Balance between HNF4a isoforms in the carbohydrate-lipid metabolic switch

摘要 肝细胞核因子4α(HNF4α)是肝脏特异性基因表达的主要调节因子，受 两个启动子(P1和P2)，它们驱动被称为HNF4α1的两组HNF4α亚型的表达 和HNF4α7。HNF4α是一种已知的糖异生调节因子，在成熟期起病的糖尿病中发生突变。 年轻人(MODY1)。传统上，人们认为HNF4α1，而不是HNF4α7在正常组织中表达 在成人肝脏中，HNF4α1表达下调，HNF4α7表达上调。现在，我们实验室的研究 揭示了HNF4α7在正常成年小鼠肝脏中的一个以前未被描述的作用-参与昼夜节律的一个 脂肪和碳水化合物代谢的变化。更具体地说，HNF4α1似乎是 糖异生作用而HNF4α7是酮体发生的驱动因素：我们认为HNF4α水平的改变 白天的同工异构体在两者之间翻转分子开关。我们的初步数据还显示，HNF4α7 是雌性小鼠循环酮体水平升高所必需的。AMP激活的蛋白激酶 AMPK是一种能量敏感酶，已被证明在体外可以磷酸化HNF4α1，但在体内和 在HNF4上，α7是未知的。SIRT1是一种脱乙酰酶，与AMPK一起调节血糖和血脂 新陈代谢。HNF4α1已知是乙酰化的，我们的初步数据表明HNF4α7而不是HNF4α1 与SIRT1交互。在这里，我们建议使用HNF4α1表达(α1HMZ)和HNF4α7表达外显子 交换小鼠(α7HMZ)以确定HNF4α亚型在两者之间的切换的生理功能 糖异生和酮生，并表征性别对这些功能的影响。在目标1中，我们将 确定间歇性禁食和生酮饮食是否会增加肝脏中HNF4α7的水平，以及 这种增加是发生在所有肝细胞中，还是只发生在一小部分肝细胞中。我们将确定HNF4α7的后果 对基因表达的影响。肾和肠也将被探索。在目标2中，我们将确定AMPK是否 途径以不同的方式作用于HNF4HNF4α亚型，帮助翻转代谢开关。磷酸化 将探索AMPK和SIRT1的脱乙酰基作用。最后，在目标3中，我们将确定雌激素是否 途径影响雌性小鼠的HNF4HNF4α亚型，并决定代谢转换的后果。 我们令人信服的初步数据表明，HNF4α亚型参与了 糖异生和酮生为这一日常发生的基本代谢过程提供了新的线索。 在进食和禁食的条件下。这一提议的结果不仅将阐明分子 这种转换背后的机制，以及性别是如何影响这种机制的。拟议的研究包括 可能影响我们对许多代谢性疾病的理解，包括糖尿病、肥胖症、脂肪肝 疾病和癌症。最后，鉴于酮体是大脑的燃料来源，我们的结果 可能会产生更广泛的影响，包括对痴呆症等神经疾病的影响。

Abstract Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of liver-specific gene expression, is regulated by two promoters (P1 and P2) which drive expression of two groups of HNF4α isoforms referred to as HNF4α1 and HNF4α7. HNF4α is a known regulator of gluconeogenesis and mutated in maturity onset diabetes of the young one (MODY1). Conventionally, it was thought that HNF4α1, but not HNF4α7, is expressed in the normal adult liver, while HNF4α1 is downregulated and HNF4α7 is upregulated in liver cancer. Now, research in our lab reveals a previously undescribed role for HNF4α7 in the normal adult mouse liver – one involved in the diurnal variations of lipid and carbohydrate metabolism. More specifically, HNF4α1 appears to be a major driver of gluconeogenesis while HNF4α7 is a driver of ketogenesis: we propose that alterations in the levels of the HNF4α isoforms during the day flip the molecular switch between the two. Our preliminary data also show that HNF4α7 is required for increased levels of circulating ketone bodies in female mice. AMP-Activated Protein Kinase (AMPK), an energy-sensing enzyme, has been shown to phosphorylate HNF4α1 in vitro, but effects in vivo and on HNF4α7 are not known. SIRT1 is a deacetylase that works with AMPK to regulate glucose and lipid metabolism. HNF4α1 is known to be acetylated and our preliminary data suggest that HNF4α7 but not HNF4α1 interacts with SIRT1. Here, we propose to use HNF4α1-expressing (α1HMZ) and HNF4α7-expressing exon swap mice (α7HMZ) to determine the physiological function of the HNF4α isoforms in the switch between gluconeogenesis and ketogenesis, and to characterize the impact of sex on those functions. In Aim 1, we will determine whether intermittent fasting and a ketogenic diet increase the levels of HNF4α7 in the liver, and whether the increase occurs in all hepatocytes, or just a subset. We will determine the consequences of HNF4α7 on gene expression. Kidney and intestines will also be explored. In Aim 2, we will determine whether the AMPK pathway acts in a differential fashion on the HNF4α isoforms to help flip the metabolic switch. Phosphorylation by AMPK and deacetylation by SIRT1 will be explored. Finally, in Aim 3, we will determine whether the estrogen pathway impacts the HNF4α isoforms in female mice and determine the consequences for the metabolic switch. Our compelling preliminary data that the HNF4α isoforms are involved in the switch between gluconeogenesis and ketogenesis shed new light on this basic metabolic process that occurs on a daily basis and under conditions of feeding and fasting. The results from this proposal will illuminate not only the molecular mechanism underlying the switch but also how that mechanism is impacted by sex. The proposed studies have the potential to impact our understanding of numerous metabolic diseases, including diabetes, obesity, fatty liver disease and cancer. Finally, given the fact that ketone bodies serve as a source of fuel for the brain, our results could have a broader impact, including on neurological diseases, such as dementia.