Folic Acid Alleviates Diet-induced Fatty Liver Injury via Regulation of Sulfur Amino Acid Metabolism and Cellular Stress-responsive Pathways = 엽산은 황 아미노산 대사 조절과 세포 스트레스 반응 경로를 통해식이 유발 지방간 손상을 완화한다

다국어 초록 (Multilingual Abstract)

Folic acid, a water-soluble B vitamin, plays a critical role in maintaining liver health by participating in one-carbon metabolism, DNA synthesis, and methylation reactions. Although recent studies suggest that folic acid improves fatty liver, its underlying mechanisms remain poorly understood. To clarify its protective role in vivo, a mouse model of diet-induced fatty liver injury was established using a high-fat methionine-defined and choline-deficient (HFMCD) diet. Male ICR mice were fed the HFMCD diet for 10 days and orally administered folic acid (2 or 10 mg/kg). Folic acid supplementation restored hepatic methionine and glutathione levels, thereby normalizing sulfur amino acid metabolism and promoting both structural and functional recovery of the liver. In a supporting in vitro experiment, treatment of HepG2 cells with folic acid significantly and dose-dependently reduced intracellular lipid accumulation induced by free fatty acids (FFA), confirming its lipid-lowering effect at the cellular level. Mechanistically, folic acid reactivated autophagy through modulation of sequestosome-1 (p62) and the mammalian target of rapamycin (mTOR), while attenuating ferroptosis by regulating ferritin heavy chain 1 (FTH1) and heme oxygenase 1 (HMOX1). Collectively, these findings demonstrate that folic acid ameliorates diet-induced fatty liver injury by rebalancing sulfur amino acid metabolism and alleviating cellular stress-responsive pathways, thereby promoting hepatocyte survival and maintaining hepatic metabolic homeostasis.

국문 초록 (Abstract)

엽산은 단일 탄소 대사, DNA 합성, 메틸화 반응에 참여하여 간 건강을 유지하는 데 중요한역할을 한다. 최근 연구에 따르면 엽산이 지방간을 개선한다는 사실이 밝혀졌지만, 엽산의근본적인 메커니즘은 아직 잘 알려져 있지 않다. 생체 내 보호 역할을 명확히 하기 위해 고지방메티오닌 제한 및 콜린 결핍 (HFMCD) 식이를 사용하여 식이 유발 지방간 손상에 대한 마우스모델을 확립하였다. 수컷 ICR 마우스에게 10 일 동안 HFMCD 식이를 공급하고 엽산 (2 또는 10
mg/kg)을 경구 투여하였다. 엽산의 공급은 간 메티오닌과 글루타티온 수치를 회복시켜 황아미노산 대사를 정상화하고 간의 구조적 및 기능적 회복을 촉진하였다. 이를 뒷받침하는 in vitro
실험에서, 유리 지방산 (FFA)에 의해 유도된 세포 내 지질 축적 상태에서 엽산을 처리한 결과, 세포 수준에서 지질의 축적이 현저히 감소하는 것을 확인하였다. 메커니즘적으로 엽산은세퀘스토좀-1 (p62)과 포유류 라파마이신 표적 (mTOR)의 조절을 통해 자가포식을 재활성화하는한편, 페리틴 중쇄 1 (FTH1)와 헴 산화효소 1 (HMOX1)을 조절하여 페롭토시스를 완화하였다. 종합적으로, 엽산은 황 아미노산 대사 균형을 재조정하고 세포 스트레스 반응 경로를 완화하며식이 유발 지방간 손상을 개선하여 간세포 생존을 촉진하고 간 대사 항상성을 유지함을입증하였다.

목차 (Table of Contents)

• 1. Introduction 13
• 1.1. Diet-induced fatty liver injury 13
• 1.2. Folic acid-related metabolic pathways in the liver 14
• 1.3. Sulfur amino acid metabolism and lipid metabolism in the liver 15
• 1.4. Cellular stress-responsive pathways in liver 16
• 1.5. Purpose of study 17
• 2. Materials and Methods 19
• 2.1. Animals and treatment 19
• 2.2. Serum biochemical analysis 19
• 2.3. Extraction of lipid content in liver tissues 20
• 2.4. Determination of MDA and TG levels 20
• 2.5. Determination of serine, glycine, methionine and taurine in the liver 21
• 2.6. Determination of SAM and SAH in the liver 21
• 2.7. Determination of betaine and cysteine in the liver 22
• 2.8. Determination of GSH in the liver 22
• 2.9. Hematoxylin and eosin staining 22
• 2.10. Cell culture and preparation of cell extract 23
• 2.11. Cell viability assay 23
• 2.12. Oil Red O staining 24
• 2.13. Western blot analysis 25
• 2.14. Statistical analysis 25
• 3. Results 27
• 3.1. Folic acid does not cause hepatic toxicity in mice 27
• 3.2. Folic acid modulates one-carbon metabolism and transmethylation cycle 29
• 3.3. Folic acid enhances transsulfuration pathway 31
• 3.4. Folic acid attenuates HFMCD-induced liver enlargement and injury 33
• 3.5. Folic acid restores one-carbon metabolism and transmethylation cycle 35
• 3.6. Folic acid alleviates HFMCD-induced hepatic lipid accumulation 37
• 3.7. Folic acid reduces FFA-induced lipid accumulation in HepG2 cells 39
• 3.8. Folic acid normalizes transsulfuration pathway in HFMCD-fed mice 41
• 3.9. Folic acid improves oxidative and ER stress responses 43
• 3.10. Folic acid improves autophagy and ferroptosis responses 45
• 4. Discussion 47
• 5. References 57
• 6. Abstract (in Korean) 65