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Contents lists available at ScienceDirect
Neurochemistry International
journal homepage: www.elsevier.com/locate/neuint
Phospholipase C-related catalytically inactive protein
regulateslipopolysaccharide-induced hypothalamic
inflammation-mediated anorexiain miceYosuke Yamawakia,b, Satomi
Shirawachia, Akiko Mizokamic, Kanako Nozakid, Hikaru Itod,e,Satoshi
Asanoa, Kana Ouea,f, Hidenori Aizawad, Shigeto Yamawakig, Masato
Hiratah,Takashi Kanematsua,i,∗a Department of Cellular and
Molecular Pharmacology, Division of Basic Life Sciences, Graduate
School of Biomedical and Health Sciences, Hiroshima University,
1-2-3Kasumi, Minami-ku, Hiroshima, 734-8553, Japanb Laboratory of
Advanced Pharmacology, Daiichi University of Pharmacy, 22-1
Tamagawa-cho, Minami-ku, Fukuoka, 815-8511, Japanc OBT Research
Center, Faculty of Dental Science, Kyushu University, Fukuoka,
812-8582, Japand Department of Neurobiology, Division of Basic Life
Sciences, Graduate School of Biomedical and Health Sciences,
Hiroshima University, 1-2-3 Kasumi, Minami-ku,Hiroshima, 734-8551,
Japane Center for Experimental Animals, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japanf
Department of Dental Anesthesiology, Division of Applied Life
Sciences, Graduate School of Biomedical and Health Sciences,
Hiroshima University, Hiroshima, 734-8553,Japang Department of
Psychiatry and Neurosciences, Graduate School of Biomedical and
Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku,
Hiroshima, 734-8551, Japanh Oral Medicine Research Center, Fukuoka
Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japani
Department of Cell Biology and Pharmacology, Faculty of Dental
Science, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka,
812-8582, Japan
A R T I C L E I N F O
Keywords:AKTAnorexiaHypothalamusInflammationPRIPSTAT3
A B S T R A C T
Peripheral lipopolysaccharide (LPS) injection induces systemic
inflammation through the activation of the in-hibitor of nuclear
factor kappa B (NF-κB) kinase (IKK)/NF-κB signaling pathway, which
promotes brain dys-function resulting in conditions including
anorexia. LPS-mediated reduction of food intake is associated
withactivation of NF-κB signaling and phosphorylation of the
transcription factor signal transducer and activator
oftranscription 3 (STAT3) in the hypothalamus. We recently reported
phospholipase C-related catalytically in-active protein (PRIP) as a
new negative regulator of phosphatidylinositol 3-kinase/AKT
signaling. AKT regulatesthe IKK/NF-κB signaling pathway; therefore,
this study aimed to investigate the role of PRIP/AKT signaling
inLPS-mediated neuroinflammation-induced anorexia.
PRIP gene (Prip1 and Prip2) knockout (Prip-KO) mice
intraperitoneally (ip) administered with LPS exhibitedincreased
anorexia responses compared with wild-type (WT) controls. Although
few differences were observedbetween WT and Prip-KO mice in
LPS-elicited plasma pro-inflammatory cytokine elevation,
hypothalamic pro-inflammatory cytokines were significantly
upregulated in Prip-KO rather than WT mice. Hypothalamic AKT andIKK
phosphorylation and IκB degradation were significantly increased in
Prip-KO rather than WT mice, in-dicating further promotion of
AKT-mediated NF-κB signaling. Consistently, hypothalamic STAT3 was
furtherphosphorylated in Prip-KO rather than WT mice. Furthermore,
suppressor of cytokine signaling 3 (Socs3), anegative feedback
regulator for STAT3 signaling, and cyclooxogenase-2 (Cox2), a
candidate molecule in LPS-induced anorexigenic responses, were
upregulated in the hypothalamus in Prip-KO rather than WT mice.
Pro-inflammatory cytokines were upregulated in hypothalamic
microglia isolated from Prip-KO rather than WT mice.
Together, these findings indicate that PRIP negatively regulates
LPS-induced anorexia caused by pro-in-flammatory cytokine
expression in the hypothalamus, which is mediated by AKT-activated
NF-κB signaling.Importantly, hypothalamic microglia participate in
this PRIP-mediated process. Elucidation of
PRIP-mediatedneuroinflammatory responses may provide novel insights
into the pathophysiology of many brain dysfunctions.
https://doi.org/10.1016/j.neuint.2019.104563Received 3 March
2019; Received in revised form 9 September 2019; Accepted 2 October
2019
∗ Corresponding author. Department of Cell Biology and
Pharmacology, Faculty of Dental Science, Kyushu University,
Fukuoka, 812-8582, Japan.E-mail address:
[email protected] (T. Kanematsu).
Neurochemistry International 131 (2019) 104563
Available online 04 October 20190197-0186/ © 2019 The Authors.
Published by Elsevier Ltd. This is an open access article under the
CC BY license (http://creativecommons.org/licenses/BY/4.0/).
T
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1. Introduction
Infectious diseases induce peripheral inflammation, which
caneventually spread to the central nervous system (CNS). This
causesneuroinflammation accompanied by upregulation of
pro-inflammatorycytokines in the brain. The spread of inflammation
leads to brain dys-function, manifesting in symptoms such as fever,
sleep disorders, de-pression-like behavior, and anorexia (Bluthe et
al., 2000; Dantzer et al.,2008; Hart, 1988; Kelley et al.,
2003).
Anorexia is a common symptom of infectious diseases (Grunfeld
andFeingold, 1992; Hart, 1988). A transient reduction in food
intake in-hibits pathogen proliferation owing to decreased nutrient
availability(Murray and Murray, 1979). However, chronic suppression
of foodintake induces malnutrition and may impair the host immune
system(Grinspoon and Mulligan, 2003). Hence, elucidation of the
mechanismunderlying infectious disease-induced anorexia is
important for im-proving the nutritional status of affected
individuals and quality of life.
Peripheral lipopolysaccharide (LPS) administration in mice
iswidely used to establish a mouse model of fever and other
brain-mediated illness responses. LPS activates toll-like receptor
4 (TLR4)-mediated nuclear factor kappa B (NF-κB) signaling via
myeloid differ-entiation primary-response protein 88
(MyD88)-dependent or in-dependent pathways, resulting in the
production of pro-inflammatorycytokines and chemokines (Akira and
Takeda, 2004). Peripheral in-jection of LPS induces the elevation
of hypothalamic pro-inflammatorycytokines and anorexia (Jang et
al., 2010), whereas the deficiency ofMyD88 inhibits LPS-induced
anorexia and downregulates hypothalamicpro-inflammatory cytokines
(Ogimoto et al., 2006). Hence, peripheralLPS administration-induced
elevation of hypothalamic pro-in-flammatory responses is essential
in LPS-induced anorexia.
NF-κB, a transcription factor, is a regulator of genes involved
ininflammation and innate immunity, including interleukin-1 beta
(IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha
(TNF-α). NF-κB is regulated by an inhibitor of NF-κB kinase (IKK)
downstream of theLPS-stimulated TLR4/MyD88 pathway (Akira and
Takeda, 2004), andinhibition of NF-κB signaling abolishes
LPS-induced anorexia (Janget al., 2010). Appetite is also regulated
by signal transducer and acti-vator of transcription 3 (STAT3)
activation in the hypothalamus (Vaisseet al., 1996). We previously
reported that peripheral LPS administrationin mice induces IL-6
expression in the hypothalamus, activating Januskinase (JAK) via a
transmembrane receptor, gp130, and phosphor-ylating STAT3, leading
to anorexia (Yamawaki et al., 2010). Thesefindings suggest that
NF-κB and STAT3 activity are fundamentally in-volved in LPS-induced
anorexia.
LPS-stimulated TLR4/MyD88 mediates the activation of the
phos-phatidylinositol 3-kinase (PI3K)/AKT signaling pathway,
activatingIKK/NF-κB and elevating pro-inflammatory cytokines, and
the
produced pro-inflammatory cytokines activate PI3K/AKT and
NF-κBsignaling per se (Ojaniemi et al., 2003; Ozes et al., 1999).
However, it isunknown whether PI3K-induced AKT signaling regulates
IKK-inducedNF-κB signaling and pro-inflammatory cytokine production
in the hy-pothalamus in an LPS-treated animal model. AKT signaling
is activatedthrough PI3K-mediated conversion of
phosphatidylinositol 4,5-bispho-sphate [PI(4,5)P2], an integral
signaling molecule and a minor com-ponent of cellular membranes,
into phosphatidylinositol 3,4,5-trispho-sphate [PI(3,4,5)P3].
Cytosolic inactive AKT is recruited to themembrane and engages
PI(3,4,5)P3 by interacting with the PH domain.This leads to
phosphorylation of T308 and S473 by phosphoinositide-dependent
protein kinase 1 and mechanistic target of rapamycin com-plex 2,
respectively, resulting in maximal activation. PI(4,5)P2 is
alsoconverted to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] by
phospholi-pase C (PLC) (Bunney and Katan, 2010). We recently
reported that PLC-related catalytically inactive protein (PRIP),
which loses the enzymaticactivity of PLC, modulates the metabolism
of PI(4,5)P2 to PI(3,4,5)P3and regulates PI3K-mediated AKT
signaling (Asano et al., 2017). ThePLC enzyme-dead molecule PRIP
was originally identified as an Ins(1,4,5)P3-binding protein with
domain organization similar to that ofPLCδ1 (Kanematsu et al.,
1992, 1996, 2000). Although we identifiedthe roles of PRIP in
Ins(1,4,5)P3-mediated intracellular Ca2+ signaling(Harada et al.,
2005; Takeuchi et al., 2000), its involvement in PI(4,5)P2-mediated
cell signaling remains unclear. Because PI3K/AKT sig-naling is
correlated with IKK/NF-κB signaling (Bai et al., 2009; Ozeset al.,
1999), PRIP may regulate LPS-mediated inflammatory responsesand
appetite. Therefore, in the present study, we examined the
phos-pholipid signaling-regulated AKT/NF-κB pathway and
inflammatoryresponses in the hypothalamus and elucidated PRIP
involvement in theregulation of anorexia using LPS-administered
Prip knockout mice.
2. Materials and methods
2.1. Animals
Experiments were conducted using 10–15-week-old male mice
withsimilar body weight. Two mammalian homologs of PRIP, PRIP1
andPRIP2, are also known as PLC-like 1 (PLCL1) and PLCL2,
respectively(Kanematsu et al., 1996; Uji et al., 2002). Prip1
(Plcl1)-KO and Prip2(Plcl2)-KO mouse strains were mated to produce
Prip1 and Prip2 doubleknockout (Prip-KO) mice and their
corresponding wild-type (WT) mice,as described previously
(Kanematsu et al., 2002; Mizokami et al.,2007). Briefly,
heterozygous (Prip1+/−, Prip2+/−) mice, both of whichwere
backcrossed to a parental C57BL/6 strain at least 11 times
(N11),were mated to generate a Prip-KO strain and a corresponding
WT strain.Each strain of littermates was mated inter se, and
Prip-KO or WThomozygotes were obtained. To obtain the required
number of
Abbreviations
CNS central nervous systemCox2 cyclooxygenase-2ELISA
enzyme-linked immunosorbent assayGAPDH glyceraldehyde phosphate
dehydrogenaseIba1 ionized calcium-binding adapter molecule 1IκB
inhibitor of NF-κBIKK inhibitor of NF-κB kinaseIL-1β interleukin-1
betaIL-6 interleukin-6Ins(1,4,5)P3 inositol 1,4,5-trisphosphateip
intraperitonealJAK Janus kinaseLPS lipopolysaccharideMyD88 myeloid
differentiation primary-response protein 88
NF-κB nuclear factor kappa BNIK NF-κB-inducing kinasePI(3,4,5)P3
phosphatidylinositol 3,4,5-trisphosphatePI(4,5)P2
phosphatidylinositol 4,5-bisphosphatePI3K phosphatidylinositol
3-kinasePLC phospholipase CPLCL1 PLC-like 1PRIP PLC-related
catalytically inactive proteinPrip-KO Prip1 and Prip2 double
knockoutqPCR quantitative real-time polymerase chain reactionRT-PCR
reverse-transcription polymerase chain reactionSocs3 suppressor of
cytokine signaling 3STAT3 signal transducer and activator of
transcription 3TLR4 toll-like receptor 4TNF-α tumor necrosis factor
alphaWT wild-type
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experimental mice, we mated each strain of mice inter se, and
micebeyond the 8 generation were used for the experiments. Mice
werereared in a pathogen-free facility at 22 °C–24 °C, with a 12-h
light/darkcycle (lights on at 8:00 a.m., lights off at 8:00 p.m.)
at HiroshimaUniversity in Japan, and they were fed a normal
laboratory diet andwater ad libitum. This study was approved by the
Animal Care and UseCommittees of Hiroshima University (permission
number: A14-189-1–3), Kyushu University (permission numbers:
A19-174 and 26–99),and Daiichi University of Pharmacy (permission
numbers: R01-017 and2019-002) and was performed in accordance with
the Guide for AnimalExperimentation Regulation of Hiroshima
University, Kyushu Uni-versity, and Daiichi University of
Pharmacy.
2.2. LPS administration
An intraperitoneal (ip) injection of LPS (100 μg/kg, 055:B5;
Sigma-Aldrich, St. Louis, MO, USA) was at 5 mL/kg. Mice were
euthanized viadecapitation under anesthesia with pentobarbital (50
mg/kg, ip), andthe brain was rapidly dissected out. The
hypothalamus was rapidly
dissected using mouse brain stereotaxic coordinates; a
3-mm-thickcoronal brain slice was obtained with brain matrices (EM
Japan, Tokyo,Japan; antero-posterior −3.0 mm to the bregma) and
placed in ice-coldPBS; the hypothalamus was then cut in pieces with
3 mm of width(medial-lateral ± 1.5 mm) with 2 mm of thickness from
the ventralside. The samples were snap-frozen in liquid nitrogen
and stored at−80 °C.
2.3. Antibodies
The primary antibodies used were as follows:
anti-phospho-STAT3(Tyr705; #9131, 1:1000), anti-STAT3 (#4904,
1:1000), anti-phospho-AKT (Thr308; #2965, 1:1000), anti-phospho-AKT
(Ser473; #4060,1:1000), anti-AKT (#2920, 1:1000), anti-phospho-IKK
α/β (Ser176,Ser180; #2697, 1:1000), anti-IKKα (#2682, 1:1000),
anti-IKKβ(#2678, 1:1000), and anti-inhibitor of NF-κB (IκB) (#4814,
1:1000)antibodies were purchased from Cell Signaling Technology,
(Danvers,MA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase
(GAPDH;#MAB374, 1:1000) antibody was purchased from Merck
Millipore
Fig. 1. High-sensitivity phenotype of lipopoly-saccharide
(LPS)-induced anorexia in Prip-KOmice. (A) Schematic timeline
representing theexperimental design and food intake measure-ment.
Mice were habituated to a novel environ-ment for 5 days followed by
an intraperitoneal (ip)saline injection (dummy) twice. The mice
weresubjected to saline ip injection (control) on day 7followed by
LPS ip injection (100 μg/kg) on day 8.(B and C) Measurement of body
weight every 4 hafter saline and LPS injection (B) and analysis
ofbody weight change over 24 h (C). (D and E)Cumulative food intake
over 24 h (D) and for each4-h period (E) are shown. Cont,
saline-treatedmice; LPS, LPS-treated mice; WT, wild-type mice;KO,
Prip-knockout mice. Gray solid color in B andE indicates the dark
phase (from 8:00 p.m. to 8:00a.m.). Data are presented as the mean
± standarderror of the mean (n = 24 for each group).*p < 0.05
between the indicated groups,†p < 0.05 versus the WT saline
value at each timepoint, ‡p < 0.05 versus the KO saline value
ateach time point (ANOVA with Tukey's honestlysignificant
difference post-hoc comparison).
Y. Yamawaki, et al. Neurochemistry International 131 (2019)
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(Darmstadt, Germany). Anti-PRIP1 and anti-PRIP2 polyclonal
anti-bodies (1:1000) were developed previously (Kanematsu et al.,
2002;Mizokami et al., 2007). Horseradish peroxidase-conjugated
anti-rabbitIgG (#AP132P, 1:10000) and anti-mouse IgG (#AP124P,
1:10000)secondary antibodies were purchased from Merck
Millipore.
2.4. Measurement of food intake
WT and Prip-KO mice were housed individually before
experiments,with the first day of isolation set as day 0. Saline (5
mL/kg, ip) wasinjected on days 5 and 6 for habituation to
injection. Food intake wasmanually measured every 4 h, after a
saline ip injection at 4:00 p.m. onday 7, followed by an LPS
injection (100 μg/kg, ip) at 4:00 p.m. on day8 (Fig. 1A).
2.5. Preparation of isolated hypothalamic microglia
Microglia were isolated using the MACS system (Miltenyi
Biotec,Teterow, Germany) as previously described (Yamawaki et al.,
2018).Briefly, the hypothalamus was obtained from WT or Prip-KO
mice (5mice per group). Pooled hypothalami were minced in Hank's
balancedsalt solution (Nacalai Tesque, Kyoto, Japan) and then
enzymaticallydigested using a neural tissue dissociation kit
(Miltenyi Biotec) for35 min at 37 °C. Tissue debris was eliminated
with a 70-μm cell strainer,and myelin was eliminated using Myelin
Removal Beads II (MiltenyiBiotec). The obtained single cells were
magnetically labeled withCD11b micro beads II (Miltenyi Biotec) and
loaded onto a MACScolumn (Miltenyi Biotec), and CD11b-positive
cells were isolated asmicroglia. RNA was extracted from the
obtained microglia, using anRNA isolation kit (Arcturus PicoPure
RNA Isolation Kit; Thermo FisherScientific, Waltham, MA, USA).
2.6. Quantitative real-time polymerase chain reaction (qPCR)
analysis
Tissue samples were homogenized in Sepasol RNA I Super G(Nacalai
Tesque) at 10,000 rpm with a polytron homogenizer, and totalRNA was
isolated in accordance with the manufacturer's protocol.cDNA was
synthesized from 0.5 μg of total RNA in a final volume of10 μL,
using the ReverTra Ace qPCR RT Master Mix with gDNA removerkit
(Toyobo, Osaka, Japan) with a thermal cycler (T Professional
BasicGradient 96; Biometra, Göttingen, Germany). Gapdh was
considered theinternal control. For qPCR analysis, two-step qPCR
(Thunderbird SYBRqPCR Mix; Toyobo) was performed with a PikoReal
96-well system(Thermo Fisher Scientific). The cycling protocol was
as follows: DNApolymerase activation at 95 °C for 1 min, followed
by denaturation at95 °C for 15 s and annealing/extension at 60 °C
for 1 min, for 40 cycles.Gene expression was normalized to that of
Gapdh mRNA in the samesamples, using the 2−ΔΔCt method. qPCR was
performed using the fol-lowing primers: Gapdh, forward
5′-AGGTCGGTGTGAACGGATTTG-3′,reverse 5′-GTAGACCATGTAGTTGAGGTCA-3′;
Il-6, forward 5′-ACAACCACGGCCTTCCCTACTT-3′, reverse
5′-CACGATTTCCCAGAGAACATGTG-3′; Il-1b, forward 5′-
AACCTGCTGGTGTGTGACGTTC-3′, reverse5′-CAGCACGAGGCTTTTTTGTTGT-3′;
Tnf-a, forward 5′-GGGGCCACCACGCTCTTCTGTC-3′, reverse 5′-
TGGGCTACAGGCTTGTCACTCG-3′;Suppressor of cytokine signaling 3
(Socs3), forward 5′-GAGATTTCGCTTCGGGACTA-3′, reverse
5′-GCTGGTACTCGCTTTTGGAG-3′; cycloox-ygenase-2 (Cox2), forward
5′-CCACTTCAAGGGAGTCTGGA-3′, reverse5′-AGTCATCTGCTACGGGAGGA-3′,
ionized calcium-binding adaptermolecule 1 (Iba1), forward
5′-TGGTCCCCCAGCCAAGA-3′, reverse5′-CCCACCGTGTGACATCCA-3′; Prip1,
5′-TGAGAATGGGGAAGAAAGTT-3′, reverse 5′-TCTATGGCTTCTCGTAAGGG-3′;
Prip2, 5′-ACTGTGGCTATGTTCTTCGA-3′, reverse
5′-TTTGATGTGAAGCAACTGAG-3′.
2.7. Reverse-transcription PCR (RT-PCR) analysis
RT-PCR analysis was performed using a PCR master mix
(QuickTaq
HS Dye Master Mix; Toyobo) with a thermal cycler (T Professional
BasicGrafient96; Biometra). The primer sequences were the same as
thoseused for qPCR. The cycling protocol was as follows: DNA
polymeraseactivation at 94 °C for 2 min, followed by denaturation
at 94 °C for 30 s,and annealing/extension at 55 °C for 30 s, for 35
cycles (Prip1, Prip2,and Iba1) or 20 cycles (Gapdh). After PCR, the
products were mixedwith an intercalator (UltraPower DNA Safedye;
Gellex International,Tokyo, Japan) and separated on a 2% agarose
gel in 1 × Tris-acetate-EDTA buffer. Amplified products were
captured with a gel imagingsystem (Atto Corporation, Tokyo,
Japan).
2.8. Western blotting
Lysates were prepared using a lysis buffer (20 mM Tris-HCl, pH
7.4,150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 0.1% sodium
deox-ycholate, and 0.1% SDS) containing a protease inhibitor
cocktail(#25955, Nacalai Tesque) and a phosphatase inhibitor
cocktail(#07574, Nacalai Tesque). The samples were centrifuged at
20,600×gfor 30 min at 4 °C, and the supernatants were harvested.
Protein con-centration was determined with a protein assay
bicinchoninate kit(#06385, Nacalai Tesque). The samples were
fractionated using SDS-PAGE and electro-transferred onto a
polyvinylidene fluoride mem-brane. Membranes were subsequently
blocked with 5% skimmed milkfor 3 h (for phospho-STAT3 detection)
or for 1 h (other antibodies),followed by incubation with each
primary antibody overnight at 4 °C.Antibodies were diluted with 2%
skimmed milk. The membranes werewashed with Tris-buffered saline
with Tween 20 and incubated with arespective horseradish
peroxidase-conjugated secondary antibody forprimary antibody
recognition. Immunoreactivity was detected with
anelectrochemiluminescence reagent (ECL; Promega, Fitchburg, WI,
USA)using the ImageQuant LAS 4000 mini imager (GE Healthcare,
Chicago,IL, USA). The density of immunoreactive bands was measured
usingImage J 1.50v software (Wayne Rasband; NIH, Bethesda, MD,
USA).
2.9. Enzyme-linked immunosorbent assay (ELISA) for plasma
cytokinequantification
Up to 150 μL blood was sampled from the orbital plexus of WT
andPrip-KO mice under isoflurane anesthesia before and 1 h after
LPS(100 μg/kg, ip) administration. Plasma prepared from the blood
sam-ples with EDTA was assayed for IL-1β (#KE10003,
Proteintech,Rosemont, IL, USA), IL-6 (#431307, Biolegend, San
Diego, CA, USA),and TNF-α (#430907, Biolegend) with ELISA kits in
accordance withthe manufacturer's instructions.
2.10. Statistical analysis
JMP 8.0.2 (SAS, Cary, NC, USA) was used for statistical
analyses.Data are expressed as mean ± standard error of the mean
values.Student's t-test or ANOVA with Tukey's honestly significant
differencepost-hoc comparison was used. A p-value less than 0.05
was consideredstatistically significant.
3. Results
3.1. Prip-KO mice showed a high-sensitivity phenotype to
LPS-inducedanorexia
To investigate whether PRIP deficiency in mice affects
LPS-inducedanorexia, we measured ad libitum food intake every 4 h
for 24 h aftersaline ip injection (control) followed by the
measuring of food intakeevery 4 h after LPS (100 μg/kg, ip)
injection (Fig. 1A). Body weight wasmeasured during the food intake
measurement (Fig. 1B). Mean weightchange was similar between
Prip-KO and WT mice after the saline in-jection on day 7; however,
significant weight decrease was observed inthe two genotypes after
LPS administration on day 8 compared with the
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respective saline controls on day 7 (Fig. 1C). Notably, greater
weightloss was observed in Prip-KO mice than in WT mice (p =
0.0192). Thesaline-administered WT and Prip-KO mice showed similar
cumulative24-h food intake (Fig. 1D) and food intake for each 4-h
period (Fig. 1E).In contrast, after the subsequent LPS injection,
the total 24-h food in-take decreased significantly by
approximately 65% and 46% in WT andPrip-KO mice, respectively,
compared with food intake in the respectivesaline controls (day 7).
Importantly, food intake reduction in Prip-KOmice was significantly
higher than that in WT mice (Fig. 1D;p = 0.0019). Moreover,
differences were observed in the food intake onday 8 during the 4–8
h and 8–12 h periods after LPS injection(p = 0.029 and p <
0.001, respectively; Fig. 1E), suggesting that Prip-KO mice are
susceptible to LPS-mediated inflammatory responses.
3.2. Higher gene expression of pro-inflammatory cytokines in
Prip-KOhypothalamus
LPS-induced anorexia is implicated in the elevation of
hypothalamicpro-inflammatory cytokines (Jang et al., 2010; Ogimoto
et al., 2006;Wisse et al., 2007). Therefore, we analyzed the gene
expression of IL-1β, IL-6, and TNF-α in the hypothalamus by qPCR.
The expression of Il-1b, Il-6, and Tnf-a was upregulated in the
both genotypes at 2 h afterperipheral LPS injection. However, the
gene expression in Prip-KO micewas significantly higher than that
in WT mice at 2 h (Il-1b, Il-6, and Tnf-a; p = 0.0016, p <
0.0001, and p < 0.0001, respectively) after LPSadministration
(Fig. 2). These results suggest that over-expression
ofpro-inflammatory cytokines occurred in the hypothalamus in an
earlyphase (~2 h) of peripherally administered LPS-induced
neuroin-flammation, which causes a severe anorectic phenotype in
Prip-KOmice.
3.3. Similar plasma levels of peripheral inflammatory biomarkers
in WTand Prip-KO mice
Peripheral inflammation induces brain dysfunction (Dantzer et
al.,2008); therefore, differences in the degree of peripheral
inflammatoryresponses may influence brain inflammation.
Pro-inflammatory cyto-kine mRNAs were upregulated 2 h after LPS ip
injection in the hy-pothalamus in Prip-KO rather than WT mice (Fig.
2). Therefore, wequantified cytokine levels (IL-1β, IL-6, and
TNF-α) in peripheral bloodvia ELISA, 1 h after LPS (100 μg/kg, ip)
injection. The plasma levels ofIL-1β and IL-6 were slightly but not
significantly increased in WT ratherthan Prip-KO mice; in contrast,
that of TNF-α was slightly but not sig-nificantly increased in
Prip-KO rather than WT mice (Table 1). Thesedata suggest that
peripheral inflammation in WT and Prip-KO mice si-milarly
influences the CNS.
3.4. PRIP deficiency enhanced the NF-κB pathway and
AKTphosphorylation in the hypothalamus
NF-κB is required for induction of a several inflammatory
genes,
including Il-1b, Il-6, and Tnf-a (Liu et al., 2017). The
LPS-inducedproduction of these pro-inflammatory cytokines in the
hypothalamuscauses anorexia (Jang et al., 2010). IκB is
phosphorylated by IKK, whichinduces the subsequent ubiquitination
and degradation of IκB, followedby promotion of NF-κB activation
(Karin and Ben-Neriah, 2000). IKK isactivated by AKT as well as
NF-κB-inducing kinase (NIK) and trans-forming growth factor
β-activated kinase in an immune challenge(Akira and Takeda, 2004;
Ozes et al., 1999). Thus, the PI3K/AKTpathway may affect
LPS-induced inflammatory responses in the CNS.To investigate the
involvement of NF-κB signaling in PRIP-regulatedPI3K/AKT signaling,
we analyzed IKKα/β phosphorylation and IκBdegradation in the
hypothalamus obtained from LPS-treated mice.IKKα/β phosphorylation
in Prip-KO mice was significantly higher thanthat in WT mice (Fig.
3A and B; p = 0.0056 versus total IKKα andp = 0.0016 versus total
IKKβ). Consistently, IκB level was significantlydownregulated (Fig.
3A and C; p = 0.0033). The phosphorylation ofAKT at T308 and S473,
which is required for full activation of AKT(Manning and Toker,
2017), increased significantly in the Prip-KO hy-pothalamus,
compared with that in the WT hypothalamus (Fig. 3A andD; p = 0.0129
and p = 0.0038, respectively). These findings indicatethat PRIP
deficiency enhances the activation of AKT-mediated NF-κBsignaling
in the hypothalamus.
3.5. PRIP deficiency upregulates STAT3 phosphorylation and Socs3
andCox2 gene expression in the hypothalamus
Hypothalamic STAT3 phosphorylation is associated with
hypo-phagia, and peripheral LPS administration promotes
pro-inflammatorycytokine expression followed by STAT3
phosphorylation in the hy-pothalamus with a reduction in food
intake (Vaisse et al., 1996;Yamawaki et al., 2010). STAT3 is
activated by many cytokines, in-cluding IL-6, IL-1β, and TNF-α, and
a major IL-6-driven signalingpathway involves JAK-dependent STAT3
activation (Aggarwal et al.,2009). We examined STAT3
phosphorylation using LPS-administered
Fig. 2. The gene expression of pro-in-flammatory cytokines is
upregulated in Prip-KO hypothalamus after peripheral
lipopoly-saccharide (LPS) injection. Time-dependentchanges in the
expression of Il-1b, Il-6, and Tnf-a inthe hypothalamus of
wild-type (WT) and Prip-KOmice after intraperitoneal LPS injection
(100 μg/kg). Gene expression was evaluated with qPCRmethods. Data
are presented as the mean ±standard error of the mean (n = 5). *p
< 0.05
between the indicated groups, †p < 0.05 versusthe WT control
value at each time point,‡p < 0.05 versus the Prip-KO control
value ateach time point (ANOVA with Tukey's honestlysignificant
difference post-hoc comparison).
Table 1Plasma cytokine levels in wild-type and Prip-knockout
mice.
Genotype Control LPS WT vs KO in LPS
IL-1β WT 6.8 ± 2.9 43.0 ± 12.4* n.s.(pg/mL) KO 5.7 ± 2.9 20.7 ±
3.9IL-6 WT 0.03 ± 0.03 15.8 ± 4.3* n.s.(ng/mL) KO 0.12 ± 0.06 10.7
± 0.9*TNF-α WT 1.92 ± 0.8 1116.9 ± 250.2* n.s.(pg/mL) KO 2.59 ± 0.4
1537.8 ± 207.5*
Plasma cytokine levels in wild-type (WT) and Prip-KO (KO) mice
were quanti-fied before (control) and 1 h after intraperitoneal
injection of 100 μg/kg oflipopolysaccharide (LPS). Data are
presented as mean ± standard error of themean values (n = 5 for
each group). *p < 0.05, the LPS group vs the corre-sponding
control group; n.s. (not significant), WT value vs KO value in
LPStreatment (ANOVA with Tukey's honestly significant difference
post-hoc com-parison).
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Prip-KO and WT hypothalamus. STAT3 phosphorylation was
sig-nificantly increased in both genotypes 2 h–4 h (WT mice) or 2
h–8 h(Prip-KO mice) after peripheral LPS injection (Fig. 4A and B).
The in-crease in phosphorylation in Prip-KO mice was significantly
higher thanthat in WT mice at 2 h, 4 h, and 8 h (Fig. 4B; p =
0.0015, p < 0.001,and p = 0.023, respectively). LPS
administration did not alter PRIP1and PRIP2 expression levels in
the hypothalamus (Fig. 4C). However,the hypothalamic STAT3
activation upregulated the gene expression ofthe downstream
signaling molecule SOCS3, a negative feedback reg-ulator for STAT3
signaling, in Prip-KO mice (2–8 h after LPS adminis-tration) and WT
mice (2–4 h after LPS administration) (Fig. 4D). Im-portantly, the
upregulation in Prip-KO hypothalamus was greater at 4 h(p = 0.0396)
and 8 h (p = 0.0830) after LPS administration than thosein WT
mice.
The expression of Cox2, an important player in LPS-mediated
an-orexia (Lugarini et al., 2002), is regulated by NF-κB and STAT3
sig-naling (D'Acquisto et al., 1997; Rummel et al., 2006).
Therefore, toexamine the downstream activation of NF-κB and STAT3
signaling, wenext investigated hypothalamic Cox2 expression. The
expression ofCox2 was significantly increased during 2–8 h after
peripheral LPS in-jection in the two phenotypes. Cox2 expression in
Prip-KO mice wassignificantly higher than that in WT mice at 2 h
and 8 h (Fig. 4E;p = 0.0057 and p = 0.0002 at 4 h and 8 h,
respectively) or substantiallyhigh at 4 h, suggesting that the
neuroinflammation response is moresevere in Prip-KO hypothalamus
than in WT mice. These data indicatethat a severe anorexia
phenotype in Prip-KO mice depends on NF-κB
and STAT3 acceleration-dependent signaling in the
hypothalamus.
3.6. High expression of pro-inflammatory cytokines in
Prip-KOhypothalamic microglia
The hypothalamus is composed of many different cell types,
in-cluding microglia, and activated microglia are involved in
aggravatingneuroinflammation. To investigate microglial
contribution in the ac-celerated inflammatory responses in Prip-KO
hypothalamus, we ana-lyzed the expression of Iba1, a marker of
active microglia. Iba1 ex-pression increased 24 h after LPS
administration, and the elevation wasmore prominent in Prip-KO
hypothalamus than that in WT mice(Fig. 5A), suggesting that
peripheral LPS application intensely activatesPrip-deficient
microglia. We next examined pro-inflammatory cytokineexpression in
hypothalamic microglia isolated from LPS-treated miceusing the MACS
system. The isolated microglia expressed Prip1 andPrip2 genes (Fig.
5B). Pro-inflammatory cytokine expression was higherin Prip-KO
hypothalami than those in WT mice (Fig. 5C; Il-1b, Il-6, andTnf-a;
p = 0.0078, p = 0.0096, and p = 0.0004, respectively).
4. Discussion
Anorexia is a hallmark of systemic inflammation, and
inflammation-associated anorexia is primarily a result of cytokine
action in the CNS. Itis known that peripheral injection of LPS
induces anorexia with ele-vation of pro-inflammatory cytokines,
including IL-1β, IL-6, and TNF-α
Fig. 3. PRIP deficiency upregulates NF-κB sig-naling mediated by
AKT-IKK signaling.Hypothalamus samples were obtained 2 h afterthe
intraperitoneal injection of saline (cont) orLPS (100 μg/kg). (A)
Western blotting was per-formed using indicated antibodies.
Representativeblot images are shown. GAPDH was used as aloading
control. (B–D) Quantitation of combinedphosphorylation of IKKα and
IKKβ was analyzedagainst total IKKα and IKKβ levels detected
usingthe corresponding pan-antibodies, respectively,and expressed
as p-IKK/IKKα and p-IKK/IKKβ (B).Quantitation of immunodensity in
the degradationof IκB and phosphorylation levels of AKT (T308and
S473) was analyzed against GAPDH (C) andtotal AKT (D),
respectively. Data are presented asthe mean ± standard error of the
mean (n = 5for each group). *p < 0.05 between the
indicatedgroups. ‡p < 0.05 versus the Prip-KO controlvalue
(ANOVA with Tukey's honestly significantdifference post-hoc
comparison).
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in peripheral and CNS. In addition, the peripheral or
in-tracerebroventricular injections of pro-inflammatory cytokines
IL-1βand TNF-α cause anorexia in rodents (Elander et al., 2007;
Fantino andWieteska, 1993; Harden et al., 2008; Michie et al.,
1989). Therefore,circulating cytokines in the body and de novo
production of cytokines inthe CNS are widely viewed as mediators of
inflammatory anorexia.Inflammatory responses through PI3K/AKT
signaling, which are acti-vated by LPS, IL-1β, and TNF-α, may
promote NF-κB activation andanorexia in an animal model (Bai et
al., 2009; Ojaniemi et al., 2003;Ozes et al., 1999). In this study,
we examined whether PRIP, a newmolecule negatively regulating
phosphatidylinositol metabolism-de-pendent AKT signaling, is
involved in inflammation-associated anor-exia, and elucidated that
deficiency in PRIP increases pro-inflammatorycytokine expression in
hypothalamic microglia and promotes LPS-mediated anorectic response
in mice.
Pro-inflammatory cytokines in peripheral blood are potent
media-tors that link the periphery and CNS in LPS ip
injection-elicited braininflammation and anorexia; consequently,
upregulation of hypotha-lamic pro-inflammatory cytokines causes
LPS-induced anorexia. In ourexperiments, levels of circulating
pro-inflammatory cytokines (IL-1β,IL-6, and TNF-α) did not differ
between WT and Prip-KO mice 1 h afterLPS injection. However, mRNA
levels of pro-inflammatory cytokines
(IL-1β, IL-6, and TNF-α) were significantly higher in the
hypothalamusin Prip-KO rather than WT mice 2 h after LPS injection.
These datasuggest that local hypothalamic inflammatory responses
vary in Prip-KO mice. Therefore, we investigated the role of PRIP
in anorexia causedby LPS-induced inflammation in the hypothalamus,
a key brain regionregulating food intake.
Inflammatory stimuli, such as LPS and pro-inflammatory
cytokines,induce inflammatory responses through PI3K-activated AKT
signaling,which regulates NF-κB activation (Ozes et al., 1999;
Ojaniemi et al.,2003). AKT phosphorylates and activates two
subtypes of IKKα andIKKβ, leading to IκB degradation and NF-κB
activation (Bai et al., 2009;Ouyang et al., 2006; Vandermoere et
al., 2005). The transcription factorNF-κB is activated downstream
of LPS and pro-inflammatory cytokines(Taniguchi and Karin, 2018)
which results in inflammatory responseand anorexia after LPS
administration (Jang et al., 2010). NF-κB, whoseactivation is
regulated by the IKK-mediated degradation of IκB, pro-motes the
transcriptional activity of various pro-inflammatory cyto-kines,
including IL-1β, TNF-α, and IL-6 (Libermann and Baltimore,1990; Luo
and Zheng, 2016). We observed that IKK phosphorylation-induced IκB
degradation increased in Prip-KO hypothalamus and en-hanced AKT
signaling activation, indicating that the upregulation
ofpro-inflammatory cytokines in the Prip-KO hypothalamus resulted
from
Fig. 4. Prip deficiency upregulates STAT3 sig-naling in the
hypothalamus. (A–C) Time-de-pendent changes in the phosphorylation
levels ofSTAT3 (A and B) and the expression of PRIP1 andPRIP2 (A
and C) in the hypothalamus of wild-type(WT) and Prip-KO mice after
intraperitoneal (ip)lipopolysaccharide (LPS) injection (100
μg/kg).Western blotting was performed using the in-dicated
antibodies. Representative blot images areshown (A). Results of
quantitative immunodensityexpressed as changes in phospho-STAT3
againsttotal STAT3 levels (B) and of PRIP1 and PRIP2levels in WT
hypothalamus against the level ofcorresponding GAPDH, a loading
control (C). Dataare presented as the mean ± standard error of
themean (n = 5). (D and E) Results of qPCR analysesfor Socs3 and
Cox2 expression in the hypotha-lamus. Data are presented as the
mean ±
standard error of the mean (n = 5 for eachgroup). *p < 0.05
between the indicated groups,†p < 0.05 versus the WT control
value at eachtime point, ‡p < 0.05 versus the Prip-KO
controlvalue at each time point (ANOVA with Tukey'shonestly
significant difference post-hoc compar-ison).
Y. Yamawaki, et al. Neurochemistry International 131 (2019)
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increased NF-κB signaling. Prip-KO mice exhibited higher
expression ofpro-inflammatory cytokines in the hypothalamus 2 h
after LPS periph-eral injection compared with that in WT mice,
suggesting that regionalinflammation responses in Prip-KO
hypothalamus were upregulated.Therefore, we conclude that the
deficiency of PRIP, a negative regulatorof AKT signaling via
PI3K-mediated PI(3,4,5)P3 production (Asanoet al., 2017), shows
increased LPS-induced AKT and IKKα/β phos-phorylation followed by
increased NF-κB-regulated inflammatory re-sponses, thus causing a
worse anorexia phenotype than that of WTmice.
Although pro-inflammatory cytokine expression in WT
hypotha-lamus increased 2 h after peripheral LPS administration
(Fig. 2), adistinct activation of IKK-IκB degradation-mediated
NF-κB signalingwas not observed between the groups of
saline-administration (control)and LPS-administration in WT mice
(Fig. 3). It is reported that LPS ipinjection (200 μg/kg) induces
anorexia via activation of IKK-NF-κBsignaling in the WT
hypothalamus (Jang et al., 2010). However, weused a dose of 100
μg/kg LPS (ip) to induce peripheral inflammation inthis study,
which may be too low to detect a change in IKK-NF-κBsignaling in
western blotting analysis.
In addition to NF-κB activation, hypothalamic STAT3 activation
isinvolved in appetite. Peripheral LPS injection increases the
expressionof IL-6 as well as IL-1β and TNF-α in the hypothalamus
(Jang et al.,2010) and induces hypothalamic STAT3 activation (Hosoi
et al., 2004).The central inhibition of NF-κB signaling prevents
LPS-induced anor-exia (Jang et al., 2010), whereas the central
inhibition of the JAK-STATpathway fails to prevent anorexia (Damm
et al., 2013). Furthermore,the central inhibition of NF-κB
decreases IL-6 expression in the
hypothalamus (Jang et al., 2010), and intraventricular injection
of IL-6accelerates IL-1β injection-mediated inhibition of food
intake in rats(Harden et al., 2008). IL-6 activates gp130/JAK
pathway and phos-phorylates STAT3 in the hypothalamus, thereby
regulating food intake(Takeda et al., 1999; Vaisse et al., 1996).
In contrast, deficiency of IL-1β, IL-6, or TNF-α does not protect
against LPS-induced anorexia(Arsenijevic et al., 2000; Fantuzzi et
al., 1996; Fattori et al., 1994),although MyD88 deficiency
completely abolishes LPS-induced anorexiaand prevents hypothalamic
pro-inflammatory cytokine elevation(Ogimoto et al., 2006; Yamawaki
et al., 2010). These data suggest thatLPS-activated NF-κB signaling
is essential for an anorectic effectthrough the production of
pro-inflammatory cytokines, and IL-6-acti-vated STAT3 signaling is
needed to exacerbate these anorectic effects.Thus, pro-inflammatory
cytokine production is involved in LPS-inducedanorectic responses.
In our study, STAT3 phosphorylation in the hy-pothalamus of Prip-KO
mice was higher than that in WT mice 4–8 hafter LPS ip injection.
The expression of pro-inflammatory cytokines at2 h was markedly
increased in Prip-KO hypothalamus compared withthat in WT mice, and
augmented hypophagia was observed during4–12 h. Furthermore, the
expression of Socs3, a negative feedbackregulator for STAT3
signaling that is positively regulated by STAT3activation (Carow
and Rottenberg, 2014), was increased in Prip-KOhypothalamus 4–8 h
after LPS ip injection. These time-course studiesindicate that
STAT3 phosphorylation regulates the duration of
in-flammation-induced anorectic effects. Taken together, the
LPS-in-ducible appetite-suppressive phenotype in Prip-KO mice may
be at-tributed to alterations in AKT signaling, which regulates
NF-κB-mediated pro-inflammatory cytokine expression signaling,
followed bySTAT3 signaling-mediated anorectic responses.
Microglia, the brain-resident immune cells, are emerging as
centralplayers in the regulation of CNS inflammation. We show that
isolatedhypothalamic microglia expressed more pro-inflammatory
cytokines2 h after peripheral LPS injection in Prip-KO mice than
those in WTmice, although the higher expression of Iba1, a marker
of activatedmicroglia, was observed in Prip-KO hypothalamus 24 h
after peripheralLPS injection. Because Iba1 expression in the brain
is delayed despitemicroglial activation in response to peripheral
LPS injection (Yamawakiet al., 2018), Prip-KO microglia may have a
higher ability for producingpro-inflammatory cytokines than WT
microglia 2 h after LPS injectionbecause of their abnormal AKT
signaling. However, further studies arerequired to investigate the
Prip-KO microglia-induced inflammation inLPS-induced anorexia.
Together, the present results indicate that Pripdeficiency enhances
pro-inflammatory cytokine-induced brain in-flammation by activating
the AKT/NF-κB pathway in hypothalamicmicroglia. This, in turn,
increases pro-inflammatory cytokine-mediatedSTAT3 phosphorylation.
This sequential signal activation induces theup-regulation of
molecules regulating anorexia, such as COX2 in thehypothalamus,
resulting in a severe anorexia phenotype in Prip-KOmice.
PRIP has several binding partners other than Ins(1,4,5)P3 and
PI(4,5)P2; these are GABAA receptor associated protein (Kanematsu
et al.,2002), GABAA receptor β subunit (Terunuma et al., 2004),
phos-phorylated AKT (Fujii et al., 2010), and PP1 and PP2A
(Kanematsuet al., 2006; Yoshimura et al., 2001). PRIP exerts its
physiologicalfunctions by binding to these proteins and modulating
their functions.Prip-KO mice exhibited an anti-obesity phenotype in
spite of having ahigher food intake than WT mice. We determined
that this phenotyperesults from a higher energy expenditure rate in
Prip-KO mice than inWT mice (Okumura et al., 2014; Oue et al.,
2016, 2017). Energy ex-penditure in non-shivering thermogenesis is
controlled by PRIP-de-pendent recruitment of protein phosphatase
activity to the lipid dropletmembrane in brown adipocytes. Thus, we
do not believe that LPS-in-duced acute anorectic responses in
Prip-KO mice are derived from thePRIP-regulating peripheral
non-shivering thermogenic pathway. How-ever, PRIP binds to
phosphorylated AKT and regulates the intracellulartrafficking of
GABAA receptor-containing secretary vesicles (Fujii et al.,
Fig. 5. Gene expression of pro-inflammatory cytokines is
upregulated inPrip-KO hypothalamic microglia after peripheral
lipopolysaccharide in-jection. (A) Gene expression of Iba1 in WT
and Prip-KO hypothalami 2 h and24 h after saline (cont) or
lipopolysaccharide (LPS) injections. (B) Expression ofPrip1, Prip2,
and Iba1 genes in isolated hypothalamic microglia (Microglia)
wasexamined by reverse-transcription polymerase chain reaction.
Hypothalamuswas used as a positive control. Iba1 and Gapdh were
used as a microglial markerand an internal control, respectively.
Control lane (no template) indicates anegative control. (C)
Pro-inflammatory cytokine expression was evaluated withqPCR methods
using isolated hypothalamic microglia from WT and Prip-KOmice 2 h
after LPS injection. Data are presented as the mean ± standard
errorof the mean (n = 5). *p < 0.05 between the indicated groups
(Student's t-test).
Y. Yamawaki, et al. Neurochemistry International 131 (2019)
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2010). Therefore, a PRIP/phospho-AKT complex potentially
modulatesthe AKT/IKK/NF-κB signaling pathway. Further studies are
required toclarify the role of PRIP in the interrelation between
AKT and NF-κBsignaling pathways in the hypothalamus.
This study shows that PRIP represses the neuroinflammatory
re-sponse via constitutive inhibition of PI3K/AKT signaling in the
brain.Our findings implicate PRIP as a novel regulator of
inflammatory brainresponses. Currently, no interventions are
available to completelycontrol the neuroinflammation that leads to
brain dysfunction in pa-tients with infectious diseases. Our
findings provide evidence indicatingthat a unique molecule, PRIP,
regulates neuroinflammation. A betterunderstanding of this process
might aid the development of ther-apeutics against
infection-induced inflammation in the brain.
Funding
This work was supported by JSPS KAKENHI (grant numbers15K19731
and 17K11670 to Y.Y., 17H01595 and 17K19766 to M.H.,16K11503 to
T.K.) and the Strategic Research Program for BrainSciences from
Japan Agency for Medical Research and Development(AMED; grant
number 16dm0107093h0001 to S.Y.).
Author contributions
Y.Y. designed the study, performed the experiments, and drafted
themanuscript. S.S., M.A., K.N., H.I., and H.A. performed some
experi-ments. S.A. and K.O. contributed reagents and analytical
tools. S.Y. andM.H. helped conduct the study. T.K. conceived and
coordinated thestudy and wrote the manuscript. All authors have
read and approvedthe final manuscript.
Declaration of competing interest
None.
Acknowledgments
We wish to acknowledge the Institute of Laboratory Animal
Science(Hiroshima University) for their support in conducting all
the animalexperiments involved in the current study.
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Phospholipase C-related catalytically inactive protein regulates
lipopolysaccharide-induced hypothalamic inflammation-mediated
anorexia in miceIntroductionMaterials and methodsAnimalsLPS
administrationAntibodiesMeasurement of food intakePreparation of
isolated hypothalamic microgliaQuantitative real-time polymerase
chain reaction (qPCR) analysisReverse-transcription PCR (RT-PCR)
analysisWestern blottingEnzyme-linked immunosorbent assay (ELISA)
for plasma cytokine quantificationStatistical analysis
ResultsPrip-KO mice showed a high-sensitivity phenotype to
LPS-induced anorexiaHigher gene expression of pro-inflammatory
cytokines in Prip-KO hypothalamusSimilar plasma levels of
peripheral inflammatory biomarkers in WT and Prip-KO micePRIP
deficiency enhanced the NF-κB pathway and AKT phosphorylation in
the hypothalamusPRIP deficiency upregulates STAT3 phosphorylation
and Socs3 and Cox2 gene expression in the hypothalamusHigh
expression of pro-inflammatory cytokines in Prip-KO hypothalamic
microglia
DiscussionFundingAuthor
contributionsmk:H1_23AcknowledgmentsReferences