The metabolic basis for impaired bile acid synthesis in malnutrition

营养不良造成全球一半的儿童死亡。严重的营养不良会干扰肝脏的合成 白蛋白、补体和凝血因子以及胆汁酸（BA）。BA缺乏损害养分吸收 和生长，并通过包括法尼醇-X-受体（FXR）在内的核受体改变信号传导， 过程的范围。我们使用出生后早期营养不良的小鼠模型， 营养不良导致FXR活化减少和FXR靶基因表达减少 包括凝血因子。由此产生的营养不良引起的凝血功能障碍可导致儿童死亡。是 尚不清楚营养不良时BA合成受损的原因。我们公布的数据和新的初步数据 表明营养不良损害了经典代谢酶（CYP 7A 1）的活性（而不是表达）。 由于必需辅因子血红素的消耗，导致BA合成途径。我们提出了新的证据， 恢复血红素增加BA的合成。我们现在试图了解为什么血红素合成在体内受损 营养不良血红素是由三羧酸（TCA）循环产物和需要铁的酶产生的。 硫（Fe-S）簇用于稳定性。Fe-S簇合物来源于含Fe和S的氨基酸 (AAs)，其存在于膳食蛋白质中或通过转硫作用产生。像大多数低蛋白饮食一样，我们的老鼠 营养不良的饮食缺乏含硫的氨基酸。小鼠表现出转硫作用的表达减少， 和Fe-S簇依赖的TCA循环酶，表明含硫的AA和Fe-S簇依赖的TCA循环酶缺乏， 集群导致营养不良的肝功能障碍。事实上，我们通过维持BA合成减少了50%， 肝细胞在低AA培养基中，我们恢复BA生产，通过添加含S的AA（但不是其他AA）。 因此，我们推测，营养不良时含硫氨基酸的缺乏通过干扰BA的合成而损害BA的合成。 TCA循环功能、血红素生物合成和CYP 7A 1活性。我们的具体目标是：1）在AA中表征- 通过定量标记葡萄糖通过TCA循环的流量， 测量血红素化前后Fe-S簇依赖性TCA循环酶的表达水平和活性 2）通过测定BA合成，确定含S AA和血红素在BA合成中的作用， 肝细胞维持在低AA培养基中，并以高通量方式用AA和 血红素和使用腺相关病毒或siRNA来操纵转硫作用和Fe-S簇的水平。 依赖酶;和3）确定新的营养治疗剂是否可以拯救BA合成， 营养不良的小鼠喂养营养不良的饮食补充含硫的氨基酸，然后定量TCA 原代肝细胞中的循环功能、血红素合成和BA产生。预期成果包括 阐明了营养不良、TCA循环和血红素稳态与BA合成之间的新联系。 这项研究意义重大，因为它有可能开发一种可扩展的基于AA的治疗方法来恢复肝脏 综合功能和治疗儿童营养不良。

Malnutrition contributes to half of all global child deaths. Severe malnutrition interferes with the liver’s synthesis of albumin, complement and coagulation factors, and bile acids (BAs). BA deficiency impairs nutrient absorption and growth and alters signaling through nuclear receptors including farnesoid-X-receptor (FXR) to impact a wide range of processes. Using a mouse model of early postnatal malnutrition, we reported that decreased BA synthesis in malnutrition causes decreased FXR activation and decreased expression of FXR target genes including coagulation factors. The resulting malnutrition-induced coagulopathy can cause child mortality. It is not known why BA synthesis is impaired in malnutrition. Our published data and new preliminary data indicate malnutrition impairs the activity (not expression) of the rate-determining enzyme (CYP7A1) in the classic pathway of BA synthesis due to depletion of the essential cofactor heme. We present novel evidence that restoring heme increases BA synthesis. We now seek to understand why heme synthesis is impaired in malnutrition. Heme is generated from tricarboxylic acid (TCA) cycle products and enzymes that require iron- sulfur (Fe-S) clusters for stability. Fe-S clusters are derived from both Fe and S-containing amino acids (AAs), which are found in dietary protein or generated by transsulfuration. Like most low-protein diets, our mouse malnourishing diet is deficient in S-containing AAs. The mice exhibit decreased expression of transsulfuration and Fe-S cluster-dependent TCA cycle enzymes, suggesting that deficiency of S-containing AAs and Fe-S clusters drives liver dysfunction in malnutrition. Indeed, we decreased BA synthesis by 50% by maintaining hepatocytes in low-AA media, and we restored BA production by adding S-containing AAs (but not other AAs). Thus, we hypothesize that deficiency of S-containing AAs in malnutrition impairs BA synthesis by disrupting TCA cycle function, heme biosynthesis, and CYP7A1 activity. Our Specific Aims are to 1) Characterize in AA- deficient hepatocytes TCA cycle dysfunction by quantifying flux of labeled glucose through the TCA cycle and measuring expression levels and activity of Fe-S cluster-dependent TCA cycle enzymes before and after heme treatment; 2) Define the role of S-containing AAs and heme in BA synthesis by measuring BA synthesis by hepatocytes maintained in low-AA media and treated in a high-throughput manner with combinations of AAs and heme and using adeno-associated virus or siRNA to manipulate levels of transsulfuration and Fe-S cluster- dependent enzymes; and 3) Determine whether a novel nutritional therapeutic can rescue BA synthesis in malnutrition by feeding mice the malnourishing diet supplemented by S-containing AAs, then quantifying TCA cycle function, heme synthesis, and BA production in primary hepatocytes. Expected outcomes include elucidation of a novel link between malnutrition, TCA cycle and heme homeostasis, and BA synthesis. The research is significant because of its potential to develop a scalable AA-based therapeutic to restore liver synthetic function and treat malnutrition in children.