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

我们通过将具有围绕Gs-α外显子1的loxP重组位点的Gs-α floxed等位基因杂合子的雌性与具有巢蛋白启动子-cre重组酶转基因的雄性交配，产生了中枢神经系统中来自母体等位基因的Gs-α表达中断的小鼠（mBGsKO）。通过正反交产生在中枢神经系统中具有类似的父系等位基因（pBGsKO）上的Gs-α表达丧失的小鼠。pBGsKO小鼠具有正常的存活率和总体表型，对葡萄糖或能量代谢或血清脂质没有影响，如通过多种实验方法（体重和组成、器官重量、血清化学和激素、葡萄糖和胰岛素耐受性测试、代谢率和食物摄入测量）所确定的。相比之下，mBGsKO发展为重度肥胖伴糖尿病、重度胰岛素抵抗和高血糖症。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发展，表明对葡萄糖代谢的影响与肥胖无关。在mBGsKO小鼠中的进一步研究表明，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（UCP 1））表达减少的结果，对食物摄入没有主要影响。我们假设mBGsKO小鼠可能缺乏黑皮质素系统刺激交感神经系统活动和能量消耗的能力。为了验证这一假设，急性食物摄入和能量消耗的反应，黑皮质素激动剂（MTII）进行了测量。在pBGsKO小鼠和对照之间没有差异，并且对MTII抑制mBGsKO小鼠食物摄入的能力几乎没有影响。然而，与对照相比，MTII刺激能量消耗的能力在mBGsKO小鼠中显著降低。此外，mBrGsKO小鼠具有受损的饮食诱导的产热和降低的心率和血压。总体而言，这些结果证实了中枢神经系统中的Gs-alpha通路是代谢的关键调节剂，并且小鼠（以及最有可能的Albright遗传性骨营养不良患者）中的母体Gs-alpha突变是由中枢神经系统中一个或多个位点中的Gs-alpha印迹引起的。原位杂交研究表明，GS-α是印记在下丘脑室旁核（PVN），一个已知的网站黑皮质素的行动和代谢调节。最近，我们使用Sf 1-cre检查了腹内侧下丘脑（VMH）中Gs-alpha缺失的小鼠，发现对常规饮食没有重大影响，但对饮食诱导的肥胖有一定的抵抗力。使用Sim 1-cre的PVN特异性Gs-alpha缺陷小鼠（在其他几个位点也丢失了Gs-alpha）对能量平衡和葡萄糖代谢显示出非常轻微的影响，在雄性中更为突出，但没有达到mBrGsKO小鼠的程度。与mBrGsKO不同，母体PVN-Gs-alpha敲除小鼠在产热方面没有表现出缺陷，表明Gs-alpha介导了其他脑区的产热途径。 我们研究了葡萄糖可兴奋的POMC（前阿黑皮素）神经元中Gs-alpha缺失的小鼠。结果显示它们是高血糖的，胰岛素水平降低，表明中枢葡萄糖感知缺陷导致胰腺β细胞胰岛素分泌缺陷。β细胞功能似乎没有自主缺陷。这些小鼠也是皮质功能减退的，可能是由于ACTH（促肾上腺皮质激素）神经元（也是POMC神经元）缺乏Gs-alpha，因此不能对CRH（促肾上腺皮质激素释放激素）产生反应。POMC神经元中组成型活性Gs-alpha突变体的初步结果显示对葡萄糖代谢的相反作用（改善葡萄糖耐量） 我们最近使用不同的转基因cre小鼠系进行了其他神经元或区域特异性Gs-alpha敲除，并排除了几个区域和神经元细胞类型，包括下丘脑腹内侧、POMC神经元、脊髓中间外侧角，作为Gs-alpha缺乏导致肥胖和/或葡萄糖耐受不良的部位。下丘脑腹内侧区Gs-alpha缺乏可保护小鼠免于饮食诱导的肥胖和对高脂饮食的瘦素抵抗。我们还通过立体定位注射AAV-cre病毒到GS-α固定小鼠的不同脑区来敲除特定脑区的GS-α。最近的研究结果表明，Gs-alpha印记在背内侧下丘脑（DMH）强烈有助于父母的起源特异性的影响，观察与Gs-alpha突变的能量平衡和黑皮质素受体4-Gs-alpha信号转导DMH介导的刺激产热和能量消耗。在下丘脑室旁核（PVN），Gs-alpha介导黑皮质素的心血管效应，但不影响摄食量。最近的研究表明，在DMH中Gs-alpha的homozygoua缺失表明该信号通路在食物摄入调节、能量平衡、产热、心血管调节和瘦素信号传导中发挥重要作用。许多这些影响，包括食物摄入调节的缺陷，可能是由于瘦素信号传导受损。 我们还敲除了表达MC 4 R的细胞中的Gs-alpha，并证实这些细胞中的Gs-alpha信号传导对于能量平衡至关重要，并且在表达MC 4 R的神经元亚群中的Gs-alpha印记导致Gs-alpha突变的母体特异性代谢效应。这些细胞中Gs-alpha表达的完全丧失也揭示了Gs-alpha信号在调节食物摄入中的重要作用。

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.