KR20230172930A - Composition for preventing or treating of obesity comprising peptides derived from Neuronal growth regulator 1(NEGR1) - Google Patents

Abstract

The present invention relates to a composition for preventing or treating obesity comprising a peptide derived from nerve cell growth regulator 1 (NEGR1) as an active ingredient. Specifically, the peptide derived from nerve cell growth regulator 1 (NEGR1) interacts with CD36 to prevent or treat obesity. Obesity can be prevented or treated by reducing the transport or accumulation of fatty acids in fat cells or liver cells.

Description

Composition for preventing or treating obesity comprising peptides derived from Neuronal growth regulator 1 (NEGR1)}

The present invention relates to a composition for preventing or treating obesity comprising a peptide derived from nerve cell growth regulator 1 (NEGR1) as an active ingredient, and more specifically, a composition derived from nerve cell growth regulator 1 (NEGR1) that interacts with CD36. It relates to a composition for preventing or treating obesity containing it as an active ingredient.

Obesity is the most common disease worldwide, characterized by an abnormal increase in intracellular fat accumulation. Obesity can cause high blood sugar, high blood pressure, hyperlipidemia, cardiovascular disease, respiratory disease, joint disease, reproductive disease, fatty liver, etc., and some cases of cancer have also been reported. Obesity can be caused by excessive food intake, endocrine diseases, genetic factors, mental factors, and drugs. It occurs when energy consumption is low compared to the nutrients consumed chronically, so excess energy is accumulated in the form of body fat. do. In particular, long-chain fatty acids (LCFA), which are most of the fat consumed through regular meals, are absorbed into the body through complex digestion and absorption processes, have a slow metabolic rate, and have a high body fat accumulation rate. LCFA not only contributes to cellular metabolic energy production and storage but also has hormone-like properties that regulate gene expression (Doege, H., and A. Stahl., 2006). Several groups of proteins are known to uptake LCFAs, including fatty acid translocase/cluster of differentiation 36 (CD36/FAT), fatty acid transport protein (FATP), and plasma membrane fatty acid 48 binding protein (FABPpm). Among these, cluster of differentiation 36 (CD36) in particular plays an important role in LCFA uptake in metabolic tissues such as adipose tissue, cardiomyocytes, and skeletal muscle cells and is known to contribute more than 50% of fatty acid uptake in these tissues. There is (Hao, J. W., et al., 2020).

CD36 is an integral membrane protein found on the surface of many vertebrate cells. It is also known as FAT (fatty acid translocase), SCARB3, GP88, gpIV (glycoprotein IV), and gpIIIb (glycoprotein IIIb). It plays a role in bringing it into the cell. CD36 is a class B-2 scavenger receptor (SR-B2) present on the cell surface and is an integrated membrane protein with a hairpin-shaped membrane topology and two transmembrane domains (Glatz, J. F. C. , and J. Luiken, 2018). Additionally, CD36 is associated with membrane lipid rafts. Lipid rafts are regions rich in glycoproteins or lipids, such as cholesterol, glycosphingolipid, and membrane-bound receptors, that exist on cell membranes and play an important role in signal transduction. CD36 is also ubiquitously expressed in a variety of mammalian cell types, including skeletal and cardiac myocytes, macrophages, endothelial cells, adipocytes, and intestinal epithelial cells, and is also known to be primarily associated with lipid metabolism and innate immunity. In particular, the cellular fatty acid uptake rate is mainly controlled by the amount of CD36 protein on the cell surface. CD36 performs various functions depending on its tissue location, and in human heart and skeletal muscle, CD36 is recognized as a major fatty acid transporter that supplies energy sources to cells. In adipocytes, CD36 mainly contributes to LCFA uptake and also regulates lipid storage processes, and is considered a marker of human adipocyte precursors, where high CD36 levels also imply a high potential for triglyceride accumulation.

Meanwhile, Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, is a glycosylphosphatidylinositol (GPI) anchored membrane protein that mediates neuronal communication and synapse formation. This protein is strongly associated with membrane lipid rafts using a glycosylphosphatidylinositol anchor. NEGR1 functions as a cell-binding factor that plays an important role in neuronal recognition and neurite outgrowth (Kim, H., et al., 2014). NEGR1 consists of three C2-type immunoglobulin domains localized to the extracellular part of the cell membrane. Genome-wide association studies (GWAS) have shown that genetic variants in NEGR1 are associated with a variety of human diseases, including obesity, intellectual disability, schizophrenia, depression, and Alzheimer's disease (Ni, H. , et al., 2018). Several genome-wide studies have reported that NEGR1 mutations are associated with body weight regulation, and NEGR1-deficient mice have significantly enlarged white adipose tissue (WAT) with increased cell size, liver fat accumulation, and skeletal muscle. Atrophy was observed (Joo, Y., et al., 2019).

However, the role of NEGR1 in intracellular cholesterol transport and intracellular storage of lipids is unclear, and in particular, there is no research on the interaction of NEGR1 with CD36 and the role of NEGR1 and CD36 in related human diseases, especially obesity.

Yao, C. H., R. Fowle-Grider, N. G. Mahieu, G. Y. Liu, Y. J. Chen, R. Wang, M. Singh, G. S. Potter, R. W. Gross, J. Schaefer, S. L. Johnson, and G. J. Patti. 2016. Exogenous Fatty Acids Are the Preferred Source of Membrane Lipids in Proliferating Fibroblasts. Cell Chem Biol 23: 483-493. Doege, H., and A. Stahl. 2006. Protein-mediated fatty acid uptake: novel insights from in vivo models. Physiology (Bethesda) 21: 259-268. Hao, J. W., J. Wang, H. Guo, Y. Y. Zhao, H. H. Sun, Y. F. Li, X. Y. Lai, N. Zhao, X. Wang, C. Xie, L. Hong, X. Huang, H. R. Wang, C. B. Li, B. Liang, S. Chen, and T. J. Zhao. 2020. CD36 facilitates fatty acid uptake by dynamic palmitoylation-regulated endocytosis. Nat Commun 11:4765. Glatz, J. F. C., and J. Luiken. 2018. Dynamic role of the transmembrane glycoprotein CD36 (SR-B2) in cellular fatty acid uptake and utilization. J Lipid Res 59: 1084-1093. Kim, H., J. S. Hwang, B. Lee, J. Hong, and S. Lee. 2014. Newly Identified Cancer-416 Associated Role of Human Neuronal Growth Regulator 1 (NEGR1). Journal of Cancer 5: 598-608. Ni, H., M. Xu, G. L. Zhan, Y. Fan, H. Zhou, H. Y. Jiang, W. H. Lu, L. Tan, D. F. Zhang, Y. G. Yao, and C. Zhang. 2018. The GWAS Risk Genes for Depression May Be Actively Involved in Alzheimer's Disease. Journal of Alzheimer's disease : JAD 64: 1149-457 1161. Joo, Y., H. Kim, S. Lee, and S. Lee. 2019. Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass. Int J Obes (Lond) 43: 1769-472 1782. Kim, H., Y. Chun, L. Che, J. Kim, S. Lee, and S. Lee. 2017. The new obesity associated protein, neuronal growth regulator 1 (NEGR1), is implicated in Niemann-Pick disease Type C (NPC2)-mediated cholesterol trafficking. Biochem Biophys Res Commun 482: 1367-1374.

The purpose of the present invention is to provide a composition for preventing or treating obesity containing a peptide derived from nerve cell growth regulator 1 (NEGR1) as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing or treating obesity containing a peptide derived from nerve cell growth regulator 1 (NEGR1) as an active ingredient.

The peptide derived from Neuronal growth regulator 1 (NEGR1) may be a peptide consisting of one or more amino acid sequences shown in SEQ ID NOs: 5, 6, 7, 9, and 10.

The peptide derived from Neuronal growth regulator 1 (NEGR1) or the peptide represented by SEQ ID NOs: 5, 6, 7, 9, and 10 interacts with cluster of differentiation 36 (CD36). You can. At this time, CD36 may interact with one region of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4.

The peptide may reduce the transport or accumulation of fatty acids in adipocytes or hepatocytes, but is not limited thereto.

A composition for preventing or treating obesity containing the nerve cell growth regulator 1 (NEGR1)-derived peptide as an active ingredient may be provided as a pharmaceutical composition.

The “prevention” refers to all actions that suppress or delay the onset of obesity by administering the composition, and “treatment” refers to all actions that improve or benefit the symptoms of obesity by administering the peptide or composition.

In the present invention, the "administration" means introducing a predetermined substance into a patient by any appropriate method, and the administration route of the composition is not particularly limited thereto, but is any general route by which the composition can reach the target in vivo. It can be administered through, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. However, since peptides are digested when administered orally, it is desirable for oral compositions to be coated with the active agent or formulated to protect them from decomposition in the stomach.

In the present invention, “obesity” is a state in which there is excessive adipose tissue in the body, and is defined as obesity if the body obesity index (weight (kg) divided by the square of height (m)) is 25 or more. Obesity is usually caused by energy imbalance when excessive intake of nutrients compared to energy consumption over a long period of time. Obesity is a metabolic disease that affects the entire body, increasing the likelihood of developing diabetes and hyperlipidemia, increasing the risk of developing sexual dysfunction, arthritis, and cardiovascular disease, and in some cases, it is also associated with the development of cancer.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, excipient, or diluent. In the present invention, “pharmaceutically acceptable” means a sufficient amount to produce a therapeutic effect and no side effects, including the type of disease, the patient's age, weight, health, gender, patient's sensitivity to the drug, and administration. It can be easily determined by a person skilled in the art according to factors well known in the medical field, such as route, method of administration, frequency of administration, treatment period, combination, or drugs used simultaneously.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The carrier is not particularly limited thereto, but for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. may be used. In the case of injections, Buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. Examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, preservatives, etc. may be additionally included.

In the present invention, the pharmaceutical composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, aerosols, external preparations, suppositories, and sterilized injection solutions according to conventional methods. It can be used interchangeably.

The composition is formulated into a unit dosage form suitable for administration into the patient's body, preferably a preparation useful for the administration of peptide medicines, according to a method commonly used in the pharmaceutical field, using an administration method commonly used in the art. administered by oral, cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal, or rectal routes. It may be administered via parenteral administration routes, but is not limited to these.

In addition, the peptide can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and to increase stability or absorption, carbohydrates such as glucose, sucrose or dextran, and ascorbic acid ( Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as drugs.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 500 mg/kg/day. A preferred dosage is 0.1 mg/kg/day to 200 mg/kg/day, and a more preferred dosage is 1 mg/kg/day to 200 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be administered alone or in combination with other pharmaceutical agents showing obesity prevention or treatment effects. Pharmaceutical agents showing the effect of preventing or treating obesity include, but are not limited to, GLP-1 receptor agonists, leptin receptor agonists, DPP-IV inhibitors, Y5 receptor antagonists, MCH (Melaninconcentrating hormone) receptor antagonists, Y2/3 receptor agonist, MC3/4 receptor agonist, gastric/pancreatic lipase inhibitor, 5HT2c agonist, β3A receptor agonist, Amylin receptor agonist, Ghrelin antagonist, and/or a ghrelin receptor antagonist, etc.

The present invention also provides a health functional food for preventing or improving obesity containing a peptide derived from neuronal growth regulator 1 (NEGR1) as an active ingredient.

The NEGR1-derived peptide of the health functional food for preventing or improving obesity may be a peptide consisting of one or more amino acid sequences represented by SEQ ID NOs: 5, 6, 7, 9, and 10.

The health functional food contains preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, and most preferably 0.001 to 50% by weight, based on the total weight of the entire food, of peptide derived from Neuronal growth regulator 1 (NEGR1). Can be added at 0.001 to 10% by weight.

The health functional food includes the form of tablets, capsules, pills, or liquid, and foods to which the extract of the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and health products. There are functional foods, etc.

The present invention is a peptide derived from Neuronal growth regulator 1 (NEGR1), which has anti-obesity activity and consists of one of the amino acid sequences shown in SEQ ID NOs: 5, 6, 7, 9, and 10. provides.

The peptide may reduce the transport or accumulation of fatty acids in adipocytes or hepatocytes.

The peptide derived from Neuronal growth regulator 1 (NEGR1) is preferably a peptide consisting of the amino acid sequence represented by SEQ ID NO: 10.

The present invention also provides a nucleotide sequence encoding a peptide derived from Neuronal growth regulator 1 (NEGR1) and consisting of the amino acid sequence shown in SEQ ID NO: 10.

The present invention relates to a composition for preventing or treating obesity comprising a peptide derived from nerve cell growth regulator 1 (NEGR1) as an active ingredient. Specifically, the peptide derived from nerve cell growth regulator 1 (NEGR1) interacts with CD36 to prevent or treat obesity. Obesity can be prevented or treated by reducing the transport or accumulation of fatty acids in fat cells or liver cells.

Figure 1 is a diagram showing that CD36 expression is upregulated in white adipose tissue (WAT) of Negr1 ^-/- mice. (A) Weight of epididymal WAT from wild type (WT) and Negr1 ^−/− mice (*, p < 0.05.) (B) Expression levels of CD36, FABPpm and FATP1 in epididymal WAT of WT and Negr1 ^−/− mice (* **, p < 0.001.) (C) Relative mRNA expression level in WAT of Negr1 ^−/− mice compared to WT mice. (D) CD36 gene expression in WAT, liver, and skeletal muscle (SkM) of WT and Negr1 ^−/− mice (E) Relative mRNA expression level in WAT of Negr1 ^−/− mice compared to WT mice. (Error bars are ± SD; *, p <0.05; **, p < 0.01.)
Figure 2 is a diagram showing that NEGR1 interacts with CD36. (A) Cell lysates from 293T cells transfected with HA-CD36 and GFP-NEGR1 plasmids were coimmunoprecipitated (IP) between CD36 and NEGR1 with anti-GFP antibody, followed by immunoprecipitation with anti-HA and anti-GFP antibodies. Blotting (IB) (*, IgG) (B) Cell lysates of 293T cells transfected with HA-CD36 and GFP-NEGR1 plasmids were subjected to IP with anti-HA antibody, followed by IB with anti-HA and anti-GFP antibodies. (C) IP with SKOV-3-FLAG-NEGR1 stable cells using anti-FLAG antibody followed by IB with anti-FLAG and anti-CD36 antibodies (D) Binding assay using soluble NEGR1-Fc protein. SKOV-3 cells were incubated with culture medium from 293T cells expressing NEGR1-Fc or Fc (control) for 2 hours, and then cells were immunostained with anti-human Fc and anti-CD36 antibodies. (Bar = 20 μm). (E) In situ PLA results with SKOV-3-FLAG-NEGR1 cells using Duolink proximity ligation assay (PLA) technology. After treatment with anti-CD36 and anti-FLAG antibodies for 2 h, cells were incubated with PLA probes (anti-mouse (-) and anti-rabbit (+), respectively) for 1 h, and actin polymers of cells were blotted with Alexa Fluor. Stained with 488 phalloidin. (Bar = 10 μm)
Figure 3 shows the results of in situ proximity ligation assay (PLA) without cell permeabilization using Duolink PLA technology. SKOV-3-FLAG-NEGR1 cells were incubated with anti-CD36 and anti-FLAG antibodies followed by incubation with PLA probe for 1 h. (Bar = 10 μm)
Figure 4 is a diagram showing the schematic diagram and results of an experiment for determining the important region of NEGR1-CD36 interaction. (A) Schematic diagram of CD36 and NEGR1 structures. Potential N-glycosylation sites are indicated by filled circles (B) Schematic of the deletion construct of CD36 extracellular domain (ECD) in GST-fused form and transfected into 293T cells with FLAG-NEGR1 plasmid, after GST pulldown, with anti-FLAG. Detection of co-fractionated NEGR1 using IB with antibodies. (TM, transmembrane; CLESH-1, CD36, LIMP-2, Emp sequence homology; MA, membrane associated; ProR, proline-rich). (C) Schematic of the deletion construct of NEGR1 in GST-fused form and transfection with HA-CD36 plasmid into 293T cells, GST pulldown, and detection of co-fractionated CD36 using IB with anti-HA antibody.
Figure 5 is a diagram confirming the coexistence of NEGR1 and CD36 in the plasma membrane. (A) Immunofluorescence microscopy of SKOV-3 transiently transfected with FLAG-NEGR1 and HA CD36 plasmids. Cells were incubated with anti-FLAG and anti-HA antibodies, followed by anti-rabbit Cy3 and anti-mouse FITC secondary antibodies. Imaging was performed using an Olympus BX51 microscope (bar = 20 μm). (B) SKOV-3-FLAG-NEGR1 stable cells double immunostained with anti-FLAG (red) and anti-CD36 (green) antibodies. (C) Lipid raft fractionation performed using 293T cells and transfected with the HA-CD36 plasmid along with GFP control (left) or GFP-NEGR1 (right), followed by membrane fractionation by suspension method using an Optiprep gradient. CD36 and NEGR1 were visualized with anti-HA and anti-GFP antibodies, respectively. Flotillin-1 used as a lipid raft marker. (SE, short exposure; LE, long exposure).
Figure 6 is a diagram showing the effect of NEGR1 expression on CD36 protein levels in WAT and liver. (A) SDS-PAGE of equal amounts of total protein (30 μg) from 293T cells transiently transfected with HA-CD36 and GFP-NEGR1 plasmids and detection of CD36 protein using anti-HA antibody. The intensity of each CD36 band in the image was quantified using ImageJ software. (B) Comparison of endogenous CD36 levels in empty vector or NEGR1 expressing 3T3-L1-NEGR1-FLAG stable cells. CD36 was detected by IB with anti-CD36 (****, p < 0.0001.) (C) Measurement of CD36 protein levels in WT and Negr1 ^−/− MEFs. WT and NEGR1-deficient MEFs were incubated under normal culture conditions (-OA) or in the presence of 0.1mM oleic acid (+OA) for 24 hours and then subjected to IB. Endogenous CD36 and NEGR1 were visualized with specific antibodies against each protein.
Figure 7 shows the results of examining CD36 protein levels in WAT and liver of Negr1 ^-/- mice. (A) Measurement of CD36 protein levels in epididymal adipose tissue of WT and Negr1 ^−/− mice. IB performed with anti-CD36 antibody, intensity of bands quantified using ImageJ and normalized using β-actin expression level (*, p < 0.05) (B) WAT tissue sections from WT or Negr1 ^−/− mice. Using immunohistochemical analysis. Tissue sections were incubated with anti-CD36 antibody followed by FITC anti-mouse secondary antibody. (Bar = 50 μm). (C) CD36 expression in the liver of WT and Negr1 ^−/− mice. Band intensity was determined using ImageJ software. (*, p < 0.05) (D) Comparison of CD36 expression in liver tissues of WT and Negr1 ^−/− mice using immunohistochemical analysis with anti-CD36 antibody. Imaging was performed using an Olympus BX51 microscope.
Figure 8 shows the results of examining the fatty acid uptake rate and cellular ROS level of WT and Negr1 ^-/- mouse adipocytes. (A) Fatty acid uptake rate in primary adipocytes obtained from epididymal adipose tissue of WT and Negr1 ^−/− mice. Measure fluorescence signal using a fluorescence microplate reader FlexStation 3 at 485/515 nm using the free fatty acid absorption assay kit until 40 min (bar = 100 μm). Imaging was performed using an Olympus BX51 microscope (B) Investigation of fat accumulation in WT and Negr1 ^−/− mice. Epididymal-derived primary adipocytes from WT and Negr1 ^−/− mice were cultured with 0.1mM oleic acid for 24 h, and then cells were stained with BODIPY 493/503 (bar = 50 μm). (C) Examination of cellular ROS levels in WT and Negr1 ^−/− MEFs, pre-incubated for 24 h in conditions with or without H ₂ O ₂ or oleic acid using the DCFDA/H2DCFDA - Cellular ROS Assay Kit. Fluorescence signal measured at 485/515 nm. (D) Determination of activated AMPK levels in WT and Negr1 ^−/− MEFs. AMPK and p-AMPK levels were detected by IB after culturing cells for 24 hours in the presence of 0.1mM oleic acid or no oleic acid.
Figure 9 is a schematic diagram summarizing the results of an experiment on the association between NEGR1 and CD36. In NEGR1-deficient cells, CD36 mRNA and protein expression increases, and fatty acid absorption and adipocyte size increase, and the amount of ROS also increases.

Through prior research, the inventor group of the present invention discovered that the human NEGR1 gene is generally down-regulated in various human tumor tissues, and in this process, it was confirmed that NEGR1 is involved in intracellular cholesterol transport and intracellular storage of lipids. In Negr1-deficient mice, white adipose tissue (WAT) was significantly enlarged and cell size was also increased. Additionally, increased liver fat accumulation and skeletal muscle atrophy were observed in Negr1-deficient mice. This supports that Negr1 is involved in lipid transport. Based on these prior studies, the present inventors came up with the present invention in the process of identifying the role of NEGR1 in intracellular lipid transport and obesity.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein is provided to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

< Example 1. Confirmation of increased CD36 expression in adipose tissue of Negr1- deficient mice ( Negr1 ^-/- )>

In a previous study, it was confirmed that the epididymal WAT of Negr1 ^-/- mice was enlarged compared to WT mice. Accordingly, we investigated which fatty acid transporter functions in uptake of LCFA from the cell membrane of Negr1-related adipose tissue.

1.1 Negr1 Deficient mice ( Negr1 ^-/- ) of white fat ( WAT ) check the increase

Negr1 ^−/− mice were provided by the Texas A&M Institute for Genomic Medicine (College Station, TX, USA) and were incubated in a controlled environment at 22–24°C and 55% humidity before and during the experiment for 12 days. They were bred and maintained in a time light/dark cycle. All animal procedures were approved by the Animal Care Committee of Seoul National University Animal Hospital.

Negr1 ^-/- and WT mice at 10 weeks of age were dissected, epididymal white adipose tissue was removed, and the weight was measured and shown in Figure 1A. As shown in Figure 1A, the epididymal WAT of Negr1 ^−/− mice is approximately 1.4-fold compared to the wild type. increased ( p =0.026).

1.2 LCFA Fatty acids involved in absorption transporter check

To identify the fatty acid transporters involved in the uptake of LCFA from the cell membrane of adipose tissue in association with Negr1, three major transporters, CD36, a membrane-bound fatty acid binding protein, were identified in epididymal WAT of WT and Negr1 ^−/− mice. The expression levels of binding protein (FABPpm) and Fatty Acid Transport Protein 1 (FATP1) were examined by quantitative real-time polymerase chain reaction (Quantitative-RT-PCR).

ren, Germany)를 사용하여 조직 샘플에서 전체 RNA를 추출하고 SuperScript III 역전사 키트(Invitrogen)를 사용하여 cDNA로 변환했다. 이후 정량적 중합효소 연쇄 반응(qPCR)을 이용하여 각 수송체의 발현량을 측정하였다. 이때 사용한 CD36에 대한 특정 프라이머는 정방향: 5-GCATGAGAATGCCTCCAAACA-3, 역방향: 5-CGGAACTGTGGGCTCATTG-3이었으며, 글리세르알데히드 3-포스페이트 탈수소효소(GAPDH)를 표준화를 위한 참조 유전자로 사용하였다. 각 발현량은 GAPDH를 참조 유전자로 사용하여 상대 발현으로 계산하여 나타내었다. 도 1B에서 보는 바와 같이, CD36의 발현 수준은 FABPpm 및 FATP1의 발현 수준보다 현저히 높았으며, 이는 CD36이 지방 조직에서 LCFA를 흡수하는 데에 중요한 역할을 할 수 있음을 시사했다. 도 1C에는 Negr1^-/- 마우스에서의 각 지방산 수송체의 발현량을 WT 마우스와 비교하여 나타내었다. 도 1C에서 보는 바와 같이, CD36 발현만이 WT 마우스에 비해 Negr1^-/- 마우스에서 유의하게 증가(1.3배, p=0.0007)하였으며, FABPpm 및 FATP1은 WT 및 Negr1^-/- 마우스에서 발현량에 유의한 차이를 나타내지 않았다.The expression levels of CD36, FABPpm, and FATP1 were measured using total RNA obtained from epididymal WAT of WT and Negr1 ^−/− mice, and are shown in Figure 1B. NuecleoSpin RNA extraction kit (Macherey-Nagel, D 117

ren, Germany) and converted to cDNA using the SuperScript III Reverse Transcription Kit (Invitrogen). Afterwards, the expression level of each transporter was measured using quantitative polymerase chain reaction (qPCR). The specific primers for CD36 used at this time were forward: 5-GCATGAGAATGCCTCCAAACA-3, reverse: 5-CGGAACTGTGGGCTCATTG-3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene for standardization. Each expression level was calculated and expressed as relative expression using GAPDH as a reference gene. As shown in Figure 1B, the expression level of CD36 was significantly higher than that of FABPpm and FATP1, suggesting that CD36 may play an important role in uptake of LCFA in adipose tissue. Figure 1C shows the expression level of each fatty acid transporter in Negr1 ^-/- mice compared with WT mice. As shown in Figure 1C, only CD36 expression was significantly increased (1.3-fold, p = 0.0007) in Negr1 ^−/− mice compared to WT mice, and FABPpm and FATP1 had significant expression levels in WT and Negr1 ^−/− mice. did not show any difference.

1.3 Confirmation of CD36 expression level according to tissue

To investigate CD36 expression in tissues other than WAT, quantitative RT-PCR was performed using mRNA from WAT, liver, and gastrocnemius (GA) tissue, a skeletal muscle, in the same manner as the above experiment, and the results are shown in Figure 1D. . As shown in Figure 1D, expression of CD36 was approximately 3-fold greater in WAT than in liver or GA muscle. Additionally, as shown in Figure 1E, the expression level of CD36 in Negr1 ^−/− mice was increased approximately 1.4-fold ( p =0.0033) in WAT and liver tissue compared to WT mice, but decreased (1.6-fold) in GA muscle. , p =0.024).

Through the above results, it was confirmed that the increased fat mass in Negr1 ^-/- mice was related to CD36, a major LCFA transporter in adipocytes.

< Example 2. NEGR1 and Check the interaction of CD36>

2.1 NEGR1 and Confirmation of binding of CD36

Based on the initial finding that NEGR1 and CD36 both contain large extracellular regions and are associated with membrane lipid rafts in the plasma membrane, we investigated the possibility of their molecular interaction.

First, cDNA of the CD36 gene was obtained from the total RNA of 293T cells, and an HA-tagged CD36 construct (pcDNA3-HA-CD36) was constructed using the pcDNA3-HA plasmid. In addition, pEGFP-C1-NEGR1 tagged with membrane-targeted enhanced green fluorescent protein (EGFP) was constructed using the pEGFP-C1 vector (Clontech) according to the method of Kim et al. (2014). Afterwards, pcDNA3-HA-CD36 and pEGFP-C1-NEGR1 plasmids were transfected into 293T cells, and then their cell lysates were subjected to co-immunoprecipitation (IP) using an anti-GFP antibody. Immunoblotting (IB) was performed using anti-HA. As a result, as shown in Figure 2A, it was confirmed that CD36-HA did not appear in the IgG-rich control group, but was present in the NEGR1-rich fraction. In addition, Figure 2B shows that NEGR1-GFP was co-fractionated by CD36 when the same experiment was performed using an anti-HA antibody, confirming that there is an interaction between NEGR1 and CD36.

Based on the interaction between NEGR1 and CD36 confirmed above, IP was performed using SKOV-3-FLAG-NEGR1 stable cells to determine intracellular molecular interactions.

First, the 3×FLAG sequence between the signal sequence (amino acids 1–39) and the remaining sequence (40–314) of the NEGR1 gene was subcloned with AflII and -FLAG-NEGR1 was prepared. Afterwards, the pcDNA4-FLAG-NEGR1 plasmid was transfected into SKOV-3 cells, and then selected with zeocin (50 μg/ml, Invitrogen) to obtain SKOV-3-FLAG-NEGR1 stable cells. The cell lysate of the obtained SKOV-3-FLAG-NEGR1 stable cells was subjected to IP using an anti-FLAG antibody and immunoblotting was performed using an anti-CD36 antibody, which is shown in Figure 2C.

As shown in Figure 2C, when IP was performed with an anti-FLAG antibody, a highly glycosylated form of CD36 was observed in the NEGR1 fraction by IB using an anti-CD36 antibody. In other words, it was confirmed that there is an interaction between NEGR1 and CD36, consistent with the 293T cell results.

2.2 NEGR1 and Confirmation of cellular localization of binding of CD36

To clarify that the NEGR1-CD36 interaction occurs at the extracellular surface of the cell membrane, an in situ binding assay was performed using the human Fc fused secreted form of NEGR1.

It is known that when a desired protein is fused to the Fc of an immunoglobulin using genetic recombination technology, the target protein is expressed and secreted at a high level. After culturing 293T cells transfected with NEGR1-Fc or Fc control plasmids fusing NEGR1 to Fc, the culture medium was collected. The collected NEGR1-Fc or Fc-containing culture medium was incubated with SKOV-3 cells for 2 hours, then each cell was washed and then co-immunostained with anti-human Fc antibody (red) and CD36 antibody (green). The results are shown in Figure 2D. As shown in Figure 2D, little Fc signal was observed in control cells treated with Fc-containing medium. In contrast, a strong Fc signal was observed in NEGR1-Fc treated cells, which overlapped with the CD36 signal. Through this, it was confirmed that NEGR1 interacts with CD36 on the cell surface.

Proximity Ligation Assays (PLA) of KOV-3-FLAG-NEGR1 cells using Duolink® In Situ PLA were used to reconfirm that NEGR1-CD36 interaction occurs at the cell membrane.

Assays were performed on SKOV-3-NEGR1-FLAG cells using Duolink PLA reagent (Sigma-Aldrich) according to the manufacturer's protocol. SKOV-3-FLAG-NEGR1 cells prepared in Example 2.1 were fixed, permeabilized with 0.1% Triton X-100, and then incubated with anti-CD36 and anti-FLAG antibodies for 2 hours. Next, cells were incubated with PLA probes (anti-mouse (-) and anti-rabbit (+), respectively) and Alexa Fluor 488 phalloidin (Invitrogen) for 1 h. Afterwards, the cells were incubated with the ligation solution for 30 minutes and the amplification solution for 2 hours, and then the cells were mounted with a mounting solution containing DAPI. Imaging was performed using an Olympus BX51 microscope or a Leica TCS SP5 AOBS confocal microscope equipped with a 63x inverted NX oil lens and is shown in Figure 2E. At this time, Phalloidin staining was used to examine cell morphology. As shown in Figure 2E, while treatment with a single antibody did not provide any signal, a clear fluorescent signal was detected in cells treated with both antibodies, indicating that these proteins are in close proximity.

The in situ proximity ligation analysis of KOV-3-FLAG-NEGR1 cells was performed in the same manner as above under non-permeable conditions, and the results are shown in Figure 3. As shown in Figure 3, when treated with both anti-CD36 and anti-FLAG antibodies, a strong fluorescence signal could be observed. This showed that NEGR1-CD36 interaction occurs at the cell membrane.

< Example 3. NEGR1 and determination of the binding area of CD36 protein>

As it was confirmed above that NEGR1 and CD36 proteins interact, the region where NEGR1 and CD36 proteins bind was determined. Figure 4A shows a schematic diagram of CD36 and NEGR1 structures. Potential N-glycosylation sites are indicated by filled circles. NEGR1 has a relatively simple structure containing three consecutive C2-type immunoglobulin-like domains (C2-type Ig-like domains), whereas the extracellular domain of CD36 is CLESH (CD36, LIMP-2, Emp sequence homology). Contains no clearly separated domains except domain (93-155) and proline-rich domain (ProR, 243-375) and contains a hydrophobic patch that recesses into the plasma membrane, lipid binding amino acid sequence 127-279 It is predicted to contain pockets.

To determine the protein region responsible for CD36-NEGR1 interaction, glutathione (GST)-fused CD36 deletion construct CD36 ECD (extracellular domain (ECD), containing 30-439 a.a., SEQ ID NO. 1), N1 (30-439 a.a.), as shown in Figure 4B. Containing 125 a.a, SEQ ID NO: 2), N2 (containing 30-242 a.a, SEQ ID NO: 3), and N3 (containing 30-375 a.a, SEQ ID NO: 4) were generated as shown in Table 1 below.

sequence number Sequence name amino acid sequence One N1 GDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGK DNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRNYIVPILWLNETGTIGDEKANMF RSQVTGKIN 2 N2 GDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFV 3 N3 GDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGK DNISKVAIIDTYKGKRRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDI 4 N4 GDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGK DNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKN

The GST-fused CD36 deletion mutant was transfected into 293T cells together with the pcDNA4-FLAG-NEGR1 plasmid, and binding to NEGR1 was confirmed by GST pull-down analysis, as shown in Figure 4B. Immunoprecipitation and GST pull-down analysis were performed using 1 μg of antibody or glutathione-Sepharose 4B beads (GE Healthcare, Piscataway, NJ, USA) by applying the method of Kim, H., et al. (2017).

As a result of GST pull-down analysis, as shown in Figure 4B, the mutant CD36 ECD containing most of the extracellular region of CD36 bound to NEGR1. Additionally, N1, the extracellular region preceding the unique hydrophobic patch of CD36 (covering 30–125 a.a.), successfully interacted with NEGR1. However, N2, a larger structure containing a hydrophobic region (covering 30–242 a.a.), lost its binding activity with NEGR1. Even after long-term reaction, NEGR1 signal could not be detected in the N2-rich fraction. In contrast, the NEGR1 signal was again detected in the fraction containing N3 (including 30-375 a.a) containing three disulfide bonds, confirming that it had binding activity with NEGR1.

On the other hand, we determined the NEGR1 domain participating in CD36 binding. As shown in Figure 4C, GST-fused NEGR1 deletion constructs D1-3 (containing 40-323 a.a, SEQ ID NO: 5), D1-2 (containing 40-215 a.a, SEQ ID NO: 6), and D2-3 (containing 141-323 a.a) Including, SEQ ID NO. 7), D1 (including 40-136 a.a, SEQ ID NO. 8), D2 (including 141-215 a.a, SEQ ID NO. 9), and D3 (including 226-323 a.a, SEQ ID NO. 10) in Table 2 below Produced together.

sequence number Sequence name amino acid sequence 5 D1-3 FPWAAVDNMMVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTVNEGTNVTLTCLATGKPEPSISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDASFPDVRKVKVVVNFAPTIQEIKSGTVTPGRSGLIRC EGAGVPPPAFEWYKGEKKLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNPPSTAQYGI 6 D1-2 FPWAAVDNMMVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTVQVPPKIYDISNDMTVNEGTNVTLTCLATGKPEPSISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDASFPDV 7 D2-3 IYDISNDMTVNEGTNVTLTCLATGKPEPSISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDASFPDVRKVKVVVNFAPTIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKKLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNPPSTAQYGI 8 D1 FPWAAVDNMMVRKGDTAVLRCYLEDGASKGAWLNRSSIIFAGGDKWSVDPRVSISTLNKRDYSLQIQNVDVTDDGPYTCSVQTQHTPRTMQVHLTV 9 D2 IYDISNDMTVNEGTNVTLTCLATGKPEPSISWRHISPSAKPFENGQYLDIYGITRDQAGEYECSAENDASFPDV 10 D3 TIQEIKSGTVTPGRSGLIRCEGAGVPPPAFEWYKGEKKLFNGQQGIIIQNFSTRSILTVTNVTQEHFGNYTCVAANKLGTTNASLPLNPPSTAQYGI

293T cells were transfected with each NEGR1 mutant and pcDNA3-HA-CD36 plasmid prepared above, and the results of GST pull-down analysis are shown in Figure 4C. As shown in Figure 4C, the D3 domain showed excellent binding activity to CD36, while the D1 and D2 domains showed no binding affinity to CD36. D1-2 also cofractionated with CD36, possibly due to sequence similarity between the Ig-like domains.

Taking the above results together, it was confirmed that the N-terminal region of CD36 and the C-terminal D3 domain of NEGR1 play an important role in their association.

< Example 4. NEGR1 and CD36 co. localization Confirm>

4.1 SKOV -3 cells NEGR1 and CD36 co. localization

Immunofluorescence staining was performed to determine whether NEGR1 and CD36 coexisted in cells. First, SKOV-3 cells were co-transfected with HA-CD36 and FLAG-NEGR1 plasmids and immunostained with anti-HA and anti-FLAG antibodies, and the results are shown in Figure 5A. SKOV-3 cells transiently transfected with FLAG-NEGR1 and HA-CD36 plasmids were cultured with or without Triton-X-100 treatment. This was followed by incubation with anti-FLAG and anti-HA antibodies, followed by anti-rabbit Cy3-conjugate and anti-mouse FITC secondary antibodies. Imaging analysis was performed using an Olympus BX51 microscope. As shown in Figure 5A, when the cells were permeabilized with 0.1% Triton- It can be seen that it co-localizes in endomembrane systems such as cotton. Meanwhile, in non-permeabilized cells without 0.1% Triton-X-100, NEGR1 and CD36 were detected at the cell membrane, especially at the cell border (bottom of Figure 5A).

4.2 SKOV -3-FLAG- NEGR1 Endogenous in stable cells NEGR1 and CD36 confirmation

To observe the behavior of endogenous NEGR1 and CD36, immunofluorescence microscopy was performed using SKOV-3-FLAG-NEGR1 stable cells with or without Triton-X 100 treatment and the results were reported. It is shown in Figure 5B. As shown in Figure 5B, the fluorescence signals of the two proteins confirmed that NEGR1 and CD36 were colocalized both in intracellular organelles (+Triton-X 100) and on the external surface of the plasma membrane (-Triton-X 100).

< Example 5. NEGR1 and binding analysis in rafts of CD36>

Next, 293T cells transfected with HA-CD36 and GFP-NEGR1 plasmids were subjected to a suspension selection experiment using OptiPrep ^™ gradient. After transfection of 293T cells with HA-CD36 plasmid together with GFP or GFP-NEGR1, membrane fractionation was performed by suspension method using Optiprep gradient. Each transfected cell lysate was adjusted to 32% OptiPrep and sequentially overlaid with 24% and 20% iodixanol solutions. Afterwards, centrifugation was performed at 76,000 × g at 4°C for 18 hours, and the fractions were separated from the top. A total of 12 fractions were obtained by designating and collecting fraction 1. CD36 and NEGR1 were visualized with anti-HA and anti-GFP antibodies, respectively, and flotillin-1 was used as a raft marker.

As a result, as shown in Figure 5C, flotillin-1 was detected in cells co-transfected with HA-CD36 and EGFP control vector. It was confirmed that fractions 3 and 4 contained rafts. Additionally, in the raft fraction, CD36 was not detected at short exposure (SE), but was detected only at long exposure (LE), confirming that only a very small amount of CD36 protein was detected in the raft fraction. In contrast, for cells co-transfected with HA-CD36 and GFP-NEGR1, CD36 was identified in the raft fraction even at short exposures. These results suggest that NEGR1 interacts with CD36 in lipid rafts.

< Example 6. of NEGR1 Effects on cellular CD36 protein levels>

We investigated whether NEGR1 affects CD36 protein levels.

6.1 In transfected 293T cells NEGR1 Effects on CD36 protein levels

After co-transfection of HA-CD36 and GFP-NEGR1 plasmids at different concentrations into 293T cells, the same amount of total protein (30 μg) was loaded on SDS-PAGE and CD36 protein was detected using anti-HA antibody. . The intensity of each CD36 band in the image was quantified using ImageJ software (right).

As shown in Figure 6A, HA-CD36 protein appeared as two major bands with different glycosylation states, and the intensity of both bands decreased as NEGR1 expression increased. In particular, the expression of the shorter band (around 60 kDa), which is the immature form, was found to decrease more significantly as NEGR1 increased than the highly glycosylated mature form (around 85 kDa).

6.2 3T3-L1- NEGR1 -In FLAG stable cells NEGR1 Effects on CD36 protein levels

To confirm this again, the CD36 protein level was evaluated by immunoblotting in control cells (cells transfected with vector only, WT) and 3T3-L1-NEGR1-FLAG stable cells using an anti-CD36 antibody. As shown in Figure 6B, the CD36 level of 3T3-L1-NEGR1-FLAG stable cells overexpressing NEGR1 was reduced to less than 46% compared to control cells (WT).

6.3 NEGR1 ^-/- In MEF Determination of CD36 protein levels

The expression of CD36 according to NEGR1 levels was examined in Mouse Embryonic Fibroblasts (MEF) obtained from day 13.5 embryos of NEGR1-deficient (NEGR1 ^-/- ) mice (Kim, H. et al., 2017). Control cells and NEGR1 ^−/− MEFs were precultured for 24 hours in medium with or without 0.1mM oleic acid, and then CD36 protein levels were assessed by immunoblotting using an anti-CD36 antibody. Endogenous CD36 and NEGR1 were visualized with anti-CD36 and anti-NEGR1 antibodies, respectively. As shown in Figure 6C, it was confirmed that the CD36 protein level was significantly increased in NEGR1-/- MEFs compared to control MEF cells, regardless of the addition of oleic acid, a representative fatty acid. This shows that NEGR1 negatively regulates the CD36 protein level. was able to prove.

6.4 NEGR1 ^-/- Determination of CD36 protein levels in mice

6.4.1 NEGR1 ^-/- mouse In WAT Determination of CD36 protein levels

Next, we examined CD36 protein levels in epididymal WAT of Negr1 ^−/− mice. Control (WT) mice and Negr1 ^−/− mice were dissected, epididymal WAT was removed, and immunoblotting was performed. Immunoblotting was performed with anti-CD36 antibodies and anti-NEGR1, and CD36 expression levels in epididymal WAT of control (WT) mice and Negr1 ^−/− mice were quantified using ImageJ and normalized to Vinculin as shown in Figure 7A. indicated. As shown in Figure 7A, the CD36 protein level in epididymal WAT of Negr1 ^−/− mice increased approximately 1.3-fold compared to WT mice.

The expression level of CD36 in epididymal WAT of WT mice and Negr1 ^−/− mice was confirmed by immunostaining. WAT tissue sections were immunostained with anti-CD36 antibody, and the results are shown in Figure 7B. As shown in Figure 7B, it was confirmed that the CD36 expression level of epididymal WAT of Negr1 ^-/- mice was increased compared to that of epididymal WAT of WT mice.

6.4.2 NEGR1 ^-/- Determination of CD36 protein levels in mouse liver

CD36 protein levels were confirmed in the liver tissues of control (WT) mice and Negr1 ^-/- mice in the same manner as in Example 6.4.1, and the results are shown in Figures 7C and 7D. As shown in Figure 7C and Figure 7D, the CD36 expression level of Negr1 ^-/- mice in immunoblotting ( Figure 7C ) was approximately 2.2 times the signal in WT mice, and staining of tissue sections ( Figure 7D ) also showed that Negr1 ^-/- It was confirmed that the CD36 protein level in mouse liver tissue was higher than that in control (WT) mice.

Through these results, it was confirmed that CD36 protein level was increased in NEGR1-deficient cells.

< Example 7. NEGR1 Fatty acid absorption and active oxygen ( ROS ) Check the impact on the level>

7.1 NEGR1 ^-/- Fatty acid absorption in mice

Based on the above results of increased CD36 expression in NEGR1 ^-/- cells, we evaluated whether the fat accumulation seen in NEGR1 ^-/- mice was associated with increased fatty acid uptake associated with CD36.

Epididymal WAT was removed from control (WT) mice and NEGR1 ^-/- mice to isolate primary adipocytes, and then analyzed using the free fatty acid uptake assay kit (Fatty Acid Uptake Assay Kit, ab176768, Fluorometric, Abcam, UK) according to the manufacturer's method. Fatty acid absorption rate was measured according to. Fluorescence signals were measured for 40 minutes using a fluorescence microplate reader FlexStation 3 at 485/515 nm and are shown in Figure 8A. Imaging was performed using an Olympus BX51 microscope. As shown in Figure 8A, adipocytes from NEGR1 ^−/− mouse epididymal WAT showed ~1.5-fold higher fatty acid uptake than WT ( p =0.000008).

Additionally, primary adipocytes from WT and NEGR1 ^−/− mice were incubated in the presence of 0.1mM oleic acid for 24 h, and lipid droplets were visualized by BODIPY 493/503 staining, as shown in Figure 8B. NEGR1 ^−/− Adipocytes from mice showed higher lipid content than those from WT mice. Through this, it was confirmed that fatty acid absorption and storage were increased in NEGR1 ^-/- mice.

7.2 NEGR1 ^-/- MEF's ROS level investigation

Considering that fatty acid utilization is often associated with the generation of cellular oxidative stress, we determined the levels of reactive oxygen species (ROS) in NEGR1 ^−/− MEFs. WT and NEGR1 ^-/- MEFs were cultured with H ₂ O ₂ 0, 100, 150, and 200 μM, or oleic acid (OA) 0, 50, and 100 μM for 24 hours, then DCFDA/H2DCFDA-Cellular ROS Assay. Kit (ab113851, Abcam, UK) was used to examine ROS levels according to the manufacturer's protocol. The fluorescence signal was measured at 485/515 nm, and the results are shown in Figures 8C and 8D.

As shown in Figures 8C and 8D, the ROS levels of WT and NEGR1 ^-/- MEFs did not differ under normal conditions (H ₂ O ₂ 0 μM), but when H ₂ O ₂ or oleic acid was added, NEGR1 ^{- /-} ROS levels in MEFs increased more than those in WT cells. This difference gradually increased as the concentration of H ₂ O ₂ or oleic acid increased, reaching ~1.2 times ( p = 0.00002) at 150 μM H ₂ O ₂ and even more significantly at 100 μM oleic acid. These results suggest that NEGR1-deficient cells have higher levels of oxidative stress.

7.3 NEGR1 ^-/- MEF's AMP-activated protein kinase kinase , AMPK) activity investigation

Since AMPK activation is closely related to fatty acid utilization and cellular oxidative stress, we examined AMPK activation in NEGR1 ^−/− MEFs. In the same manner as in Example 7.2, 0 or 0.1mM oleic acid was added to the culture medium of WT and Negr1 ^-/- MEF and incubated for 24 hours, followed by immunoblotting using anti-CD36 antibody, anti-pAMPK antibody, and anti-AMPK antibody. Detected. As shown in Figure 8E, under normal culture conditions, p-AMPK levels in NEGR1 ^−/− MEFs were lower than those in WT cells despite elevated CD36 protein levels, and when cultured with 0.1mM oleic acid, p-AMPK levels in NEGR1 −/− MEFs were lower than those in WT cells. -AMPK levels were all upregulated. However, the activated AMPK ratio (pAMPK/AMPK) was still low in NEGR1 ^−/− MEFs and was inversely proportional to CD36 levels. Collectively, the above results suggest that AMPK activation was attenuated in NEGR1-depleted cells despite increased oxidative stress.

As seen above, the present invention focused on the fact that adipose tissue increased in NEGR1-deficient mice, and in particular, both mRNA and protein levels of CD36 increased in liver and adipose tissue. In the process of studying the effects of NEGR1 and CD36, NEGR1 The interaction between CD36 and CD36 was identified for the first time. In addition, the amino acid sequences of each NEGR1 and CD36 involved in the interaction between NEGR1 and CD36 were confirmed for the first time.

That is, CD36 is an integral membrane protein containing a hairpin shape with both N- and C-termini inside the cell, as well as CD36 ECD (30-439 a.a), as well as NEGR1 with a small N-terminal fragment N1 (30-125 a.a). It was shown that it is sufficient for interaction with .

Additionally, studies of NEGR1 sequences participating in CD36 binding showed GST-fused NEGR1 deletion constructs D1-3(40-323 a.a), D1-2(40-215 a.a), D2-3(141-323 a.a), and D1(40-136). a.a), D2 (141-215 a.a) and D3 (226-323 a.a), the D3 fragment shows excellent binding activity to CD36 and can regulate CD36-related diseases through interaction with CD36 with only 98 amino acid sequences. was confirmed. In particular, it can be used to prevent or treat obesity by reducing the transport and accumulation of fatty acids related to CD36 through the use of NEGR1.

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Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu 145 150 155 160 Val Pro Tyr Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro Tyr Asn 165 170 175 Asn Thr Ala Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile 180 185 190 Ser Lys Val Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg Asn Leu Ser 195 200 205 Tyr Trp Glu Ser His Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser 210 215 220 Phe Pro Pro Phe Val Glu Lys Ser Gln Val Leu Gln Phe Phe Ser Ser 225 230 235 240 Asp Ile Cys Arg Ser Ile Tyr Ala Val Phe Glu Ser Asp Val Asn Leu 245 250 255 Lys Gly Ile Pro Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala 260 265 270 Ser Pro Val Glu Asn Pro Asp Asn Tyr Cys Phe Cys Thr Glu Lys Ile 275 280 285 Ile Ser Lys Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser Lys Cys 290 295 300 Lys Glu Gly Arg Pro Val Tyr Ile Ser Leu Pro His Phe Leu Tyr Ala 305 310 315 320 Ser Pro Asp Val Ser Glu Pro Ile Asp Gly Leu Asn Pro Asn Glu Glu 325 330 335 Glu His Arg Thr Tyr Leu Asp Ile Glu Pro Ile Thr Gly Phe Thr Leu 340 345 350 Gln Phe Ala Lys Arg Leu Gln Val Asn Leu Leu Val Lys Pro Ser Glu 355 360 365 Lys Ile Gln Val Leu Lys Asn Leu Lys Arg Asn Tyr Ile Val Pro Ile 370 375 380 Leu Trp Leu Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Asn Met 385 390 395 400 Phe Arg Ser Gln Val Thr Gly Lys Ile Asn 405 410 <210> 2 <211> 125 <212> PRT <213> Homo sapiens <400> 2 Gly Asp Leu Leu Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu 1 5 10 15 Glu Gly Thr Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val 20 25 30 Tyr Arg Gln Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met 35 40 45 Met Asn Ser Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr Thr Tyr 50 55 60 Arg Val Arg Phe Leu Ala Lys Glu Asn Val Thr Gln Asp Ala Glu Asp 65 70 75 80 Asn Thr Val Ser Phe Leu Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser 85 90 95 Leu Ser Val Gly Thr Glu Ala Asp Asn Phe Thr Val Leu Asn Leu Ala 100 105 110 Val Ala Ala Ala Ser His Ile Tyr Gln Asn Gln Phe Val 115 120 125 <210> 3 <211> 242 <212> PRT <213> Homo sapiens <400> 3 Gly Asp Leu Leu Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu 1 5 10 15 Glu Gly Thr Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val 20 25 30 Tyr Arg Gln Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met 35 40 45 Met Asn Ser Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr Thr Tyr 50 55 60 Arg Val Arg Phe Leu Ala Lys Glu Asn Val Thr Gln Asp Ala Glu Asp 65 70 75 80 Asn Thr Val Ser Phe Leu Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser 85 90 95 Leu Ser Val Gly Thr Glu Ala Asp Asn Phe Thr Val Leu Asn Leu Ala 100 105 110 Val Ala Ala Ala Ser His Ile Tyr Gln Asn Gln Phe Val Gln Met Ile 115 120 125 Leu Asn Ser Leu Ile Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg 130 135 140 Thr Leu Arg Glu Leu Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu 145 150 155 160 Val Pro Tyr Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro Tyr Asn 165 170 175 Asn Thr Ala Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile 180 185 190 Ser Lys Val Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg Asn Leu Ser 195 200 205 Tyr Trp Glu Ser His Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser 210 215 220 Phe Pro Pro Phe Val Glu Lys Ser Gln Val Leu Gln Phe Phe Ser Ser 225 230 235 240 Asp Ile <210> 4 <211> 375 <212> PRT <213> Homo sapiens <400> 4 Gly Asp Leu Leu Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu 1 5 10 15 Glu Gly Thr Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val 20 25 30 Tyr Arg Gln Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met 35 40 45 Met Asn Ser Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr Thr Tyr 50 55 60 Arg Val Arg Phe Leu Ala Lys Glu Asn Val Thr Gln Asp Ala Glu Asp 65 70 75 80 Asn Thr Val Ser Phe Leu Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser 85 90 95 Leu Ser Val Gly Thr Glu Ala Asp Asn Phe Thr Val Leu Asn Leu Ala 100 105 110 Val Ala Ala Ala Ser His Ile Tyr Gln Asn Gln Phe Val Gln Met Ile 115 120 125 Leu Asn Ser Leu Ile Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg 130 135 140 Thr Leu Arg Glu Leu Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu 145 150 155 160 Val Pro Tyr Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro Tyr Asn 165 170 175 Asn Thr Ala Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile 180 185 190 Ser Lys Val Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg Asn Leu Ser 195 200 205 Tyr Trp Glu Ser His Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser 210 215 220 Phe Pro Pro Phe Val Glu Lys Ser Gln Val Leu Gln Phe Phe Ser Ser 225 230 235 240 Asp Ile Cys Arg Ser Ile Tyr Ala Val Phe Glu Ser Asp Val Asn Leu 245 250 255 Lys Gly Ile Pro Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala 260 265 270 Ser Pro Val Glu Asn Pro Asp Asn Tyr Cys Phe Cys Thr Glu Lys Ile 275 280 285 Ile Ser Lys Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser Lys Cys 290 295 300 Lys Glu Gly Arg Pro Val Tyr Ile Ser Leu Pro His Phe Leu Tyr Ala 305 310 315 320 Ser Pro Asp Val Ser Glu Pro Ile Asp Gly Leu Asn Pro Asn Glu Glu 325 330 335 Glu His Arg Thr Tyr Leu Asp Ile Glu Pro Ile Thr Gly Phe Thr Leu 340 345 350 Gln Phe Ala Lys Arg Leu Gln Val Asn Leu Leu Val Lys Pro Ser Glu 355 360 365 Lys Ile Gln Val Leu Lys Asn 370 375 <210> 5 <211> 283 <212> PRT <213> Homo sapiens <400> 5 Phe Pro Trp Ala Ala Val Asp Asn Met Met Val Arg Lys Gly Asp Thr 1 5 10 15 Ala Val Leu Arg Cys Tyr Leu Glu Asp Gly Ala Ser Lys Gly Ala Trp 20 25 30 Leu Asn Arg Ser Ser Ile Ile Phe Ala Gly Gly Asp Lys Trp Ser Val 35 40 45 Asp Pro Arg Val Ser Ile Ser Thr Leu Asn Lys Arg Asp Tyr Ser Leu 50 55 60 Gln Ile Gln Asn Val Asp Val Thr Asp Asp Gly Pro Tyr Thr Cys Ser 65 70 75 80 Val Gln Thr Gln His Thr Pro Arg Thr Met Gln Val His Leu Thr Val 85 90 95 Gln Val Pro Pro Lys Ile Tyr Asp Ile Ser Asn Asp Met Thr Val Asn 100 105 110 Glu Gly Thr Asn Val Thr Leu Thr Cys Leu Ala Thr Gly Lys Pro Glu 115 120 125 Pro Ser Ile Ser Trp Arg His Ile Ser Pro Ser Ala Lys Pro Phe Glu 130 135 140 Asn Gly Gln Tyr Leu Asp Ile Tyr Gly Ile Thr Arg Asp Gln Ala Gly 145 150 155 160 Glu Tyr Glu Cys Ser Ala Glu Asn Asp Ala Ser Phe Pro Asp Val Arg 165 170 175 Lys Val Lys Val Val Val Val Asn Phe Ala Pro Thr Ile Gln Glu Ile Lys 180 185 190 Ser Gly Thr Val Thr Pro Gly Arg Ser Gly Leu Ile Arg Cys Glu Gly 195 200 205 Ala Gly Val Pro Pro Pro Ala Phe Glu Trp Tyr Lys Gly Glu Lys Lys 210 215 220 Leu Phe Asn Gly Gln Gln Gly Ile Ile Gln Asn Phe Ser Thr Arg 225 230 235 240 Ser Ile Leu Thr Val Thr Asn Val Thr Gln Glu His Phe Gly Asn Tyr 245 250 255 Thr Cys Val Ala Ala Asn Lys Leu Gly Thr Thr Asn Ala Ser Leu Pro 260 265 270 Leu Asn Pro Pro Ser Thr Ala Gln Tyr Gly Ile 275 280 <210> 6 <211> 175 <212> PRT <213> Homo sapiens <400> 6 Phe Pro Trp Ala Ala Val Asp Asn Met Met Val Arg Lys Gly Asp Thr 1 5 10 15 Ala Val Leu Arg Cys Tyr Leu Glu Asp Gly Ala Ser Lys Gly Ala Trp 20 25 30 Leu Asn Arg Ser Ser Ile Ile Phe Ala Gly Gly Asp Lys Trp Ser Val 35 40 45 Asp Pro Arg Val Ser Ile Ser Thr Leu Asn Lys Arg Asp Tyr Ser Leu 50 55 60 Gln Ile Gln Asn Val Asp Val Thr Asp Asp Gly Pro Tyr Thr Cys Ser 65 70 75 80 Val Gln Thr Gln His Thr Pro Arg Thr Met Gln Val His Leu Thr Val 85 90 95 Gln Val Pro Pro Lys Ile Tyr Asp Ile Ser Asn Asp Met Thr Val Asn 100 105 110 Glu Gly Thr Asn Val Thr Leu Thr Cys Leu Ala Thr Gly Lys Pro Glu 115 120 125 Pro Ser Ile Ser Trp Arg His Ile Ser Pro Ser Ala Lys Pro Phe Glu 130 135 140 Asn Gly Gln Tyr Leu Asp Ile Tyr Gly Ile Thr Arg Asp Gln Ala Gly 145 150 155 160 Glu Tyr Glu Cys Ser Ala Glu Asn Asp Ala Ser Phe Pro Asp Val 165 170 175 <210> 7 <211> 182 <212> PRT <213> Homo sapiens <400> 7 Ile Tyr Asp Ile Ser Asn Asp Met Thr Val Asn Glu Gly Thr Asn Val 1 5 10 15 Thr Leu Thr Cys Leu Ala Thr Gly Lys Pro Glu Pro Ser Ile Ser Trp 20 25 30 Arg His Ile Ser Pro Ser Ala Lys Pro Phe Glu Asn Gly Gln Tyr Leu 35 40 45 Asp Ile Tyr Gly Ile Thr Arg Asp Gln Ala Gly Glu Tyr Glu Cys Ser 50 55 60 Ala Glu Asn Asp Ala Ser Phe Pro Asp Val Arg Lys Val Lys Val Val 65 70 75 80 Val Asn Phe Ala Pro Thr Ile Gln Glu Ile Lys Ser Gly Thr Val Thr 85 90 95 Pro Gly Arg Ser Gly Leu Ile Arg Cys Glu Gly Ala Gly Val Pro Pro 100 105 110 Pro Ala Phe Glu Trp Tyr Lys Gly Glu Lys Lys Leu Phe Asn Gly Gln 115 120 125 Gln Gly Ile Ile Ile Gln Asn Phe Ser Thr Arg Ser Ile Leu Thr Val 130 135 140 Thr Asn Val Thr Gln Glu His Phe Gly Asn Tyr Thr Cys Val Ala Ala 145 150 155 160 Asn Lys Leu Gly Thr Thr Asn Ala Ser Leu Pro Leu Asn Pro Pro Ser 165 170 175 Thr Ala Gln Tyr Gly Ile 180 <210> 8 <211> 96 <212> PRT <213> Homo sapiens <400> 8 Phe Pro Trp Ala Ala Val Asp Asn Met Met Val Arg Lys Gly Asp Thr 1 5 10 15 Ala Val Leu Arg Cys Tyr Leu Glu Asp Gly Ala Ser Lys Gly Ala Trp 20 25 30 Leu Asn Arg Ser Ser Ile Ile Phe Ala Gly Gly Asp Lys Trp Ser Val 35 40 45 Asp Pro Arg Val Ser Ile Ser Thr Leu Asn Lys Arg Asp Tyr Ser Leu 50 55 60 Gln Ile Gln Asn Val Asp Val Thr Asp Asp Gly Pro Tyr Thr Cys Ser 65 70 75 80 Val Gln Thr Gln His Thr Pro Arg Thr Met Gln Val His Leu Thr Val 85 90 95 <210> 9 <211> 74 <212> PRT <213> Homo sapiens <400> 9 Ile Tyr Asp Ile Ser Asn Asp Met Thr Val Asn Glu Gly Thr Asn Val 1 5 10 15 Thr Leu Thr Cys Leu Ala Thr Gly Lys Pro Glu Pro Ser Ile Ser Trp 20 25 30 Arg His Ile Ser Pro Ser Ala Lys Pro Phe Glu Asn Gly Gln Tyr Leu 35 40 45 Asp Ile Tyr Gly Ile Thr Arg Asp Gln Ala Gly Glu Tyr Glu Cys Ser 50 55 60 Ala Glu Asn Asp Ala Ser Phe Pro Asp Val 65 70 <210> 10 <211> 97 <212> PRT <213> Homo sapiens <400> 10 Thr Ile Gln Glu Ile Lys Ser Gly Thr Val Thr Pro Gly Arg Ser Gly 1 5 10 15 Leu Ile Arg Cys Glu Gly Ala Gly Val Pro Pro Pro Ala Phe Glu Trp 20 25 30 Tyr Lys Gly Glu Lys Lys Leu Phe Asn Gly Gln Gln Gly Ile Ile Ile 35 40 45 Gln Asn Phe Ser Thr Arg Ser Ile Leu Thr Val Thr Asn Val Thr Gln 50 55 60 Glu His Phe Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly Thr 65 70 75 80 Thr Asn Ala Ser Leu Pro Leu Asn Pro Pro Ser Thr Ala Gln Tyr Gly 85 90 95Ile

Claims (10)

A composition for preventing or treating obesity comprising a peptide derived from neuronal growth regulator 1 (NEGR1) as an active ingredient. According to paragraph 1,
The peptide derived from Neuronal growth regulator 1 (NEGR1) is a method for preventing obesity, characterized in that it is a peptide consisting of one or more amino acid sequences among the amino acid sequences represented by SEQ ID NOs: 5, 6, 7, 9, and 10, or Therapeutic composition. According to claim 1 or 2,
A composition for preventing or treating obesity, wherein the peptide interacts with cluster of differentiation 36 (CD36). According to claim 1 or 2,
A composition for preventing or treating obesity, wherein the peptide reduces the transport or accumulation of fatty acids in adipocytes or hepatocytes. A health functional food for preventing or improving obesity containing a peptide derived from neuronal growth regulator 1 (NEGR1) as an active ingredient. According to clause 5,
The peptide derived from Neuronal growth regulator 1 (NEGR1) is a method for preventing obesity, characterized in that it is a peptide consisting of one or more amino acid sequences among the amino acid sequences represented by SEQ ID NOs: 5, 6, 7, 9, and 10, or Health functional food for improvement. A peptide derived from neuronal growth regulator 1 (NEGR1), which has anti-obesity activity and consists of one of the amino acid sequences shown in SEQ ID NOs: 5, 6, 7, 9, and 10. In clause 7,
The peptide is a peptide with anti-obesity activity, characterized in that it reduces the transport or accumulation of fatty acids in adipocytes or hepatocytes. In clause 7,
The peptide derived from Neuronal growth regulator 1 (NEGR1) is a peptide with anti-obesity activity consisting of the amino acid sequence represented by SEQ ID NO: 10. A peptide derived from Neuronal growth regulator 1 (NEGR1), a nucleotide sequence encoding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 10.