Towards a better understanding of the molecular mechanisms regulating liver metabolism in response to fasting

肝脏是调节动物全身新陈代谢的中心器官。作为对禁食的反应，肝细胞启动了几个分解代谢过程，以支持大脑和周围组织的功能。尽管对调节肝脏对禁食的代谢适应的分子机制的理解已经有了很大的提高，但肝细胞如何触发这些分解代谢过程以匹配营养状态仍然不完全。我们假设，调控禁食反应的关键因素仍有待确定。为了识别这些新的调控因子，我们利用了过去成功使用的一种简单方法来识别控制脂肪细胞发育的蛋白质。简单地说，我们使用了肝细胞系(FAO)，并准备了平板，其中每孔一个细胞通过连续稀释分离。从这些平板中，我们扩增了FAO细胞的新克隆培养物，并测量了每个新品系的葡萄糖产量，作为它们在禁食时触发分解代谢的自然潜力的指示器。有趣的是，我们观察到一些克隆系产生低量葡萄糖(Low)，而另一些克隆系产生大量葡萄糖(High)。为了确定导致这种差异反应的基因，我们进行了微阵列分析，比较了Low和High品系。类羟基类固醇脱氢酶2(Hsdl2)是在分解代谢的肝细胞中高表达的基因。HSDL2是一种特性不佳的蛋白质。我们的初步实验表明，禁食可以高度诱导小鼠肝脏Hsdl2的表达。在培养的肝细胞中，撤除模拟禁食状态的血清也增加了Hsdl2的表达。为了证实这些发现，我们观察到胰岛素是调节禁食反应的主要参与者，它强烈抑制了FAO细胞中Hsdl2的表达。综上所述，这些结果表明，HSDL2被禁食激活，因此可能在这一过程中发挥作用。基于我之前在肝脏、新陈代谢、细胞信号和细胞生物学方面的工作和专业知识，这项研究计划旨在提高对控制肝脏对禁食反应的分子机制的理解。这项提案未来5年的目标是：1)确定HSDL2的定位和代谢功能。2)定义控制HSDL2活性的翻译后修饰。3)明确肝脏HSDL2缺失/过表达对体内代谢的影响。4)确定其他调节肝脏新陈代谢的蛋白质以响应禁食。研究控制肝脏代谢的分子机制对禁食的反应将提高我们对在营养缺乏时触发的确保生存的生物过程的理解。除了提高肝脏生理学的基本知识外，该项目还可能揭示控制哺乳动物细胞新陈代谢的一般途径。

The liver is a central organ that regulates systemic metabolism in animals. In response to fasting, hepatocytes turn on several catabolic processes to support the function of the brain and peripheral tissues. Although the comprehension of the molecular mechanisms regulating the metabolic adaptation of the liver to fasting has greatly improved, how exactly hepatocytes trigger these catabolic processes to match the nutritional state is still incompletely characterized. We hypothesize that key players regulating the response to fasting still remain to be identified. In order to identify these new regulators, we took advantage of a simple approach that we successfully used in the past to identify proteins controlling fat cell development. Briefly, we used a hepatocyte cell line (FAO) and prepared plates in which a single cell per well was isolated by serial dilutions. From these plates, we amplified new clonal cultures of FAO cells and measured glucose production of each new line as an indicator of their natural potential to trigger catabolism upon fasting. Interestingly, we observed that some clonal lines produced low amounts of glucose (Low) while others produced a lot (High). In order to identify the genes responsible for this differential response, we performed microarray analyzes to compared Low and High lines. Hydroxysteroid dehydrogenase type-like 2 (Hsdl2), was identified as a gene highly expressed in in catabolic hepatocytes. HSDL2 is a poorly characterized protein. Our preliminary experiments revealed that hepatic Hsdl2 expression is highly induced by fasting in mice. In cultured hepatocytes, withdrawal of the serum, which mimics a fasting state, also increased the expression of Hsdl2. Confirming these findings, we observed that insulin, which is a major player in regulating the fasting response, strongly repressed the expression of Hsdl2 in FAO cells. Together, these results indicate that HSDL2 is activated by fasting and may thus play a role in this process. Building upon my previous work and expertise on the liver, metabolism, cell signaling and cell biology, this research program aims to improve the understanding of the molecular mechanisms controlling the hepatic response to fasting. The objectives of this proposal for the next 5 years are to: 1) Define the localization and the metabolic functions of HSDL2. 2) Define the post-translational modifications controlling the activity of HSDL2. 3) Define the metabolic impacts of hepatic HSDL2 loss/overexpression in vivo. 4) Identify additional proteins regulating liver metabolism in response to fasting. Studying the molecular mechanisms controlling liver metabolism in response to fasting will improve our understanding of the biological processes triggered to ensure survival when nutrients are scarce. In addition to improving the basic knowledge of liver physiology, this project could potentially reveal general pathways controlling metabolism in mammalian cells.