[보고서]비만억제활성 생물소재의 고속탐색 및 단백체 분석을 통한  비만특이 바이오마커 발굴과 응용

윤종원

비만억제활성 생물소재의 고속탐색 및 단백체 분석을 통한 비만특이 바이오마커 발굴과 응용
High throughput screening of anti-obesity biomaterials and discovery of obesity-specific biomarkers by proteome analysis and their application 원문보기

보고서 정보
주관연구기관	대구대학교 DaeGu University
연구책임자	윤종원
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2013-02
과제시작연도	2012
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201300034283
과제고유번호	1345171775
DB 구축일자	2013-12-21
키워드	비만억제물질,캪사이신,키토산올리고당,단백질체,비만마커단백질,바이오마커,비만저항성,성별특이성,비만과 암Anti-obesity compounds,Capsaicin,Chitosan-oligosaccharides,Proteome,Obesity marker proteins,Biomarker,Obesity resistance,Gender dimorphism,Obesity and cancer

초록 ▼

연구의 목적 및 내용
● 식/약용버섯, 동충하초, 약용식물 등으로부터 비만억제 물질 탐색
● 키토산 올리고당의 항비만활성 규명 및 단백체 분석을 통한 작용기전 해석
● 캪사이신의 항비만효과 검정 및 에너지대사 촉진관련 마커단백질 다수 발굴
● 비만이 잘 유도되는 쥐와 비만에 대한 저항성을 나타내는 실험쥐의 혈액과 주요 대사조직의 단백질체 분석을 통한 비만 원인 단백질 탐색
● 성별에 따라 차이나는 (gender-dependent) 비만관련 바이오마커 발굴 및 분자기작연구
● 비만이 폐암유병률에 미치는 영향 및 촉진인자 단백질 발굴
연구결과
(1) 천연 생물소재로 부터 비만억제물질 추출: 표고, 상황버섯 등 5종의 식,약용버섯 및 300여종 이상의 각종 약용식물로부터 추출물 300여종을 제조, 비만억제활성을 평가하여 차가버섯 등 3종의 비만억제소재 확보.
(2) 키토산올리고당 (CO)의 비만억제 효과 및 분자메커니즘 연구: 키토산 올리고당을 식이한 비만마우스(ob/ob)의 주요 조직의 단백질체를 분석하여 CO가 어떤 경로로 비만억제 활성을 나타내었는가에 대한 분자메커니즘을 규명함.
(3) 캪사이신 (capsaicin)의 비만 억제능 규명 및 실험동물의 단백질체 분석을 통한 분자메커니즘 규명: 비만 억제 메커니즘의 하나인 에너지소모 촉진과 관련한 새로운 마커 단백질을 탐색하고자 정상쥐에 고지방식 을식이하고 캪사이신을 경구 투여하여, 비만 관련 주요조직인 지방조직, 간, 근육의 단백질체 비교 분석을 통해 에너지 소모와 관련된 20종 이상의 새로운 단백질을 규명하였음.
(4) 비만이 잘 유도되는 쥐와 비만에 대한 저항성을 나타내는 실험쥐의 혈액과 주요 대사조직의 단백질체 분석을 통한 비만 원인 단백질 탐색: 고지방식이 결과, 비만이 잘 유도되는 쥐 (OP)와 비만에 대한 저항성을 가지는 쥐 (OR)를 분류하고, 혈액 및 대사조직의 단백질체를 비교 분석하여, 다수의 비만 특이마커 단백질 발굴.
(5) 성별에 따라 차이나는 비만관련 바이오마커 발굴 및 분자기작연구: OP와 OR 쥐의 조직 (지방조직, 근육, 간)과 혈장을 이용하여 비만에 대해 성에 따라 다른 단백질체를 비교분석한 결과, 상이한 발현양상을 나타내는 단백질을 다수 발견하였고, 성별 특이 (gender-dimorphic) 비만분자기작을 제시하였음.
(6) 비만이 폐암발생에 미치는 영향 및 마커단백질 발굴: C57BL/6N 마우스에 LLC (Lewis lung cancer) 세포를 주사하여 폐암을 유발시키고 혈장 단백질체 분석을 수행하여 14종의 마커단백질을 확인. 비만이 암발생을 촉진하는 혈액마커단백질 4종을 발견함.
연구결과의 활용계획
● 차가버섯으로부터 insulin sensitizer 물질 분리⟶인슐린대체물질 (insulin mimicker)로 응용가능성을 제시하였음.
● 키토산올리고당의 비만억제활성과 주요 작용기전을 세계최초로 규명함.
● 캪사이신의 비만 억제 효과를 확인하였을 뿐만 아니라, 단백질체 분석을 통해 비만에 대한 캪사이신의 작용기전을 최초로 제시하였음. 이러한 결과를 통해 캪사이신의 항비만 효과에 대한 단백질체학적 관점에서의 이해와 비만억제제로서의 캪사이신에 대한 약리학적 활용이 가능할 것으로 기대함.
● OP와 OR rats간의 상이한 단백질 발현양상을 최초로 단백질체 분석을 통해 확인하였으며, 이러한 단백질들은 심화된 기능연구를 통해 비만 바이오마커로 활용될 수 있을 것으로 기대됨.
● 비만에 대해 성에 따라 상이한 단백질 양상을 다양한 조직과 혈장에서 단백질체학 연구를 통해 처음으로 확인하였으며 확인된 마커단백질들은 표적 비만치료제 개발 분야 등 비만 바이오 마커로의 활용가능성을 기대함.

Abstract ▼

Purpose&contents
● Screening of compounds with anti-obesity activity from mushrooms and medicinal plants
● Unraveling anti-obesity effects of chitosan-oligosaccharides and its molecular action by proteomic approach
● Evaluation of anti-obesity activity of capsaicin and discovery of marker proteins involved in stimulation of energy metabolism
● Discovery od obesity marker proteins by proteomic analysis of blood and metabolic tissues in obesity-prone and obesity-resistant rats
● Discovery of gender-dimorphic obesity marker proteins and studies about their molecular mechanism
● Discovery of synergistic obesity marker proteins in the cause of lung cancer
Result
(1) Preparation of anti-obesity biomaterials from edible/medicinal mushrooms and entomopathogenic fungi: We obtained hot-water extracts from 5 mushrooms and tested their anti-obesity activity in 3T3-L1 adipocytes. In addition, over 300 crude compounds were obtained from numerous medicinal plants.
(2) Studies on anti-obesity effects of chitosanoligosaccharides (CO) and molecular mechanism: We investigated body weight reduction and lipid metabolism in CO-fed ob/ob mice, and elucidated molecular mechanism of CO by proteomic analysis of adipocytes to suggest mechanism of anti-obesity effects of CO.
(3) Studies on anti-obesity effects and molecular action of capsaicin in many rat tissues using proteomic analysis: We investigated anti-obesity effects of capsaicin in rats fed with a HFD to discover novel biomarkers involved in energy expenditure by proteomic approach of tissues including adipose tissue, muscle, and liver.
(4) Studies on causative protein markers of obesity in obesity-prone (OP) and obesity-resistant (OR) rats by proteomic approach: We successfully discovered numerous marker proteins determining OP and OR phenotypes by differential proteome analyses of blood and key metabolic tissues of OP and OR rats.
(5) Discovery of biomarkers depended on gender in various tissues and plasma of OP and OR rats by proteomic analysis: To discover gender-dimorphic obesity markers, differential proteome analyses were performed in plasma and major metabolic tissues (adipose tissue, muscle and liver) of male and female rats of OP and OR rats.
(6) Effects of obesity on development of lung cancer: We discovered a total of 14 blood marker proteins involved in stimulatory effects on lung cancer using proteomic analysis in mouse model injected Lewis lung cancer.
Expected Contribution
● Our studies not only confirmed anti-obesity effects of capsaicin but also for the first time verified action mechanism of capsaicin in obesity using proteomic analysis, suggesting possible pharmacological application of capsaicin as an obesity-suppressant.
● Our data first identified differential expression pattern of proteins between OP and OR rats by proteomic approach, suggesting that these proteins may be used as biomarkers of obesity through a further functional study of each protein.
● Discovered gender-dependent biomarkers via various tissues and plasma proteome analysis can contribute to understanding obesity pathophysiology, and suggest new direction for finding causative proteins of obesity.

목차 Contents

중견연구자지원사업(핵심연구) 최종보고서 ... 1
목차 ... 2
연구계획 요약문 ... 3
연구결과 요약문 ... 4
한글요약문 ... 4
SUMMARY ... 5
연구내용 및 결과 ... 6
1. 연구개발과제의개요 ... 6
2. 국내외 기술개발 현황 ... 6
3. 연구수행 내용 및 결과 ... 8
4. 목표달성도 및 관련분야에의 기여도 ... 60
5. 연구결과의 활용계획 ... 61
6. 연구과정에서 수집한 해외과학기술정보 ... 62
7. 주관연구책임자 대표적 연구실적 ... 63
8. 참고문헌 ... 63
9. 연구성과 ... 67
10. 기타사항 ... 83

표/그림 (97)

표 (A) After treatment of cells with PC, Oil Red O staining was performed on day 6 of differentiation. Fat droplets in adipocytes were stained red, and were then monitored by microscope and photographed (200X). (B) Toxicity of 3T3-L1 preadipocytes by PC was tested in various concentrations for 24 h using the MTT assay. (C) Inhibitory effect of PC and berberine on triglyceride accumulation of 3T3-L1 cells was determined by TG content assay on day 7. Data are representative of two independent experiments and presented as mean ±SD. Student’s t test was employed (*p<0.05, **p<0.01, ***p<0.001).
표 (A) PPARg, C/EBPα, FABP4, LPL, CD36, and SCD2 was determined using quantitative real-time RT-PCR. Relative mRNA levels of each gene were normalized to those of GAPDH. (B) Adipogenic protein levels of PPARγ, FABP4, and GLUT4 in response to PC treatment analyzed by Western blot analysis. Data are representative of two independent experiments. Total RNA was extracted from cells treated with PC and without PC (75 ug/mL) on the 5th day of differentiation. mRNA expression of Data are presented as means ±SD, and Student’s t test was employed (**p<0.001).
표 . Cells were treated with various concentrations of MC (5, 10, 20, 50 and 100 μg/ml) under the condition of differentiation induction and then lipid droplets were stained by Oil Red O. Magnification, 200X.
표 Triglyceride content in 3T3-L1 cells 8 days after induction of differentiation. Data shown are representative of three experiments.
표 MC increases protein expression levels that are critical for adipocyte phenotype.
표 Cytotoxicity of MC in preconfluent 3T3-L1 preadiopcytes. Values represent mean ±SD (n=8) (*p<0.05, **p<0.01).
표 Morus alba extract (MC) induces an increase in expression levels of proteins that are critical for an adipocyte phenotype.
표 Effects of Morus alba extract (MC) on PPARγ ligand activity. Luciferase activities were measured after transfection with two plasmids. Cells were treated for 24 h with Rosiglitazone or MC at various concentrations 16 h after transfection (A). Western blot image of PPARγ (B). Data shown are representative results of three experiments. **p<0.01, ***p<0.001.
표 Effects of Morus alba extract (MC) on expression levels of adipogenic genes during adipogenesis. After two days of post confluence (day 0), 3T3-L1 preadipocytes were treated with MC in the presence of differentiation mixtures. Total RNAs were harvested from the cells at day 2 (A) or 4 (B) and mRNA levels of the indicated gene were measured by real-time quantitative RT-PCR. Data are expressed as mean ±SD. (n=3). *p<0.05, **p<0.01.
표 (A) Inhibitory effects of SWPP2-65 on lipid droplet formation. (B) Cytotoxicity test of SWPP2-65. (C) Triglyceride content of 3T3-L1 cells differentiated for 7 days with or without SWPP2-65 treatment.
표 . Effect of water extracts (IOWE) from Inonotus obliquus on adipogenesis of 3T3-L1 adpocytes (A) Cells were treated with various dose of IOWE for 4 days of 0.17 μM insulin, where positive control was treated with 1.7 μM insulin (INS). Oil red O staining was performed at day 8. (B) Cytotoxicity of IOWE in preconfluent 3T3-L1 preadiopcytes (C) Triglyceride content in 3T3-L1 cells 8 days after induction of differentiations. Positive control (con+) was treated with 1.7 μM insulin (INS) and negative control (con-) was treated without both insulin and IOWE. Data shown are representative of three experiments.
표 Effects of water extracts (IOWE) from Inonotus obliquuson on gene expression patterns during adipogenesis. Two-day post confluent 3T3-L1 preadipocytes (day 0) were treated with 10 mg/ml of IOWE in the absence of insulin for 4 days. Cells treated with 1.7 μM insulin were used as control. Total RNAs were prepared from 3T3-L1 cells at day 6 and mRNA level of the indicated gene were measured by real-time quantitative RT-PCR. (A) mRNA expression of adipogenic transcription factors PPARγ, C/EBPα. (B) mRNA expression of PPARγ target genes LPL, CD36, and aP2. The mean values of the results are shown with the SD (n=3). (C) The water extracts (IOWE) from Inonotus obliquus increased PPARγ and GLUT4 protein expression in adipocytes. The data shown are representative of three experiments
표 Luciferase reporter assay. 3T3-L1 cells were transfected with two plasmids. Twelve hours after transfection, cells were treated with rosiglitazone or IOWE at indicated concentrations for 24 h. Values represent means SD (n=3). Data shown are representative of three experiments
표 Fat droplets in adipocytes in control and chitosan oligosaccharides (CO)-treated 3T3-L1 cells stained by Oil Red O at day 5 (left two panels). Effect of chitosan oligosaccharides (CO) on Oil Red O stain (right panel A) and triglyceride content (right panel B) in 3T3-L1 cells. Preadipocytes were cultured in growth medium until they reached confluency and quiescent cells were incubated in DM (differentiation medium) and post-DM with or without chitosan oligosaccharides for 8 days.
표 Effects of chitosan oligosaccharides (CO) of different molecular weights on triglyceride content in 3T3-L1 cells.
표 2D gel electrophoresis of total protein extracted from the cell: (A) pre-adipocyte and (B) mature adipocyte showing up-regulated (ARP1 actin B, M6PRBP, Ribonuclease and Predicted gene) and down-regulated (MPI and PRPS) proteins.
표 Differential expression levels of proteins in pre and mature adipocytes. For protein nomenclatures, see the Abbreviations section. Values are mean ±SEM of three independent experiments, and are significant at *p<0.05, **p<0.001 when compared to preadipocytes.
표 (Left figure) Effect of chitosan oligosaccharides (CO) on mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) (panel A) and CCAAT/enhancer binding protein (C/EBP)α (panel B) analyzed by RT-PCR.
표 (Left) Effects of chitosan oligosaccharides (CO) on diet intake (A) and body weight gain (B) in the ob/ob mice. Values are mean ±SEM and difference between control and CO-treated group (n=6). Asterisk indicates statistically significant (*p<0.05 and **p<0.01) when compared to Control. (Right) Effects of chitosan oligosaccharides (CO) on blood glucose levels (A) and change in area under the curve (△AUC) of intra-peritoneal glucose tolerance test(IPGTT) (B) performed in the ob/ob mice. Values are mean ±SEM and difference between control and CO-treated group (n=6). Asterisk indicates statistically significant (*p<0.05 and **p<0.01) when compared to Control.
표 (Left) A representative 2-DE gel image of silver-stained plasma proteins in ob/obmice. Differentially regulated proteins were marked with arrows together with identified major ob/ob mice plasma proteins (also see Table 6). (Right) Zoom-in gel images and their volume density for altered levels of five plasma proteins with or without chitosan oligosaccharides (CO) treatment. Values are mean ±SEM of three independent experiments. Asterisk indicates statistically significant (*p<0.05 and **p<0.01) when compared to Control.
표 (Left) A representative 2-DE gel image of silver-stained WAT proteins in ob/ob mice. Differentially regulated (>two-fold) proteins were marked with arrows. (Right) Zoom-in gel images for the proteins of interest showing their differential levels with or without CO treatment.
표 Proteomic approach에 의한 키토산올리고당 (CO)의 항비만 메커니즘 제안
표 Capsaicin-treated rats showed decreased weight-gain and adiposity. (A) Effects of capsaicin on body weight profiles of rats fed with high fat diet (HFD) with or without capsaicin treatment. Data are mean ±SE (n=6 per group). (B) Representative images of H HFD+Cap: High fat diet group with capsaicin feeding; HFD-Cap: High fat diet group without capsaicin feeding.
표 The representative 2-DE gel images of silver-stained proteins of WAT (left). Arrows indicate differentially regulated proteins. Zoom-in gel images (A) and their volume density (B) for altered levels of five white adipose tissue proteins in Normal, HFD-Cap, HFD+Cap. Spot density was digitized with software and mean ±SEM of three independent experiments. Asterisk indicates statistically significant (*p<0.05,**p<0.01) when compared to normalgroup. Nor: Normal; HFD+Cap: High fat diet group with capsaicin feeding; HFD-Cap: High fat diet group without capsaicin feeding.
표 Effects of capsaicin on fold change in mRNA expressions of thermogenic factors in WAT analyzed by quantitative real-time PCR (A) Capsaicin treatment increased the expression of thermogenic and β -oxidation relative genes in WAT. (B) Effects of capsaicin on fold change in mRNA expressions of thermogenic factors and obesity-related gene in WAT analyzed by quantitative real-time PCR. Spot densities were digitized with software and mean a ±SEM of three independent experiments, and are significant at *p<0.05, **p<0.001 when compared to normal group.
표 Effect of capsaicin in WAT of HFD-fed rats on protein expression levels of proteins associated with fatty acid oxidation and energy expenditure. Nor: Normal; HFD+Cap: High fat diet group with capsaicin feeding; HFD-Cap: High fat diet group without capsaicin feeding.
표 A representative 2-DE gel image of silver-stained liver proteins of rats. Differentially regulated proteins were indicated with arrows.
표 Zoom-in gel images and their volume density for altered levels of rat liver proteins with (+Cap) or without (-Cap) capsaicin treatment. Asterisk indicates statistically significant (*p<0.05 and **p<0.01) when compared to HFD-Cap.
표 Western blot analysis for liver protein levels of UCP2, FAS, p-AMPK, p-ACC, and CPT-1 in rats fed with HFD with (+Cap) or without (-Cap) capsaicin treatment. Asterisk indicates statistically significant (*p<0.05 and **p<0.01) when compared to HFD-Cap.
표 Representative H&E staining of liver tissue in rats fed with HFD with (+Cap) or without (-Cap) capsaicin treatment. (A) Normal, normal-diet, (B) HFD-Cap, rats fed with HFD (45% kcal) without capsaicin treatment, and (C) HFD+Cap, rats fed with HFD (45% kcal) with capsaicin treatment (10 mg/kg) in two days for 2 weeks. Liver tissues were stained with hematoxylin and eosin (ⅹ400). Large vacuoles indicated by arrows were formed by fat accumulation in liver via the process of steatosis.
표 Representative 2-DE gel image of silver-stained gastrocnemius muscle proteins of rats. Differentially regulated proteins are indicated with circles and arrows together with identified major proteins.
표 Zoom-in gel images and their volume density for altered levels of six muscle proteins in HFD-fed rats with or without capsaicin (Cap). Asterisk indicates statistical significance (*p<0.05) when compared to HFD control.
표 Detection of PHB, MM-CK, and Glo1 by Western blotting to validate results of proteomic analysis. Nor: normal-diet; HFD: high fat-diet; Cap: capsaicin.
표 Effects of capsaicin administration on expression of proteins associated with energy expenditure in rat skeletal muscle. Nor: normal-diet; HFD: high fat-diet; Cap: capsaicin.
표 Effects of capsaicin treatment in L6 myotube cells on expression levels of proteins associated with energy expenditure. (A) Various concentrations (10, 50, 100, and 200 μM) of capsaicin were treated after incubation of the cell for 1 hr, (B) 100 μM of capsaicin was treated in L6 cells for 1, 2, 4, and 8 hr, respectively. Con: control; Cap: capsaicin.
표 A proposed scheme for potential effects of capsaicin on skeletal muscle in HFD-induced obese rats. ATPase, ATP synthase; FA, fatty acid; FAS, fatty acid synthase; NEFA, non-esterified fatty acid; TCA cycle, tricarboxylic acid cycle; TRPV1, transient receptor potential vanilloid 1 (capsaicin receptor). Arrows and T indicate positive and negative effects, respectively.
표 Phenotype of OP and OR rats fed an HFD. (A) Body weight profiles and (B) total weight gain of normal rats fed an LFD as well as OP and OR rats fed an HFD. They were weighed every alternative day for the 56-day duration of the study. (C) Total food intake per total body weight of rats in normal, OP, and OR rats. (D) Levels of leptin and insulin in plasma of normal, OP, and OR rats, measured using ELISA. Data was presented as mean ±SEM in six rats per group and were estimated using the ANOVA test. Asterisk indicates statistical significance (*p<0.05 and **p<0.01) between each group.
표 Adipose tissue weight of obesity-prone and obesity-resistant rats. n=6 per group; *p<0.01, **p<0.001, Error bars represent means ±SEM.
표 Confocal merged images from epididymal WAT and interscapular BAT from Nor, OP and OR co-stained with anti-UCP1 (red) and anti-caveolin 1 (green) antibodies. Representative image shown and similar results from 6 independent rats. The scale bar = 50 μm.
표 A representative 2-DE gel image of silver-stained BAT and WAT proteins of rats.
표 Proteins of BAT showing differential expression among three experimental groups. Each scatterplot was made from individual protein density of six rats in each group as a function of body weight (left three columns) and bar graphs were constructed from average protein density of six rats in each group (right column). Data are presented as mean values ±SEM. of volume density (%) of target spot in each group. They were estimated using ANOVA test and Pearson’s linear correlation test by SPSS program and the p values represent the significance of expression level between each group.
표 Proteins of WAT showing differential expression among three experimental groups. Each Scatter-plot was made from individual protein density of six rats in each group as a function of body weight (left three columns) and bar graphs were constructed from average protein density of six rats in each group (right column). Data are presented as mean values ±SEM. of volume density (%) of target spot in each group. They were estimated using the ANOVA test and Pearson’s linear correlation test by the SPSS program and p values represent the significance of expression level between each group.
표 Validation of proteomic data for some proteins of interest (A) and differential expression patterns of key thermogenic and lipogenic proteins (B) in normal, OP, and OR rats by immunoblot analysis. Full names of proteins are presented in the abbreviations section.
표 Altered expressions of many proteins were associated with obesity-resistance in BAT (upper figure) and WAT (lower figure) of OR rats fed a high fat diet. Up- and down-regulated proteins were marked in black and white circle, respectively.
표 Integrative clustering analysis. Expression profiles of the 91 differentially expressed genes in BAT of the OP and OR groups. A green to red color palette is used to represent expression levels, relative to the mean expression level of the control group, with green representing down-expression and red representing up-expression. Black indicates missed or excluded data.
표 Integrative clustering analysis. Expression profiles of the 53 differentially expressed genes in WAT of the OP and OR groups. A green to red color palette is used to represent expression levels, relative to its mean expression level of the control group, with green representing down-expression and red representing up-expression. Black indicates missed or excluded data.
표 Differentially expressed genes (2􋽧fold change) in white adipose tissue (WAT) of normal (NOR), obesity-prone (OP), and obesity resistant (OR)
표 Expression of fatty acid oxidation and lipid synthesis-related genes (1.5􋽧fold change) in adipose tissue of normal (NOR), obesityprone (OP), and obesity-resistant (OR) rats
표 . Distribution of differentially expressed genes (2􋽧fold changes) from microarrays according to their molecular functions or biological process in BAT and WAT of OP and OR rats.
표 Validation of microarray results from BAT and WAT of the experimental groups by real-time RT-PCR analysis. Results are expressed as fold changes normalized to GAPDH gene expression and represent the mean ±SEM of triplicate independent experiments. *p<0.05, **p<0.001 for significance.
표 Representative 2-DE gel image of silver-stained gastrocnemius muscle proteins of rats. Differentially regulated proteins are indicated by arrows together with identified major proteins in Table 4. Data are representative of at least four independent experiments.
표 Proteins showing differential expression in rat gastrocnemius muscle in normal, OP, and OR rats
표 Expression Patterns of Proteins Determining Fastand Slow-Twitch Muscle Types of Rats before and after HFD Feeding
표 Comparison of expression patterns of various proteins determining fast- and slow-muscle types in normal, OP, and OR rats. (A) Zoom-in gel images for altered levels of muscle fiber marker proteins. (B) Western blot analysis of muscle fiber markers. Band density was digitized with software, and mean ±SE of three independent experiments are shown; statistical significance was determined by t-test where p-values are *p<0.05 and **p<0.01.
표 Differential expression levels of skeletal muscle proteins in normal, OP, and OR rats. Zoom-in gel images and their volume density for expression patterns of five muscle proteins. Spot density was digitized with software, and mean ±SE of four experiments are shown, *p<0.05. Group normal versus Group OP and Group OP versus Group OR.
표 Effects of HFD on expression of UCPs and some antioxidant proteins (A) as well as mitochondrial contents (B) in normal, OP, and OR rats. (A) Band density was digitized with software, and mean ±SE of three independent experiments are shown for each group, where values not sharing a common letter are statistically different by least-significance difference post-hoc comparison (p<0.05). (B) Relative mitochondrial copy number in skeletal muscle as determined by quantitative PCR (n=4, for each group). Statistical significance was determined by t-test, where p-values are *p<0.05 and **p<0.01.
표 Changes in expression of proteins associated with energy expenditure and lipid metabolism in rat skeletal muscle. Band density was digitized with software, and mean ±SE of three independent experiments are shown. Statistical significance was determined by t-test, where p-values are *p<0.05 and **p<0.01.
표 Representative 2-DE gel image of silver-stained liver proteins of rats.
표 List of identified differentially expressed liver proteins in normal, OP, and OR rats fed a high-fat diet
표 Significantly increased liver proteins in OP rats compared to control and OR rats. Zoom-in-gel images of each protein are shown with their average expression levels in three rats in each group. Data are presented as mean values ±SEM. of volume density (%) of target spot in each group. They were estimated using the ANOVA test and the p values represent the significance of expression level between each group.
표 Significantly decreased liver proteins in OP rats compared to control and OR rats. Zoom-in-gel images of each protein were shown with their average expression levels in three rats in each group. Data are presented as mean values ±SEM of volume density (%) of target spot in each group. They were estimated using the ANOVA test and the p values represent the significance of expression level between each group.
표 Validation of proteomic data for some proteins of interest (A) and differential expression patterns of seven metabolic liver proteins (B) in normal, OP, and OR rats by immunoblot analysis. Band density was calculated by ImageMaster 2D software V4.95 and relative intensity (%) demonstrated that values of target proteins were normalized to those of β-actin. Asterisk indicates statistically significant (*p<0.05 and **p<0.01).
표 A representative silver-stained 2-DE gel image of rat plasma proteome. Differentially regulated proteins in each group and major rat plasma proteins were marked with arrows. Proteins of numbers in gels are listed in Table 7.
표 Identified differentially regulated and major plasma proteins in normal, OP, and OR rats through proteomic analysis
표 Differential protein levels of plasma proteins in OP and normal/OR rats. Line graphs refer to protein density of six individual rats as a function of body weight (left three) and bar graphs stand for average levels of six rats. These data were identified by 2-DE combined with MALDI-TOF-MS, presenting a volume density (%) of each protein spot in 2-DE gels. Data were exhibited as mean values ±SEM of volume density of target protein spot in six rats per group. They were estimated using ANOVA and Pearson’'s linear correlation test by SPSS program and the p values represent the significance of expression level between each group.
표 Differential protein levels of plasma proteins in OP and normal/OR rats. Left three figures are zoom-in images of representative gels and right bar graphs were produced from average protein levels of six rats. Data were exhibited as mean values ±SEM of volume density (%) of the target spot in each group. They were estimated using the ANOVA test and the p values represent the significance of expression level between each group.
표 Differential protein levels of plasma proteins in OP and normal/OR rats. Line graphs were produced from protein density of six individual rats as a function of body weight. Data were exhibited as mean values ±SEM of volume density (%) of target spot in each group. They were estimated using the ANOVA test and Pearson’'s linear correlation test by SPSS program and the p values represent the significance of expression level between each group.
표 Validation of differentially regulated plasma proteins inOP and normal/OR rats by immunoblot analysis. Levels of five identified proteins from 2-DE analysis (A) together with eight important plasma proteins (B and C) were established. Data are representative of two independent experiments.
표 Summary of verified plasma proteins by proteomic approach in OP and OR rats. Physiological function of each protein mentioned here indicates the most representative role of each protein in its cellular function.
표 Body weight (A), total body weight gain (B), total food intake (C), and food efficiency (D) in male and female rats fed a ND and an HFD. Data are presented as mean ±SEM in seven rats per group and were estimated using the ANOVA test. *p<0.05, **p<0.01; †p<0.05 ††p<0.01.
표 Plasma biochemical parameters in each group 1)
표 Representative silver-stained 2-DE gel images of rat BAT proteome in ND and HFD rats. Differentially regulated proteins in each group are marked with arrows.
표 (A) Validation of differentially regulated BAT proteins in male (M) and female (F) rats in response to HFD by immunoblot analysis. (B) Differentially regulated BAT proteins of metabolic importance in male (M) and female (F) rats in response to HFD by immunoblot analysis.
표 Representative silver-stained 2-DE gel images of rat subcutaneous, inguinal, and abdominal WAT proteome in HFD rats. Proteins showing gender differences are marked with arrows.
표 Differentially expressed WAT proteins showing gender differences in both ND and HFD rats with higher protein abundances in males and females. Data are expressed as mean values ±SD of the volume density (%) of the changed spot in 3 individual gels using pooled WAT (3 types).
표 Differentially expressed WAT proteins showing gender differences in both ND and HFD rats with opposite protein abundances between the genders. Data are expressed as mean values ±SD of the volume density (%) of the changed spot in 3 individual gels using pooled WAT (3 types).
표 Validation of proteomic data of WAT using immunoblot analysis.
표 Representative silver-stained 2-DE gel images of rat muscle proteome in soleus and gastrocnemius muscles. Differentially regulated proteins in each group are marked with circles.
표 Scatter plots comparing global type-specific muscle protein expression profiles between males vs. females in ND (A) and HFD (B). These plots were created using all spots that appeared in the three gels of each group. X- and Y-axis represent volume (%) of each protein spot.
표 Graphical presentation of the statistical evaluation of contractile protein abundance of soleus (sol) and gastrocnemius muscles (gas) between the genders (males vs. females) and diets (ND vs. HFD). White bars are for normal diet and black bars are for HFD. *p<0.05, **p<0.01; †p<0.05 ††p<0.01.
표 Differentially regulated muscle proteins of metabolic importance in male (M) and female (F) rats in response to HFD by immunoblot analysis. Sol: soleus muscle, Gas: gastrocnemius muscle. White bars are for normal diet and black bars are for HFD. The Y-axes of the bar graphs refer to protein density normalized by β-actin. *p<0.05, **p<0.01; †p<0.05 †† p<0.01.
표 Representative silver-stained 2-DE gel images of liver proteome of male and female rats fed ND or HFD. Differentially regulated proteins in each group are marked with arrows.
표 Differentially expressed liver proteins showing gender difference in both ND and HFD rats with higher protein levels in males (A) and females (B). Data are exhibited as mean values ±SEM of volume density (%) of the changed spot. *p<0.05, **p<0.01; †p<0.05 ††p<0.01.
표 Differentially expressed liver proteins showing opposite gender difference between ND and HFD rats. Data are exhibited as mean values ±SEM of volume density (%). *p<0.05, **p<0.01; †p<0.05 ††p<0.01.
표 Western blot analyses for validations of differentially regulated liver proteins from proteomic findings (A), and for some metabolically important liver proteins (B). Levels of 4 identified proteins from 2-DE analysis (A) together with 4 important liver proteins of metabolic importance (B) were established.
표 A representative silver-stained 2-DE gel image of rat plasma proteome. Differentially regulated proteins in each group are marked with arrows.
표 Differentially expressed plasma proteins showing gender difference. Data are exhibited as mean values ±SEM of volume density (%) of the changed spot in three individual gels. (p);precursor *p<0.05, **p<0.01; † p<0.05 ††p<0.01.
표 Validation of differentially regulated plasma proteins in male (M) and female (F) rats in response to HFD by immunoblot analysis. Levels of six identified proteins from 2-DE analysis (A) together with three important plasma proteins (B) were established.
표 Summary of differential regulation of plasma proteins between male and female rats fed either a low fat diet (LFD) or a high fat diet (HFD). NA stands for no alteration. (p), precursor. *Expression patterns of RBP4, adiponectin and resistin were investigated by immunoblot analysis.
표 Scatter plots comparing global protein regulation profiles between males vs. females (A) or OP vs. OR rats (B). Regulation of plasma proteins of males or OP rats was plotted against those of the females or OR rats and these plots were drawn using all spots appeared in three gels of each group. Spots with volume (%) of five and over were excluded from scatter plots. Upper and the lower diagonal lines show the 1.5-fold regression lines. X- and Y-axis represent volume (%) of each spot in the normal, OP, and OR groups of males and females.
표 Phenotypes of C57BL/6N mice fed ND or HFD with or without injection of LLC cells. (A) Time profiles and (B) total changes in body weight, fat mass (C, D), tumor growth (E, F), and expression of PCNA in tumors (G, scar bar = 10 m). Data are presented as mean ±SD of six mice per group. Statistical significance between ND and HFD mice was determined by ANOVA test, where p value is *p<0.01.
표 A representative silver-stained 2DE gel image of mouse plasma proteome. Differentially regulated proteins were marked with arrows.
표 Scatter plots comparing global protein regulation profiles between controls and tumor-bearing mice fed a ND or HFD (upper two plots). Low abundant proteins were separately presented in the zoom-in plots (lower two plots). Regulation of plasma proteins of ND or control mice was plotted against those of the HFD or tumor-bearing mice, and these plots were drawn using all spots that appeared in the three gels of each group. X and Y axis represent volume (%) of each spot in the ND and HFD group of controls and mice with tumors. Red spot, tumor>control (p0.05). For abbreviation of each protein name, see abbreviations. (p); precursor.
표 Synergistically regulated plasma proteins by HFD treatments (Group I). Data are expressed as the mean values ±SD of volume density (%) of the changed spots in three individual gels from six mice. Statistical significance between ND and HFD mice was determined by ANOVA test, where p value is *p<0.05 and **p<0.01. Significances between controls and tumor mice were represented by †p<0.05 and ††p<0.01. (p); precursor.
표 Differentially regulated proteins only in tumor-bearingmice (Group II). Data are expressed as mean values ±SD of volume density (%) of the changed spots in three individual gels from six mice. Significances between controls and tumor mice were represented by †p<0.05 and ††p<0.01. (p); precursor.
표 Validation of proteomic data by immunoblot (panel A) and ELISA (panel B) analysis. (A) Levels of five identified proteins from 2DE analysis were investigated three times independently using pooled plasma sample from six mice per each group. Band density was calculated using the ImageMaster 2D software version 4.95, and relative intensity (%) were the values of target proteins after being normalized to beta-actin levels. (B) Levels of four identified proteins from 2DE analysis were investigated three times independently using individual plasma sample from six mice per each group. Statistical significance between ND and HFD mice was determined by ANOVA test, where p value is *p<0.05 and **p<0.01. Significances between controls and tumor mice were represented by †p<0.05 and ††p<0.01.
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