Nonalcoholic Steatohepatitis: Natural History, Pathogenesis and Therapy

非酒精性脂肪性肝病（NAFLD）的特征是脂肪在肝细胞中的积累，伴随炎症和不同程度的细胞损伤和纤维化。当存在细胞损伤和纤维化时，该疾病有可能进展并被称为非酒精性脂肪性肝炎（NASH），其可导致肝硬化、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已成为一种非常常见的疾病，估计在美国影响高达30%的个体。不幸的是，它通常被忽视，正如我们最近的发现所证明的那样，在5年的时间里，在NIH临床中心作为健康志愿者参加研究的受试者中，有28%可能患有潜在的NAFLD。因此，明确需要了解疾病的病理生理学及其治疗。 我们对NAFLD的关注有三个方面：首先，我们的目标是确定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中脂肪积累和损伤的生理学，特别是因为它涉及到口腔热量负荷的处理。 在一项随机安慰剂对照试验中，维生素E已被证明是NASH的有效疗法。奇怪的是，维生素E治疗不仅导致损伤减少（被认为反映了其抗氧化作用），而且还通过未知的机制与肝脏脂肪减少有关。此外，维生素E治疗NASH的最佳剂量目前尚不清楚。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的体外模型，以绘制受维生素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）研究确定了与肝脂肪增加或肝酶升高相关的单核苷酸多态性（SNP），可能反映了非酒精性脂肪性肝病（NAFLD）。我们启动了一项研究，以调查这些SNPs是否与NAFLD患者的组织学严重程度相关。在NASH临床研究网络和美国国立卫生研究院临床中心研究中招募的1117名（894名成人/223名儿童）患有组织学证实的NAFLD的个体在GWA研究中对与肝脂肪或肝酶相关的SNP进行基因分型。我们证实了PNPLA 3基因中rs738409 G等位基因与脂肪变性的相关性，并首次描述了其与组织学严重程度的相关性。在儿科患者中，高危rs738409 G等位基因与疾病的早期表现相关。我们还描述了10号染色体基因座的SNP与NASH纤维化严重程度之间的未知关联。 同样，我们证明了在调整年龄、性别和BMI后，羟基类固醇（17）脱氢酶13（HSD 17 B13）、RAR相关孤儿受体（RORA）和蛋白磷酸酶1、调节亚基3B（PPP 1 R3 B）基因附近或基因中的SNP与NAFLD的组织学特征的相关性。RORA（一种参与控制昼夜节律和代谢功能的核受体）附近的深入基因分型表明，与NAFLD相关的SNP位于4种剪接变体中的2种（变体2和3）的推定启动子区，而不是上游的SNP其他变体，这表明不同亚型相对表达的改变影响肝脏中的脂肪积累。 在标准条件下，细胞系中RORA敲除不影响肝脏脂质蓄积的程度。然而，当细胞超载营养物质（高葡萄糖，高脂肪酸培养基），细胞内脂肪的量显着减少敲低，主要是通过减少的平均大小的脂滴。在脂肪细胞中观察到类似的效果。我们已经建立了小鼠肝脏特异性Rora敲除，并通过该敲除证明了RORA对肝脏脂肪生成的重要作用。 关于HSD 17 B13，我们知之甚少。我们发现它主要在肝脏中表达，并与脂滴共定位，但其底物和生理作用尚不清楚。基因区域的深入基因分型证明了基因中编码和剪接位点SNP与NAFLD的相关性，证实了这种酶在NASH发病机制中的可能作用。我们确定了HSD 17 B13作为类维生素A代谢中的关键酶的作用，并确定了与NAFLD相关的遗传变异对其酶活性也至关重要。我们目前正在研究HSD 17 B13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密切相关，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实时呼吸测试设备结合标记的口服脂肪酸，以证明与对照组相比，NAFLD受试者的脂肪酸氧化速率降低。同样，我们利用代谢组学方法来确定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，旨在阐明肝脏在转录组学、脂质组学水平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.