Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, California, USA.
Biophys J. 2010 Apr 21;98(8):1385-95. doi: 10.1016/j.bpj.2009.12.4308.
The liver plays a central role in maintaining whole body metabolic and energy homeostasis by consuming and producing glucose and fatty acids. Glucose and fatty acids compete for hepatic substrate oxidation with regulation ensuring glucose is oxidized preferentially. Increasing fatty acid oxidation is expected to decrease lipid storage in the liver and avoid lipid-induced insulin-resistance. To increase hepatic lipid oxidation in the presence of glucose, we previously engineered a synthetic glyoxylate shunt into human hepatocyte cultures and a mouse model and showed that this synthetic pathway increases free fatty acid beta-oxidation and confers resistance to diet-induced obesity in the mouse model. Here we used ensemble modeling to decipher the effects of perturbations to the hepatic metabolic network on fatty acid oxidation and glucose uptake. Despite sampling of kinetic parameters using the most fundamental elementary reaction models, the models based on current metabolic regulation did not readily describe the phenotype generated by glyoxylate shunt expression. Although not conclusive, this initial negative result prompted us to probe unknown regulations, and malate was identified as inhibitor of hexokinase 2 expression either through direct or indirect actions. This regulation allows the explanation of observed phenotypes (increased fatty acid degradation and decreased glucose consumption). Moreover, the result is a function of pyruvate-carboxylase, mitochondrial pyruvate transporter, citrate transporter protein, and citrate synthase activities. Some subsets of these flux ratios predict increases in fatty acid and decreases in glucose uptake after glyoxylate expression, whereas others predict no change. Altogether, this work defines the possible biochemical space where the synthetic shunt will produce the desired phenotype and demonstrates the efficacy of ensemble modeling for synthetic pathway design.
肝脏通过消耗和产生葡萄糖和脂肪酸，在维持全身代谢和能量稳态方面发挥着核心作用。葡萄糖和脂肪酸通过调节作用竞争肝脏底物氧化，以确保优先氧化葡萄糖。增加脂肪酸氧化有望减少肝脏中的脂质储存，并避免脂质引起的胰岛素抵抗。为了在存在葡萄糖的情况下增加肝脏中的脂质氧化，我们之前在人肝细胞培养物和小鼠模型中设计了一种合成乙醛酸支路，并表明该合成途径增加了游离脂肪酸的β氧化，并赋予了小鼠模型对饮食诱导肥胖的抵抗力。在这里，我们使用集成建模来破译对肝脏代谢网络的扰动对脂肪酸氧化和葡萄糖摄取的影响。尽管使用最基本的基本反应模型对动力学参数进行了采样，但基于当前代谢调节的模型并没有轻易地描述乙醛酸支路表达产生的表型。尽管这不是决定性的，但这一初步的负面结果促使我们探索未知的调控机制，发现苹果酸通过直接或间接作用抑制己糖激酶 2 的表达。这种调控可以解释观察到的表型（增加脂肪酸降解和减少葡萄糖消耗）。此外，该结果是丙酮酸羧化酶、线粒体丙酮酸转运蛋白、柠檬酸转运蛋白和柠檬酸合酶活性的函数。这些通量比的某些子集预测在表达乙醛酸后脂肪酸增加和葡萄糖摄取减少，而其他子集预测没有变化。总的来说，这项工作定义了合成支路可能产生所需表型的可能生化空间，并展示了集成建模在合成途径设计中的有效性。
