Study on the physiological roles of novel ketone body-utilizing enzyme in the action of endocrine disrupting chemicals

乙酰乙酰辅酶A合成酶（AACS，EC 6.2.1.16）是一种新型的酮体利用酶，其生理作用被认为与辅酶A转移酶不同。为了鉴定AACS基因的调控区，将几个缺失构建体与荧光素酶基因融合并在3 T3-L1细胞中表达。通过缺失-335 ~-3碱基，启动子活性基本消失。点突变研究表明GC盒基序（-87 ~-74）和C/EBP家族识别序列（-196 ~-183）是重要的顺式调控元件。由于我们先前的研究发现AACS在雄性大鼠的各种脂肪生成组织，特别是白色脂肪组织（WAT）中高度表达，因此AACS可能与雄性和雌性之间的代谢差异有关。事实上，我们发现雌激素增强AACS mRNA在ST-13细胞中的表达。为了进一步研究AACS在WAT中的性别差异，我们比较了雄性和雌性大鼠WAT中AACS和各种因子的mRNA表达水平。在整个组织中，雄性皮下WAT中AACS mRNA的表达水平高于雌性，而在肠系膜WAT中没有观察到这种差异。辅酶A转移酶、ATP依赖性柠檬酸裂解酶（ACL）和乙酰辅酶A羧化酶-1（ACC-1）mRNA在两种组织中均无显著性差异。接下来，我们检测了AACS mRNA在前脂肪细胞、小尺寸（约100μm）脂肪细胞和大尺寸（超过200μm）成熟脂肪细胞中的表达水平。在雄性和雌性WAT中，AACS mRNA主要在小型脂肪细胞中表达，并且相似。而在大体积脂肪细胞中，AACS mRNA在雄性皮下WAT中的表达水平明显高于雌性。这些发现提出了AACS在男性皮下脂肪细胞过度生长中发挥重要作用的可能性。

Acetoacetyl-CoA synthetase (AACS, EC 6.2.1.16) is a novel ketone body-utilizing enzyme, the physiological role of which is regarded to be different from that of CoA transferase. In order to identify the regulatory region of AACS gene, several deletion constructs were fused to luciferase gene and expressed in 3T3-L1 cells. By the deletion of the bases -335 to -3,promoter activity was essentially abolished. Furthermore, point mutation study revealed that GC-box motif (-87 to -74) and C/EBP family recognition sequence (-196 to -183) were important cis-regulatory elements. Since our previous studies revealed that AACS was highly expressed in various lipogenic tissues, especially white adipose tissue(WAT) in male rats, AACS seems to be related to metabolic differences between male and female. Indeed, we found that estrogen enhanced AACS mRNA expression in ST-13 cells. In order to further investigate sex-differences of AACS in WAT, we compared mRNA expression levels of AACS and various factors in WAT of male rats with those in WAT of female rats. In whole tissues, expression level of AACS mRNA was higher in male subcutaneous WAT than in female one, but in mesenteric WAT, such difference was not observed. CoA transferase, ATP-dependent citrate lyase(ACL) and Acetyl-CoA carboxylase-1(ACC-1) mRNA exhibited no significant sex-difference in either tissue. Next, we examined AACS mRNA expression levels in preadipocytes, small-size (about 100μm) adipocytes, and large-size (over 200μm) mature adipocytes. In both male and female WAT, AACS mRNA was mainly and similarly expressed in small-size adipocytes. However, in large-size adipocytes, AACS mRNA was expressed much higher level in male subcutaneous WAT than in female one. These findings raise the possibility that AACS plays important roles in overgrowth of male subcutaneous adipocytes.