synaptic
injury and microglial M1-type polarization.
PirB mediated ANGPTL8‑induced synaptic injury
and neuroinflammation

PirB is a receptor with an intracellular domain recruiting phosphatases to dephosphorylate downstream signals [46]. Therefore, we measured the phosphorylationof signals downstream of PirB in neurons and microglia.
Induced by ANGPTL8 stimulation, primary neurons
exhibited activated phosphorylation levels of the PirB
downstream proteins AKT and ERK, leading to upregulation of ROCK2 protein expression (a neurite growthinhibiting factor [47]) and inhibiting downstream cofilin
protein phosphorylation (Fig. 5A and B). Consistent withneurons, microglia also exhibited increased phosphorylation levels of AKT, ERK, P38 and P65 after stimulation with ANGPTL8 (Fig. 5G). To investigate whether
the activated signals were specifically mediated by the
ANGPTL8-PirB axis, a PirB blockade assay was performed on neurons and microglia. ANGPTL8-induced
inhibition of neuronal axon growth and microglial proinflammatory polarization were abrogated in the PirB
blockade group compared to control group, as evidenced
by increased expression of axonal growth and synaptic plasticity markers (Gap-43, Snap-25, Nfl, and Syp)
(Fig. 5C), ameliorated synaptic connectivity (Fig. 5D),and downregulation of M1 markers (Il-1β, Il-6, and Tnfα) (Fig. 5H). To further evaluate the underlying mechanisms, we used small-molecule inhibitors to suppress thephosphorylation of signals downstream of PirB (AKT,ERK, P38, and P65). The results showed that inhibiting the phosphorylation of ERK1/2 (U0126) attenuated
ANGPTL8-induced suppression of axonal growth andsynaptic connectivity, whereas inhibitors of p-AKT (MK2206) failed to produce the same results (Fig. 5E and F).
In contrast, inhibitors of p-P38 (SB203580) and p-P65(BAY 11-7082), but not p-AKT or p-ERK1/2, abrogatedthe ANGPTL8-induced production of proinflammatory
cytokines in microglia (Fig. 5I). These data suggested that
ANGPTL8-induced neuronal injury was predominantly
mediated by the ERK1/2 signaling pathway, whereas theproinflammatory polarization of microglia was mediatedby the P38 and P65 signaling pathways.
PirB−/− mice were resistant to ANGPTL8‑induced
neuroinflammation and synaptic injury

To assess whether ANGPTL8 exerts proinflammatory and synapse-damaging effects via PirB in vivo, we
injected exogenous ANGPTL8 into the hippocampusof WT mice and PirB−/− mice for 30 days (Fig. 6A).
There was no significant difference in body weightMeng et al. Journal of Neuroinflammation(2024) 21:192

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Fig. 4 ANGPTL8 induced synaptic injury and neuroinflammation. A Immunofluorescence analysis of ANGPTL8 and PirB in the hippocampal CA1
region of the indicated mice. B Immunofluorescence analysis of colocalization of ANGPTL8 and PirB in mouse neurons and microglia. C Pathway
enrichment analysis of neurons treated with rA8. D Heatmap showing differences in relative mRNA expression in neurons treated with scramble
or rA8. E Immunofluorescence staining of β-III tubulin (green) in neurons treated with scrambled ANGPTL8 or rA8 (1000 ng/ml). F mRNA expressionof axonal growth and synaptic plasticity markers in neurons treated with different concentrations of rA8 for 5 days (n = 6). Comparisons were
performed between untreated groups and treated groups. G mRNA expression of proinflammatory cytokines in microglia treated with different
concentrations of rA8 for 24 h (n = 3). Comparisons were performed between untreated groups and treated groups. H Flow cytometry of microglial
polarization after treatment with scrambled ANGPTL8 or rA8 (1000 ng/ml). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data are shownas the mean ± s.e.m. and were statistically analyzed by one-way ANOVA with Bonferroni’s test. rA8: flag-targeted recombinant ANGPTL8 protein

Meng et al. Journal of Neuroinflammation(2024) 21:192

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Fig. 5 PirB mediated ANGPTL8-induced synaptic injury and neuroinflammation. A Scheme showing the signaling pathway downstream of PirB
as indicated in the literature. B Western blot showing the phosphorylation of AKT, ERK1/2, ROCK2, and Cofilin in mouse neurons stimulatedwith scrambled ANGPTL8 or rA8 (1000 ng/ml). C mRNA expression of axonal growth and synaptic plasticity markers (Gap-43, Snap-25, Nfl, and Syp)
in mouse neurons pretreated with the indicated antibodies before rA8 stimulation (n = 6). D Immunofluorescence staining showing the synaptic
connections of neurons treated with the indicated conditions. E mRNA expression of axonal growth and synaptic plasticity markers (Gap-43,
Snap-25, Nfl, and Syp) in mouse neurons treated by the indicated conditions (n = 6). Comparisons were performed between untreated groups
and treated groups. F Immunofluorescence staining showing the synaptic connections of neurons treated with the indicated conditions. G
Western blot analysis of the phosphorylation of signaling molecules in mouse microglia stimulated with scrambled ANGPTL8 or rA8 (1000 ng/
ml). H mRNA expression of proinflammatory cytokines in mouse microglia cells pretreated with control IgG or an anti-PirB ectodomain antibody
for 30 min followed by 24-h stimulation with scrambled ANGPTL8 or rA8 (1000 ng/ml) (n = 6). I mRNA expression of proinflammatory cytokines
in mouse microglia treated with the indicated conditions (n = 6). Comparisons were performed between untreated groups and treated groups. The
data are shown as the mean ± s.e.m. and were statistically analyzed by two-tailed Student’s t test (C, H) or one-way ANOVA with Bonferroni’s test (E,I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS: nonsignificant; rA8: flag-targeted recombinant ANGPTL8 protein; MK-2206: p-AKT inhibitor;
U0126: p-ERK1/2 inhibitor; SB203580: p-P38 inhibitor; BAY11-7082: p-P65 inhibitor(sFig. 7A), glucose (sFig. 7C and D), or lipids (sFig. 7B)
levels between WT mice and PirB−/− mice after injection of exogenous ANGPTL8 into the hippocampus.

Moreover, no significant difference was observed
between ANGPTL8 protein-treated mice and controls
in distance traveled or velocity (sFig. 7E). ComparedMeng et al. Journal of Neuroinflammation(2024) 21:192

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Fig. 6 PirB−/− mice were resistant to ANGPTL8-induced neuroinflammation and synaptic injury. A Experimental scheme. B Representative
traces of paths by the indicated mice to explore a novel object (the green square represents the familiar object, and the purple square
represents the novel object). F, familiar object; N, novel object. C Recognition index of the indicated mice (n = 5 mice per group), calculated
as the difference in the amount of time spent exploring the novel and familiar objects (left) and in the frequency of novel object explorations
and familiar object explorations (right). D Traces of paths to the target hole (red) in Barnes maze probe trials for the indicated mice. E
Barnes maze acquisition and probe trials for the indicated groups (n = 5 mice per group). F Immunofluorescence staining of IBA-1 (red)and CD11b (green) in the hippocampal CA1 region of the indicated mice. G Representative images of synaptic ultrastructure (red arrow)in the hippocampus of the indicated mice. H Quantification of the number of synapses in the hippocampus of the indicated mice. I mRNA
expression of proinflammatory cytokines (Il-1β, Il-6, and Tnf-α) and axonal growth and synaptic plasticity markers (Gap-43, Snap-25, Nfl, and Syp)in the hippocampus of the indicated groups. *P < 0.05, **P < 0.01, ***P < 0.001; +P < 0.05, ++P < 0.01: WT + rA8 group vs. PirB−/− + rA8 group; #P < 0.05,##P < 0.01, ###P < 0.001: WT + Scrambled group vs. WT + rA8 group. NS, nonsignificant. The data are shown as the mean ± s.e.m. (n = 5 mice
per group) and were statistically analyzed by two-way ANOVA with Bonferroni’s test. rA8, flag-targeted recombinant ANGPTL8 protein

Meng et al. Journal of Neuroinflammation(2024) 21:192

with those injected with the control protein, WT mice
injected with the ANGPTL8 protein exhibited a lower
recognition index for the novel objects (Fig. 6B and C)
but not lower general activity (sFig. 7F); more errors in
the Barnes maze test (Fig. 6D and E); more microglia(marked by IBA-1) infiltrating the hippocampal CA1
region (Fig. 6F); higher levels of inflammatory markers(CD11b and CD16/32) (Fig. 6F; sFig. 7G); fewer synapses(Fig. 6G and H); more swollen postsynaptic membranes,
blurred synaptic sites or missing synaptic sites (Fig. 6G);
upregulation of inflammatory cytokine mRNA expression (Il-1β, Il-6, and Tnf-α); and decreased expression ofaxonal growth and synaptic plasticity markers (Gap-43,
Snap-25, Nfl, and Syp) (Fig. 6I). Notably, we observed that
PirB−/− mice had no obvious changes after ANGPTL8
injection relative to the control group (Fig. 6B–I), indicating that PirB−/− mice were resistant to ANGPTL8induced neuroinflammation and synaptic injury.

Discussion
In diabetes-associated cognitive dysfunction, the hippocampus is characterized by damaged neuronal axons
and synapses and elevated proinflammatory cytokines[48]. In the present study, elevated ANGPTL8 levels were
found in the hippocampus and CSF of mouse models
with diabetes-associated cognitive dysfunction, whichbind with their receptor (PirB) on neurons and microglia to trigger neuronal damage and neuroinflammation
through the PirB-mediated signaling pathway, indicatingthat ANGPTL8 directly causes the impairment of neurons (Fig. 7).
We and others have found elevated levels of circulating ANGPTL8 in individuals with diabetes [11,49]. The present study revealed that ANGPTL8 levels increased not only in the circulation but also in the
CSF of those afflicted by diabetes-associated cognitive
dysfunction. These results indicate that ANGPTL8 may
induce cognitive impairment in patients with diabetes.
Among patients with type 2 diabetes, not difference for
BMI, HbA1c, fasting blood glucose, and TG levels in
those with and without cognitive decline, suggests that
increased ANGPTL8 expression in the CSF of diabetic
patients with cognitive decline was not associated withbody weight or blood glucose levels. Recent studies on
diabetes-associated cognitive dysfunction have shownthat the development of cognitive dysfunction may berelated to brain insulin resistance, persistent low-grade
inflammation [50]. Therefore, we speculate that the elevation of ANGPTL8 levels in the brain may be related tothese factors, as ANGPTL8 is one of important inflammatory factors [15, 51].
Our study also found that ANGPTL8 in the CSF wasderived from brain tissue rather than crossing via the

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blood‒brain barrier from circulation. Chronic low-grade
inflammation, which occurs in diabetic conditions [24,52], leads to ANGPTL8 production in the CA1 regionof the hippocampus, where more microglial aggregation is detected in diabetic mice. In addition to theabove reasons, insulin resistance may also contribute toincreased ANGPTL8 production in brain tissue. It has
been reported that insulin resistance occurs in the brain
in individuals with diabetes and cognitive impairment[50] and causes increased production of ANGPTL8 [15,53]. In this study, ANGPTL8 levels in the brain increased
regardless of weight gain in diabetic mice. Therefore,
factors other than obesity, such as neuroinflammation,
brain insulin resistance, and a high glucose environment
in the brain, may be the main inducers for the increased
ANGPTL8 levels.
The present study also showed that depletion of
Angptl8 in the CNS prevented microglial infiltration,
protected neurons from damage, and protected cognitive
function. Moreover, no statistically significant difference
was observed in neural development, body weight, GTT,
ITT, or hippocampal TG levels between mice with depletion of Angptl8 in the CNS and their control littermatesfollowing ND or HFD feeding. These results indicate that
increased ANGPTL8 levels in the hippocampus rather
than other factors are important for the occurrence ofdiabetes-related neuroinflammation and cognitive dysfunction. This is consistent with our previous study
showing that hepatocyte-specific Angptl8 depletion
reduced hepatic inflammation [15]. This finding provides
ANGPTL8 as an innovative mechanism for diabetesassociated cognitive dysfunction.
PirB, a receptor expressed in neurons, is recognized
for its role in inhibiting neurite outgrowth and impacting nervous system plasticity [54, 55]. In our previous
studies, it was found that ANGPTL8 interacts with PirB
on macrophages [15]. This is also consistent with recent
findings highlighting that ANGPTL8 binds to PirB on
cardiac cells and hepatocytes [56, 57]. In this study, we
identified the colocalization of ANGPTL8 and PirB in
both neurons and microglia. Notably, previous studies
have demonstrated that PirB is involved in cognitive dysfunction through its modulation of axon outgrowth andsynaptic plasticity [17, 26].
Considering that local secretion and diffusion of cells
result in a concentration gradient of secreted proteinsaround the cell [58], protein concentrations in the CSF
or plasma does not accurately reflect protein concentrations in extracellular fluids [59]. Since the actual
ANGPTL8 concentration in the extracellular fluidsof the CNS was unclear, we conducted concentration
gradients to observe the effects of different ANGPTL8
concentrations, which was consistent with the previous

Meng et al. Journal of Neuroinflammation(2024) 21:192

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Hippocampus

Healthy brain

Diabetic brain withcognitive dysfunction

Diabetic brain

Up-regulate

Inflammatory
cytokines

NeuronMicroglia

Inhibit axon growth anddestabilize synapses

A8

PirB
NeuronProinflammatory
cytokine↑

M2 microglia

cytomembrane
Damaged neuron

M1 microglia

Fig. 7 Schematic showing that ANGPTL8 exaggerates diabetes-associated cognitive dysfunction by interacting with PirB on microglia and neurons.
ANGPTL8 expression is increased along with decreased expression of synaptic and axonal markers and increased expression of proinflammatory
cytokines in the hippocampus of diabetic brains with cognitive dysfunction. The colocalization of ANGPTL8 and paired immunoglobulin-likereceptor B (PirB) on the surface of neurons and microglia membranes downregulates synaptic and axonal markers of neurons and upregulates
the expression of proinflammatory cytokines in microglia, which reveals the role of ANGPTL8 in the pathogenesis of diabetes-associated cognitive
dysfunction and identifies the ANGPTL8-PirB signaling pathway as a potential target for the management or treatment of diabetes-associatedcognitive dysfunction

studies [56]. The results of mRNA sequencing revealed
that ANGPTL8 contributed to axonal injury. The present study further provided evidence that PirB mediated the actions of ANGPTL8 in inhibiting axonal
growth, reducing synaptic connectivity, and diminishing the expression levels of axonal growth and synaptic
plasticity markers (Gap-43, Snap-25, Syp, and Nfl) in
hippocampal neurons. These results are consistent withprior research, which has shown that PirB is related toreduced axon growth markers in neurodegenerative
disease [26, 37, 39].
Moreover, this study delved into the role of
ANGPTL8 in CNS inflammation. We found a substantial upregulation of M1 markers in microglia after
ANGPTL8 treatment, accompanied by an increased
M1 population and a decreased M2 population. Given
the pivotal role of microglia-mediated inflammationin diabetes-associated cognitive dysfunction [60], thisMeng et al. Journal of Neuroinflammation(2024) 21:192

finding implies that ANGPTL8, as a proinflammatory
factor secreted from neurons and deposited in the hippocampus of the diabetic brain, may act in parallel toneurons and microglia in injuring synaptic plasticity,
promoting neuroinflammation, and impairing cognitive
performance and is thus involved in the development
of diabetes-associated cognitive dysfunction. These
results support the viewpoint that ANGPTL8 acts as
a proinflammatory factor in pathologic conditions [15,51, 61, 62].
For the molecular mechanisms underlying neuronal damage induced by ANGPTL8, primary neurons
exhibited activated signaling pathways (AKT and ERK)
downstream from PirB after ANGPTL8 stimulation, subsequently leading to the activation of ROCK2 signaling,of which upregulation disrupts axon growth and synaptic
stability and impairs spatial learning and memory [47].
In addition, ANGPTL8 treatment induced an increasein the activity of cofilin, reflected by its lower phosphorylation status. As a downstream effector of ROCK, highcofilin activity can inhibit the extension of neurites [17,47]. Moreover, ANGPTL8-induced destruction of neuronal network structure and damage to axons and synapses were eliminated by blocking PirB or inhibiting its
downstream signals. These findings are consistent withprevious studies indicating that blocking PirB reverses
the inhibition of neurite outgrowth [54] and that PirB
may be involved in the modulation of synaptic plasticity
through ROCK2/cofilin signaling [17, 26, 63, 64].
Similarly, to elucidate the underlying mechanisms of
ANGPTL8-induced neuroinflammation, we detected
inflammatory signals and found that the NF-κB, AKT,ERK1/2, and P38 signaling pathways in microglia
strongly responded to ANGPTL8 stimulation. Moreover, ANGPTL8-induced inflammation was abrogated by
PirB blockade with neutralizing antibodies and inhibitorsof P38 and P65, further indicating that ANGPTL8-associated signals are downstream of PirB. These results are
consistent with previous studies showing that PirB regulates the phosphorylation of AKT, ERK1/2, P38, and P65[56].
Furthermore, PirB−/− mice were used to further confirm the role of PirB in vivo in this study. The results
showed that knockout of PirB reduced neuroinflammation, alleviated neuronal damage, and improved cognitive dysfunction induced by ANGPTL8. These findings
shed mechanistic light on previous studies suggestingthat PirB participates in a key pathological process of thenervous system, specifically axonal regeneration inhibition in mediating cognitive decline [16, 17]. This suggeststhat targeting ANGPTL8-PirB axis signaling may provide
a therapeutic strategy for diabetes-associated cognitive
dysfunction.

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Nevertheless, there are several limitations to consider
in this study. First, the molecular mechanism by which
the expression of ANGPTL8 in neurons is increased
should be identified. Second, we did not perform studies
in female mice. Moreover, our study focused on the plasticity of the neural structure, but whether such neuromorphological alterations are accompanied by functionalchanges remains to be identified in our next work.
Collectively, ANGPTL8-PirB is an important signaling
axis for the pathogenesis of diabetes-associated cognitive
dysfunction via crosstalk between neurons and microglia.
It is possible that increased ANGPTL8 levels in the diabetic hippocampus result in the exacerbation of neuronaldamage, the induction of microglial polarization, and theenhancement of neuroinflammation. Thus, blocking thebinding of ANGPTL8 to PirB may be a promising strategy for the management of diabetes-associated cognitive
dysfunction that reduces inflammation and preserves
neuronal function. In conclusion, this study reveals apreviously unrecognized role of ANGPTL8 in the pathogenesis of diabetes-associated cognitive dysfunction andidentifies the ANGPTL8-PirB signaling pathway as apotential target for the management of diabetes-associated cognitive dysfunction in the future.
Abbreviations
T2D	Type 2 diabetes mellitus
ANGPTL8	Angiopoietin-like protein 8
PirB	Paired immunoglobulin-like receptor B
RIFL	Refeeding induced in fat and liver
LILRB	Leukocyte immunoglobulin-like receptor B
CNS	Central nervous system
MMSE	Mini-Mental State Examination
MoCA	Montreal cognitive assessment
MCI	Mild cognitive impairment
CSF	Cerebrospinal fluid
WT	Wild-type
Angptl8Nestin CKO	CNS-specific Angptl8-depletioncKO	Conditional knockout
gRNA	Guide RNA
PirB−/−	PirB knockout
HFD	High-fat diet
ND	Normal dietrA8	Recombinant ANGPTL8 proteinmin	Minute
GTT​	Glucose tolerance test
ITT	Insulin tolerance test
LPS	LipopolysacchariderA8-flag	Flag-targeted recombinant ANGPTL8 protein
i.p.	IntraperitoneallyqPCR	Quantitative polymerase chain reaction
ELISA	Enzyme-linked immunosorbent assay
TG	TriglyceridepAbs	Primary antibodies
mAb	Monoclonal antibody
PBS	Phosphate-buffered saline
DAPI	4’,6-Diamidino-2-phenylindole
PFA	Paraformaldehyde
PLL	Poly-l-lysine
DMEM/F12	
Dulbecco’s modified Eagle’s medium/nutrient mixture
F-12
FBS	Fetal bovine serum
FC	Fold change

Meng et al. Journal of Neuroinflammation(2024) 21:192

BSA	Bovine serum albumin
RT‒qPCR	Real-time quantitative PCR
PVDF	Polyvinylidene fluoride
TBST	Tris-buffered saline containing Tween-20
BMI	Body mass index
Gap-43	Growth-associated protein-43
Snap-25	Synaptosomal-associated protein 25
Nfl	Neurofilament light chain
Syp	Synaptophysin
LTP	Long-term potentiation
HbA1c	Glycated hemoglobin

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03183-8.
Supplementary Material 1.
Supplementary Material 2.
Supplementary Material 3.
Acknowledgements
We would like to thank all the participants in the study.
Author contributions
X.M., D.L., Y.Y. and X.Y. participated in the research design. X.M., D.L., and R.K.
conducted the experiments. Y.X., L.P., Y.G., P.Y., P.L., H.Z., L.H., Y.Z., B.M., Y.H., L.X.,
J.X., X.L., W.L., Y.C., S.Z. contributed to data analysis and interpretation. X.M. and
D.L. drafted the paper. X.Y. and Y.Y. supervised the study and revised the paper.
All authors contributed to the paper and approved the final manuscript.
Funding
This work was supported by Grants from the National Natural Science
Foundation of China (82270880, 81974109 and 81570740).
Availability of data and materials
All data needed to evaluate the conclusions in the manuscript are present in
the manuscript and the supplementary Materials. All methods and protocols
used are included in the main manuscript or supplementary files. All reagents,
antibodies, and resources are listed in the main manuscript. The data sets used
and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Declarations
Ethics approval and consent to participate
The study protocols of participants (approval number: TJ-IRB20230452) wereapproved by the Committee on Human Research at Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology. All animal
protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Model Animals of Tongji Hospital, Huazhong Universityof Science and Technology. The animals received humane care according to
the criteria outlined in the Guide for the Care and Use of Laboratory Animals
prepared by the National Academy of Sciences and published by the National
Institutes of Health.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Division of Endocrinology, Department of Internal Medicine, Tongji Hospital,
Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430030, China. 2 Branch of National Clinical Research Center for Metabolic Diseases, Wuhan, Hubei, China. 3 Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,

Page 19 of 21

Wuhan, China. 4 Computer Center, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, China.
Received: 22 January 2024 Accepted: 22 July 2024

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