Hepatocyte PPARgamma regulated mechanisms in NAFLD and lipid homeostasis

摘要-非酒精性脂肪肝（NAFLD）包括非酒精性脂肪肝（NAFL/脂肪变性） 非酒精性脂肪性肝炎（NASH）虽然单纯性脂肪变性可能是“良性”的，但它是一种独立的风险 NASH发展的因素。噻唑烷二酮类（TZD）是用于减少脂肪变性的PPARγ激动剂， NASH糖尿病患者然而，TZD对脂肪变性的影响是有限的，没有任何纤维化的解决。 TZD的抗脂肪变性作用可能是由于在人类和小鼠模型中降低了胰岛素抵抗。 然而，TZD介导的肝细胞PPARγ活化可能抵消TZD的抗脂肪生成作用 并防止纤维化的任何减少。这是基于以下观察结果：1）肝脏PPARγ 显著增加NAFLD; 2）TZD以肝细胞PPARγ依赖性方式加重脂肪变性， 3）临床上TZD的抗脂肪生成作用仅限于降低胰岛素抵抗。到 迄今为止，肝细胞PPARγ调节的脂质代谢对NAFLD的影响知之甚少。因此 我们已经用腺相关病毒（Adeno-associated viral，ADV）敲低了成年小鼠肝细胞中的PPARγ（>99.5%）。 仅在肝细胞中表达Cre重组酶载体（AAV 8-TBGp-Cre）。肝细胞PPARγ缺失 减少饮食诱导的脂肪变性，因此在SA#1研究中，建议确定 肝细胞PPARγ可预防饮食诱导的NAFLD。我们假设Cd 36介导的FA的减少 在肝细胞PPARγ缺失后观察到的摄取和/或Mogat 1介导的FA再酯化对于 减少脂肪变性和随后的NASH发展。为了验证这一假设，成年PPARγfl/fl小鼠 将被喂食已知诱导脂肪变性的高脂肪、高胆固醇、高蔗糖饮食（HF-HC-HSD）（8周， 饮食）和NASH（27周饮食）。肝细胞PPARγ将被敲低，而无或有恢复。 Cd 36（FA易位酶）或Mogat 1（单酰基甘油酰基转移酶）。肝脏FA摄取，MOGAT活性，FA 三酰基甘油（TAG）、二酰基甘油（DAG）和单酰基甘油（MAG）的组成]，基因表达 并评估肝脏和全身代谢病理生理学。虽然肝细胞PPARγ的丢失 减少脂肪变性，它导致餐后血脂异常。因此，在SA#2研究中，建议确定 肝细胞PPARγ缺失后促进餐后血脂异常的机制。我们 假设与肝细胞PPARγ特异性缺失相关的血脂异常是由于肠 通过给小鼠喂食含有不可吸收脂肪的饮食或口服给药后测试脂肪吸收 放射性标记的TAG在泰洛沙泊的存在下。我们还假设组织特异性TAG的变化 在肝细胞PPARγ丧失后摄取受损，将通过评估肝脏、心脏、肌肉和 通过口服管饲法或静脉内递送的放射性标记的TAG的脂肪组织摄取（以排除肠内代谢的变化） 摄取）。这一项目的成果将在实地产生影响，因为这些成果将确定以下方面的作用： 肝细胞PPARγ及其下游机制：1）饮食诱导的脂肪变性的发展， NASH; 2）NASH中代谢功能的失调; 3）肝脏脂质组成对NASH的贡献。 胰岛素敏感性和肝脏疾病进展;和4）通过控制脂质稳态来调节脂质稳态 肠脂质吸收和/或组织特异性餐后TAG清除。这些结果可能导致 开发阻断肝细胞PPARγ的前列腺脂肪变性作用的药物，可单独使用或与 与其他疗法联合使用以预防和逆转NASH。

Abstract – Non-alcoholic fatty liver disease (NAFLD) encompasses non-alcoholic fatty liver (NAFL/steatosis) to non-alcoholic steatohepatitis (NASH). Although simple steatosis can be “benign”, it is an independent risk factor for NASH development. Thiazolidinediones (TZD) are PPARγ agonists used to reduce steatosis in diabetics with NASH. However, the impact of TZD on steatosis is limited, without any resolution of fibrosis. The antisteatotic actions of TZD may be due to reduced insulin resistance in both humans and mouse models. However, TZD-mediated activation of hepatocyte PPARγ may offset the putative antisteatogenic effects of TZD and prevent any reduction in fibrosis. This is based on the following observations: 1) Hepatic PPARγ dramatically increases in NAFLD; 2) TZD exacerbate steatosis in a hepatocyte PPARγ-dependent fashion in mice; and 3) Clinically, the antisteatogenic effect of TZD is limited to the reduction in insulin resistance. To date, the impact of hepatocyte PPARγ-regulated lipid metabolism on NAFLD is poorly understood. Therefore we have knocked-down hepatocyte PPARγ (>99.5%) in adult PPARγfl/fl mice using an adeno-associated viral vector that express Cre recombinase only in hepatocytes (AAV8-TBGp-Cre). Loss of hepatocyte PPARγ reduced diet-induced steatosis, therefore in SA#1 studies are proposed to determine how loss of hepatocyte PPARγ prevents diet-induced NAFLD. We hypothesize that the reduction in Cd36-mediated FA uptake and/or Mogat1-mediated FA re-esterification, observed after loss of hepatocyte PPARγ, are critical for the reduction in steatosis and subsequent development of NASH. To test this hypothesis, adult PPARγfl/fl mice will be fed a high fat, high cholesterol, high sucrose diet (HF-HC-HSD) known to induce steatosis (8 weeks of diet) and NASH (27 weeks of diet). Hepatocyte PPARγ will be knocked down, without or with restoration of Cd36 (FA translocase) or Mogat1 (Monoacylglycerol acyltransferase). Hepatic FA uptake, MOGAT activity, FA composition of triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG)], gene expression and liver and systemic metabolic pathophysiology will be assessed. Although loss of hepatocyte PPARγ reduced steatosis, it led to postprandial dyslipidemia. Therefore in SA#2 studies are proposed to determine the mechanism(s) that promotes postprandial dyslipidemia after loss of hepatocyte PPARγ. We hypothesize that dyslipidemia associated with specific loss of hepatocyte PPARγ is due to enhanced intestinal fat absorption which will be tested by feeding mice a diet containing a nonabsorbable fat or after oral delivery of radio-labeled TAG in the presence of tyloxapol. We also hypothesize that changes in tissue-specific TAG uptake is impaired after loss of hepatocyte PPARγ, which will be tested by assessing liver, heart, muscle and adipose tissue uptake of radio-labeled TAG delivered by oral gavage or iv (to factor out changes in intestinal uptake). The results derived from this project will have an impact in the field because they will define the role of hepatocyte PPARγ and its downstream mechanisms in: 1) the development of diet-induced steatosis and NASH; 2) the dysregulation of metabolic function in NASH; 3) the contribution to hepatic lipid composition to insulin sensitivity and liver disease progression; and 4) the regulation of lipid homeostasis by controlling intestinal lipid absorption and/or tissue-specific postprandial TAG clearance. These results could lead to the development of drugs that block prosteatotic actions of hepatocyte PPARγ that could be used alone or in combination with other therapies to prevent and reverse NASH.