Nonalcoholic Steatohepatitis: Natural History, Pathogenesis and Therapy

非酒精性脂肪性肝炎：自然史、发病机制和治疗

• 批准号: 10250247
• 负责人: Yaron Rotman
• 金额: $ 128.56万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10250247
• 关键词: 4 hydroxynonenal; Adipocytes; Adult; Affect; Agonist; Alcoholic Liver Diseases; Alleles; Animal Model; Antioxidants; Breath Tests; Carbohydrates; Cell Line; Cell model; Cells; Circadian Rhythms; Cirrhosis; Clinical; Clinical Trials; Code; Collaborations; Devices; Diet; Disease; Drug Industry; Eating; Energy Metabolism; Enrollment; Enzymes; Etiology; Fatty Acids; Fatty Liver; Fatty acid glycerol esters; Fibrosis; Food Energy; Functional disorder; GLP-I receptor; Generations; Genes; Genetic study; Genotype; Glucose; Hepatic; Hepatocyte; Histologic; Histology; Hydroxysteroids; In Vitro; Individual; Inflammation; Injury; Knock-out; Knockout Mice; Label; Lead; Light; Lipids; Liver; Liver diseases; Malignant neoplasm of liver; Metabolic; Metabolism; Methods; Modality; Modeling; Morbidity - disease rate; Mus; Natural History; Nuclear Receptors; Nutrient; Oral; Orphan; Oxidative Stress; Oxidoreductase; Pathogenesis; Pathway interactions; Patients; Physiological; Physiology; Play; Population; Promoter Regions; Protein Isoforms; Proteins; RNA Splicing; Retinoids; Role; Sampling; Severities; Single Nucleotide Polymorphism; Site; Standardization; Study models; Testing; Time; United States; United States National Institutes of Health; Up-Regulation; Variant; Vitamin E; adduct; cell injury; clinical center; cohort; effective therapy; experimental study; fatty acid oxidation; genetic analysis; genetic association; genetic variant; genome wide association study; healthy volunteer; high risk; improved; in vivo; interest; knock-down; lipid biosynthesis; liver transplantation; metabolomics; mortality; mouse model; new therapeutic target; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel therapeutics; pediatric patients; randomized placebo controlled trial; receptor; response; therapeutic target; transcriptomics

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 identify and refine effective treatments for the disorder. 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. 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. These findings generated remarkable interest in the pharmaceutical industry and several HSD17B13 inactivating agents are being explored 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 tested it under several dietary conditions mimicking NAFL, NASH and alcoholic liver disease. Curiously, we did not identify protection from injury for any of those models, suggesting that inter-species differences may exist. We are currently exploring those differences to further shed light on the potential therapeutic targeting of HSD17B13. In a similar genetic analysis, we identified associations of SNPs near RAR-related orphan receptor (RORA) with NAFLD histology. 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. 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. In collaboration with colleagues at NCI, 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. 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. 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 Semaglutied, a GLP-1 receptor agonist.

非酒精性脂肪肝病（NAFLD）的标志是肝细胞中脂肪的积累，伴有炎症和细胞损伤和纤维化的变化程度。当存在细胞损伤和纤维化时，该疾病可能会进展，并被称为非酒精性脂肪性肝炎（NASH），这可能导致肝硬化，肝癌，发病率和死亡率。纳什的病因尚不清楚，也没有批准的治疗方式。 NAFLD已成为一种极为普遍的疾病，估计会影响美国30％的人。不幸的是，我们最近的发现表明，在5年的时间里，有28％的受试者成为NIH临床中心研究的健康志愿者可能有28％的受试者可能在NAFLD的基础。因此，显然需要了解该疾病的病理生理及其治疗。 我们对NAFLD的关注是三个方面：首先，我们旨在通过使用遗传研究和细胞或动物模型来识别和表征在NAFLD在NAFLD发挥作用的关键基因，并确定其适合性作为治疗靶点。其次，我们旨在识别和完善对该疾病的有效治疗方法。我们的第三个重点是肝脏脂肪积累和损伤的生理，尤其是与口服热量负荷处理有关的生理。 基因组广泛的缔合（GWA）研究确定了与肝脂肪升高或肝脏升高有关的单核苷酸多态性（SNP），大概反映了非酒精性脂肪肝病（NAFLD）。我们开始了一项研究，以研究这些SNP是否与大量NAFLD患者队列中的组织学严重程度相关。我们证实了PNPLA3基因中rs738409g等位基因与脂肪变性的关联，并首先描述了其与组织学严重程度的关联。在小儿患者中，高危卢比的等位基因与较早的疾病相关。 同样，我们发现了羟基固醇附近或基因中的几个SNP（17）脱氢酶13（HSD17B13）与NAFLD的组织学特征的关联。基因区域的深入基因分型表明，基因中的编码和剪接位点SNP与NAFLD的关联，证实了该蛋白质在NASH发病机理中的可能作用。 HSD17B13是一种酶，我们发现它主要在肝脏中表达并与脂质液滴共定位。我们确定HSD17B13参与性类维生素性代谢，并且导致其酶促功能丧失的遗传变异与NAFLD的严重程度降低相关。这些发现对制药行业产生了巨大的兴趣，正在探索几种HSD17B13灭活剂作为NASH的潜在疗法。 尽管有希望的遗传关联和药物兴趣，但HSD17B13在体内的实际生理作用以及其失活导致保护NASH相关损伤的机制仍然未知。为了提高我们对HSD17B13S角色的理解，我们建立了HSD17B13敲除小鼠模型，并在模仿NAFL，NASH和酒精性肝病的几种饮食条件下对其进行了测试。奇怪的是，我们没有确定任何模型中的伤害保护，这表明可能存在种间差异。我们目前正在探索这些差异，以进一步阐明HSD17B13的潜在治疗靶向。 在类似的遗传分析中，我们确定了与RAR相关的孤儿受体（RORA）与NAFLD组织学附近的SNP的关联。 RORA附近的深度基因分型，RORA是一种参与控制昼夜节律和代谢功能的核受体，表明与NAFLD相关的SNP位于4种剪接变体的2个启动子的区域中，与其他变体的SNP相反，在相对表达中，依赖于这些不同的表达的依次是不同的，这是相对表达的。在标准条件下，细胞系中的RORA敲除不会影响肝脂质积累的程度。但是，当细胞用养分过载（高葡萄糖，高脂肪酸培养基）时，细胞内脂肪的量会随着敲低而显着降低，主要是通过脂质液滴的平均大小降低。在脂肪细胞中也有类似的作用。我们已经建立了一个鼠肝特异性的Rora敲除，这证明了Rora在产生肝脏脂肪中的重要作用。 维生素E已在一项随机安慰剂对照试验中显示为NASH的有效疗法。奇怪的是，维生素E的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。我们将一项临床机械试验与体外实验结合在一起，以确定维生素E的作用机理。我们确定了一种关键的细胞途径，通过该途径，氧化应激（NAFLD的一个共同特征）通过上调从头开始脂肪生成而增加肝脏脂肪，并确认维生素E通过其抗氧化剂的活性阻止了这种途径的激活。与NCI的同事合作，我们能够建立一种自动化方法，以量化肝4-羟基烯烯（4-HNE）加合物，这是氧化应激诱导的损害的标志，并确认了其与维生素E试验样品的适用性。 由于NAFLD与食物摄入和能量代谢相关，因此我们正在进行临床试验，以评估脂肪肝脏的营养的处理和命运。与对照组相比，我们使用了呼气实时呼气测试装置与标记的脂肪酸结合使用，以证明NAFLD受试者的脂肪酸氧化速率降低。同样，我们利用一种代谢组学方法来确定NAFLD受试者对标准化餐的挑战的反应。最后，我们目前正在进行一项临床试验，旨在阐明在转录组，脂质组水平上对口服碳水化合物负荷的肝反应，并确定对GLP-1受体激动剂的响应的预测规则。