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（Acetoacetyl-CoA synthetase, EC 6.2.1.16）是一种新型的酮体利用酶，其生理作用被认为不同于辅酶a转移酶。为了确定AACS基因的调控区域，我们将几个缺失构建体与荧光素酶基因融合，并在3T3-L1细胞中表达。由于-335 ~ -3碱基的缺失，启动子活性基本上被消除了。此外，点突变研究发现GC-box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。由于我们之前的研究发现AACS在雄性大鼠的各种脂质组织中都有高表达，尤其是白色脂肪组织（WAT），因此AACS似乎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了ST-13细胞中AACS mRNA的表达。为了进一步研究WAT中AACS的性别差异，我们比较了雄性大鼠与雌性大鼠WAT中AACS及各因子的mRNA表达水平。在全组织中，男性皮下WAT中AACS mRNA的表达水平高于女性，而在肠系膜WAT中则无差异。辅酶a转移酶、atp依赖性柠檬酸解酶（ACL）和乙酰辅酶a羧化酶-1(ACC-1) mRNA在两种组织中均无显著的性别差异。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。在男性和女性WAT中，AACS mRNA主要在小尺寸脂肪细胞中表达，并且表达相似。然而，在大尺寸脂肪细胞中，AACS mRNA在男性皮下WAT中的表达水平远高于女性皮下WAT。这些发现提出了AACS在男性皮下脂肪细胞过度生长中起重要作用的可能性。

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.