Kaempfer S, Blackham M, Christiansen M, Wu K, Cesar D, Vary T, Hellerstein M K
Department of Nutritional Sciences, University of California, Berkeley 94720.
Am J Physiol. 1991 Jun;260(6 Pt 1):E865-75. doi: 10.1152/ajpendo.1991.260.6.E865.
We measured the contribution of glucose to hepatic cytosolic acetyl-CoA in vivo in rats and compared it with the phosphorylation state of a potentially regulatory enzyme complex [pyruvate dehydrogenase (PDH)]. Xenobiotic probes were used to sample hepatic cytosolic acetyl-CoA [acetylated sulfamethoxazole (SMX)] and UDP-glucose (glucuronidated acetaminophen) in vivo during [U-14C]glucose infusions. Percent active (dephosphorylated) form of PDH (PDHa) was determined on freeze-clamped liver. First, we confirmed using liver cell elutriation that acetylation of SMX occurs in parenchymal hepatocytes. Next, the fraction of cytosolic acetyl-CoA derived from [14C]glucose in vivo was shown to depend on dietary state. Specific activity of acetyl-CoA relative to plasma glucose or hepatic UDP-glucose was lower after 48 h fasting than after overnight fasting, and glucose refeeding (25 mg.kg-1.min-1 iv) maximally increased [14C]-glucose fractional contribution to acetyl-CoA within 2 h in the overnight-fasted but not in the prolonged fasted group. Hepatic PDHa demonstrated a similar but not identical pattern. The isotopic and enzymatic parameters showed significant correlations (r2 = 0.61 in 48-h fasted-refed group, r2 = 0.28 in overnight-fasted refed group), although [14C]glucose contribution to acetyl-CoA increased disproportionately compared with PDHa as refeeding progressed. The indirect pathway of UDP-glucose synthesis correlated inversely with the fractional contribution of glucose to acetyl-CoA. In summary, the fraction of hepatic acetyl-CoA derived from glucose in vivo is influenced by acute and chronic dietary factors and is only partially explained by PDHa. Regulation of the carbon source of hepatic acetyl-CoA in vivo and interactions suggested by these results (e.g., glucose-fatty acid cycle; branch-point regulation of glucose recycling) can be addressed in a quantitative fashion using this experimental framework.
我们在大鼠体内测量了葡萄糖对肝细胞质乙酰辅酶A的贡献，并将其与一种潜在调节酶复合物[丙酮酸脱氢酶（PDH）]的磷酸化状态进行了比较。在[U-14C]葡萄糖输注期间，使用外源性探针在体内对肝细胞质乙酰辅酶A[乙酰化磺胺甲恶唑（SMX）]和UDP-葡萄糖（葡萄糖醛酸化对乙酰氨基酚）进行采样。通过对冷冻钳夹的肝脏测定PDH的活性（去磷酸化）形式百分比（PDHa）。首先，我们通过肝细胞淘洗法证实SMX的乙酰化发生在实质肝细胞中。接下来，体内源自[14C]葡萄糖的细胞质乙酰辅酶A部分显示取决于饮食状态。禁食48小时后，相对于血浆葡萄糖或肝脏UDP-葡萄糖的乙酰辅酶A比过夜禁食后更低，并且葡萄糖再喂养（25mg·kg-1·min-1静脉注射）在2小时内使过夜禁食组而非长期禁食组中[14C]葡萄糖对乙酰辅酶A的分数贡献最大程度增加。肝脏PDHa表现出相似但不完全相同的模式。同位素和酶学参数显示出显著相关性（48小时禁食-再喂养组中r2 = 0.61，过夜禁食-再喂养组中r2 = 0.28），尽管随着再喂养的进行，[14C]葡萄糖对乙酰辅酶A的贡献与PDHa相比不成比例地增加。UDP-葡萄糖合成的间接途径与葡萄糖对乙酰辅酶A的分数贡献呈负相关。总之，体内源自葡萄糖的肝脏乙酰辅酶A部分受急性和慢性饮食因素影响，并且仅部分由PDHa解释。使用该实验框架可以定量方式研究体内肝脏乙酰辅酶A碳源的调节以及这些结果所暗示的相互作用（例如，葡萄糖-脂肪酸循环；葡萄糖循环的分支点调节）。
