Saddik M, Gamble J, Witters L A, Lopaschuk G D
Department of Pediatrics, University of Alberta, Edmonton, Canada.
J Biol Chem. 1993 Dec 5;268(34):25836-45.
The role of acetyl-coenzyme A carboxylase (ACC) in regulating fatty acid oxidation was investigated in isolated fatty acid perfused working rat hearts. Overall fatty acid oxidation rates were determined by addition of 1.2 mM [3H]palmitate to the perfusate of hearts in which the endogenous triglyceride pool was prelabeled with [14C]palmitate. Rates of both exogenous and endogenous fatty acid oxidation were measured by simultaneous measurement of 3H2O and 14CO2 production, respectively. A second series of hearts were perfused under similar conditions except that [U-14C]glucose was present in the perfusate for measurement of glucose oxidation rates. Addition of dichloroacetate (DCA, 1 mM) to the perfusate resulted in a dramatic stimulation of glucose oxidation (a 411% increase), with a parallel decrease in fatty acid oxidation (from 305 +/- 51 to 206 +/- 40 nmol/g dry weight.min.unit work). DCA treatment increased the contribution of glucose oxidation to ATP production from 7.1 to 30.6%, while decreasing the contribution of overall fatty acid oxidation from 92.9 to 69.4%. Tissue levels of malonyl-CoA in hearts treated with DCA were higher compared to controls (14.0 +/- 0.6 and 10.0 +/- 0.7 nmol/g dry weight, respectively) and were negatively correlated (r = -0.85) with overall fatty acid oxidation rates. Acetyl-CoA levels were also significantly higher in DCA-treated hearts, and a positive correlation (r = 0.88) was seen between myocardial acetyl-CoA and malonyl-CoA levels. This suggests that DCA treatment increased the supply of acetyl-CoA for ACC. Western blots revealed the presence of both the 280-kDa (ACC-280) and the 265-kDa (ACC-265) isoforms of ACC in cardiac tissue, with a predominance of ACC-280. The activity of ACC extracted from hearts was similar in both groups when assayed under optimal conditions of acetyl-CoA and citrate. However, using affinity purified ACC, it was demonstrated that heart ACC (predominantly ACC-280) had a higher Km for acetyl-CoA than ACC isolated from white adipose tissue (predominantly ACC-265). We conclude that ACC is an important regulator of fatty acid oxidation in the heart and that acetyl-CoA supply is a key determinant of heart ACC-280 activity. As acetyl-CoA levels increase, ACC-280 is activated resulting in an increase in malonyl-CoA inhibition of fatty acid oxidation.
在分离的灌注脂肪酸的工作大鼠心脏中研究了乙酰辅酶A羧化酶（ACC）在调节脂肪酸氧化中的作用。通过向预先用[14C]棕榈酸标记内源性甘油三酯池的心脏灌注液中添加1.2 mM [3H]棕榈酸来测定总体脂肪酸氧化速率。分别通过同时测量3H2O和14CO2的产生来测量外源性和内源性脂肪酸氧化速率。第二组心脏在相似条件下进行灌注，不同之处在于灌注液中存在[U-14C]葡萄糖以测量葡萄糖氧化速率。向灌注液中添加二氯乙酸（DCA，1 mM）导致葡萄糖氧化显著增加（增加411%），同时脂肪酸氧化平行下降（从305±51降至206±40 nmol/g干重·分钟·单位功）。DCA处理使葡萄糖氧化对ATP产生的贡献从7.1%增加到30.6%，同时使总体脂肪酸氧化的贡献从92.9%降至69.4%。与对照组相比，用DCA处理的心脏中丙二酰辅酶A的组织水平更高（分别为14.0±0.6和10.0±0.7 nmol/g干重），并且与总体脂肪酸氧化速率呈负相关（r = -0.85）。DCA处理的心脏中乙酰辅酶A水平也显著更高，并且心肌乙酰辅酶A和丙二酰辅酶A水平之间呈正相关（r = 0.88）。这表明DCA处理增加了ACC的乙酰辅酶A供应。蛋白质免疫印迹显示心脏组织中存在280 kDa（ACC-280）和265 kDa（ACC-265）两种ACC同工型，以ACC-280为主。在乙酰辅酶A和柠檬酸的最佳条件下测定时，两组从心脏中提取的ACC活性相似。然而，使用亲和纯化的ACC表明，心脏ACC（主要是ACC-280）对乙酰辅酶A的Km值高于从白色脂肪组织中分离的ACC（主要是ACC-265）。我们得出结论，ACC是心脏中脂肪酸氧化的重要调节因子，并且乙酰辅酶A供应是心脏ACC-280活性的关键决定因素。随着乙酰辅酶A水平的增加，ACC-280被激活，导致丙二酰辅酶A对脂肪酸氧化的抑制作用增加。
