Characterization of Insulin-Dependent Glucose Transporters in a Teleost Fish Model, the Goby Gillichthys mirobilis

胰腺激素胰岛素的最基本的生物学作用之一是促进细胞从血液和细胞外空间摄取葡萄糖和其他燃料。 胰岛素调节的葡萄糖转运蛋白系统的发展可以说是一种关键的生理适应，其支持吸热（"温血"）动物（如哺乳动物）中细胞和组织的高能量需求。 相反，在外温脊椎动物中，胰岛素调节代谢的这一方面和其他方面假设不太发达。 目前对胰岛素调节的细胞葡萄糖转运的理解几乎完全依赖于对哺乳动物胰岛素调节的GLUT-4葡萄糖转运蛋白的研究。 相比之下，该系统在吸热生理学进化"之前"的状态（例如，在鱼类中）尚不清楚，部分原因是缺乏变温脊椎动物中胰岛素缺乏的可用模型，以便于对该系统进行实验分析。 拟开展的研究的目的是：（1）在分子和细胞水平上表征硬骨鱼胰岛素调节的葡萄糖转运蛋白系统，（2）阐明其在该脊椎动物葡萄糖稳态调节中的生理作用，以及（3）确定相对于哺乳动物存在的可能的进化差异。 虎鱼，Gillichthys mirabilis，是一个特别有用的模型，为此类研究，因为它可以通过实验操作到胰岛素缺乏的状态，让人想起1型糖尿病。 先前对该模型的研究表明，胰岛素缺乏型虾虎鱼表现出高血糖水平和骨骼肌中胰岛素调节的葡萄糖转运活性的明显损害，而胰岛素替代疗法纠正了这些代谢缺陷。 此外，胰岛素治疗刺激体内葡萄糖清除和体外肌肉葡萄糖转运。 因此，假设与哺乳动物GLUT-4相关的胰岛素调节的葡萄糖转运蛋白存在于硬骨鱼的肌肉中，并且在葡萄糖调节中起重要作用，尽管差异可能反映了由变温鱼与恒温鱼代表的不同代谢策略。 在支持这一假设，最近的分子克隆数据在虾虎鱼和其他硬骨鱼种已经确定了至少两个不同的cDNA相关的哺乳动物GLUTs 1 - 4。& 本研究的主要目的是：1）从虾虎鱼肌肉中分离、克隆和鉴定GLUTcDNA，主要是GLUT4和GLUT1，并开发特异性的分子探针（cDNA，抗血清），用于随后的生化分析; 2）确定胰岛素对虾虎鱼肌肉葡萄糖转运系统的特异性调节作用，在基因表达水平和细胞对胰岛素的生理反应水平上;以及3）表征胰岛素和拮抗其作用的激素的相互作用（所谓的反调节激素）在调节肌肉GLUT系统和肝脏代谢，以促进对这种鱼的葡萄糖调节生理学的更全面的理解。 这些研究应该提供一个新的生物学视角和了解激素胰岛素的基本作用。

One of the most fundamental biological roles of the pancreatic hormone insulin is to promote cellular uptake of glucose and other fuels from the blood and extracellular spaces. The development of an insulin-regulated glucose transporter system is arguably a key physiological adaptation that supports the heightened energetic demands of cells and tissues in endothermic ("warm-blooded") animals, such as mammals. Conversely, in ectothermic vertebrates, this and other aspects of insulin-regulated metabolism are hypothetically less developed. Current understanding of insulin-regulated cellular glucose transport has been dependent almost entirely on studies of the insulin-regulated GLUT-4 glucose transporter of mammals. By contrast, the status of this system "before" the evolution of an endothermic physiology (e.g., in fishes) is not understood, due in part to the lack of available models of insulin deficiency in ectothermic vertebrates to facilitate experimental analysis of the system. The objectives of the proposed studies are (1) to characterize a teleost fish insulin-regulated glucose transporter system at the molecular and cellular levels, (2) to elucidate its physiological role in the regulation of glucose homeostasis in this vertebrate, and (3) to identify the likely evolutionary differences relative to that which exist in mammals. The gobiid fish, Gillichthys mirabilis, is a particularly useful model for such studies, since it can be experimentally manipulated into a state of insulin-deficiency reminiscent of type-1 diabetes mellitus. Previous work on this model indicates that insulin-deficient gobies exhibit high blood glucose levels and a pronounced impairment of insulin-regulated glucose transport activity in skeletal muscle, while insulin replacement therapy corrects these metabolic deficiencies. In addition, insulin treatment stimulates glucose clearance in vivo and muscle glucose transport in vitro. It is therefore hypothesized that an insulin-regulated glucose transporter, related to mammalian GLUT-4, exists in muscle of this teleost fish and plays an important role in glucoregulation, albeit with differences likely to reflect the different metabolic strategies represented by the ectothermic fish versus endotherms. In support of this hypothesis, recent molecular cloning data in the goby and some other teleost fish species have identified at least two different cDNAs related to mammalian GLUTs 1 & 4. The following three specific aims are proposed: 1) to isolate, clone, and characterize GLUT cDNAs from goby muscle, principally putative GLUT4 and GLUT 1, and to develop specific molecular probes (cDNA, antisera) for use in subsequent biochemical analyses; 2) to define insulin's specific regulatory actions on the muscle glucose transporter system in the goby, both at the level of gene expression and the level of the cell-physiological response to insulin; and 3) to characterize the interaction of insulin and the hormones that antagonize its actions (so-called counterregulatory hormones) in regulating the muscle GLUT system and hepatic metabolism, in order to advance a more integrative understanding of glucoregulatory physiology in this fish. These studies should provide a novel biological perspective and understanding of a fundamental action of the hormone insulin.