Nonalcoholic Steatohepatitis: Natural History, Pathogenesis and Therapy

非酒精性脂肪性肝病(NAFLD)的特点是脂肪在肝细胞内堆积，并伴有炎症和不同程度的细胞损伤和纤维化。当出现细胞损伤和纤维化时，疾病有可能进展，被称为非酒精性脂肪性肝炎(NASH)，可导致肝硬化、肝癌、发病率和死亡率。NASH的病因尚不清楚，也没有批准的治疗方法。NAFLD已经成为一种极其常见的疾病，据估计，在美国，高达30%的人会受到影响。不幸的是，它通常没有被认识到，正如我们最近发现的那样，在NIH临床中心的研究中，在5年的时间里，28%的健康志愿者可能有潜在的NAFLD。因此，显然有必要了解该病的病理生理学及其治疗方法。 我们对NAFLD的关注有三个方面：第一，我们的目标是识别和完善这种疾病的有效治疗方法。其次，我们的目标是通过使用遗传学研究和细胞或动物模型来识别和表征在NAFLD发病机制中起作用的关键基因。我们的第三个重点是肝脏脂肪积累和损伤的生理学，特别是与处理口服卡路里负荷有关的生理学。 在一项随机的安慰剂对照试验中，维生素E已被证明是治疗NASH的有效方法。奇怪的是，维生素E的治疗不仅减少了损伤(被认为反映了它的抗氧化效果)，而且还通过一种未知的机制与肝脏脂肪的减少有关。此外，目前尚不清楚治疗NASH的最佳维生素E剂量。我们进行了一项临床机制试验，以确定维生素E的作用机制及其最佳剂量，目前正在分析这项研究的数据和补充体外模型，以绘制维生素E影响的途径以及它们是否依赖于其抗氧化能力。 全基因组关联(GWA)研究发现了与肝脏脂肪增加或肝酶升高相关的单核苷酸多态(SNPs)，推测这可能反映了非酒精性脂肪性肝病(NAFLD)。我们在一大批NAFLD患者中发起了一项研究，以调查这些SNPs是否与组织学严重程度有关。在GWA研究中，1117名(894名成人/223名儿童)在NASH临床研究网络和美国国立卫生研究院临床中心的研究中登记了组织学证实的NAFLD的SNPs基因，这些SNPs与肝脏脂肪或肝酶相关。我们证实了PNPLA3基因中rs738409G等位基因与脂肪变性的关联，并首次描述了其与组织学严重程度的关联。在儿科患者中，高危rs738409G等位基因与较早出现疾病相关。我们还描述了迄今为止未知的10号染色体SNPs与NASH纤维化严重程度之间的关联。 同样，在调整了年龄、性别和体重指数后，我们发现羟类固醇(17)脱氢酶13(HSD17B13)、RAR相关孤儿受体(RORA)和蛋白磷酸酶1调节亚基3B(PPP1R3B)基因附近或基因中的SNP与NAFLD的组织学特征有关。RORA是一种参与控制昼夜节律和代谢功能的核受体，其附近的深入基因分型表明，与NAFLD相关的SNPs位于4个剪接变体中的2个(变体2和3)的假定启动子区，而不是其他变体的上游，这表明不同亚型的相对表达的变化影响肝脏中的脂肪积累。 在标准条件下，细胞系中的RORA基因敲除不影响肝脏脂质聚集的程度。然而，当细胞因营养过重(高葡萄糖、高脂肪酸介质)而显著减少时，细胞内脂肪的数量显著减少，主要是通过减少脂滴的平均大小。在脂肪细胞中也发现了类似的影响。我们已经建立了小鼠肝脏特异的RORA基因敲除，并通过它证明了RORA在肝脏脂肪生成中的重要作用。 人们对HSD17B13知之甚少。我们发现它主要在肝脏表达，并与脂滴共定位，但其底物和生理作用尚不清楚。对该基因区域的深入基因分型表明，该基因中的编码和剪接位点SNPs与NAFLD有关，证实了该酶在NASH发病机制中的可能作用。我们确定HSD17B13是维甲酸代谢中的一个关键酶，并确定与NAFLD相关的基因变异对其酶活性也是必不可少的。我们目前正在研究HSD17B13在基因敲除动物模型中的作用。 由于NAFLD与食物摄入和能量代谢有着复杂的关系，我们正在进行临床试验，以评估脂肪肝对营养物质的处理和命运。我们使用BreathID实时呼气测试设备结合一种标记的口服脂肪酸来证明与对照组相比，NAFLD受试者的脂肪酸氧化率降低。同样，我们利用代谢组学的方法来确定NAFLD受试者对标准化膳食挑战的反应。最后，我们目前正在进行一项临床试验，旨在从转录和脂类水平阐明肝脏对口服碳水化合物负荷的反应，并确定对一类新型药物GLP-1受体激动剂的反应的预测规则。

Nonalcoholic fatty liver disease (NAFLD) is marked by accumulation of fat in liver cells with accompanying inflammation and variable degrees of cell injury and fibrosis. When cell injury and fibrosis are present, the disease has a potential to progress and is referred to as nonalcoholic steatohepatitis (NASH), which can lead to cirrhosis, liver cancer, morbidity and mortality. The etiology of NASH is not clear nor is there an approved treatment modality for it. NAFLD has become an extremely common disorder, estimated to affect up to 30% of individuals in the US. Unfortunately, it commonly goes unrecognized, as demonstrated by our recent finding that over a 5-year period, 28% of subjects enrolled as healthy volunteers to studies at the NIH Clinical Center were likely to have underlying NAFLD. As such, there is a clear need to understand the pathophysiology of the disease and its treatment. Our focus on NAFLD is three-fold: first, we aim to identify and refine effective treatments for the disorder. Secondly, we aim to identify and characterize key genes that play a role in the pathogenesis of NAFLD through the use of genetic studies and cell- or animal models. Our third focus is on the physiology of fat accumulation and injury in the liver, especially as it relates to handling of oral caloric load. Vitamin E has been shown in a randomized placebo-controlled trial to be an effective therapy for NASH. Curiously, treatment with vitamin E resulted not only in a decrease in injury (thought to reflect its antioxidant effect) but was also associated with a decrease in liver fat, through an unknown mechanism. Furthermore, the optimal dose of vitamin E to treat NASH is currently unknown. We conducted a clinical mechanistic trial to determine the mechanism of action of vitamin E and its optimal dose and are currently analyzing data from this study and complementary in vitro models to map the pathways affected by vitamin E and whether they are dependent on its antioxidant capacity. Genome wide association (GWA) studies identified single nucleotide polymorphisms (SNPs) that are associated with increased hepatic fat or elevated liver enzymes, presumably reflecting nonalcoholic fatty liver disease (NAFLD). We initiated a study to investigate whether these SNPs are associated with histological severity in a large cohort of NAFLD patients. 1117 (894 adults/223 children) individuals enrolled in NASH-Clinical Research Network and National Institutes of Health Clinical Center studies with histologically-confirmed NAFLD were genotyped for SNPs that are associated with hepatic fat or liver enzymes in GWA studies. We confirmed the association of the rs738409G allele in the PNPLA3 gene with steatosis and were first to describe its association with histological severity. In pediatric patients, the high-risk rs738409G allele was associated with an earlier presentation of disease. We also described a hitherto unknown association between SNPs at a chromosome 10 locus and the severity of NASH fibrosis. Similarly, we demonstrated associations of SNPs near or in the genes for hydroxysteroid (17) dehydrogenase 13 (HSD17B13), RAR-related orphan receptor (RORA) and protein phosphatase 1,regulatory subunit 3B (PPP1R3B) with histological features of NAFLD, after adjustment for age, gender and BMI. In-depth genotyping near RORA, a nuclear receptor involved in control of circadian rhythm and metabolic functions, showed that SNPs that are associated with NAFLD are located in the putative promoter region of 2 of the 4 splice variants (variants 2 and 3) as opposed to SNPs upstream of other variants, suggesting that alternation in the relative expression of the different isoforms affects fat accumulation in the liver. RORA knock-out in cell lines did not affect the degree of hepatic lipid accumulation under standard conditions. However, when cells were overloaded with nutrients (high glucose, high-fatty acids medium), the amount of intracellular fat significantly decreased with knock-down, predominantly through a decrease in the average size of the lipid droplets. A similar effect was seen in adipocytes. We have established a murine liver-specific Rora knock-out and through that demonstrate the important role of RORA on the generation of liver fat. Little is known about HSD17B13. We found it to be predominantly expressed in the liver and to colocalize with lipid droplets, but its substrate and physiological roles are unknown. In-depth genotyping of the gene region demonstrated associations of coding and splice-site SNPs in the gene with NAFLD, confirming a possible role for this enzyme in the pathogenesis of NASH. We identified a role for HSD17B13 as a key enzyme in retinoid metabolism and determined that genetic variants that are associated with NAFLD are also essential to its enzymatic activity. We are currently studying the effects of HSD17B13 in a knock-out animal model. As NAFLD is intricately related to food intake and energy metabolism, we are undertaking clinical trials to evaluate the handling and fate of nutrients by the fatty liver. We used the BreathID real-time breath test device in combination with a labeled orally-delivered fatty acid to demonstrate a decrease in the rate of fatty acid oxidation in subjects with NAFLD compared to controls. Similarly, we utilized a metabolomic approach to identify the response of NAFLD subjects to a standardized meal challenge. Finally, we are currently performing a clinical trial, aimed to elucidate the hepatic response to an oral carbohydrate load at the transcriptomic, lipidomic levels, and to identify prediction rules for response to a novel class of medications, GLP-1 receptor agonists.