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Neurochemistry International

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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/).

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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

Y. Yamawaki, et al. Neurochemistry International 131 (2019) 104563

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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).


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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).


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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).


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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

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