Role of Gs-alpha in regulation of adipose tissue growth and function

我们通过aP2-cre重组酶转基因小鼠与在gs - α外显子1周围有loxP重组位点的floxed gs - α小鼠重复交配，产生了脂肪组织gs - α缺失小鼠（FGsKO小鼠）。迄今为止的结果表明，FGsKO小鼠的存活率较低，线性生长下降，特别是那些脂肪组织中Gs-alpha表达严重降低的小鼠。虽然FGsKO小鼠的胰岛素样生长因子1 （IGF1）水平降低了50%，但这些影响的原因尚不清楚。在这组小鼠中，白色脂肪组织（WAT）垫几乎不存在。来自FGsKO胚胎的成纤维细胞在已知诱导脂肪形成的药物存在下显著减少了成脂转化。这些体内和体外实验结果证实了gs - α对正常脂肪形成至关重要。存活下来的FGsKO小鼠的相对脂肪量也减少了，WAT垫更小，脂肪细胞更小，每个细胞的脂质含量更少。相反，肩胛间棕色脂肪组织（BAT）垫比正常大，细胞脂质储存增加，单眼分布更明显。BAT的这种组织学模式与代谢激活减少相一致，这可能是由于交感神经系统无法通过β -肾上腺素能/ gs - α途径刺激脂肪分解。与此一致的是，FGsKO小鼠BAT中PGC-1、解偶联蛋白1 （uncoupling protein 1, UCP1）等脂质代谢相关基因的表达显著降低。与对照组相比，FGsKO小鼠的血糖和胰岛素水平都较低，并且在常规和高脂肪饮食中葡萄糖耐量和胰岛素敏感性都有所改善，这与它们的瘦型一致。两种形式的适应性产热，寒冷和饮食诱导的产热，是通过增加交感神经系统活动介导的。FGsKO小鼠不耐冷，冷诱导的产热作用可能明显受损，因为置于寒冷环境中的FGsKO小鼠不能维持体温，也不能提高BAT中UCP1的表达。这与已知BAT在冷诱导产热中的作用是一致的，并且FGsKO小鼠的结果是BAT不能被交感神经刺激激活，尽管它们的交感神经活动（根据尿儿茶酚胺水平测定）显着增加。相反，饮食诱导的生热作用得以维持，事实上FGsKO小鼠的生热作用高于正常水平，这是基于FGsKO小鼠在高脂肪饮食中没有增加体重，并且在高脂肪饮食中显著增加了它们的能量消耗。这些结果表明，低温和饮食诱导的产热可以发生在不同的组织中，我们认为肌肉是这些小鼠饮食诱导的产热的主要部位。为了进一步研究这一点，我们观察了对照小鼠在急性感冒或高脂肪饮食后的交感神经活动和pgc -1 α诱导。高脂肪饮食只增加了骨骼肌的交感神经活动，而心脏、肝脏和棕色脂肪没有变化。相反，急性冷暴露后所有组织的交感神经活动增加。冷暴露后BAT中pgc -1 α被显著诱导，高脂饮食后pgc -1 α被显著诱导。最后，这些结果以及杂合子FGsKO小鼠的结果表明，脂肪组织不是母体等位基因上的种系gs - α突变导致严重肥胖和胰岛素抵抗的位点。我们目前正在培育一种Ucp1-cre转基因小鼠，如果成功的话，我们将能够培育出蝙蝠特异性的Gs-alpha小鼠。此外，我们正在尝试使用其他cre系（如脂联素-cre转基因小鼠）产生更多的wat特异性gs - α敲除模型。

We generated mice with Gs-alpha deficiency in adipose tissue (FGsKO mice) by repeated matings of aP2-cre recombinase transgenic mice with floxed Gs-alpha mice which have loxP recombination sites surrounding Gs-alpha exon 1. Results to date show that FGsKO mice had poor survival and decreased linear growth, particularly those mice in which Gs-alpha expression in adipose tissue was severely reduced. The cause of these effects are unclear, although insulin-like growth factor 1 (IGF1) levels were reduced by 50% in FGsKO mice. In this subset of mice, white adipose tissue (WAT) pads were almost absent. Fibroblasts from FGsKO embryos had significantly reduced adipogenic conversion in the presence of agents known to induce adipogenesis. These in vivo and in vitro results confirm that Gs-alpha is critical for normal adipogenesis. FGsKO mice which survived had also had reduced relative fat mass with smaller WAT pads and smaller adipocytes with less lipid content per cell. In contrast, the interscapular brown adipose tissue (BAT) pads were larger than normal and the cells had increased lipid stores with a more unilocular distribution. This histological pattern in BAT is consistent with reduced metabolic activation presumably due to an inability of the sympathetic nervous system to stimulate lipolysis via beta-adrenergic/Gs-alpha pathways. Consistent with this, the expression of PGC-1, uncoupling protein 1 (UCP1) and other genes associated with lipid metabolism were markedly reduced in BAT from FGsKO mice. FGsKO mice were hypoglycemic and hypoinsulinemic relative to controls and had improved glucose tolerance and insulin sensitivity on both regular and high-fat diets, consistent with their lean phenotype. Two forms of adaptive thermogenesis, cold- and diet-induced thermogenesis, are mediated by increased sympathetic nervous system activity. FGsKO mice are cold intolerant and cold-induced thermogenesis is proabably markedly impaired as FGsKO mice placed in a cold environment do not maintain their body temperature or raise their expression of UCP1 in BAT. This is consistent with the known role for BAT in cold-induced thermogenesis and the results in FGsKO mice that BAT fails to be activated by sympathetic stimulation despite the fact that their sympathetic activity, as determined by urine catecholamine levels, was markedly increased. In contrast, diet-induced thermogenesis is maintained and in fact greater than normal in FGsKO mice based upon the observations that FGsKO mice fail to gain weight on a high-fat diet and have markedly increase their energy expenditure on a high-fat diet. These results suggest that cold- and diet-induced thermogenesis can occur in separate tissues and we propose that muscle is the main site for diet-induced thermogenesis in these mice. To further examine this we looked at sympathetic activity and PGC-1alpha induction after either acute cold or high fat diet in control mice. In response to high fact diet sympathetic activity only increased in skeletal muscle, with no change in heart, liver, or brown fat. In contrast sympathetic activity increased in all tissues after acute cold exposure. PGC-1alpha was markedly induced in BAT after cold exposure and much less so after high fat diet. Finally, these results as well as results in heterozygous FGsKO mice suggest that adipose tissue is not the site whereby germline Gs-alpha mutations on the maternal allele lead to severe obesity and insulin resistance. We are presently generating a Ucp1-cre transgenic mice which if successful will allow us to generated BAT-specific Gs-alpha mice. In addition we are trying to generate more WAT-specific Gs-alpha knockout models using other cre lines such as adiponectin-cre transgenic mice.