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Role of Abnormal L-Serine Metabolism by Lowered Expression of 3-Phosphoglycerated Dehydrogenase in Fatty Liver Disease : 지방간 발생에서 3-Phosphoglycerated Dehydrogenase 발현 억제에 의한 L-Serine 대사 이상의 역할

DC Field Value Language
dc.contributor.advisor이병훈-
dc.contributor.author심우철-
dc.date.accessioned2018-05-28T16:51:52Z-
dc.date.available2021-04-13T02:14:27Z-
dc.date.issued2018-02-
dc.identifier.other000000151438-
dc.identifier.urihttps://hdl.handle.net/10371/140953-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2018. 2. 이병훈.-
dc.description.abstractFatty liver disease is early-stage liver disease that fat makes up more than 5% of the organs weight. Fatty liver is a reversible state and benign, but without proper treatments, it can lead to liver dysfunction. In this study, we propose that 3-phosphoglycerate dehydrogenase (PHGDH), which is a rate-limiting enzyme in serine biosynthesis, can affect lipid metabolism by regulating L-serine pool in fatty liver disease.
Previous study showed that hepatic L-serine is decreased in chronically ethanol-fed rats. Based on the result, L-serine was treated in alcoholic fatty liver model and it reversed ethanol-induced fatty liver by metabolizing homocysteine via methionine synthase (MS) and cystathionine β-synthase (CβS). L-serine also increased intracellular NAD+ and silent information regulator 1 (SIRT1) activity via lactate dehydrogenase (LDH). L-serine increased mitochondrial gene expression, mass and function by deacetylated PGC-1α.. Increased SIRT1 activity by L-serine ameliorated lipid accumulation and insulin resistance in vitro.
PHGDH and L-serine were found to be significantly lowered in chronic ethanol diet and high-fat diet fatty liver model. Free fatty acids and ethanol also decreased PHGDH expression in vitro. Diminished synthesis of L-serine led to increase in abnormal sphingolipids and ceramides in PHGDH-KO MEF cells and alcoholic fatty liver model. GEO analysis of hepatitis patients revealed phgdh gene expression was diminished. Serum L-serine of fatty liver patients was also down-regulated and negatively correlated with MRI fat fraction, serum ALT and triglyceride (TG). Increased synthesis of L-serine by PHGDH gain of function reversed lipid accumulation in various cells by increasing intracellular NAD+ and SIRT1 activity.
PHGDH is found to be positively regulated by nuclear factor like 2 (NRF2) at both transcriptional and translational levels. Fatty liver disease model showed the decreased expression of NRF2 and increased NRF2 activity reversed free fatty acid-induced decrease in PHGDH expression.
In conclusion, PHGDH plays an important role in regulating lipid metabolism by synthesizing L-serine in the liver. This study showed that PHGDH can be used as a therapeutic target for hepatosteatosis and L-serine has a potential for curing fatty liver disease.		-
dc.description.tableofcontentsI. Introduction 1
1.1 Fatty liver 1
1.2. L-serine 4
1.3. Homocysteine toxicity and role of homocysteine in fatty liver 7
1.4. Homocysteine metabolism 9
1.5. SIRT1 11
1.6. De novo serine synthesis 13
1.7. Regulation of PHGDH 15
1.8. The aim of study 16
II. Materials and Methods 17
2.1. L-serine effect on alcoholic fatty liver 17
2.1.1. Cell culture 17
2.1.2. Nile red assay 17
2.1.3. Animal experiments 18
2.1.3.1. Binge ethanol study 18
2.1.3.2. Chronic ethanol feeding study 18
2.1.4. Histopathologic evaluation 19
2.1.5. Serum biochemistry 19
2.1.6. TG analysis 19
2.1.7. Determination of sulfur amino acids and metabolites 20
2.1.8. RNA interference 20
2.1.9. Statistical analysis 20
2.2. L-serine effect on SIRT1 activity 21
2.2.1. Cell culture 21
2.2.2. NAD+/NADH measurement 21
2.2.3. PGC-1α deacetylation assay 22
2.2.4. RNA interference 22
2.2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) 22
2.2.6. Mitotracker Red staining 23
2.2.7. Mitochondrial DNA quantification 23
2.2.8. ATP measurements 23
2.2.9. Oxygen consumption ratio (OCR) measurements 23
2.2.10. Nile red assay 24
2.2.11. Western blot analysis 24
2.2.12. Membrane fraction 24
2.2.13. Statistical analysis 25
2.3. Down-regulation of PHGDH in fatty liver disease 25
2.3.1. Animal experiments 25
2.3.1.1. Chronic ethanol feeding study 26
2.3.1.2. High-fat diet study 26
2.3.1.3. Methionine-choline deficient diet study 26
2.3.2. qRT-PCR 27
2.3.3. Primary hepatocyte isolation 27
2.3.4. Western blot analysis 27
2.3.5. Clinical data from fatty liver disease patients 28
2.3.6. Cell culture 28
2.3.7. NAD+/NADH measurement 28
2.3.8. PGC-1α deacetylation assay 29
2.3.9. Nile red assay and TG analysis 29
2.3.10. Transient transfection and RNA interference 30
2.3.11. Statistical analysis 30
2.4. The mechanism of regulating PHGDH 30
2.4.1. Bisulfite conversion 30
2.4.2. Histone H3 and H4 acetylation assay 31
2.4.3. Western blot analysis 31
2.4.4. Statistical analysis 31
III. Results 33
3.1. L-serine reverses alcoholic fatty liver 33
3.1.1. L-serine decreased ethanol-induced lipid accumulation 33
3.1.2. L-serine inhibited homocysteine by MS and CβS-dependent homocysteine metabolism 38
3.2. L-serine up-regulates SIRT1 activity 42
3.2.1. L-serine increases intracellular NAD+ by lactate dehydrogenase and SIRT1 activity 42
3.2.2. L-serine up-regulates mitochondrial mass and function 45
3.3. PHGDH, a rate-limiting enzyme in de novo serine synthesis is down-regulated in fatty liver disease 49
3.3.1. Hepatic L-serine synthesizing enzyme expression and L-serine level in vivo and in vitro dsease model 49
3.3.2. Sphingolipids and ceramides level was affected in fatty liver disease 52
3.3.3. Hepatic L-serine synthesizing enzyme expression and L-serine level in clinical patients 56
3.3.4. Up-regulation of L-serine and PHGDH function reverses lipid metabolism 60
3.4. Regulation of PHGDH is mediated by NRF2 63
3.4.1. PHGDH is not regulated by epigenetic modification 63
3.4.2. PHGDH is regulated by NRF2 67
IV.Discussion 71
V. Conclusion 76		-
dc.formatapplication/pdf-
dc.format.extent3981833 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectL-Serine-
dc.subjectHomocysteine-
dc.subject3-Phosphoglycerate dehydrogenase-
dc.subjectSIRT1-
dc.subjectLipid metabolism-
dc.subjectFatty liver-
dc.subject.ddc615-
dc.titleRole of Abnormal L-Serine Metabolism by Lowered Expression of 3-Phosphoglycerated Dehydrogenase in Fatty Liver Disease-
dc.title.alternative지방간 발생에서 3-Phosphoglycerated Dehydrogenase 발현 억제에 의한 L-Serine 대사 이상의 역할-
dc.typeThesis-
dc.description.degreeDoctor-
dc.contributor.affiliation약학대학 약학과-
dc.date.awarded2018-02-
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