IL-15 receptor-alpha and IL-15 action in aging skeletal muscle and adipose tissue

描述(由申请人提供)： 衰老的特征是骨骼肌量减少(肌萎缩症)和内脏脂肪组织沉积增加。这些身体成分的变化导致虚弱、胰岛素抵抗和心血管疾病，这些都是老年退伍军人群体的主要健康问题。白介素15(IL-15)是一种肌源性细胞因子，对幼年小鼠的肌肉质量、脂肪质量和胰岛素敏感性具有良好的作用。在随意喂养的啮齿动物中，循环中的IL-15水平随着年龄的增长而下降，但不是卡路里限制的啮齿动物，这表明IL-15活性的下降导致了与年龄相关的身体成分的变化。然而，尽管IL-15对年轻小鼠的身体成分有有利的影响，但与对照组相比，老年小鼠的IL-15过度表达会导致脂肪质量增加和肌肉质量减少。IL-15的分泌和生物活性受IL-15与多种形式的IL-15α受体亚单位(IL-15R1)的不同结合所调节，IL-15R1可以是膜结合形式(mBIL-15R1)，也可以是两种可溶性形式(sIL-15R1)之一。后者可以以组织特有的方式不同地影响IL-15的分泌和活性。IL-15与sIL-15R1变异体的结合也可以不同地抑制肿瘤坏死因子-1(TNF-1)的信号传递，肿瘤坏死因子-1是另一种参与肌肉萎缩和胰岛素抵抗的细胞因子。在人类受试者中，已经描述了IL-15R1基因中的几个单核苷酸多态(SNPs)，它们影响肌肉发达、肥胖和胰岛素敏感性。在骨骼肌中，IL-15R1的选择性剪接mRNA变体(剪接的sIL-15R1)的表达随着年龄的增长而下降，这表明IL-15/剪接的sIL-15R1复合体的减少导致了IL-15的水平和活性随年龄的下降。IL-15和IL-15R1相互作用的分子生物化学是复杂的、组织特异性的，在骨骼肌或脂肪组织中还没有得到很好的研究，在衰老系统中也没有得到很好的研究。因此，这项研究将检验整体假设，即IL-15对年轻和老年小鼠的身体成分具有相反的影响，这是由于剪接的sIL-15R1表达减少，而IL-15与分子上不同的、蛋白质分解脱落的sIL-15R1形式的结合增加。这项研究将利用逆转录病毒载体在原代和永生化的小鼠骨骼肌细胞培养中过表达IL-15和IL-15R1变体，以确定这些分子在肌肉中IL-15分泌和生物活性中的作用。IL-15/sIL-15R1复合变异体在小鼠永生化成脂培养中的作用也将被确定。IL-15/sIL-15R1复合变异体在骨骼肌成肌和成脂培养中对TNFR1介导的肿瘤坏死因子-1信号的影响也将被确定。在体内，创新的和翻译的质粒电穿孔技术将被用来在年轻和正常衰老的小鼠中诱导IL-15、sIL-15R1变异体和可能的超激活性的IL-15/sIL15R1融合蛋白的系统表达。这些分子在高脂饮食、骨骼肌减少和TNFR1介导的骨骼肌核凋亡反应中对身体成分和胰岛素敏感性的影响将被确定。该项目的具体目标是：1.确定IL-15R1变体在骨骼肌成肌和成脂培养中对IL-15稳定性、分泌和生物活性的作用。2.确定IL-15R1变异体对体内肌肉分泌IL-15、身体成分和胰岛素敏感性的影响。3.检测IL-15/sIL-15R1融合蛋白在幼龄和老年小鼠肌肉和脂肪组织中的生物活性。 这些研究将提供机制信息，这些信息可以被用来从药理学上调节身体成分以及老年人骨质疏松症和肥胖的病理后果。

DESCRIPTION (provided by applicant): Aging is characterized by reduced skeletal muscle mass (sarcopenia) and increased visceral adipose tissue deposition. These changes in body composition result in frailty, insulin resistance, and cardiovascular disease, which are major health problems in the aging Veteran population. Interleukin-15 (IL-15) is a muscle-derived cytokine with favorable effects on muscle mass, fat mass, and insulin sensitivity in young mice. Circulating IL-15 levels decline with age, in ad lib-fed, but not calorie- restricted rodents, suggesting declines in IL-15 activity contribute to age-associated changes in body composition. However, while IL-15 has favorable effects on body composition in young mice, IL-15 overexpression in aging mice results in increased fat mass and reduced muscle mass compared to controls. IL-15 secretion and bioactivity are modulated by differential association of IL-15 with several forms of the IL-15 alpha receptor subunit (IL-15R1), which can appear as a membrane-bound form (mbIL-15R1) or as one of two soluble forms (sIL-15R1). The latter can differentially affect IL-15 secretion and activity in a tissue- specific manner. Association of IL-15 with sIL-15R1 variants can also differentially inhibit signaling by tumor necrosis factor-1 (TNF-1), another cytokine involved in muscle wasting and insulin resistance. In human subjects, several single-nucleotide polymorphisms (SNPs) have been described in the IL-15R1 gene which impact muscularity, adiposity, and insulin sensitivity. In skeletal muscle, expression of the alternatively-spliced mRNA variant of IL-15R1 (spliced sIL-15R1) declines with age, suggesting a decrease in IL-15/spliced sIL-15R1 complexes leads to age-associated declines in IL-15 levels and activity. The molecular biochemistry of IL-15 and IL-15R1 interactions are complex, tissue-specific, and have not been well-studied in skeletal muscle or adipose tissue, nor in aging systems. Therefore, this study will test the overall hypothesis that IL-15 has opposing effects on body composition in young and old mice due to decreased expression of spliced sIL-15R1 and increased association of IL-15 with the molecularly distinct, proteolytically-shed sIL-15R1 form. The proposed study will utilize retroviral vectors to overexpress IL-15 and IL-15R1 variants in mouse primary and immortalized skeletal myogenic cultures, in order to define the roles of these molecules in IL-15 secretion and bioactivity in muscle. The effects of IL-15/sIL-15R1 complex variants in mouse immortalized adipogenic cultures will also be determined. The effects of IL-15/sIL-15R1 complex variants on TNFR1- mediated TNF-1 signaling in skeletal myogenic and adipogenic cultures will be determined as well. In vivo, the innovative and translational technique of plasmid electroporation into skeletal muscle (EPM) will be used to induce systemic expression of IL-15, sIL-15R1 variants, and a putative superagonistic IL-15/sIL15R1 fusion protein, in young and normally-aging mice. The effects of these molecules on body composition and insulin sensitivity in response to high-fat feeding, sarcopenia, and TNFR1-mediated skeletal myonuclear apoptosis will be determined. The Specific Aims of the project are: 1. Determine the role of IL-15R1 variants in IL-15 stability, secretion and bioactivity in skeletal myogenic and adipogenic cultures. 2. Determine the role of IL-15R1 variants on IL-15 secretion from muscle, body composition, and insulin sensitivity in vivo. 3. Determine the bioactivity of an IL-15/sIL-15R1 fusion protein in muscle and adipose tissue in young and aging mice. These studies will provide mechanistic information which could be exploited pharmacologically to modulate body composition and the pathological consequences of sarcopenia and adiposity in the elderly.