Greg Nicholson

Project: The Insulin-regulated Glucose Transporter System in an Ectothermic Vertebrate: Studies in the Diabetic Teleost Fish, Gillichthys mirabilis.

Summary:

In endothermic vertebrates, insulin is important on a minute-to-minute basis in providing cells with an adequate level of metabolic fuel, via its acute activation of cellular nutrient uptake. In mammals, insulin stimulates the translocation of intracellular vesicles containing transmembrane glucose transporters (Glut4) from the trans-Golgi region to the plasma membrane where they are then active. In ectothermic vertebrates, on the other hand, this metabolic regulatory machinery is expected to be less well developed, given the lack of an evolved endothermic physiology. We hypothesize that the evolution of endothermy cannot have occurred without substantial co-evolution of the insulin-regulated Glut4 system, so as to provide cells and tissues operating at higher, endothermic metabolic activity with the necessary fuels to support that activity.

The model of insulin-dependent diabetes mellitus (IDDM) in the goby, unique to the Long Beach Endocrinology laboratory, is being utilized to study the insulin-regulated glucose transport system "before" the evolution of endothermy. Similar to that in mammalian models, in vitro insulin treatment of goby muscle explants results in a significant (pC-2-deoxyglucose (¹⁴C-2-DG) within 20 min; however, this increase in transport is only ~2-fold, as compared with rat tissue under similar conditions (10-20-fold), suggesting that while the goby has an insulin-recruitable glucose transporter system, it does not appear to be as active as that in endotherms. Importantly, in gobies with IDDM, muscle ¹⁴C-2-DG transport becomes resistant to insulin action, similar to the situation in mammals in which the Glut 4 glucose transporter gene is down-regulated with insulin deficiency. In addition, purified goby muscle plasma membranes have been shown to bind ³H-cytochalaisin-B, a competitive inhibitor of glucose transport. This binding occurs in a specific manner, can be competitively inhibited in the presence of glucose, and appears to be due to a 45-55 kDa protein present in skeletal muscle plasma membranes. Our continuing research on this system are aimed at further characterizing this goby insulin-regulated glucose transport system, both at the biochemical/molecular as well as microstructural levels.

Introduction:

Research in our laboratory has demonstrated that the goby, upon surgical removal of its pancreatic endocrine organ (called "isletectomy", or "Ix"), exhibits the symptoms of a full- blown state of IDDM. This symptomatology includes hyperglycemia, ketonemia, glycosuria, and others, all of which can be normalized upon insulin replacement therapy (see Kelley, 1993). In addition, such diabetic gobies show a direct correlation between the degree of their metabolite imbalances (e.g., elevated serum glucose) and their food consumption rate; i.e., diabetic gobies are incapable of assimilating an increased nutrient load in the absence of insulin. As insulin replacement therapy corrected these impairments, we originally hypothesized that insulin-regulated glucose transport mechanisms, homologous (or possibly analagous) to those in peripheral tissues of mammals, may exist in this ectothermic teleost fish.

In mammals, glucose transport is accomplished by up to six homologous transmembrane facilitative glucose transporters, termed Glut1 to Glut 7 (the Glut 6 gene is not translated), each with distinct kinetics and tissue distribution (see review, Mueckler, 1994). Among the Glut isoforms, the transporter most important in post-prandial whole body glucose uptake in response to insulin is Glut 4. Expressed predominantly in muscle and adipose tissues, Glut 4 responds to elevated insulin via a rapid and reversible increase in glucose transport (Charron et al., 1989). This acute insulin regulation of Glut 4 is independent of de novo protein synthesis. Rather, a rapid increase in the V_max of glucose transport is accomplished through a rapid redistribution of Glut 4 proteins from intracellular membrane compartments to the cell surface where they are active (Cushman and Wardzala, 1980). In addition to its acute effects, insulin also regulates Glut 4 gene expression (Birnbaum, 1989). Thus, with insulin deficiency (e.g., with IDDM) a peripheral insulin resistance occurs via a down-regulation of Glut 4 transporters.

Whether a comparable glucose transporter system exists in ectothermic vertebrates has not been known. Earlier studies on teleost fish demonstrated that insulin injections can increase uptake of radiolabled glucose (and amino acids) into peripheral tissues in vivo (Ince and Thorpe, 1976; Ablett et al., 1981). However, in vitro studies to characterize insulin's direct mechanism(s) of action have never been attempted. In our studies of the goby system, we have demonstrated that insulin can activate muscle glucose transport activity in vitro and, importantly, that muscle from diabetic gobies becomes resistant to insulin action. Continuing studies, described in part on this webpage, aim to characterize further the insulin-regulated glucose transporter system of the goby, using both biochemical as well as microstructural approaches.