Post-Developmental Adipocyte Autophagy in Control of Insulin-Glucose Homeostasis

描述（由申请人提供）：肥胖、代谢综合征和2型糖尿病的特征是胰岛素抵抗影响多个组织（如肝脏、肌肉和脂肪）。最近的研究表明，细胞内自噬在维持胰岛素抵抗发病机制相关组织的代谢健康中起着重要作用。自噬是细胞内溶酶体运输途径，维持能量稳态并抵消多种形式的细胞应激。此外，自噬是细胞中线粒体降解和更新的唯一机制。自噬作用于骨骼肌、肝脏、胰腺细胞和下丘脑，维持胰岛素-葡萄糖稳态；此外，肝脏的脂肪分解需要自噬。先前的研究考察了自噬在脂肪组织中的作用，利用小鼠模型，在胚胎发生过程中脂肪细胞自噬受损。这些小鼠很瘦，没有饮食引起的肥胖，表现出增强的胰岛素敏感性和葡萄糖耐受不良。这些观察结果导致人们猜测，抑制脂肪细胞的自噬可能为治疗或预防肥胖及其代谢后果提供了一种有希望的策略。然而，尽管具有明显有利的代谢表型，胚胎自噬受损的小鼠并不健康：它们的死亡率增加（12周龄时为40%），脂肪组织中出现炎症和巨噬细胞浸润。由于这些小鼠的脂肪细胞发育和脂肪组织库形成的极端改变，它们不是评估成熟脂肪细胞自噬发育后作用的适当模型。我们假设自噬在成熟脂肪细胞中起着重要的保护作用，脂肪细胞自噬的发育后损伤会通过既定的信号机制诱导细胞内应激和胰岛素抵抗；此外，我们预测“自噬应激”将导致脂肪细胞释放独特的循环因子，通过直接作用于其他器官（如肝脏、肌肉和胰腺β细胞），驱动全身胰岛素抵抗和葡萄糖耐受不良。该项目的总体目标是：(i)通过确定脂肪细胞自噬调节成人全身代谢的特定靶组织和分子作用，确定脂肪细胞自噬在体内胰岛素抵抗和葡萄糖耐受不良发病机制中的作用；（ii）确定成熟脂肪细胞中自噬的正常调节和代谢功能，而不考虑自噬在脂肪细胞发育/分化中的作用。我们对小鼠进行了基因工程改造，使自噬可以在成年后通过他莫昔芬诱导的基因损伤，在强烈脂肪形成的发育时期后被关闭。“成熟脂肪细胞自噬敲除小鼠”的初步研究结果表明，自噬不调节肥胖，但对健康的胰岛素-葡萄糖稳态是必要的。在拟议的工作中，我们将这些观察扩展到组织特异性机制的系统评估，通过该机制，成熟脂肪细胞自噬损伤导致小鼠体内胰岛素抵抗和葡萄糖耐受不良（Aim #1）；我们将利用培养的原代脂肪细胞祖细胞来研究脂肪形成过程中自噬损伤的时间如何影响体外脂肪细胞的代谢表型（Aim #2）。

DESCRIPTION (provided by applicant): Obesity, Metabolic Syndrome, and Type 2 diabetes are characterized by insulin resistance affecting multiple tissues (e.g., liver, muscle, and fat). Recent studies determined that intracellular autophagy plays an important role maintaining the metabolic health of tissues involved in the pathogenesis of insulin resistance. Autophagy is the intracellular lysosomal trafficking pathway that maintains energy homeostasis and counteracts multiple forms of cellular stress. In addition, autophagy is the sole mechanism for mitochondrial degradation and turnover in cells. Autophagy functions in skeletal muscle, liver, pancreatic beta cells, and hypothalamus to maintain insulin-glucose homeostasis; in addition, autophagy is required for lipolysis in liver. Prior studies examining the role of autophagy in adipose tissue utilized mouse models in which adipocyte autophagy was impaired during embryogenesis. These mice were lean and protected from diet-induced obesity, and exhibited enhanced insulin sensitivity and glucose intolerance. Such observations led to speculations that inhibiting autophagy in adipocytes might provide a promising strategy for treating or preventing obesity and its metabolic consequences. However, despite their apparently favorable metabolic phenotype, the mice with embryonic impairment of autophagy were not healthy: they exhibit increased rates of death (40% at 12 weeks of age) and inflammation and macrophage infiltration in adipose tissue. Due to the extreme alterations of adipocyte development and adipose tissue depot formation in these mice, they are not adequate models for assessing the post-developmental role of autophagy in the mature adipocyte. We hypothesize autophagy plays an important, protective function in mature adipocytes, and post-developmental impairment of adipocyte autophagy will induce intracellular stress and insulin-resistance via established signaling mechanisms; in addition, we predict "autophagic stress" will cause release of unique circulating factors from adipocytes that drive systemic insulin-resistance and glucose intolerance via direct effects on other organs (e.g., liver, muscle, and pancreatic beta cells). The Overall Objectives of this project are: (i) to determine the role of adipocyte autophagy in the pathogenesis of insulin-resistance and glucose intolerance in vivo by defining specific target tissues and molecular actions through which adipocyte autophagy regulates systemic metabolism in the adult; and (ii) to define the normal regulation and metabolic functions of autophagy in mature adipocytes, independently of the role of autophagy in adipocyte development/differentiation. We genetically engineered mice so that autophagy can be turned off in adulthood, after the developmental period of intense adipogenesis, via a tamoxifen- inducible genetic impairment. Preliminary findings with "mature adipocyte autophagy knockout mice" indicate autophagy does not regulate adiposity but is necessary for healthy insulin-glucose homeostasis. In the proposed work we will extend these observations to a systematic assessment of the tissue specific-mechanisms through which impairment of autophagy in mature adipocytes causes insulin resistance and glucose intolerance in mice in vivo (Aim #1); and we will make use of cultured primary adipocyte progenitor cells to study how the timing of autophagy impairment during adipogenesis influences the metabolic phenotype of adipocytes in vitro (Aim #2).