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：首先，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发病机制中发挥作用的关键基因，并确定它们作为治疗靶点的适用性。其次，我们的目标是了解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。第三，我们希望确定和完善针对这种疾病的有效治疗方法。 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。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。 同样，我们发现羟基类固醇(17)脱氢酶13(HSD17B13)基因附近或基因中的几个SNP与NAFLD的组织学特征有关。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，证实了该蛋白在NASH发病机制中的可能作用。HSD17B13是一种酶，我们发现它主要在肝脏中表达，并与脂滴共存。我们发现HSD17B13参与类维A酸代谢，导致其酶功能丧失的基因变异与NAFLD严重程度的降低有关。我们进一步表征了蛋白质的结构和其中对其功能至关重要的结构域。这些发现引起了制药业的极大兴趣，几种HSD17B13灭活剂正在人体中作为治疗NASH的潜在疗法进行研究。 尽管HSD17B13具有良好的遗传关联和药理作用，但HSD17B13在体内的实际生理作用以及其失活导致对NASH相关损伤的保护机制仍不清楚。为了加深对HSD17B13基因作用的理解，我们建立了Hsd17B13基因敲除小鼠模型，目前正在多种条件下对其进行研究，旨在复制人类的表型。我们的小鼠和细胞模型使我们能够准确地确定HSD17B13丢失保护肝脏免受损伤的机制，以及它与其他代谢特征的关联。 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标记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。我们最近研究了摄入液体混合餐后的非酒精性脂肪肝受试者。我们在多个时间点采集了血浆样本，并利用脂质组学方法和平行的小鼠模型来确定餐后肝二酰甘油(DAG)包装为极低密度脂蛋白(VLDL)颗粒的增加，这是NAFLD所独有的。最后，我们目前正在进行另一项研究，在口服碳水化合物负荷前后采集NAFLD受试者的肝脏样本，旨在阐明肝脏转录和脂体反应以及对肝脏内胰岛素信号的影响。 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。我们结合了临床机制试验和体外实验来确定维生素E的作用机制。我们确定了氧化应激(NAFLD的共同特征)通过上调肝脏新生脂肪生成来增加肝脏脂肪的关键细胞途径，并证实了维生素E通过其抗氧化活性阻止了这一途径的激活。我们能够建立一种自动化的方法来量化肝脏4-羟基壬烯醛(4-HNE)加合物，这是氧化应激诱导损伤的标志，并通过维生素E试验的样本证实了它的适用性。 在目前的一项试验中，患有NAFLD的受试者接受Semagluide治疗，Semagluide是一种胰升糖素样肽1(GLP-1)受体激动剂，先前已证明对NASH有效。本研究旨在阐明赛马路德治疗NASH的作用机制及其对肝脏脂质体的影响，并找出临床疗效的早期预测因素。

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 characterize key genes that play a role in the pathogenesis of NAFLD through the use of genetic studies and cell- or animal models and identify their suitability as therapeutic target. Second, we aim to understand the physiology of fat accumulation and injury in the liver, especially as it relates to handling of oral caloric load. Third, we hope to identify and refine effective treatments for the disorder. 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. 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. Similarly, we discovered an association of several SNPs near or in the gene for hydroxysteroid (17) dehydrogenase 13 (HSD17B13) with histological features of NAFLD. 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 the protein in the pathogenesis of NASH. HSD17B13 is an enzyme that we found to be predominantly expressed in the liver and to colocalize with lipid droplets. We identified that HSD17B13 is involved in retinoid metabolism and that genetic variants that lead to loss of its enzymatic function are genetically associated with decreased severity of NAFLD. We further characterized the structure of the protein and the domains in it that are key to its function. These findings generated remarkable interest in the pharmaceutical industry and several HSD17B13 inactivating agents are being studied in human subjects as potential therapies for NASH. Despite the promising genetic association and pharma interest, the actual physiological role of HSD17B13 in vivo and the mechanism by which its inactivation leads to protection from NASH-associated injury are still unknown. To improve our understanding of HSD17B13s role, we established an Hsd17b13 knock-out mouse model and are currently studying it under multiple condition, aiming to replicate the human phenotype. Our mouse and cell models allow us to pinpoint the mechanism by which loss of HSD17B13 protects the liver from injury, as well as its association with other metabolic features. 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 fatty acid to demonstrate a decrease in the rate of fatty acid oxidation in subjects with NAFLD compared to controls. We recently studied NAFLD subjects after ingestion of a liquid mixed meal. We collected plasma samples at multiple time points and utilized a lipidomic approach and parallel mouse models to identify an increase in packaging of hepatic diacylglycerols (DAG) into very low density lipprotein (VLDL) particles after a meal, that is unique to NAFLD. Finally, we are currently performing an additional study, where liver samples from subjects with NAFLD are obtained before and after an oral carbohydrate load aimed to elucidate the hepatic transcriptomic and lipidomic response as well as effects on intra-hepatic insulin signaling. Vitamin E has been shown in a randomized placebo-controlled trials 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. We combined a clinical mechanistic trial with in vitro experiments to determine the mechanism of action of vitamin E. We identified a key cellular pathway by which oxidative stress (a common feature of NAFLD) increases liver fat through upregulation of hepatic de novo lipogenesis and confirmed that vitamin E blocks the activation of this pathway through its antioxidant activity. We were able to establish an automated method to quantify hepatic 4-hydroxynonenal (4-HNE) adducts, a marker of oxidative stress-induced damage, and confirmed its applicability with samples from the vitamin E trial. In a current trial, subjects with NAFLD are treated with semaglutide, a glucagon like peptide 1 (GLP-1) receptor agonist with effectiveness against NASH previously shown. This study aims to elucidate the mechanism of action of semaglutide in treating NASH, its effects on the liver lipidome and identify early predictors of clinical response.