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

我们通过将Gs-α外显子1周围有loxP重组位点的Gs-α开花等位基因杂合子的雌性小鼠与携带巢蛋白启动子-cre重组酶转基因的雄性小鼠交配，产生了中枢神经系统(MBGsKO)母体等位基因Gs-α表达中断的小鼠。通过正反交产生类似于父亲等位基因(PBGsKO)的Gs-α在中枢神经系统中表达缺失的小鼠。通过多种实验方法(体重和成分、器官重量、血清化学和激素、葡萄糖和胰岛素耐量试验、代谢率和摄食量测量)测定，pBGsKO小鼠的存活和总表型正常，对葡萄糖或能量代谢和血脂没有影响。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。肥胖在5周后开始发展。对年轻小鼠的研究表明，胰岛素抵抗和葡萄糖耐受性在肥胖之前就开始发展，这表明对葡萄糖代谢的影响不依赖于肥胖。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消耗相关的棕色脂肪组织基因表达减少的结果，如解偶联蛋白1(UCP1)，对食物摄入量没有主要影响。我们推测，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。为了验证这一假说，我们测量了黑素皮质素激动剂(MTII)的急性食物摄入量和能量消耗反应。PBGsKO小鼠与对照组相比差异无统计学意义，对MTII抑制mBGsKO小鼠摄食能力影响不大。然而，与对照组相比，mBGsKO小鼠MTII刺激能量消耗的能力显著降低。此外，mBrGsKO小鼠还削弱了饮食诱导的产热作用，并降低了心率和血压。总体而言，这些结果证实，中枢神经系统中的Gs-α通路是新陈代谢的关键调节器，小鼠(以及最有可能是奥尔布赖特遗传性骨病患者)中的母体Gs-α突变是由于Gs-α印记在中枢神经系统的一个或多个位置造成的。原位杂交研究表明Gs-α被印记在下丘脑室旁核(PVN)，这是一个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检查了下丘脑腹侧内侧(Vmh)Gs-α缺失的小鼠，发现对正常饮食没有重大影响，但对饮食诱导的肥胖有一些抵抗。使用Sim1-cre的PVN特异性Gs-α缺乏症小鼠(在其他几个位置也丢失Gs-α)对能量平衡和葡萄糖代谢的影响非常轻微，在雄性小鼠中更为明显，但没有mBrGsKO小鼠那么严重。与mBrGsKO不同，母系PVN-Gs-α基因敲除小鼠在产热方面没有缺陷，表明Gs-α介导了其他大脑区域的生热途径。 我们研究了葡萄糖可兴奋的POMC(前阿片黑素皮质素)神经元中Gs-α缺失的小鼠。结果显示，他们血糖升高，胰岛素水平降低，这表明中枢葡萄糖感知缺陷导致胰岛β细胞胰岛素分泌缺陷。似乎在β细胞功能方面没有自主缺陷。这些小鼠也是皮质醇缺乏症，可能是由于促肾上腺皮质激素(ACTH)神经元(也是POMC神经元)缺乏Gs-α，因此不能对CRH(促肾上腺皮质激素释放激素)做出反应。POMC神经元成分活性Gs-α突变体的初步结果显示，对葡萄糖代谢(改善糖耐量)有相反的影响 我们最近利用不同的转基因cre小鼠品系进行了其他神经元或区域特异性Gs-α基因敲除，并排除了一些区域和神经细胞类型，包括下丘脑腹内侧部、POMC神经元、脊髓内侧间外侧角，作为Gs-α缺乏导致肥胖和/或葡萄糖耐量异常的部位。下丘脑腹内侧的GS-α缺乏保护小鼠免受饮食诱导的肥胖和对高脂肪饮食的瘦素抵抗的影响。我们还通过立体定向将AAV-cre病毒注射到Gs-α牙线感染小鼠的不同脑区，敲除了特定脑区的Gs-α。最近的研究结果表明，Gs-α在下丘脑背内侧(DMH)的印迹强烈地促进了Gs-α突变对能量平衡的亲本特异性影响，DMH中的黑素皮质素受体4-Gs-α信号介导了产热和能量消耗的刺激。在下丘脑室旁核(PVN)，Gs-α介导黑素皮质素的心血管效应，但不影响其对食物摄入量的影响。最近对DMH中Gs-α纯合缺失的研究表明，这一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转导中发挥重要作用。这些影响中的许多，包括食物摄取调节的缺陷，可能是由于瘦素信号受损造成的。 我们还敲除了MC4R表达细胞中的Gs-α，并证实这些细胞中的Gs-α信号对能量平衡至关重要，并且Gs-α在表达MC4R的神经元亚群中的印记导致Gs-α突变的亲本特异性代谢效应。在这些细胞中Gs-α表达的完全丧失也揭示了Gs-α信号在食物摄入调节中的重要作用。

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.