烟酰胺N-甲基转移酶在脂肪细胞分化中的作用及其表观遗传学机制研究

Adipose tissue has a complex role in whole body energy metabolism that has evolved from a simple lipid storage to an active secretory and endocrine organ. The dysfunction of adipose tissue can result in insulin resistant and its complications, type 2 diabetes, hypertriglyceridemia, low levels of high-density lipoprotein cholesterol, and hepatic steatosis. Modulating adipogenesis has emerged as a potential protective mechanism that reduces adipose tissue dysfunction. Epigenetic mechanisms in particular histone methylation play important roles in regulating gene expression and cell differentiation. Nicotinamide N-methyltransferase (NNMT) is strongly expressed in adipose tissue but its metabolic function is not well studied. NNMT methylates nicotinamide, one of the forms of vitamin B3, to produce N1-methylnicotinamide (MNA). MNA can be further oxidized by aldehyde oxidase to two related compounds N1-methyl-2-pyridone-5-carboxamide (2py) and N1-methyl-4-pyridone-3 carboxamide (4py). Riederer et al proposed that NNMT is a major methyltransferase in adipocytes. However, no loss of function studies were performed. First evidence that NNMT can affect methyl donor balance came from NNMT-expressing cancer cells. NNMT impairs the methylation potential of cancer cells by consuming methyl units from S-adenosyl methionine to create the stable metabolic product 1-methylnicotinamide. These evidences indicate that NNMT may regulate adipogenesis by affecting methyl donor balance and histone methylation. . Our recent work showed NNMT is expressed in 3T3-L1 preadipocytes and its expression transiently decreases during the expansion phase and is restored later in adipogenesis. Interestingly, we found that NNMT knockdown in 3T3-L1 cells impaired adipogenesis. The present investigation aims to extend these findings and determine the physiological significance of NNMT with respect to adipogenesis in vitro and in vivo. Using methods of ethology, cell biology and molecular biology, we will perform the lipid accumulation，lipolysis，and glucose uptake in NNMT knockdown and over expression 3T3-L1 cell line. We will also see that role of epigenetic modification (eg, SAM content, DNA methylation and histone methylation) in the regulation of NNMT on adipogenesis. We will further investigate the detailed characterizations of adipose tissue (adipocyte size and number and inflammation) in ob/ob mice maintained standard chow diet supplemented with NNMT inhibitor, N-methylnicotinamide. This approach will also provide information involving in food intake, body weight, blood chemistry (insulin, glucose and lipids) as well as insulin and glucose tolerance in the body. This study may ultimately help to identify novel therapeutic strategies aimed at ameliorating the metabolic sequelae of obesity such as cardiovascular disease and type 2 diabetes.

烟酰胺N-甲基转移酶（NNMT）以S-腺苷甲硫氨酸（SAM）为甲基供体，催化维生素B3烟酰胺的转甲基反应。我们新近研究表明，NNMT在脂肪细胞中的表达量和细胞分化程度密切相关，但其机制有待揭示。本研究拟以3T3-L1前脂肪细胞为研究对象，通过慢病毒转染降低或升高NNMT的表达，并应用药理学方法改变细胞内DNA及组蛋白的甲基化状态，观察细胞脂滴累积、脂解作用及葡萄糖摄取功能的改变，分析脂肪细胞分化标志因子（C/EBPα、C/EBPβ及PPARγ）的表达量；以ob/ob小鼠为研究对象，食物中添加NNMT抑制剂N-甲基烟酰胺，研究脂肪细胞体积、数量、脂肪组织炎症反应方面的改变，测定糖脂代谢相关指标，探讨脂肪组织SAM含量、DNA甲基化及组蛋白甲基化修饰的变化，从而明确NNMT在体内及体外对脂肪细胞分化的调控作用，并揭示表观遗传学修饰在其中扮演的角色，为肥胖及相关疾病的治疗提供有价值的研究资料。

肥胖症在全世界范围内的流行使得人们开始高度关注脂肪沉积调控机制的研究。脂肪细胞分化失常与脂肪沉积密切相关。NNMT在脂肪组织中大量表达，但其在脂肪细胞分化和代谢中的作用还研究甚少。本研究的主要目标是明确NNMT在体内及体外对脂肪细胞分化的调控作用，并揭示表观遗传学修饰在其中扮演的角色。主要的研究结果如下：1）降低NNMT的表达以及培养基中添加MNA处理均可以下调3T3-L1细胞中aP2、PPARγ、C/EBPα等促脂肪生成基因的表达并减少脂肪的累积，提示抑制NNMT的表达或活力可以阻碍3T3-L1细胞向成熟脂肪细胞的分化。2）降低NNMT的表达不影响诱导分化早期（Day 2）脂肪生成相关基因的表达，却显著降低诱导分化后期（Day 4和Day 8）C/EBPα和脂肪酸合成酶等促脂肪生成基因的表达。3）降低NNMT的表达显著增加3T3-L1细胞内SAM的水平以及细胞全蛋白中包含赖氨酸甲基化修饰的蛋白的含量；显著升高细胞全蛋白中H3K4me3和H3K9me2的修饰水平以及C/EBPα和PPARγ基因启动子区域内H3K9me2修饰水平。4）NNMT的代谢产物MNA可抑制ob/ob小鼠体重增加并减少肝脏TG含量，改善ob/ob小鼠葡萄糖耐受和胰岛素敏感性，表明MNA具有改善小鼠糖脂代谢的功能。本研究证实NNMT作为脂肪组织中主要的甲基转移酶，参与调控脂肪细胞的甲基供给平衡，进而参与脂肪细胞分化过程，为有效地预防肥胖及其代谢并发症的发生提供了新的、有潜在应用价值的研究资料。