Hepatocyte PPARgamma regulated mechanisms in NAFLD and lipid homeostasis

摘要-非酒精性脂肪性肝病(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)。尽管单纯性脂肪变性可能是“良性的”，但它是一种独立的风险。 纳什发展的因素。噻唑烷二酮类(TZD)是一种PPARγ激动剂，用于减少 糖尿病患者与纳什。然而，TZD对脂肪变性的影响是有限的，没有任何缓解纤维化的作用。 TZD的抗脂肪变性作用可能是由于降低了人类和小鼠模型的胰岛素抵抗。 然而，TZD介导的肝细胞PPARγ的激活可能抵消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 显著增加的非酒精性脂肪肝；2)TZD加剧肝细胞PPARγ依赖的脂肪变性 3)临床上，TZD的抗脂肪生成作用仅限于减轻胰岛素抵抗。至 目前，肝细胞PPARγ调节的脂代谢对非酒精性脂肪肝的影响还知之甚少。因此 我们使用腺相关病毒在成年PPARγfl/fl小鼠体内击倒了肝细胞PPARγ(&gt；99.5%)。 仅在肝细胞中表达Cre重组酶的载体(AAV8-TBGp-Cre)。肝细胞PPARγ缺失 减少饮食诱导的脂肪变性，因此在SA#1研究中建议确定如何减少 肝细胞PPARγ可预防饮食诱导的非酒精性脂肪肝。我们假设CD36介导的FA的减少 在肝细胞PPARγ缺失后观察到的摄取和/或Mogat1介导的FA再酯化是 脂肪变性的减少和NASH的后续发展。为了验证这一假设，成年pparγfl/fl小鼠 会被喂以高脂肪、高胆固醇、高蔗糖的饮食(HF-HC-HSD)，已知会导致脂肪变性(8周 饮食)和纳什(27周饮食)。肝细胞PPARγ将被击倒，无论是否恢复 CD36(脂肪酸转位酶)或Mogat1(单酰甘油酰基转移酶)。肝脏摄取FA、MOGAT活性、FA 三酰基甘油(TAG)、二酰基甘油(DAG)和单甘油酯(MAG)的组成]，基因表达 并对肝脏和全身代谢病理生理学进行评估。尽管肝细胞PPARγ的丢失 脂肪变性减少，会导致餐后血脂紊乱。因此，在SA#2中，建议进行研究以确定 肝细胞PPARγ缺失后促进餐后血脂异常的机制(S)。我们 假设与肝细胞PPARγ特异性丢失相关的血脂异常是由肠道增强引起的 脂肪吸收将通过给小鼠喂食含有不可吸收脂肪的食物或口服后进行测试 在泰洛沙普存在下的放射性标记标签。我们还假设组织特异性标签的变化 肝细胞PPARγ丢失后摄取受损，这将通过评估肝脏、心脏、肌肉和 口服或静脉注射放射性标记TAG的脂肪组织摄取(排除肠道变化 吸收)。这个项目的结果将在实地产生影响，因为它们将定义 肝细胞PPARγ及其下游机制：1)饮食诱导的脂肪变性和 NASH；2)NASH代谢功能失调；3)对肝脏脂质成分的贡献 胰岛素敏感性与肝病进展；4)通过控制 肠道脂肪吸收和/或组织特定的餐后标签清除。这些结果可能会导致 阻断肝细胞PPARγ促变作用的药物的开发 联合其他疗法预防和逆转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.