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

更好地了解禁食后调节肝脏代谢的分子机制

• 批准号: RGPIN-2020-07165
• 负责人: Laplante, Mathieu
• 金额: $ 2.4万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760591
• 关键词: Towards; better; understanding; molecular; mechanisms

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.

肝脏是调节动物全身代谢的中心器官。作为对禁食的响应，肝细胞开启几个分解代谢过程以支持脑和外周组织的功能。虽然对调节肝脏代谢适应禁食的分子机制的理解已经有了很大的提高，但肝细胞如何确切地触发这些分解代谢过程以匹配营养状态仍然没有完全表征。我们假设，调节禁食反应的关键参与者仍有待确定。为了鉴定这些新的调节剂，我们利用了一种简单的方法，我们在过去成功地使用这种方法来鉴定控制脂肪细胞发育的蛋白质。简言之，我们使用肝细胞系（FAO）并制备平板，其中通过连续稀释分离每孔单个细胞。从这些平板中，我们扩增了FAO细胞的新克隆培养物，并测量了每个新细胞系的葡萄糖产量，作为其在禁食时触发catastrophic的天然潜力的指标。有趣的是，我们观察到一些克隆系产生少量的葡萄糖（低），而其他克隆系产生大量的葡萄糖（高）。为了鉴定负责这种差异反应的基因，我们进行了微阵列分析以比较低和高品系。羟类固醇脱氢酶样2（Hsdl 2）是一种在肝细胞中高表达的基因。HSDL 2是一种特征性较差的蛋白质。我们的初步实验表明，肝脏Hsdl 2的表达是高度诱导禁食小鼠。在培养的肝细胞中，模拟禁食状态的血清的撤回也增加了Hsdl 2的表达。为了证实这些发现，我们观察到，胰岛素，这是一个主要的球员在调节空腹反应，强烈抑制Hsdl 2在FAO细胞的表达。总之，这些结果表明，HSDPA 2被禁食激活，因此可能在这一过程中发挥作用。 基于我以前在肝脏，代谢，细胞信号传导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。该提案未来5年的目标是：1）定义HSDL 2的定位和代谢功能。2)定义控制HSDL 2活性的翻译后修饰。3)定义体内肝脏HSDL 2丢失/过表达的代谢影响。4)确定其他蛋白质调节肝脏代谢响应禁食。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生物过程的理解，以确保在营养缺乏时的生存。除了提高肝脏生理学的基础知识外，该项目还可能揭示控制哺乳动物细胞代谢的一般途径。