Velázquez-Villegas Laura A, Charabati Tania, Contreras Alejandra V, Alemán Gabriela, Torres Nimbe, Tovar Armando R
Department of Physiology of Nutrition, National Institute of Medical Sciences and Nutrition Salvador Zubirán, Mexico City, Mexico; and.
National Institute of Genomic Medicine, Mexico City, Mexico.
J Nutr. 2016 Sep;146(9):1634-40. doi: 10.3945/jn.116.232868. Epub 2016 Jul 27.
Glutamine is catabolized in the liver by glutaminase 2 (GLS2). Evidence suggests that peroxisome proliferator-activated receptor α (PPARα) represses the expression of several amino acid-catabolizing enzymes, but for Gls2 this is unknown.
The aim of the study was to assess whether PPARα regulates Gls2 expression.
For 8 d, 7-9-wk-old male C57BL/6 wild-type (WT) and Ppara-null mice weighing 23.4 ± 0.5 g were fed diets with different dietary protein:carbohydrate (DP:DCH) ratios (6%:77%, 20%:63%, or 50%:33%). Liver samples were obtained after 16 h of feed deprivation or 3 h of refeeding, and microarrays were performed. Hepatic glutaminase expression was measured by quantitative polymerase chain reaction and Western blotting. Cotransfection analyses in hepatocellular carcinoma cell line (HepG2) cells with PPARα and hepatocyte nuclear factor 4α (HNF4α) expression vectors were performed.
The microarray results showed that Gls2 was the only upregulated gene in WT mice, but not in the Ppara-null mice. In the feed-deprived WT mice, the Gls2 mRNA and protein abundances in the 50%:33% group were 2.5- and 1.1-fold greater (P < 0.05), respectively, than those in the 20%:63% group, which were 2.3- and 0.4-fold greater than those in the 6%:77% group (P < 0.01). Gls2 mRNA expression in the 6%:77% group of feed-deprived Ppara-null mice was 33-fold greater than that in the same group of WT mice (P < 0.0001). GLS2 protein abundance in HepG2 cells was 78% greater than that in the controls (P < 0.0001) after HNF4α overexpression, and it was 99% greater after transfection with a short hairpin targeting PPARα.
In Ppara-null mice, Gls2 mRNA expression was greater than in WT mice, regardless of the DP:DCH ratio. In HepG2 cells overexpressing HNF4α, Gls2 expression increased, an effect repressed by overexpression of PPARα. This suggests that Gls2 depends on the PPARα/HNF4α counterregulatory transcriptional control.
谷氨酰胺在肝脏中由谷氨酰胺酶2（GLS2）进行分解代谢。有证据表明，过氧化物酶体增殖物激活受体α（PPARα）可抑制几种氨基酸分解代谢酶的表达，但对于GLS2而言，情况尚不清楚。
本研究旨在评估PPARα是否调节GLS2的表达。
将7 - 9周龄、体重23.4±0.5 g的雄性C57BL/6野生型（WT）和Ppara基因敲除小鼠喂养8天，给予不同蛋白质：碳水化合物（DP:DCH）比例（6%:77%、20%:63%或50%:33%）的饮食。在禁食16小时或再喂养3小时后采集肝脏样本，并进行微阵列分析。通过定量聚合酶链反应和蛋白质免疫印迹法检测肝脏谷氨酰胺酶的表达。在肝癌细胞系（HepG2）细胞中进行PPARα和肝细胞核因子4α（HNF4α）表达载体的共转染分析。
微阵列结果显示，GLS2是野生型小鼠中唯一上调的基因，而在Ppara基因敲除小鼠中并非如此。在禁食的野生型小鼠中，50%:33%组的GLS2 mRNA和蛋白质丰度分别比20%:63%组高2.5倍和1.1倍（P<0.05），而20%:63%组又分别比6%:77%组高2.3倍和0.4倍（P<0.01）。禁食的Ppara基因敲除小鼠6%:77%组的GLS2 mRNA表达比同组野生型小鼠高33倍（P<0.0001）。在HepG2细胞中，过表达HNF4α后GLS2蛋白质丰度比对照组高78%（P<0.0001），而用靶向PPARα的短发夹转染后则高99%。
在Ppara基因敲除小鼠中，无论DP:DCH比例如何，GLS2 mRNA表达均高于野生型小鼠。在过表达HNF4α的HepG2细胞中，GLS2表达增加，而过表达PPARα可抑制这种效应。这表明GLS2依赖于PPARα/HNF4α的反向调节转录控制。
