Excessive sugar consumption alters one-carbon metabolic kinetics

Abstract

背景 甘氨酸甲基轉移酵素 (glycine N- methyltransferase, GNMT) 是肝臟中主要酵素之一，此酵素主要的反應是將S-腺苷基甲流氨酸的甲基轉移到甘氨酸的氮原子上，產生肌氨酸以及S-腺核苷同半胱氨酸。因此GNMT可能透過影響哺乳動物細胞內DNA與蛋白質的甲基化修飾並影響基因的穩定及表現。高糖飲食提升人體許多疾病風險,包括糖尿病、心血管疾病、脂肪性肝炎等。高糖攝取對於癌症的代謝機制亦中扮演重要的角色。然而，GNMT在以葉酸為主的單碳代謝角色和高糖曝露對單碳代謝之角色尚未闡明清楚。本研究以細胞及動物的模式探討高糖暴露對於單碳代謝的影響以及其中GNMT所扮演之角色。 實驗設計在體內與體外方別以穩定同位素標定特定目標追蹤高糖攝取對體內或幫胞內重要代謝路徑之間分流的變化及動態平衡。以有無表達GNMT的細胞及動物模式分別在體內及體外模式中探討高葡萄糖及果糖攝取對於糖酵解、三羧酸循環、以及單碳路徑之間的分流影響，並進一步研究當中甘氨酸甲基轉移酵素所扮演的角色。 結果在GNMT缺失會影響細胞中的有氧糖解作用 (aerobic glycolysis)，並影響葡萄糖及果糖在三羧酸循環代謝中的利用。GNMT缺失同時會進一步影響細胞內葡萄糖利用於絲氨酸合成 (de novo serine synthesis)。此外與有表達GNMT有細胞相比, GNMT缺失會造成甘氨酸裂解系統通量改變，此系統透過粒腺體甲酸的形成而合成單碳單元。表達GNMT則會影響果糖利用包括由糖酵解的三羧酸循環及單碳代謝通量，但並不影響果糖甘氨酸裂解系統或有氧糖解作用的代謝流。在動物模型中，無論有無表達GNMT之小鼠若攝取高果糖均會干擾來自粒腺體甘氨酸裂解系統所產生之嘆單元進入胸腺嘧啶生成。此外正常小鼠攝取高葡萄糖及高果糖會促進內生性甲酸用於嘌呤生合成，然而在GNMT剔除的小鼠模式中高葡萄糖及高果糖攝取會干擾內生性甲酸用於嘌呤的生成，實驗表明高糖攝取在有無表達GNMT的模型中會影響核苷酸的形成。 結論 本研究同時以細胞及動物模式探討高糖攝取對於單碳代謝的影響，並提出甘氨酸甲基轉移酵素再醣類及能量代謝之潛在角色。 關鍵字：甘氨酸甲基轉移酵素，葡萄糖，果糖，三羧酸循環，甘氨酸裂解系統，單碳代謝

Background. Glycine N- methyltransferase (GNMT, EC2.1.1.20) is an abundant liver protein that converts S- adenosylmethionine to S- adenosylhomocysteine while generating sarcosine from glycine. GNMT is a folate binding protein commonly diminished in human hepatoma. Sugar consumption is suspected to play an important role in the pathogenesis of diabetes, cardiovascular disorders, fatty liver disease, and some forms of cancers. Acute diabetic condition induced hepatic protein abundance and activity of glycine N-methyltransferase (GNMT) and phosphatidylethanolamine N- methyltransferase (PEMT), enzymes that have important roles in regulation of methyl groups and homocysteine. The present study aims to investigate how glucose and fructose modulate glycolysis, tricarboxylic acid (TCA) cycle and one carbon metabolic kinetics in vitro and in vivo. Study design. In cell models, hepatocyte- derived cell- lines with and without GNMT expression were cultured in regular minimum essential medium alpha medium (α MEM) treated with high glucose or fructose. In vivo mouse models: Study 1 was preformed to investigate the route of glucose and fructose on glycolysis, TCA cycle and one carbon metabolism in mice with and without GNMT. Study 2 was preformed to investigate the impacts of glucose and fructose on one- carbon kinetics. Results. In the cell model, defected GNMT results in wastage of sugar by increasing anaerobic glycolysis of glucose and fructose and impairs the utilization of both sugars for TCA cycle metabolite formation. Defected GNMT promotes the utilization of glucose (but not fructose) for de novo serine synthesis. Defected GNMT activates mitochondrial formate generation via glycine cleavage system (GCS) in vitro. GNMT assists fructose utilization for TCA cycle, 1C metabolism (but not GCS) and aerobic glycolysis metabolism. In the mouse model, we demonstrated that high fructose excessive consumption inhibited thymidine synthesis from mitochondrial formate production via GCS. However, high glucose and high fructose excessive consumption promoted 10- formylTHF-dependent purine synthesis in WT mice but inhibited 10- formylTHF-dependent purine synthesis in GNMT KO mice, suggesting that high sugar excessive consumption may affect nucleotide biosynthesis. Conclusion. This study investigated the effects of glucose and fructose on one-carbon metabolism in defected GNMT model between in vitro and in vivo. Key words Glycine N- methyltransferase, glucose, fructose, TCA cycle, glycine cleavage system, one- carbon kinetics