[보고서]항비만 파이토케미칼의 약물표적으로 작용하는 후각수용체 규명 연구

박태선

보고서 정보

주관연구기관

연세대학교
Yonsei University

연구책임자

박태선

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2019-06

과제시작연도

2018

주관부처

과학기술정보통신부
Ministry of Science and ICT

등록번호

TRKO201900020667

과제고유번호

1711071352

사업명

개인기초연구(과기정통부)(R&D)

DB 구축일자

2020-05-02

키워드

후각수용체.비만.리간드.말초조직.약물표적.

표 말초초직에서 cAMP에 의해 매개되는 신호전달체계
표 α-Cedrene induces cAMP accumulation and Ca2+mobilization in olfactory sensory neuron. (a) Two-dimensional (2D) and 3D structures of α-cedrene obtained by means of the Gaussview software. (b) Olfactory sensory neurons were pretreated with 3-isobutyl-1-methylxanthine (IBMX) for 30 min to inhibit phosphodiesterases, and intracellular cAMP levels were measured after 15 min of odorant stimulation. (c–e) Intracellular Ca2+ levels were monitored every 1 s until recovery of the basal level according to confocal microscopy analysis (LSM7, Zeiss, Germany). The elevated intracellular Ca2+ concentrations of all responding cells were normalized with the control values of Ringer solution-treated cells
표 Sequence alignment of MOR23 and OR10J5. Identical residues are highlighted in blue, whereas different residues are not highlighted. Residues boxed in red represent amino acids which are predicted to point inside the binding cavity of both receptors
표 MOR23 and its human homolog, OR10J5, traffic to the cell surface when expressed in transfected Hana3A cells. (a) FLAG-M2-magnetic beads were used to capture N-terminally FLAG-tagged ORs, which were detected with an anti-FLAG antibody. (b) N-terminally FLAG-tagged ORs were labeled with an Alexa 488-conjugated secondary antibody and imaged by confocal microscopy, and (c) analyzed by flow cytometry
표 Intracellular cAMP levels were measured after α-cedrene stimulation. Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001
표 α-Cedrene reduces triglyceride concentration in HepG2 cells. (a) The percentage of viable cells was determined by an MTT assay. (b) Oil red O staining of HepG2 cells after stimulation by α-cedrene at the indicated concentration. Spectrophotometric quantification of Oil Red O-stained HepG2 cells is presented as means from three independent experiments for each group, and representative photomicrographs (×200) are shown in the left panel. Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001
표 α-Cedrene reduces triglyceride concentration in HepG2 cells via the Adcy-cAMP pathway. (a) Oil red O staining of HepG2 cells exposed to the Adcy inhibitor for 24 h with or without 100 μM α-cedrene. Significant differences between groups are indicated by asterisks. (b) HepG2 cells were exposed to the Adcy inhibitor for 24 h with or without 100 μM α-cedrene, and levels of cAMP were measured.; *P < 0.05; **P < 0.01; ***P < 0.001
표 α-Cedrene reguates the expressions of molecules involved in lipogenesis and fatty acid oxidation via the Adcy-cAMP pathway in HepG2 cells. (a) Western blot analysis of Adcy3, PKA, phosphor-CREB, phosphor-AMPK, and phosphor-HSL in HepG2 cells exposed to the Adcy inhibitor for 24 h with or without 100 μM α-cedrene. (b) RT-PCR analysis of LXRα, SREBP1c, aP2, SCD1, ACC, PPARγ, mtGPAT, and CPT-1 in HepG2 cells exposed to the Adcy inhibitor for 24 h with or without 100 μM α-cedrene. Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001
표 A reduction in OR10J5 expression by specific siRNA abrogates the lipid-lowering effect of α-cedrene in HepG2 cells. (a) OR10J5 was knocked down in HepG2 cells by siRNA transfection. RT-PCR indicates that OR10J5 expression was decreased by anti-OR10J5 siRNA. (b) Oil red O staining of HepG2 cells transfected with nontargeting (control) siRNA or anti-OR10J5 siRNA for 24 h with or without 100 μM α -cedrene
표 A reduction in OR10J5 expression by specific siRNA abrogates the beneficial effect of α-cedrene on the expressions of molecules related to lipogenesis and fatty acid oxidation in HepG2 cells. (a) Western blot analysis of Adcy3 and PKA in HepG2 cells transfected with nontargeting siRNA or anti-OR10J5 siRNA for 24 h with or without 100 μM α-cedrene. (b) RT-PCR analysis of aP2, mtGPAT, and CPT-1 in HepG2 cells transfected with nontargeting siRNA or anti-OR10J5 siRNA for 24 h with or without 100 μM α-cedrene. Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001
표 A schematic diagram illustrating the proposed mechanism by which α-cedrene regulates lipogenesis and fatty acid oxidation in hepatocytes
표 ligand-OR pair list
표 MOR23 knockdown efficiency by siRNA was monitored by semi-quantitative RT-PCR
표 A reduction in MOR23 expression by specific siRNA increases intracellular lipid accumulation in 3T3-L1 cells. Oil red O staining of 3T3-L1 cells transfected with MOR23 siRNA with or without 100 μM α-cedrene
표 A reduction in MOR23 expression by specific siRNA decreases intracellular cAMP levels in 3T3-L1 cells. cAMP level of 3T3-L1 cells transfected with MOR23 siRNA with or without 100 μM α-cedrene
표 A reduction in Adenylyl cyclases expression by Adcy inhibitor(SQ22536) increases intracellular lipid accumulation in 3T3-L1 cells. Oil red O staining of 3T3-L1 cells exposed to Adcy inhibitor for 24 h with or without 100 μM α-cedrene
표 A reduction in MOR23 expression by specific siRNA significantly impaired the cAMP signaling pathway in 3T3-L1 cells. (a) Western blot analysis of Adcy3, PKA Cα, phosphor-AMPK, and phosphor-HSL in 3T3-L1 cells transfected with MOR23 siRNA with or without 100 μM α-cedrene. (b) RT-PCR analysis of the mRNA levels of PPARγ, C/EBPa, aP2, and FAS in 3T3-L1 cells transfected with MOR23 siRNA with or without 100 μM α-cedrene
표 MOR23 depletion also decreases O2 consumption rates and abolishes the thermogenic effect of α-cedrene in 3T3-L1 cells. (a–b) The function of the mitochondrial electron respiratory complex in 3T3-L1 cells was assessed as the OCR via serial injection of 1 μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A. The OCR value (mean of the three points±s.e.m.) is graphed in b
표 MOR23 depletion also decreases thermogenenic genes in 3T3-L1 cells. (a) Western blot analysis of phosphor-CREB in 3T3-L1 cells transfected with MOR23 siRNA with or without 100 μM α-cedrene. (b) RT-PCR analysis of the mRNA levels of PGC1α, Cytc, COX4, PRDM16, Cidea, and UCP1 in 3T3-L1 cells transfected with MOR23 siRNA with or without 100 μM α-cedrene
표 A schematic diagram illustrating the proposed mechanism by which α-cedrene regulates adipogenesis, fatty acid oxidation, and thermogenesis in adipocytes
표 MOR23 knockdown efficiency by siRNA was monitored by semi-quantitative RT-PCR
표 reduction in MOR23 expression by specific siRNA decreases myogenesis in C2C12 myotubes. Gieamsa-staining of C2C12 cells transfected with MOR23 siRNA with or without 100 μM α-cedrene. A) Representative images of Giemsa-stained myoblasts at 72 h of differentiation in the presence of α-cedrene (100 μM).B) Quantification of myotube size. Independent cell culture was carried out in triplicate. At least 300 myotubes were analyzed for both treated and untreated cells. C) Fusion index of myotubes. Significant differences between groups are indicated by asterisks;**p 0.05)
표 A reduction in MOR23 expression by specific siRNA significantly decreased mRNA expression in C2C12 myotubes. Quantitative real-time PCR analysis of myogenin and desmin in C2C12 cells transfected with anti-MOR23 siRNA with or without 100 μM α-cedrene. Significant differences between groups are indicated by asterisks; *p 0.05)
표 A reduction in MOR23 expression by specific siRNA increases free fatty acid-induced atrophy in C2C12 myotubes. A) After 3 days of differentiation, C2C12 myotubes were treated with vehicle or α-cedrene (100 μM) for 24 h in palmitic acid (0.5 mM) supplemented media. Cells were then fixed with 4% paraformaldehyde and stained by Giemsa. B) Quantification of myotube size. Independent cell culture was carried out in triplicate. At least 300 myotubes were analyzed for both treated and untreated cells. C) Fusion index of myotubes. Significant differences between groups are indicated by asterisks;**p 0.05)
표 A reduction in MOR23 expression by specific siRNA increases free fatty acid-induced lipogenesis in C2C12 myotubes. A) After 3 days of differentiation, C2C12 myotubes were treated with vehicle or α-cedrene (100 μM) for 24 h in palmitic acid (0.5 mM) supplemented media. Cells were then fixed with 4% paraformaldehyde and stained by ORO. B) Total cellular triglyceride level. Significant differences between groups are indicated by asterisks;**p 0.05)
표 A reduction in MOR23 expression by specific siRNA significantly increased mRNA expression in C2C12 myotubes. Quantitative real-time PCR analysis of MAFbx, MuRF1, SREBP1c, andFAS in C2C12 cells transfected with anti-MOR23 siRNA for 24 h with or without100 μM α-cedrene. Significant differences between groups are indicated by asterisks;**p 0.05)
표 α-Cedrene induces skeletal muscle hypertrophy in chow-fed mice. A) Grip strength performance. B) Body weights. Significant differences between groups are indicated by asterisks; *p 0.05)
표 α-Cedrene induces skeletal muscle hypertrophy in chow-fed mice. A) Representative H *p 0.05)
표 α-Cedrene induces skeletal muscle hypertrophy in chow-fed mice. A) Visualization of neutral lipids by ORO analysis in the gastrocnemius muscle. B) Total triglyceride level in the gastrocnemius muscle. Significant differences between groups are indicated by asterisks; *p 0.05)
표 α-Cedrene attenuates skeletal muscle atrophy induced by high-fat diet in mice. A) Grip strength performance. B) Body weights. Significant differences between groups are indicated by asterisks; *p < 0.05; **p < 0.01
표 α-Cedrene attenuates skeletal muscle atrophy induced by high-fat diet in mice. A) Representative H *p < 0.05; **p < 0.01
표 α-Cedrene attenuates skeletal muscle atrophy induced by high-fat diet in mice. A) Visualization of neutral lipids by ORO analysis in the gastrocnemius muscle. B) Total triglyceride level in the gastrocnemius muscle. Significant differences between groups are indicated by asterisks; *p < 0.05; **p < 0.01
표 α-Cedrene regulates the expression of MOR23 and its downstream signaling molecules related to protein synthesis and protein degradation in skeletal muscle of mice. A) mRNA levels of MOR23 in the skeletal muscle tissue of mice. B) Protein levels of Adcy3 in the skeletal muscle tissue of mice. C)cAMP concentrations in the skeletal muscle tissue D)Protein levels of PKACα,p-CREB, and CREB in the skeletal muscle tissue. E) Quantitative real-time PCR analysis of PGC-1α1 in the skeletal muscle tissue. F) Protein levels of p-FoxO3 and FoxO3 in the skeletal muscle tissue. G) Quantitative real-time PCR analysis of MAFbx and MURF1 in the skeletal muscle tissue. H) Protein expression of MAFbx and MURF1 in the skeletal muscle tissue. Significant differences between groups are indicated by asterisks; *p < 0.05; **p < 0.01; ***p < 0.001
표 α-Cedrene regulates IGF-1 and myostatin signaling cascades in skeletal muscle of mice. A) Quantitative real-time PCR analysis of PGC-1α4, IGF-1, and myostatin in the skeletal muscle tissue of mice. B) Protein expression of IGF-1 and myostatin in the skeletal muscle tissue. C) Protein levels of p-AKT, AKT, p-mTORC1, mTORC1, p-S6K1, S6K1, p-4E-BP1, and 4E-BP1 in the skeletal muscle tissue. D) Protein levels of p-SMAD2,3 and SMAD2,3 in the skeletal muscle tissue. Significant differences between groups are indicated by asterisks; *p < 0.05; **p < 0.01; ***p < 0.001
표 A schematic diagram illustrating the proposed mechanism by which MOR23 increases skeletal muscle mass and strength

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항비만 파이토케미칼의 약물표적으로 작용하는 후각수용체 규명 연구
Identification of olfactory receptor isoforms specific to a phytochemical with strong anti-obesity property and could work as a novel drug target 원문보기

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항비만 파이토케미칼의 약물표적으로 작용하는 후각수용체 규명 연구 Identification of olfactory receptor isoforms specific to a phytochemical with strong anti-obesity property and could work as a novel drug target 원문보기

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