Knapik-Czajka Malgorzata
Jagiellonian University Medical College, Faculty of Pharmacy, Department of Analytical Biochemistry, Medyczna 9 Street, 30-688 Krakow, Poland.
Toxicology. 2014 Nov 5;325:107-14. doi: 10.1016/j.tox.2014.09.001. Epub 2014 Sep 2.
The rate-limiting step in branched-chain amino acids (BCAAs) disposal is catalyzed by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDH). BCKDH activity is regulated mainly by a reversible dephosphorylation (activation)/phosphorylation (inactivation) cycle catalyzed by a specific phosphatase (BDP) and kinase (BDK). Current catalytic activity of BCKDH, described as BCKDH activity state, and thus also BCAAs catabolic rate depend directly on the portion of BCKDH occurring in its active dephosphorylated form. Liver BCKDH activity state alters in response to different nutritional factors. Feeding rats a low-protein diet decreases BCKDH activity. It has been previously shown that lipid lowering drugs, fibrates upregulate liver BCKDH activity and stimulate BCAAs catabolism, especially under the condition of dietary protein deprivation. Effect of statins on liver BCKDH activity has not been studied yet. The present study was aimed at investigating the in vivo effect of simvastatin on liver BCKDH activity, as well as E1, E2 and BDP and BDK mRNA levels in rats fed with either a standard (23% protein) or a low protein (8% protein) diet. For 14 days, simvastatin (80 mg/kg b wt/day) or the vehicle (0.3% methylcellulose) were administrated orally by gavage to the treated and control groups, respectively. The actual BCKDH and total BCKDH activities were assayed spectrophotometrically prior to and following incubation with lambda phosphatase, respectively. The mRNA levels of the selected genes were quantified by means of a semi-quantitative RT-PCR. In rats fed with the low protein diet simvastatin administration reversed physiological adaptation of liver BCKDH to protein restriction and increased liver BCKDH activity state by 39% (p<0.05). Changes in BCKDH activity did not correspond to any changes in mRNA levels for BCKDH catalytic and regulatory enzymes. On the contrary, in rats fed with standard diet liver BCKDH activity state did not alter substantially in response to simvastatin administration. In conclusion, the obtained results indicate that simvastatin stimulates liver BCKDH activity and BCAAs degradation in rats fed with the low protein diet, whereas it exerts no effect on BCKDH in rats fed with the standard diet. Stimulation of liver BCKDH activity can be attributed to altered phosphorylation status of the complex (increased dephosphorylation), and it is not associated with changes in mRNA levels for complex enzymes. It is conceivable that in protein malnourished rats simvastatin effect on liver BCKDH activity and BCAAs metabolism can contribute to the myotoxicity observed during treatment with this agent.
支链氨基酸（BCAAs）代谢的限速步骤由线粒体支链α-酮酸脱氢酶复合体（BCKDH）催化。BCKDH活性主要受一种特异性磷酸酶（BDP）和激酶（BDK）催化的可逆去磷酸化（激活）/磷酸化（失活）循环调控。BCKDH的当前催化活性，即BCKDH活性状态，进而BCAAs分解代谢率直接取决于以活性去磷酸化形式存在的BCKDH的比例。肝脏BCKDH活性状态会因不同营养因素而改变。给大鼠喂食低蛋白饮食会降低BCKDH活性。此前已表明，降脂药物贝特类可上调肝脏BCKDH活性并刺激BCAAs分解代谢，尤其是在饮食蛋白质缺乏的情况下。他汀类药物对肝脏BCKDH活性的影响尚未得到研究。本研究旨在调查辛伐他汀对喂食标准（23%蛋白质）或低蛋白（8%蛋白质）饮食的大鼠肝脏BCKDH活性以及E1、E2、BDP和BDK mRNA水平的体内作用。分别通过灌胃法给治疗组和对照组大鼠口服辛伐他汀（80 mg/kg体重/天）或赋形剂（0.3%甲基纤维素），持续14天。在与λ磷酸酶孵育之前和之后，分别通过分光光度法测定实际BCKDH活性和总BCKDH活性。通过半定量RT-PCR对所选基因的mRNA水平进行定量。在喂食低蛋白饮食并给予辛伐他汀的大鼠中，肝脏BCKDH对蛋白质限制的生理适应性发生逆转，肝脏BCKDH活性状态增加了39%（p<0.05）。BCKDH活性的变化与BCKDH催化和调节酶的mRNA水平的任何变化均不对应。相反，在喂食标准饮食的大鼠中，给予辛伐他汀后肝脏BCKDH活性状态没有明显改变。总之，所得结果表明，辛伐他汀可刺激喂食低蛋白饮食的大鼠肝脏BCKDH活性和BCAAs降解，而对喂食标准饮食的大鼠的BCKDH没有影响。肝脏BCKDH活性的刺激可归因于复合体磷酸化状态的改变（去磷酸化增加），且与复合体酶的mRNA水平变化无关。可以想象，在蛋白质营养不良的大鼠中，辛伐他汀对肝脏BCKDH活性和BCAAs代谢的影响可能导致在用该药物治疗期间观察到的肌毒性。
