Role of Gs-alpha in central regulation of energy and glucose metabolism

我们将含有gs - α外显子1周围loxP重组位点的gs - α柔化等位基因的雌性杂合与含有巢蛋白启动子-cre重组酶转基因的雄性杂交，获得了中枢神经系统（mBGsKO）母系gs - α表达中断的小鼠。在父本等位基因（pBGsKO）上中枢神经系统gs - α表达类似缺失的小鼠通过反向杂交产生。通过多种实验方法（体重和组成、器官重量、血清化学成分和激素、葡萄糖和胰岛素耐量试验、代谢率和食物摄入测量）确定，pBGsKO小鼠具有正常的生存和整体表型，对葡萄糖或能量代谢或血脂没有影响。相比之下，mBGsKO出现了严重的肥胖和糖尿病，严重的胰岛素抵抗和高甘油三酯血症。5周后，肥胖开始发展。对年轻小鼠的研究表明，胰岛素抵抗和葡萄糖耐受不良在肥胖之前就开始出现，表明其对葡萄糖代谢的影响独立于肥胖。对mBGsKO小鼠的进一步研究表明，肥胖主要是交感神经系统活动和能量消耗减少以及与能量耗散相关的棕色脂肪组织基因（如解偶联蛋白1 (UCP1)）表达减少的结果，与食物摄入无关。我们假设mBGsKO小鼠可能存在黑素皮质素系统刺激交感神经系统活动和能量消耗的能力缺陷。为了验证这一假设，测量了对黑素皮质激素激动剂（MTII）的急性食物摄入和能量消耗反应。pBGsKO小鼠与对照组之间没有差异，MTII对mBGsKO小鼠的食物摄入抑制能力影响不大。然而，与对照组相比，mBGsKO小鼠的MTII刺激能量消耗的能力明显降低。此外，mBrGsKO小鼠的饮食诱导产热功能受损，心率和血压降低。总的来说，这些结果证实了中枢神经系统中的gs - α通路是代谢的关键调节因子，小鼠（很可能是奥尔布赖特遗传性骨营养不良患者）母系gs - α突变是由于中枢神经系统中一个或多个部位的gs - α印记所致。原位杂交研究表明，gs - α在下丘脑室旁核（PVN）中有印记，PVN是已知的黑素皮质素作用和代谢调节的部位。我们最近使用Sf1-cre对下丘脑腹侧内侧（VMH） gs - α缺失的小鼠进行了研究，发现对常规饮食没有重大影响，但对饮食引起的肥胖有一定的抵抗力。使用Sim1-cre的pvn特异性gs - α缺陷小鼠（在其他几个位点也丢失gs - α）对能量平衡和葡萄糖代谢的影响非常轻微，在雄性中更为突出，但不如mBrGsKO小鼠那么严重。与mBrGsKO不同，母体PVN-Gs-alpha敲除小鼠在产热方面没有缺陷，这表明Gs-alpha介导了脑其他区域的产热途径。

We generated mice with disruption of Gs-alpha expression from the maternal allele in the central nervous system (mBGsKO) by mating females heterozygous for a Gs-alpha floxed allele with loxP recombination sites surrounding Gs-alpha exon 1 to males with a nestin promoter-cre recombinase transgene. Mice with similar loss of Gs-alpha expression in the central nervous system on the paternal allele (pBGsKO) were generated with reciprocal crosses. pBGsKO mice had normal survival and overall phenotype with no effect on glucose or energy metabolism or serum lipids as determined by multiple experimental approaches (body weight and composition, organ weights, serum chemistries and hormones, glucose and insulin tolerance tests, metabolic rate and food intake measurements). In contrast, mBGsKO developed severe obesity with diabetes, severe insulin resistance, and hypertriglyceridemia. The obesity began to develop after 5 weeks. Studies in younger mice indicate that the insulin resistance and glucose intolerance began to develop prior to obesity, indicating an effect on glucose metabolism independent of obesity. Further studies in mBGsKO mice showed that the obesity was primarily the result of reduced sympathetic nervous system activity and energy expenditure and reduced expression of brown adipose tissue genes associated with energy dissipation, such as uncoupling protein 1 (UCP1), with no primary effect on food intake. We hypothesized that mBGsKO mice may be defective the ability of the melanocortin system to stimulate sympathetic nervous system activity and energy expenditure. To test this hypothesis, acute food intake and energy expenditure responses to a melanocortin agonist (MTII) were measured. There were no differences between pBGsKO mice and controls, and there was little effect on the ability of MTII to inhibit food intake in mBGsKO mice. However, the ability of MTII to stimulate energy expenditure was markedly reduced in mBGsKO mice as compared to controls. Moreover mBrGsKO mice have impaired diet-induced thermogenesis and reduced heart rate and blood pressure. Overall these results confirm that Gs-alpha pathways in the central nervous system are critical regulators of metabolism and that maternal Gs-alpha mutations in mice (and most likely Albright hereditary osteodystrophy patients) results from Gs-alpha imprinting in one or more site in the central nervous system. In situ hybridization studies showed that Gs-alpha is imprinted in the paraventricular nucleus of the hypothalamus (PVN), a known site of melanocortin action and metabolic regulation. We more recently examined mice with loss of Gs-alpha in the ventral medial hypothalamus (VMH) using Sf1-cre and see no major effects on regular diet, but some resistance to diet-induced obesity. Mice with PVN-specific Gs-alpha deficiency using Sim1-cre (also loss of Gs-alpha in a couple of other sites) show very mild effects on energy balance and glucose metabolism, more prominent in males, but not to the extent as mBrGsKO mice. Unlike mBrGsKO, maternal PVN-Gs-alpha knockout mice show no defects in thermogenesis, indicating that Gs-alpha mediates thermogenic pathways in other brain regions. We have studied mice with loss of Gs-alpha in glucose-excitable POMC (proopiomelanocortin) neurons. Results show them to be hyperglycemic with reduced insulin levels, suggesting a defect in central glucose sensing leading to a insulin secretory defect from pancreatic beta cells. There are appears to be no autonomous defects in beta-cell function. These mice are also hypocortisolemic, presumably due to the fact that the ACTH (adrenocorticotropin) neurons (which are also POMC neurons) lack Gs-alpha and therefore cannot respond to CRH (corticotropin releasing hormone). Preliminary results with constitutively-active Gs-alpha mutants in POMC neurons show opposite effects on glucose metabolism (improved glucose tolerance) We have most recently made other neuron or region-specific Gs-alpha knockouts using different transgenic-cre mouse lines and have ruled out several areas and neuronal cell-types, including the ventromedial hypothalamus, POMC neurons, interomediolateral horn of the spinal cord, as sites where Gs-alpha deficiency leads to obesity and/or glucose intolerance. Gs-alpha deficiency in the ventromedial hypothalamus protects mice from diet-induced obesity and leptin resistance in response to high fat diet. We are also knocking out Gs-alpha in specific brain regions by stereotaxic injection of AAV-cre virus into various brain regions of Gs-alpha floxed mice. Recent results show that Gs-alpha imprinting in the dorsomedial hypothalamus (DMH) strongly contributes to the parent-of-origin-specific effect on energy balance observed with Gs-alpha mutations and that melanocortin receptor 4-Gs-alpha signaling in DMH mediates the stimulation of thermogenesis and energy expenditure. In the paraventricular nucleus of the hypothalamus (PVN) Gs-alpha mediated melanocortins' cardiovascular effects but not its effects on food intake. More recent studies with homozygoua deletion of Gs-alpha in DMH show this signaling pathway to play an important role in food intake regulation, energy balance, thermogenesis, cardiovascular regulation, and leptin signaling. Many of these effects, including the defect in food intake regulation, likely result from impaired leptin signaling. We have also knocked out Gs-alpha in MC4R-expressing cells and confirmed that Gs-alpha signaling in these cells is critical for energy balance and that imprinting of Gs-alpha in a subpopulation of MC4R-expressing neurons leads to parent-of-origin-specific metabolic effects of Gs-alpha mutations. Complete loss of Gs-alpha expression in these cells also uncovers an important role of Gs-alpha signaling in regulation of food intake.