리놀레산과 리놀렌산이 한우의 지방대사에 미치는 영향 : in vitro 및 in vivo 연구

Ahmed, Belal Shah 전북대학교 일반대학원 2018 국내박사

Long-chain fatty acids (LCFAs) are fatty acids with 14 or more linearly arranged carbon atoms and may be saturated (no double bonds) or unsaturated (one or more double bonds). These fatty acids are found mostly as components of the triglycerides of edible oils and fats. The studies were conducted in vitro and in vivo. The first in vitro study was performed to elucidate the effects of unsaturated fatty acids (USFAs) such as linoleic acid (LA), oleic acid (OA), and their combination (LA+OA) on cell proliferation, apoptosis, necrosis, and the lipid metabolism related gene expression in bovine satellite cells (BSCs) isolated from bovine muscles. Cell viability was significantly increased with the LA and OA treatment. Furthermore, LA+OA enhanced cell proliferation in a dose-dependent manner (10 to 100 µM), whereas it lowered at 250 µM. In addition, a cell-cycle analysis showed that 100 µM of LA and OA markedly decreased the G0/G1 phase proportion (62.58% and 61.33%, respectively), compared to controls (68.02%), whereas the S-phase cells’ proportion was increased. The ratio of G2/M phase cells was not significantly different among the groups. Moreover, analyses with AO/EB staining showed that no apoptosis occurred. Necrosis were determined by flow cytometry using Annexin V-FITC/PI staining which revealed no early apoptosis in the cells pretreated with LA and OA, but occurred in the LA+OA group. We also analyzed the mRNA expression of lipid metabolizing genes such as PPARγ, ACOX, LPL, CPT-1, and FABP4, which were upregulated in LA and OA treated cells compared to the control group. In essence, USFAs (LA and OA) promote the cell proliferation without any apoptosis and necrosis, which might upregulate the lipid metabolism related gene expressions, and increase fatty-acid oxidation in the BSCs’ lipid metabolism. The second in vitro study was conducted to determine the effects of saturated (SFA) and unsaturated fatty acids (USFA) on triacylglycerol (TAG) content, as well as genes associated with lipid synthesis and fatty acid composition in bovine satellite cells (BSCs). Both saturated (stearic and palmitic) and unsaturated (linoleic and oleic) fatty acids stimulated the TAG accumulation at concentration 100 μM and oleate significantly increased than stearate and palmitate. Results revealed that lipid droplet formation was significantly stimulated by linoleate and oleate at 100 µM. The expressions of ATGL, CPT1 and CD36 were upregulated by LCFA treatment compared with control, which is involved in the uptake and secretion of LCFAs. All the fatty acid also increased diacylglycerol acyltransferase 2 (DGAT2) than the untreated control and significant each other (p<0.05). However, there was no significant effect on the expression of DGAT1 among the group. In fatty acid composition, the MUFA was significantly increased (p<0.05) in response to oleate and linoleate than the control. PUFA was also significantly increased (p<0.05) in addition of stearate, linoleate and oleate compared to the control group. In contrast, SAF was significantly decreased in the oleate and linoleate treated groups. After all, the results from this study help enhance our understanding of the regulatory role of LCFAs on TAG accumulation and fatty acid composition in BSCs. The last in vivo experiment was conducted to evaluate the effect of feeding LCFA-Ca salts on meat quality, blood parameters and nucleotide compounds of Hanwoo (Korean native cattle). A total of 69 animals with mean body weight of 599±5 kg were divided into three treatments as Control (n=20), Group 1 (n=25) and Group 2 (n=24) were fed 1) control diet with no added fat, 2) 50% linoleic acid and 24% oleic acid containing LCFA-Ca and 3) 26% linoleic acid and 50% oleic acid containing LCFA-Ca respectively 100g/d/animal on dry matter required basis (DMR). For meat quality, we observed highest cooking loss, shear force and lowest pH in Group 1, compared with other two groups, but not reached significant level. The CIE a* value was significantly higher in control group (p<0.05), whereas no significant differences of L* and b* among the groups. Nucleic acid related compounds IMP, ADP and inosine tended (p<0.05) to be greater in Group 2 compared to other two groups, whereas AMP was high in Control. Group 1 had the highest MDA content than other groups (p<0.05). Fatty acid compositions including SAF, PUFA and ω6/ω3 were significantly higher in Group 2 than the rest of two groups (p<0.05) but did not affect the percentage of MUFA. Serum concentration of triglycerides and calcium were greater (p<0.05) in Hanwoo steers fed LCFA-Ca than Control group. Cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) concentrations were greater in Group 2 without any significant effect. In our study up-regulation of leptin gene expression was observed in Group 1 than other groups. Other genes related to lipogenesis, LPL, FASN and SCD mRNA expressions were not significant between the treatments. However ACC, ATGL and FABP4 were significantly higher in group 1 than group 2 and HSL was higher in group 2. Therefore, it can be concluded that LCFA-Ca salts had impact on meat quality traits of Hanwoo including blood parameters, nucleotide compounds and some lipid metabolism gene expressions.

Health benefits of natural products on obesity-induced nonalcoholic fatty liver disease via regulation of lipid metabolism

AHMAD RANDY University of Science and Technology 2017 국내박사

Obesity is an excessive body fat deposition, characterized by increased lipid accumulation in adipose tissue, which may trigger metabolic disorder and further negative effects on several organs (Kopelman, 2000). In case of the liver, excessive fatty acid accumulation to the liver that is induced by obesity and metabolic disorder may cause non-alcoholic fatty liver disease (NAFLD) (Adams et al., 2005; Ahmed, 2015; Fabbrini, Sullivan, & Klein, 2010; Gholam, Flancbaum, Machan, Charney, & Kotler, 2007). Although NAFLD is a broad spectrum disease with many factors contribute to the progression of this disease, the early stage of NAFLD is still considered to be initiated by excessive hepatic lipid accumulation, which is caused by an imbalance between hepatic lipid synthesis or uptake and hepatic lipid oxidation or removal (Kawano & Cohen, 2013; Q. Liu, Bengmark, & Qu, 2010). Obesity-induced lipid accumulation in the adipose tissue can increase the free fatty acid influx to the liver, and obesity-induced insulin resistance can increase hepatic de novo lipogenesis and impair hepatic lipid oxidation, all of them are associated with and may promote NAFLD. Thus, among the strategy to prevent NAFLD are to attenuate hepatic lipid accumulation by lowering free fatty acid influx, lowering de novo lipogenesis, and increasing hepatic lipid oxidation (L. Abu-Elheiga, Matzuk, Abo-Hashema, & Wakil, 2001; Berge et al., 2005; Berk & Stump, 1999). Recent study and finding on NAFLD lead to the development of a new hypothesis to explain the progression of NAFLD, which is ‘multiple-parallel hits’ hypothesis. This ‘multiple hits’ theory reasons that initial cause and progression of NAFLD happen through different and non-parallel events of metabolic disorder such as hepatic lipid accumulation, inflammation, lipotoxicity, insulin resistance, oxidative stress (Ahmed, 2015; Musso, Cassader, & Gambino, 2016; Yilmaz, 2012). Treatment for NAFLD that can improve above mentioned multiple factors may have beneficial effects to improve NAFLD (Musso et al., 2016). Beneficial effects of natural products on NAFLD was also being an interest for its potency to improve signs of NAFLD or other NAFLD-related diseases. Several natural products were reported to alleviate symptoms of NAFLD. Silybum marianum extract was proposed to be a safe treatment for NAFLD by improving AST/ALT level in patient with hepatic steatosis as well as an antioxidants to protect the liver from peroxidation through its antioxidant activity (Cacciapuoti, Scognamiglio, Palumbo, Forte, & Cacciapuoti, 2013). (Flora, Hahn, Rosen, & Benner, 1998). Resveratrol that can be found in the skin of grapes and blueberries is a potent in vitro and in vivo antioxidant activity, and plays an improving activity on patients with NAFLD improve the patient’s steatosis grade mainly trhough improvement of inflammatory markers and hepatic cell apoptosis (de la Lastra & Villegas, 2007; Faghihzadeh, Adibi, Rafiei, & Hekmatdoost, 2014). Catechin-reach green tea extract also showed promising potency on NALFD by improving plasma markers of patient with NAFLD (Pezeshki, Safi, Feizi, Askari, & Karami, 2016). Therefore, researching in natural products can give a good opportunity to find an effective agent for NAFLD treatment. In the first chapter of this thesis, the potential activity of Calliandra calothyrsus extract (CCE) on treating NAFLD was examined in vitro. HepG2 hepatocarcinoma cells were utilized as a model for liver lipid accumulation to investigate the in vitro activity of CCE to inhibit fatty acid-induced lipid accumulation. 3T3-L1 preadipocyte cells were utilized to study the in vitro lipid lowering activity of CCE in adipose tissue via inhibition of adipocyte differentiation. At first, screening with 140 Indonesian plant extracts was conducted to test their lipid lowering activity by oil red O assay on HepG2 cells. CCE had the most potent lipid-lowering activity among Indonesian plants and CCE exhibited lipid-lowering activity by inhibiting lipid synthesis and activating lipid oxidation via activation of AMPK. On 3T3-L1 cells, CCE inhibited lipid accumulation in adipocyte model by inhibiting the differentiation of adipocyte. This in vitro activity of CCE suggests that CCE may potentially improve obesity-induced NAFLD via activation of AMPK signaling and suppression of adipocyte differentiation. In the second chapter, the effects of Ligularia fischeri extract (LFE) was examined on fatty acid-induced lipid accumulation in HepG2 model of liver cells. L. fischeri is an edible plant that grows in Japan, Korea, China, and Russia. It is traditionally used to treat several human diseases, including diseases related to hepatic function. L. fischeri and its constituents are well-known for their antioxidant activity, potent activation of hepatic antioxidant defense enzymes, and promotion of liver protection. Analysis using oil red O assay shows that LFE attenuated lipid accumulation on fatty-acid induced HepG2 model. Treatment of LFE also reduced the level of triglyceride (TG) on fatty-acid induced HepG2 model. Experiments with chemical constituents of LFE show that amongst other constituents, 3,4-dicaffeoylquinic acid (3,4-DCQA) had the most potent activity to inhibit lipid accumulation in fatty acid-induced model, and TG level was also reduced by treatment with 3,4-DCQA. Studies on detailed mechanism of LFE activity show that LFE and 3,4-DCQA lowered lipid accumulation by inhibiting lipid synthesis and activating lipid oxidation via activation of AMPK. In summary, results from this study gave evidences on the potential benefit of LFE to protect against obesity-induced NAFLD by using in vitro fatty acid-induced model in hepatocellular carcinoma HepG2 cells. In the third chapter of this thesis, the effect of LFE on obesity-induced NAFLD and other metabolic syndrome was investigated using an in vivo model. Obesity and NAFLD were induced in C57B/6J mice model by feeding the animal with high-fat diet. LFE improved obesity-induced NAFLD by lowering total body fat, body weight, and improving serum lipid profile. LFE also improved symptoms of metabolic diseases such as insulin resistance and oxidative stress. LFE decreased the level of lipid peroxidation through activation of the antioxidant defense system via Nrf2 activation. LFE attenuated lipid and TG accumulation by activating liver lipid oxidation and inhibiting lipid synthesis via activation of AMPK. In summary, results from this study gave evidences on the potential benefit of LFE to protect against progression of obesity-induced NAFLD. In present studies, the potential benefit of using natural products for improving obesity-induced NAFLD was investigated using in vitro and in vivo model. The activity of CCE to inhibit adipocyte differentiation and lipid accumulation may suggest the potency of C. callothyrsus to be used in inhibiting progression and alleviating symptoms of NAFLD. In vitro and in vivo finding shows that L. fischeri can improve NAFLD condition by lowering liver lipid accumulation through AMPK signaling, and also enhancing anti-oxidant mechanisms via Nrf2. Overall results suggest the potential benefit of natural products on improving obesity-induced NAFLD.

n-3 fatty의 유방암 예방효과에 관한 메타 분석

안남길 고려대학교 보건대학원 2011 국내석사

목적: n-3 fatty acid의 섭취는 유방암의 발생을 낮춘다고 보고되고 있다. 그러나 역학적 연구에서는 이러한 결과에 대해서 명확하게 뒷받침해주지 못하고 있다. 이에 본 연구자는 n-3 fatty acid 섭취의 유방암에 대한 예방효과를 메타분석을 통해 증명하고자 한다. 방법: 문헌검색도구로는 PubMed, Web of Science, Ovid MEDLINE, Cochrane을 이용하여 검색하였다. 그 결과 n-3 fatty acid와 유방암의 관련성에 대해서 보고한 논문 11편이 리뷰되었다. 분석은 6편의 환자-대조군 연구와 5편의 코호트 연구로 이루어졌고 기준은 다음과 같았다. ① 연구방법은 환자-대조군연구와 코호트 연구로 제한하였다. ② n-3 fatty acid섭취에 따른 유방암의 발생에 관한 위험도가 제시되어야 한다. ③ 선정된 논문에는 p-value, 신뢰구간이 제시되어야 한다. 동질성검정 결과 통합비차비의 모델로는 랜덤효과모델이 선정되었다. 각각의 레벨에서 통합비차비를 계산하는 방법으로는 inverse of variance method 가 사용되었다. 결과: n-3 fatty acid 섭취량이 높은 그룹은 낮은 그룹에 비해 유방암 발생의 위험도가 0.73(95% CI: 0.56-0.95)으로 예방효과를 보였고 통계적으로 유의하였다. n-3 fatty acid 섭취량에 따른 유방암의 발생은 약한 역상관관계가 있는 것으로 나타났다. 환자-대조군 연구에서는 n-3 fatty acid 섭취량이 높은 그룹에서 낮은 그룹에 비해 유방암의 예방에 대해서 통계적으로 유의한 결과를 제시하였다(위험도[95% CI]: 0.53[0.29-0.95]). 코호트 연구에서는 n-3 fatty acid 섭취에 따른 유방암의 예방효과는 통계적으로 유의하지 않은 결과를 나타내었다(위험도[95% CI]: 1.02[0.91-1.31]). s 결론: n-3 fatty acid 섭취에 따른 유방암에 대한 예방효과에 대해서 결론을 내리기에는 근거가 부족하며, 앞으로 더 많은 연구들이 추가되어야 할 것이다. Objectives: It was previously believed that n-3 fatty acid may decrease breast cancer. But the protective effects of n-3 fatty acid for breast cancer have proved equivocal in several epidemiological studies. This study was performed to reexamine the association between breast cancer and n-3 fatty acid using the method of meta-analysis. Methods: We retrieved the relevant reports using PubMed, Web of Science, Ovid MEDLINE and Cochrane Search. Overall, 11 cases of n-3 fatty acid intake and breast cancer were reviewed. The analysis was based on 6 case-control studies and 5 cohort studies. The criteria for quality evaluation were as follows; ① The study design was case-control and cohort. ② The overall effect size of the risk of breast cancer due to n-3 fatty acid was calculated by the relative risk. ③ The paper should have confirmed p-values and confidence intervals. The random effect model was assumed when the test for heterogeneity was significant. The summary statistic used was the common odds ratio estimated for each level of the fatty acid on the study, and this was weighted by the inverse of variance method. Results: The overall relative risk of n-3 fatty acid with breast cancer was 0.73 (95% CI: 0.56-0.95). N-3 fatty acid intake showed a weak, positive inverse association with the incidence of breast cancer. In the case-control studies, a significant protective effect was found for n-3 fatty acid (OR 0.53, 95% CI: 0.29-0.95) while in the cohort studies, a significant protective effect was not found for n-3 fatty acid (OR 1.02, 95% CI: 0.91-1.13). Conclusion: These results suggested that there is not clear evidence that high consumption of n-3 fatty acid reduces the risk of breast cancer. Further studies are required.

Metabolic Engineering of Fatty Acid and Lipid Biosynthesis in Plant Seeds

박믿음 세종대학교 대학원 2025 국내박사

Effect of intrinsic factors on quantitative and qualitative traits in Korean pig varieties

이상훈 Graduate School, Korea University 2011 국내박사

본 연구는 돼지의 품종, 성별, 근섬유 특성과 heart fatty acid binding protein 유전자 변이가 도체 형질, 육질, 지방 및 관능적 특성에 미치는 영향을 분석하기 위해 수행되었다. 먼저 품종과 성별, 근섬유 특성이 육질 및 도체 형질에 미치는 영향을 알아보기 위하여 총 1374 두(암퇘지 758두, 수퇘지 316두, 거세돈 300두)가 분석하였다(Chapter 2). 암퇘지는 거세돈과 수퇘지에 비해 등심근 면적이 유의적으로 넓은것으로 분석되었다. pH24 h, 및 전미돈육생산협회 기준 근내 지방도는 거세돈이 암컷보다 높게 나타났다. 예상대로 pH45 min 와 pH24 h는 유리육즙량과 강한 연관성을 보였다. 버크셔 품종은 다른 품종들에 비해 두꺼운 등지방을 나타내었다. 한편 듀록은 삼원 교잡종과 요크셔종에 비해 얇은 등지방 두께를 보였다. 듀록, 랜드레이스 및 삼원교잡종은 상대적으로 큰 등심근 단면적을 보였다(P < 0.001). 근섬유 특성 중 근섬유 단면적 크기는 품종별 차이를 보였는데 버크셔와 요크셔 품종이 type I 의 크기가 유의적으로 크게 분석되었다. 삼원교잡종과 요크셔 품종은 낮은 type I 근섬유 밀도를 나타내었다. 육질 측정 항목(pH45 min, pH24 h, 명도, 유리육즙량, NPPC 근내지방도), 근섬유특성, 도체형질을 이용하여 각각의 주성분 분석을 통해 주성분을 추출하였다. 결과로 거세 듀록 품종, 버크셔 암퇘지 및 거세 버크셔 품종의 특징은 높은 근육 pH, 높은 근내지방도, 낮은 유리육즙량, 상대적으로 큰 근섬유 크기로 특징지어 졌다. 메산품종은 낮은 근육 pH, 낮은 근내지방도, 높은 유리 즙량, 작은 근섬유 크기를 나타내었다. 한편 근섬유 수 및 크기로 대표되는 근섬유의 형태학적특성이 돼지의성장특성 및 육질에 미치는 영향 또한 분석 되었다 (Chapter 3).근섬유 수로 나눈 각 실험군에서 근섬유 크기는 적육 생산량과 정의 상관 관계를 보였다. 등심근 단면적은 근섬유 수 및 크기와 강한 상관관계를 나타내었다. 성장특성과 관련하여, 높은 근섬유수 및 큰 근섬유 면적을 보이는 실험군에서 전체적인 성장률이 낮게 평가 되었다. 결과로 근섬유수가 많고 중간 크기의 근섬유 면적을 나타내는 실험군은 육질의 저하가 크게 일어 나지 않았으며 근섬유 크기가 크고 근섬유 수가 작은 경우는 초기 근육 pH의 빠른 저하를 나타냈었다. 식육의 지방은 식육생산자와 소비자 모두를 고려 할 때, 육질 및 생산성에 영향을 미치는 요인중 하나이다. 따라서 식육내 근내지방도 축적 기작 및 양상에 대한 연구가 필요하다고 판단된다. 더욱이 식육의 근내지방도와 연관있는 것으로 보고 되고있는 Heart fatty acid-binding-protein(H-FABP)유전자와 근내지방도와의 연관성 구명은 몇몇 연구에서 진행 되었지만 식육내 지방산 조성과의 연관성 구명은 연구가 미미 하였다. 따라서 제한효소 절편길이 다형성을 이용한 H-FABP유전자형이 근내지방량, 육질 및 지방산 조성에 영향을 분석하였다(Chapter 4). 실험군의 등심근 근내지방도는 제한효소 절편길이 다형성을 이용하여 제한효소HinfI에 의해 구분된 유전자형과 유의적연관성이 관찰되었다. HH유전자형은 다른 유전자형에 비해 높은 근내지방도을 나타 내었다. 육질의 경우 유리육즙량만이 영향을 받는 것으로 분석되었다. 지방산포화도의 경우는 hh유전자형이 HH과Hh유전자형에 비해 높게 나타났다. MspI 제한효소로 인한 유전자형의 경우는 불포화 지방산 조성과 유의적 연관성을 나타냈다(P < 0.05) 반면 HaeIII 에 의한 유전자 형에서는 지방산 조성과 유의적 연관성을 찾아 볼 수 없었다. 마지막으로 객관적 육질 특성 항목, 근섬유 특성 및 지방산 특성이 돈육 등심근의 관능적 품질에 미치는 영향을 분석하였다(Chapter 5). 본 연구에서 사용된 근육 시료인 등심근에서는 객관적 육질 항목과 근섬유 특성 항목 조합으로 이루어 진 변수로는 관능적 특성 중 초기연도의 변이를 18.0% 정도 설명하는 것으로 분석되었다. 한편 씹힙성의 경우 19.2%를 객관적 육질 특성 항목 및 근섬유 특성 항목으로 설명되는 것으로 분석 되었다. 결과적으로 다양한 육질 특성 항목 및 근섬유 특성, 지방산 특성 항목을 이용한 다중회귀 모델은 다양한 측정 항목, 예로 나이, 일당 증체량, 골 형성 수준 등의 항목의 추가로 그 설명 정도를 높일 수 있다고 예상되는데 이는 앞서 언급 된 도체 및 생리학적 측정 항목들도 육질에 영향을 미칠 것으로 예상되기 때문이다. The overall objective of this thesis wasdesigned to investigate the effects of breed, sex, muscle fiber characteristics and heart fatty-acid-binding protein (H-FABP) gene polymorphism on meat quality, carcass traits, fat and sensory characteristics in the porcine longissimus dorsi muscle. First, to investigate the effects of breed, sex, and muscle fiber characteristics on meat quality and carcass traits, total of 1374 pigs (758 gilts, 316 boars and 300 castrated male pigs) were used (Chapter 2).Female pigs showed a larger loin area than castrated male and male pigs. Muscle pH24 h and National Pork Producers Council (NPPC) marbling score of castrated male pigs were higher than female pigs. As expected, drip loss was highly correlated to pH45 minor pH24 h. The effects of different breed on carcass traits and muscle fiber characteristics clarified. Berkshire pigs had higher back-fat thickness compared to other breeds (Duroc,(Landrace Yorkshire) Duroc (LYD) and Yorkshire had thinner back-fat thickness).Duroc, Landrace and LYD showed a largerloin area compared to other breeds (P < 0.001).In the case of size of muscle fiber, there were no significant effects of breed on mean of cross sectional area of muscle fiber(CSAF). Berskshire and Yorkshire exhibited a lager CSAF of type I fiber. LYD and Yorkshire pigs had lower density of type I. Principal components analysis was performed to illustrate the spatial relationship of the pork quality attributes evaluated and corresponding instrumental properties (pH45 min, pH24 h, L*, drip loss, NPPC marbling score) and muscle fiber characteristics and carcass traits.The castrated Duroc pigs, Berkshire gilt and castrated pigs were characterized high pH45 min, pH24 h, NPPC marbling score and low drip loss with large muscle fiber among pigs groups. The castrated Meishan pigs, Meishan boar and gilt showed tendency of low pH45 min, pH24 h, NPPC marbling score and higher drip loss with small muscle fiber compared to the pigs groups. Consequently, to illustrate these complex relationships in two dimensional spaces can be a way to understand the effects of breeds, sex and interaction between each other on meat quality, muscle fiber characteristics and carcass traits. On the other hand, in results of effects of total number of fiber (TNF) and CSAF on growth performance, muscle fiber characteristics were significantly detected (Chapter 3). The positive relationship between CSAF and the lean meat productivity within each TNF groups were observed. Loin area was positively correlated with type I, IIA and IIB fiber and cross sectional area of type I, IIA, IIB. In growth performance, High TNF and large CSAF (HL)showed slow growth rate Low TNF and small CSAF (LS), LS also showed slow growth rate during postnatal development.This result may explain that low TNF and small CSAF were related with not only decreased lean meat productivity, but also delay of early postnatal development. As results, these also suggest that lager CSAF with higher TNF guarantee the normal pH decline and larger CSAF with lower TNF induced rapid pH decline during early postmortem. Taking into consideration industry needs and consumer demands, there is a need to identify ways to control fat deposition,which may closely related to meat quality and leanness of carcass. Moreover, the polymorphisms of the H-FABP gene of Berkshire pig and the association of IMF contents, fatty acid composition, and meat quality with different H-FABP RFLP genotypes have not been well established. Therefore, the relationship between polymorphisms of the H-FABP gene and variation in intramuscular fat (IMF) content, fatty acid composition, and meat quality were evaluated in Berkshire pigs (Chapter 4.) An association between IMF content and H-FABPHinfI restriction fragment length polymorphism (RFLP) genotype was detected. The HH genotype showed the greatest IMF content among the genotype classes (P <0.05). Only drip loss was significantly different among the H-FABPHinfI RFLP genotype classes (P < 0.01). The degree of saturation of fatty acids was greater in hh-genotype pigs than HH- and Hh-genotypes. The H-FABPMspI RFLP genotype affected unsaturated fatty acid content, and the ratio of poly unsaturated fatty acids to saturated fatty acid (P < 0.05), whereas the H-FABPHaeIII RFLP genotype had no effect on fatty acid characteristics. In final (Chapter 5.), to quantify sensory quality trait variabilityof loingissimus dorsi muscle in relation to meat quality traits, muscle fiber characteristics and fatty acid composition were analyzed. In longissimus dorsi muscle, the combination of meat quality and muscle fiber characteristics allowed 18.0% of initial hardness and 19.2% of chewiness variability to be explained.Consequently, it is possible to improve this model based on the physicochemical properties by adding simple characteristics concerning animals, such as life average daily gain, bone development scoring and age.

Epigenetic regulation of lipid metabolism and T cell immunity by the chromatin architectural protein CTCF

최예은 Graduate School, Yonsei University 2019 국내박사

지방간은 전세계 사람들의 10 ~ 25 %에서 발생한다고 알려져 있으며 간에서 일어나는 대사 증후군으로 간주되는 질환입니다. 간은 유리 지방산 (FFA) 흡수에 대한 높은 수용력을 가지고 있으며 지질 대사에 중요한 역할을 합니다. CCCTC- 결합 인자 (CTCF)는 고도로 보존 된 징크 핑거 단백질이며, 전사 인자로서 가장 잘 알려져 있습니다. 이것은 전사 활성제, 억제제 또는 인핸서와 프로모터 사이의 통신을 차단하여 절연체 단백질로서 작용할 수 있습니다. CTCF 는 또한 크로마틴 도메인 경계에 붙어있는 동안 다른 전사 인자를 모집 할 수 있습니다. 이 연구에서는 간 특이성 CTCF-knockout (CTCF188 cKO) 마우스를 사용하여 간에서의 CTCF 역할을 보았습니다. CTCF 결핍 시 마우스 간조직에서의 지방의 축적과 트리글리세라이드와 콜레스테롤이 증가되어 있는 것을 관찰할 수 있었습니다. 이와 다르게 CTCF-cKO 마우스의 혈청에서 트리글리세라이드와 콜레스테롤이 감소하였습니다. 또한 간 조직의 지질 증가로 인한 지방 독성은 활성 산소종 (ROS), 세포 사멸 및 염증을 일으켰습니다. 이러한 표현형에 따라 우리는 지방간(fatty liver) 이 CTCF 결핍에 의해 발생했다는 것을 알 수 있었습니다. 지방간 (fatty liver)에서 Ctcf 영향을 조사하기 위해 전사체 (transcriptome) 분석을 진행하였습니다. CTCFcKO마우스의 표현형을 설명하기 위해 RNA-sequencing 분석을 수행하였더니, 운반체,지방산 합성 및 지방산 산화와 같은 대사에 관련한 Gene Set Enrichment Analysis (GSEA)분석 들이 나왔습니다. CTCF-cKO 마우스에서 많은 유전자들이 변하는 것을 관찰하였지만 그 중에서도 간 에서 지방산의 주요 수용체로 알려진 Cd36 의 발현이 크게 증가되어있는 것을 확인하였습니다. 간에서 지방산의 주요 수용체로 알려져있는 CD36은 cKO에서 크게 발현의 증가로 인해 지방의 축적을 일으킨다는 것을 실험을 통해 확인하였습니다. 간세포 핵 인자 4α (HNF4α)은 대사의 지질 및 분화를 포함한 간 다양한 생물학적 과정에서 중요한 역할을 합니다. 이 논문에서는 CD36의 발현을 억제 시키는 HNF4α의 전사 인자 네트워크가 cKO에서 감소하고 있는 것을 확인하였습니다. CD36와 긍정적인 상관 관계를 나타내는 PPAR (퍼옥시즘 증식 활성화 수용체) γ는 지질 리간드로 인해 활성화 됨으로써 cKO에서 고도로 농축되어 있는 것을 확인하였습니다. 따라서 CTCF 손실에 의한 HNF4α의 저해 및 PPARγ의 유도는 CD36의 유도를 나타낸다 것을 확인하였습니다. 우리의 연구는 HNF4α의 저해 및 PPARγ의 유도를 통해 cKO의 간 지질 대사를 조절하는 CTCF 능력을 발견하였습니다.앞서 CTCF가 결핍 되었을 때 지방간 (fatty liver) 이 발생되는 것을 관찰하였습니다. CTCF 가 없는 상황이 지속되었을 때 간암이 발생하는지를 확인하고자 하였습니다. 최근에 DNA 손상은 암 발생에 있어서 중요한 것으로 알려져 있습니다. DNA 손상은 자연적으로 발생 할 수 있습니다. 이는 DNA 손상 복구 시스템에 의해 고쳐지게 되지만, DNA 손상 복구 시스템에 문제가 생기게 되면 DNA 손상이 축적되게 됩니다. 이로 인해 유전적 변화와 유전자의 불안정성을 일으키게 됩니다. DNA 손상은 주로 이중가닥 끊김 (DSBs) 을 유발 합니다. 염색체 상에서 발생한 이중가닥 끊김 (DSBs) 의 경우, DNA의 결실, 전좌, 결합을 일으키게 됩니다. 이러한 현상은 유전자 재배열이라는 이름으로 암 세포에서 빈번하게 발견되는 현상입니다. 이전 보고들에 따르면 여러 암세포 안에서 CTCF 와 Cohesin 에 의해 생기는 chromosome looping anchors 부분은 DNA 끊김에 취약하다고 알려져 있습니다. CTCF 는 DNA 손상 회복 경로 중에서도 상 동성 재조합 (homologous recombination, HR) 에 중요 한 조절 인자로 알려져 있습니다. CTCF 가 유전자의 안정성을 유지하기 때문에 CTCF의 손실은 염색체의 불안정성을 증가시키고 HR-매개 복구 경로를 손상시킵니다. CTCF결핍으로 인해 생기는 HCC의 결과는 지질대사 문제뿐만 아니라 DNA 손상과 연관 관계가 있음을 추측할 수 있었습니다. CTCF 결함이 있는 마우스의 간세포에서 독소루비신에 의해 유도된 DNA 손상인자인 γ-H2AX가 유지되는 것을 확인하였습니다. 우리는 또한 CTCF 결핍 마우스가 정상적인 상태에 비해 DNA 손상 축적을 유도하는 DNA 손상 복구 경로에 결함이 있음을 확인했습니다. 디 에틸 니트로사민 (DEN) 은 알려진 발 암물질로, 돌연변이 유발 DNA 부가 물의 형성을 유도하는 DNA 알킬화제입니다. 유전독성 약물 DEN은 설치류에서 HCC 를 유도하기 위해 60년대부터 사용되어 왔으며 마우스 에서 간암을 유발하는 데 가장 널리 사용되는 화학 물질입니다. DEN 모델에서HCC 발달의 증가가 보고되었습니다. CTCF 가 결핍된 마우스는 DEN 유도된 HCC의 비율이 정상치와 비교하여 더 높게 나타났습니다. 이러한 결과로 인해 CTCF 결핍에 의한 DNA 손상이 간세포 암의 진행을 촉진 시킨다는 것을 알 수 있었습니다. 이것이 나이가 많은 CTCF 결핍 마우스가 대사 문제가 있는 간세포 암 (HCC) 에 걸린 이유 입니다.이러한 분석을 통해 간에서의 지질 대사 문제로 DNA 손상 및 복구에서 CTCF 의 기능적 역할을 확립하고 있습니다. 또한 간에서 유전자 구조화와 안정성 사이에 잠재적인 연관성을 제공하고 있습니다. 결론적으로, 간세포에서 CTCF 는 CD36 의존적으로 지방간 (fatty liver) 을 예방하고 DNA 손상 회복 및 염색체 안정성을 유도하여 HCC 진행을 예방합니다. 이러한 연구 결과를 통해 CTCF가 간의 지질 대사에 기여하고 있을 가능성이 있음을 보여주고 있습니다. 이와 관련, 간에서 CTCF의 연구는 지질 대사를 조절하여 지방간 환자에게 유용한 치료법을 제공하고 CTCF는 HCC 치료의 유망한 치료 표적 이 될 수 있다는 것을 시사합니다. Fatty liver occurs in 10%–25% of people worldwide and is considered the hepatic manifestation of the metabolic syndrome. The liver has a high capacity for free fatty acid (FFA) uptake and plays an important role in lipid metabolism. CCCTC-binding factor (CTCF) is a highly conserved zinc finger protein known as a transcription factor. It has function of a transcriptional activator, a repressor or an insulator protein that blocks the communication between enhancers and promoters. CTCF can also recruit other transcription factors while bound to chromatin domain boundaries. However, the molecular targets and mechanisms of CTCF action on liver metabolism and fatty liver remain poorly understood. In this study, I found out the CTCF role in the liver by utilizing conditional liver-specific CTCF-knockout (cKO) mice, increased level of triglyceride and cholesterol were observed lipid accumulation in CTCF-deficient mouse liver tissue. On the contrary to this, in CTCF-deficient mouse serum, level of triglyceride and cholesterol were decreased. Additionally, lipotoxicity caused by increased lipid in liver tissues. So it leads the increase level of reactive oxygen species (ROS) production, apoptosis and inflammation. According to those phenotype I could know fatty liver occurred by CTCF deficiency. To investigate the effect of CTCF on fatty liver, transcipritome analysis was performed. I performed RNA-sequencing analysis to illustrate the cKO phenotype. Term of lipid metabolism process and fatty acid metabolic genes were found by Gene Set Enrichment Analysis (GSEA) tool. I observed many lipid related genes were altered by CTCF defects. However, among those genes I found that the expression of cluster of differentiation 36 (CD36), a transporter known as a major receptor of fatty acid in liver, is greatly increased in cKO. Hepatocyte nuclear factor 4α (HNF4α) play important role in diverse biological processes of liver including metabolisms lipids and differentiation. Here, I demonstrate a transcription factor network under the control of HNF4α which is working as a repressor to CD36 were diminished in cKO. PPAR (peroxisome proliferator-activated receptors) γ which is positively correlated with CD36 were highly enriched in cKO by activated with lipid ligand. Thus, inhibition of HNF4α and induction of PPARγ by CTCF loss shows induction of CD36. Our studies provide, the ability of CTCF to coordinate hepatic lipid metabolism in cKO through inhibition of HNF4α and induction of PPARγ. I observed fatty liver when CTCF deficiency occurred earlier. I wanted to see whether CTCF defect affects the occurrence of liver cancer when the situation persisted. Recently, DNA damage is known as one of the facilitating effect on cancer development. DNA damage can be naturally occurred, however defect of DNA damage repair pathway can leads to accumulation of DNA damage. It causes genetic alteration and genomic instability. DNA damage mainly causes double strand breaks (DSBs). DSBs in the chromosome bring deletions, translocations, and fusions in the DNA. These consequences are collectively referred to as genomic rearrangements, which are commonly found in cancerous cells. Previous studies have shown that chromosome looping anchors bound by CTCF and cohesin are vulnerable to DNA breaks in various cancer cells. CTCF is an important regulator of DNA damage repair pathway, especially in homologous recombination (HR) pathway. Since CTCF maintains genome stability, loss of CTCF increases the chromosomal instability and compromises HR-mediated repair pathway. I could speculate that HCC caused by CTCF is associated with DNA damage as well as lipid metabolism problems. Primary hepatocytes from CTCF-deficient mice maintain the formation of doxorubicin-induced γ-H2AX which is the marker of DNA damage. This study further show that CTCF-deficient mice have flaw in DNA damage repair pathway, which leads to accumulation of DNA damage compared to normal states. Diethylnitrosamine (DEN) is known as carcinogen. DNA alkylating agent that leads the formation of mutagenic DNA adducts. The genotoxic drug DEN is used since the 60s to induce HCCs in rodents and it is the most widely used model to induce liver cancer in mice. A dramatic increase in HCC development was reported in the DEN model. cKO mouse reveals higher rate of DEN induced HCC compare to the normal. This indicates that DNA damage accelerate the development of HCC by CTCF defects. . Based on these results, CTCF-deficient in mice develops hepatocellular carcinoma (HCC) by damage from the metabolic problems. These analyses establish a functional role of CTCF in DNA damage and repair with lipid metabolism problem in liver. Also, it provides a potential link between genome organization and stability in liver. In conclusion, hepatocyte CTCF prevents fatty liver by regulating lipid metabolism and HCC progression by participating DNA damage repair and maintaining the chromosome stability. These findings indicates that CTCF may contribute to lipid metabolism in the liver. In this regard, the studies of CTCF in liver may provide a useful therapy for fatty liver patients by controlling lipid metabolism and CTCF may be a promising therapeutic target for the treatment HCC. 자가 면역 및 염증과 강하게 결합하는 다면 발현적인 사이토카인 인 IL-21은 다양한 면역 반응을 조절합니다. IL-21은 IL-6에 의해 CD4 + T 세포에서 강력하게 유도 될 수 있습니다; 그러나 크로마틴 수준에서 Il21 유전자의 전사 조절의 기초가 되는 메커니즘에 대해서는 거의 알려져 있지 않습니다. 여기에서는 Il21 유전자 49kb 상위에 위치하는 보존 된 논 코딩 시퀀스가 STAT3 및 NFAT 의존적으로 Il21 유전자 발현을 상향 조절 할 수 있는 인핸서를 포함하고 있는 것을 보여주었습니다. 또한, 우리는 CTCF 및 코히신에 항시 적으로 결합하는 Il21 위치의 인핸서를 차단하는 인슬레이터를 발견했습니다. Naïve CD4 + T 세포는 이러한 상위 및 하위의 CTCF 결합 부위가 상호 작용하여 DNA 루프를 형성하는 것을 보여주었습니다. 그러나 IL-21 프로모터는 Il21 위치의 모든 시스 요소와 상호 작용하고 있지는 않습니다. 대조적으로, IL-6로 인한 CD4 + T 세포의자극으로 인해 프로모터와 새로운 먼 부위의 인핸서 영역에 STAT3를 동원합니다. 이것은 크로마틴 구조의 동적 변화를 유발하고 프로모터와 규제 요소들을 공간적으로 근접시키게 됩니다. 프로모터와 거리가 먼 인핸서 영역 사이의 장거리 상호 작용은 IL-6 /STAT3 신호 전달 경로에 의존했지만, IL-21 발현이 억제 된 조절 T 세포에서는 이러한 상호작용이 사라졌습니다. 따라서, 이 연구는 Il21 유전자의 적절한 전사 조절의 기초가 되는 새로운 위상 성 크로마틴 프레임 워크를 보여주는 결과를 밝혀냈습니다. IL-21, a pleiotropic cytokine strongly linked with autoimmunity and inflammation, regulates diverse immune responses. IL-21 can be potently induced in CD4+T cells by IL-6; however, very little is known about the mechanisms underlying the transcriptional regulation of the Il21 gene at the chromatin level. Here, I demonstrated that a conserved non-coding sequence located 49 kb upstream of the Il21 gene contains an enhancer element that can upregulate Il21 gene expression in a STAT3- and NFAT-dependent manner. In addition, I identified enhancer-blocking insulator elements in the Il21 locus, which constitutively bind CTCF and cohesin. In naïve CD4+T cells, these upstream and downstream CTCF-binding sites interact with each other to make a DNA loop; however, the IL-21 promoter does not interact with any cis-elements in the Il21 locus. In contrast, stimulation of CD4+T cells with IL-6 leads to recruitment of STAT3 to the promoter and novel distal enhancer region. This induces dynamic changes in chromatin configuration, bringing the promoter and the regulatory elements in close spatial proximity. The long-range interaction between the promoter and distal enhancer region was dependent on IL-6/STAT3 signaling pathway but was disrupted in regulatory T cells, where IL-21 expression was repressed. Thus, our work uncovers a novel topological chromatin framework underlying proper transcriptional regulation of Il21 gene.

진귤 잎 유래 polymethoxyflavones 분획물의 비만 개선 효과에 대한 연구

진영준 제주대학교 대학원 2022 국내박사

Polymethoxyflavones (PMFs)는 감귤류에서만 발견되는 플라보노이드로 제주 재래귤 중에서는 진귤에서 PMFs 함량이 가장 높다고 보고된 바 있다. PMFs는 건강에 유익한 다양한 생리활성을 나타내기 때문에 PMFs를 다량 함유하는 분획물(PMFs-rich fraction, PRF)을 획득하기 위한 방법 개발이 필요하다. 본 연구에서는 진귤 과실과 잎에 함유된 PMFs 함량을 비교하여 잎이 과실보다 PMFs 함량이 3배 정도 높음을 확인하여 잎으로부터 PMFs를 다량 함유하는 분획물 PRF를 획득하였다. 또한 진귤 잎 유래 PRF의 비만 개선 효과를 in vitro와 in vivo에서 분석하였다. 본 연구의 in vitro 모델로는 3T3-L1 전구지방세포를 사용하였다. PRF를 분화 유도물질과 공동 처리하였을 때 PRF는 지방세포로의 분화를 농도 의존적으로 억제하였다. PRF는 peroxisome proliferator-activated receptor gamma (PPARγ)와 CCAAT/enhancer binding protein alpha (CEBPα) 발현을 억제함으로써 sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS), adipocyte fatty-acid-binding protein 2 (aP2)의 발현을 억제함을 확인할 수 있었다. 완전히 분화된 3T3-L1 지방세포에 PRF를 처리하면, 지방세포의 항상성 유지에 필요한 PPARγ와 CEBPα, SREBP1, FAS의 발현은 감소했지만, protein kinase A/sterol regulatory element-binding protein 1(HSL)의 활성화가 일어나 지방분해는 촉진됨을 확인할 수 있었다. 또한, 완전히 분화된 3T3-L1 지방세포에서 PRF는 AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC)의 인산화를 증가시켜 지방산화를 촉진할 수 있음을 확인하였다. In vitro에서 얻은 실험결과를 바탕으로 고지방 식이로 유도된 비만 생쥐에서 PRF의 항비만 효능을 평가하였다. 5 주령 C57BL/6 수컷 생쥐에 고지방식이를 5주 동안 공급하여 비만을 유도하였다. 고지방 식이로 유도된 생쥐는 고지방식이군(고지방식이 + 0.1% carboxymethyl cellulose), orlistat 투여군 (고지방식이 + 15.6 mg orlistat/kg of BW), PRF 투여군 (고지방식이 + 50, 100, 200 mg of PRF/kg of BW)으로 나누어 5주간 경구 투여하였다. PRF 및 orlistat 투여군의 체중 증가량, 지방조직, 비장, 신장 무게는 고지방식이군에 비해 유의적으로 낮게 나타났다. PRF 투여군은 고지방식이군에 비해 혈장 내 triglyceride (TG), low-density lipoprotein (LDL) 함량은 낮고 high-density lipoprotein (HDL) 함량은 높았다. 이 결과는 PRF 투여로 고지방식이로 유도된 지방이상혈증이 개선되었음을 나타낸다. 또한, PRF 및 orlistat 투여군은 고지방식이군에 비해 내당능이 개선된 것을 확인할 수 있었다. PRF 투여군은 공복 시 인슐린 분비량이 농도 의존적으로 감소하였고, homeostasis model assessment - insulin resistance (HOMA-IR) 분석으로 산출한 인슐린 저항성도 개선되었다. 각 실험군의 지방조직을 hematoxylin and eosin (H&E) 염색하여 비교한 결과, PRF 투여군의 지방구의 크기는 고지방식이군의 지방구 크기보다 작았다. PRF에 의한 항비만 효과의 분자적 기전을 알아보기 위해서 각 실험군의 지방조직에서 지방 대사와 관련된 단백질들의 발현 양상을 분석하였다. PRF 투여는 지방조직에 유지에 필요한 인자인 PPARγ의 발현을 감소시켰고, 지방분해를 촉진하는 HSL의 인산화를 증가시켰다. 또한 PRF 투여는 AMPK/ACC 경로를 활성화하여 지방산화를 촉진하는 것으로 나타났다. 또한 각 실험군의 간 조직을 분석한 결과, PRF는 고지방식이로 유도되는 지방간 생성을 저해하여 간 기능을 개선하는 작용을 할 것으로 사료되었다. 요약하면, 본 연구는 진귤 잎을 사용하여 효율적으로 PRF 획득하는 방법을 확립하였고, PRF는 in vitro 및 in vivo 에서 지방분해 및 지방산 산화를 촉진함으로써 항비만 효능을 나타냄을 보여주었다. Polymethoxyflavones (PMFs) are flavonoids exclusively found in citrus fruits. Among the native citrus plants in cultivated in Jeju do, Citrus sunki Hort, ex Tanaka have the highest contents of PMFs. As PMFs have diverse health-beneficial bioactivities, it is necessary to develop a method to obtain the PMF-rich fraction (PRF) for research on their industrial application. In this study, PMF contents were compared between the fruits and leaves of C. sunki. The results showed approximately three-fold higher PMFs contents in the leaves than in the fruits, and thus, we established a procedure for obtaining the PRF from the leaves. Then, the anti-obesity effect of PRF was investigated in vitro and in vivo. In this study, we used 3T3-L1 preadipocytes as in vitro model system. Upon simultaneous treatment with PRF and differentiation-inducing medium, PRF inhibited differentiation of preadipocytes into adipocytes in a concentration-dependent manner. PRF also inhibited the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (CEBPα), consequently reducing the expression of sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS), and adipocyte fatty-acid-binding protein 2 (aP2). The treatment of differentiated 3T3-L1 adipocytes with PRF led to a decline in the expressions of PPARγ, as well as CEBPα, SREBP1, and FAS, which have a role in maintaining the adipocyte homeostasis. Protein kinase A (PKA)/sterol regulatory element-binding protein 1 (HSL) was activated to promote lipolysis. In addition, in differentiated 3T3-L1 adipocytes, PRF increased the activation of AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) to promote fatty acid oxidation. Based on the in vitro results, the anti-obesity effect of PRF in mice with high-fat-diet-induced obesity was evaluated. Five-week-old C57BL/6 male mice were supplied with a high-fat-diet (HFD) for five weeks to induce obesity. The HFD-fed obese mice were divided into three groups, the control group (HFD + 0.5% carboxymethyl cellulose), orlistat group (HFD + 15.6 mg orlistat/kg of BW), and PRF group (HFD + 50, 100, or 200 mg of PRF/kg of BW), for a 5-week oral administration period. The overall weight gain, adipose tissue weights, spleen weights, and kidney weights of the PRF and orlistat groups were significantly reduced compared to those in the controls. The PRF group exhibited lower contents of plasma triglycerides (TG) and plasma low-density lipoprotein (LDL-CHO) than the control group, whereas high-density lipoprotein (HDL-CHO) content was higher than the control group. These results suggested that PRF improves the dyslipidemia induced by the HFD. In addition, the PRF and orlistat groups exhibited an improvement in glucose tolerance compared to controls. Moreover, the PRF group exhibited a decrease in the level of insulin secretion in a concentration-dependent manner upon fasting, thereby insulin resistance estimated by homeostasis model assessment-insulin resistance (HOMA-IR) were improved. A comparison of the hematoxylin and eosin-stained adipose tissue from each group showed that the size of lipid droplets was smaller in the PRF group than that in the HFD group. To identify the molecular mechanism on the anti-obesity effect of PRF, we analyzed the expressions of the proteins, which were related to lipolysis in the adipose tissues. The PRF administration reduced the expression of PPARγ, a factor required for the maintenance of adipose tissues. Furthermore, it increased the phosphorylation of HSL, which facilitates lipolysis. The PRF administration also activated the AMPK/ACC pathway to promote fatty acid oxidation. In addition, an analysis of the liver tissue of each group revealed that PRF potentially improved the liver function as it inhibited the incidence of fatty liver caused by the HFD. In summary, this study has developed an efficient method to obtain PRF from C. sunki leaves, and the PRF was found to exert an anti-obesity effect via promoting the lipolysis and fatty acid oxidation both in vitro and in vivo.

Epigenetic Regulation and Tumor-Suppressive Role of a Liver-Specific Long Non-Coding RNA via Notch Pathway Inhibition in Hepatocellular Carcinoma

장세하 아주대학교 일반대학원 2026 국내석사

In chapter 1, Long non-coding RNAs (lncRNAs) have emerged as critical regulators in the pathogenesis of hepatocellular carcinoma (HCC), yet the mechanisms governing liver-specific lncRNAs remain largely unexplored. In this study, we identified and characterized a novel liver-specific lncRNA, designated as Liver-specific Upregulated Non-coding RNA regulator (LUNAR), which plays a key tumor-suppressive role in HCC progression. Through a comprehensive analysis of stage-specific transcriptomic datasets, LUNAR was discovered to be a liver-enriched transcript whose expression is progressively diminished during hepatocarcinogenesis. Integrative validation using multiple patient cohorts, in vitro migration and invasion assays, and in vivo orthotopic xenograft models demonstrated that restoring LUNAR expression markedly inhibits metastatic behaviors without affecting proliferation. Mechanistically, this anti-metastatic function is mediated through selective suppression of the NOTCH signaling pathway, while its downregulation in tumors is driven by promoter hypermethylation leading to epigenetic silencing. Collectively, these findings define LUNAR as a liver-specific, epigenetically regulated tumor suppressor lncRNA with strong diagnostic and prognostic potential, providing new insights into HCC metastasis and offering a promising target for future therapeutic intervention. In chapter 2, this study first demonstrated that Odoribacter splanchnicus (O. splanchnicus)–conditioned media dose-dependently reduced free fatty acid– induced lipid accumulation in HepG2 and Hepa1-6 cells, indicating a direct anti-steatotic effect in vitro. Consistent with these findings, supplementation with O. splanchnicus markedly alleviated Western diet–induced hepatic steatosis, inflammation, and fibrosis in mice. Mechanistically, O. splanchnicus restored the expression of key fatty acid oxidation regulators, including PPAR- α, ACOX1, and CPT1A, supporting its role in enhancing lipid metabolic homeostasis. Together with our observation that O. splanchnicus abundance was significantly reduced in MASLD patients compared with healthy controls, these results suggest that O. splanchnicus exerts hepatoprotective and anti-steatotic effects and may represent a promising microbial therapeutic candidate for MASLD management.

Molecular Analysis of FAD2 Genes and the Development of High Oleic Oilseed Rape (Brassica napus L.) by Metabolic Engineering of FAD2 Gene

이경렬 성균관대학교 일반대학원 2013 국내박사

Plant FAD2 catalyzes the synthesis of linoleic acid from oleic acid through desaturation at 12th carbon position. It is essential enzyme to be alive in low temperature and which is responsible for the synthesis of precursor of jasmonic acid, second messenger molecule. FAD2 gene is also necessary to modify genetically the fatty acid composition of seed oil as human wants. To increase the oleic acid content of seed oil, it is required the seed-specific suppression of FAD2 gene expression. Moreover, oil crop with high linoleic acid is favorable to accumulate high amount of unusual fatty acids useful food and industry, such as GLA and SDA, vernolic acid and α-eleostearic acid, in seed. Oilseed rape has high seed oil content and high seed yield, but which contains low linoleic acid content. When it is used for the production of high amount of these unusual fatty acids, additional FAD2 gene should be introduced into it. Therefore, FAD2 genes were cloned from Vitis labrusca and Brassica napus for the research on modification of linoleic acid content. Sequence analysis of deduced amino acid of two functional VlFAD2 genes and three BnFAD2 genes demonstrated characteristics similar to those of ER-localized fatty acid desaturases. Phylogenetic analysis and tissue-specific RT-PCR showed that two species have FAD2 genes expressed as constitutive or seed-type genes, respectively. Complementation analysis demonstrated that introduction of two VlFAD2 genes can restore the PUFA proportion in fad2 mutant Arabidopsis. Except BnFAD2-3 gene, yeasts harboring three BnFAD2 genes, respectively, synthesize linoleic acid. These results indicated that two functional VlFAD2 genes may be responsible for the high level of linoleic acid in seed and three BnFAD2 genes are functional, however, BnFAD2-3 gene is not functional by mutation. B. napus FAD2 genes are originated different two FAD2 genes from B. rapa and B. oleracea, respectively. Oleic acid content of oilseed rapes transformed three vectors with suppression manner of antisense, RNAi or antisense+RNAi, respectively, was up to 85% of total fatty acid in seed oil. Three events stably inherited the high oleic trait were selected. Analysis of T-DNA integration site showed that two of three events contained no vector backbone sequence. Two events, AS9A and HPAS29, are considered to be suitable for GM assessment even though more analyses such as genomic Southern and genotyping are required. It is thought that these high oleic oilseed rape events could be used to manufacture high temperature frying oil and high quality biodiesel.

Fatty acid double layer coated iron-based nanoparticles with stable mobility and selective reactivity to hydrophobic contaminants in subsurface

우희수 서울대학교 대학원 2018 국내박사

Injection of reactive iron-based nanoparticles (Fe NPs) to the DNAP sources in the subsurface is one of the emerging efficient technologies as an alternative to predominant treatment technologies. The small size of reactive nanomaterials (e.g., 10 ~ 100 nm) can offer the potential of being derivable in the contaminated subsurface, and thus, potentially eliminate the need for excavation. However, due to their high surface energy, Fe NPs tend to rapidly agglomerate in water forming micro to millimeter scale aggregates or flocs, which weaken their mobility in groundwater. Moreover, commonly encountered groundwater solutes such as anions, cations, and dissolved organic matters can affect the reactivity of Fe NPs negatively. In this study, fatty acid double layer mimic lipid bilayer, constituent of cell membrane, was used as a stabilizer of Fe NPs to achieve stable mobility and selective reactivity toward chlorinated hydrocarbons and the method for coating the fatty acid double layer onto the surface of Fe NP was developed. Fatty acid double layer coated Fe NPs (PO-OFe) were prepared through two sequential steps: 1) primary hydrophobic layer preparation via thermal decomposition method with oleic acid; 2) secondary hydrophilic layer by potassium oleate. The mas of coated fatty acid was approximately 50% of total particle mass and zeta potential of PO-OFe has reached the great extent of – 115 mV, indicated that aqueous stability and superior mobility of PO-OFe can be obtained by electrostatic and steric repulsive forces induced from the fatty acid double layer. PO-OFe remained stable in aqueous phase in sedimentation test although unmodified Fe NPs were deposited on the bottom of the test cell only in 2 h, indicating fatty acid double layer prohibit nanoparticle from agglomeration. Consistent with the sedimentation teste results, no deposition was detected in 1-D column tests, the mobility of which is even better than that of CMC-Fe which are the modified Fe NPs by one of the mostly used modification for Fe NP stabilization in remediation application. Due to non-polar zone in fatty acid double layer onto the surface of PO-OFe, hydrophilic dissolved groundwater solutes as well as a reducible contaminant (nitrate) rarely affected the reactivity of PO-OFe toward carbon tetrachloride, a model chlorinated solvent, at an insignificant level, suggesting that PO-OFe have selective reactivity. Nitrate, that is well known as a reducible hydrophilic contaminant by zero valent iron, was not transformed by the contact with PO-OFe and the reaction rate of PO-OFe was slower than those of unmodified or CMC modified Fe NPs. These are attributable to the presence of non-polar zone in fatty acid double layer that can block hydrophilic molecules from permeation through the layer and require hydrophobic contaminants to be diffused through the layer onto the surface of Fe NPs. The selective reactivity will keep the fatty acid double layer coated iron nanoparticles reactive. Therefore, the greater longevity of PO-OFe can be expected especially in the geochemical condition of high concentration of groundwater solutes. Remedial action using the reactants with the longevity and great mobility can offer the chance to clean-up the site where as-used technologies cannot be economically applied. Fatty acid double layer coated Fe NPs may be a suitable remediation alternative for these sites.