Hollenberg A N, Susulic V S, Madura J P, Zhang B, Moller D E, Tontonoz P, Sarraf P, Spiegelman B M, Lowell B B
Division of Endocrinology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1997 Feb 21;272(8):5283-90. doi: 10.1074/jbc.272.8.5283.
The ob gene product, leptin, is a major hormonal regulator of appetite and fat cell mass. Recent work has suggested that the antidiabetic agents, the thiazolidinediones (TZ), which are also high affinity ligands of peroxisome proliferator-activated receptor-gamma (PPARgamma), inhibit leptin expression in rodents. To examine the effects of this class of drug on the leptin gene in adipocytes we performed Northern analysis on primary rat adipocytes cultured in the presence or absence of TZ. TZ reduced leptin mRNA levels by 75%. To determine whether this effect was mediated at the transcriptional level, we isolated 6510 base pairs of 5'-flanking sequence of the leptin promoter and studied reporter constructs in primary rat adipocytes and CV-1 cells. Sequence analysis demonstrated the presence of a consensus direct repeat with a 1-base-pair gap site between -3951 and -3939 as well as a consensus CCAAT/enhancer binding protein (C/EBP) site between -55 and -47. Our functional analysis in transfected primary rat adipocytes demonstrates that, despite the presence of a canonical direct repeat with a 1-base-pair gap site, TZ alone decreases reporter gene expression of leptin promoter constructs ranging from -6510 to +9 to -65 to +9. In CV-1 cells, which contain endogenous PPARgamma, TZ treatment alone had little effect on these constructs. However, TZ treatment did inhibit C/EBPalpha-mediated transactivation of the leptin promoter. This down-regulation of leptin reporter constructs mapped to a -65 to +9 promoter fragment which binds C/EBPalpha in gel-mobility shift assays but does not bind PPARgamma2 alone or as a heterodimer with 9-cis-retinoic acid receptor. Conversely, the promoter (-5400 to +24 base pairs) of the aP2 gene, another adipocyte-specific gene, was induced 7.3-fold by TZ. Co-transfection with C/EBPalpha minimally stimulated the aP2 promoter from basal levels but notably blocked activation by TZ. These data indicate that PPARgamma and C/EBPalpha can functionally antagonize each other on at least two separate promoters and that this mechanism may explain the down-regulation of leptin expression by thiazolidinediones.
肥胖基因（ob基因）的产物瘦素是食欲和脂肪细胞量的主要激素调节因子。最近的研究表明，抗糖尿病药物噻唑烷二酮类（TZ）也是过氧化物酶体增殖物激活受体γ（PPARγ）的高亲和力配体，它能抑制啮齿动物的瘦素表达。为了研究这类药物对脂肪细胞中瘦素基因的影响，我们对在有或无TZ存在的情况下培养的原代大鼠脂肪细胞进行了Northern分析。TZ使瘦素mRNA水平降低了75%。为了确定这种效应是否在转录水平介导，我们分离了瘦素启动子5'侧翼序列的6510个碱基对，并在原代大鼠脂肪细胞和CV-1细胞中研究了报告基因构建体。序列分析表明，在-3951至-3939之间存在一个有1个碱基对间隔位点的共有直接重复序列，以及在-55至-47之间存在一个共有CCAAT/增强子结合蛋白（C/EBP）位点。我们在转染的原代大鼠脂肪细胞中的功能分析表明尽管存在一个有1个碱基对间隔位点的典型直接重复序列，但单独使用TZ会降低瘦素启动子构建体（范围从-6510至+9到-65至+9）的报告基因表达。在含有内源性PPARγ的CV-1细胞中，单独使用TZ处理对这些构建体几乎没有影响。然而，TZ处理确实抑制了C/EBPα介导的瘦素启动子的反式激活。瘦素报告基因构建体的这种下调定位于一个-65至+9的启动子片段，该片段在凝胶迁移率变动分析中与C/EBPα结合，但不单独与PPARγ2结合，也不与9-顺式视黄酸受体形成异二聚体结合。相反，另一个脂肪细胞特异性基因aP2基因的启动子（-5400至+24个碱基对）被TZ诱导了7.3倍。与C/EBPα共转染从基础水平上对aP2启动子的刺激最小，但显著阻断了TZ的激活作用。这些数据表明，PPARγ和C/EBPα在至少两个不同的启动子上可以在功能上相互拮抗，并且这种机制可能解释了噻唑烷二酮类药物对瘦素表达的下调作用。
