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

Gs-α 在能量和葡萄糖代谢中枢调节中的作用

• 批准号: 10248141
• 负责人: Lee Weinstein
• 金额: $ 70.84万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10248141
• 关键词: Acute; Agonist; Alleles; Area; Beta Cell; Blood Pressure; Body Composition; Body Weight; Brain region; Brown Fat; CRH gene; Cardiovascular system; Cell physiology; Cells; Chemistry; Corticotropin; Corticotropin-Releasing Hormone; Couples; Cyclic AMP; Defect; Development; Diabetes Mellitus; Diet; Disease; Eating; Energy Metabolism; Enterobacteria phage P1 Cre recombinase; Exons; Female; Food Energy; Food Intake Regulation; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; Generations; Genes; Genetic Recombination; Germ-Line Mutation; Glucose; Glucose Intolerance; Gs alpha mutations; Heart Rate; High Fat Diet; Hormones; Horns; Human; Hyperglycemia; Hyperlipidemia; Hypertension; Hypertriglyceridemia; Hypothalamic structure; Impairment; In Situ Hybridization; Injections; Insulin; Insulin Resistance; Knock-out; Knockout Mice; Leptin; Leptin resistance; Lipids; LoxP-flanked allele; Malignant Neoplasms; Measurement; Measures; Mediating; Melanocortin 4 Receptor; Metabolic; Metabolism; Middle Hypothalamus; Morbid Obesity; Mus; Mutation; Nerve; Neuraxis; Neurons; Obesity; Organ Weight; Parents; Partner in relationship; Pathway interactions; Patients; Phenotype; Play; Population; Prevalence; Pro-Opiomelanocortin; Pseudohypoparathyroidism; Regulation; Resistance; Role; Serum; Signal Pathway; Signal Transduction; Site; Spinal Cord; Structure of beta Cell of islet; Sympathetic Nervous System; System; Testing; Therapeutic Agents; Thermogenesis; Tissues; Transgenes; Transgenic Organisms; Virus; cell type; energy balance; glucose metabolism; glucose tolerance; imprint; improved; insulin tolerance; loss of function mutation; male; metabolic phenotype; metabolic rate; mouse model; mutant; nestin protein; novel therapeutics; paraventricular nucleus; promoter; receptor; reduced food intake; response; uncoupling protein 1

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.

我们通过与GS-Alpha的FloxErele配合了女性杂合子，从而与GS-Alpha杂合子配对中枢神经系统（MBGSKO）中的GS-Alpha表达，从而与GS-Alpha Exon 1与GS-Alpha Exon围绕GS-Alpha EXON与NESTIN促进者促进蛋白促进者重新组合酶变成型GS-Alpha重新组合站点进行了交配。用相互的杂交产生了中枢神经系统（PBGSKO）中中枢神经系统中GS-Alpha表达相似的小鼠。 PBGSKO小鼠的生存率正常和总体表型，对多种实验方法（体重和组成，器官重，血清化学和激素，葡萄糖和胰岛素耐受性测试，代谢率和食物摄入量测量）确定，对葡萄糖或能量代谢或血清脂质无影响。相比之下，MBGSKO患有严重的糖尿病，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。在年轻小鼠中的研究表明，胰岛素抵抗和葡萄糖不耐症在肥胖之前开始发展，这表明对葡萄糖代谢产生了独立于肥胖的影响。在MBGSKO小鼠中的进一步研究表明，肥胖症主要是交感神经系统活性和能量消耗降低以及与能量耗散相关的棕色脂肪组织基因的表达降低的结果，例如解开蛋白1（UCP1），没有对食物摄入的主要作用。我们假设MBGSKO小鼠可能是黑色素皮质系统刺激交感神经系统活动和能量消耗的能力的缺陷。为了检验这一假设，测量了对黑色皮质素激动剂（MTII）的急性食物摄入和能量消耗的反应。 PBGSKO小鼠和对照之间没有差异，对MTII抑制MBGSKO小鼠的食物摄入的能力几乎没有影响。但是，与对照组相比，MBGSKO小鼠中MTII刺激能量消耗的能力显着降低。此外，MBRGSKO小鼠还损害了饮食引起的热生成，心率和血压降低。总的来说，这些结果证实，中枢神经系统中的GS-Alpha途径是代谢的关键调节剂，并且小鼠（最有可能是奥尔布赖特遗传性骨do症患者）的母体GS-Alpha突变是由GS-Alpha在中枢神经系统中一个或多个部位烙印的GS-Alpha所致。原位杂交研究表明，GS-Alpha印在下丘脑（PVN）的室室核中，该核（PVN）是黑色素质素作用和代谢调节的已知部位。最近，我们使用SF1-CRE检查了腹侧下丘脑（VMH）中GS-ALPHA的小鼠，但对常规饮食没有重大影响，但对饮食诱发的肥胖症的耐药性没有重大影响。使用SIM1-CRE（在其他几个地点中丧失GS-Alpha）具有PVN特异性GS-Alpha缺乏症的小鼠对能量平衡和葡萄糖代谢显示出非常轻微的影响，在男性中更为突出，但在MBRGSKO小鼠中并非如此。与MBRGSKO不同，母体PVN-GS-Alpha基因敲除小鼠在热发生中没有缺陷，表明GS-Alpha介导其他大脑区域的热途径。 我们研究了在葡萄糖可启动的POMC（propiomelanocortin）神经元中丧失GS-Alpha的小鼠。结果表明，它们具有高血糖，胰岛素水平降低，这表明中央葡萄糖传感的缺陷导致胰腺β细胞的胰岛素分泌缺陷。 Beta细胞函数中似乎没有自主缺陷。这些小鼠也是低皮质血症的，可能是由于ACTH（肾上腺皮质激素）神经元（也是POMC神经元）缺乏GS-Alpha的事实，因此无法对CRH做出反应（皮质肾上腺激素释放激素）。 POMC神经元中组成型GS-Alpha突变体的初步结果对葡萄糖代谢显示相反的作用（提高了葡萄糖耐受性） 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.腹侧下丘脑的GS-Alpha缺乏可保护小鼠免受饮食诱导的肥胖和瘦素耐药性，以响应高脂肪饮食。我们还通过立体定位将AAV-CRE病毒注射到GS-Alpha Floxed小鼠的各个大脑区域中，从而在特定的大脑区域中淘汰了GS-Alpha。最近的结果表明，背侧下丘脑（DMH）中的GS-Alpha强烈促进了Origin特定的特异性影响对GS-Alpha突变观察到的能量平衡的影响，而黑素性受体受体4-GS-ALPHA信号在DMH中介导了DMH介导了热生成和能量实例的刺激。在下丘脑（PVN）GS-Alpha介导的黑色素皮质素的心血管效应但没有其对食物摄入的作用的情况下。 DMH中GS-Alpha的纯量化删除的最新研究表明，这种信号传导途径在食物摄入调节，能量平衡，热生成，心血管调节和瘦素信号传导中发挥重要作用。其中许多作用，包括食物摄入调节的缺陷，可能是瘦素信号传导受损所致。 我们还淘汰了表达MC4R的细胞中的GS-Alpha，并确认这些细胞中的GS-Alpha信号对于能量平衡至关重要，并且在表达MC4R的神经元的亚群中，GS-Alpha的烙印会导致gs-alpha突变的gs-offress-tark targression extermants extrapers-alpha。这些细胞中GS-Alpha表达的完全丧失还发现了GS-Alpha信号在调节食物摄入中的重要作用。