Fish and mammalian glut4 traffic characteristics: an evolutionary perspective on the importance of glut4 protein motifs for trafficking

Abstract

Glucose transporters (GLUTs) are extremely important for glucose metabolism. Glucose transporters uptake glucose from blood stream into the cells where it can be metabolized. Among the glucose transporters family, GLUT4, which is solely expressed in muscle and adipose tissues, displays a unique feature as it can change its cellular distribution within minutes in response to insulin to regulate glucose uptake. Therefore, the study of GLUT4 cellular trafficking is fundamental to understand its functioning and to deepen our knowledge on glucose homeostasis. In this work, we utilized a GLUT4 fish variant, brown trout GLUT4, to study GLUT4 trafficking and the role of GLUT4 protein motifs in this process, in 3T3-L1 adipocytes. We observed that, in comparison to mammalian GLUT4 (RatGLUT4), brown trout GLUT4 (BtGLUT4) had a much weaker translocation to the plasma membrane in response to insulin which was in part due to a slower cellular trafficking (exocytosis and endocytosis) and to a poor targeting to the GLUT4 storage vesicles responsible for “holding” GLUT4 inside the cell in the absence of insulin; these vesicles represent the main pool of insulin-responsive GLUT4. In this thesis we also studied the most common GLUT4 endocytic routes. We analyzed the contribution of the clathrin-mediated and the cholesterol-dependent endocytic pathways for RatGLUT4 and BtGLUT4 internalization. We observed that whilst RatGLUT4 internalizes through both the clathrin-mediated and the cholesterol-dependent pathways, BtGLUT4 only utilizes the former. It has been suggested that in adipocytes, the main cholesterol-dependent internalization pathway is the caveolar route. The internalization through this pathway is mediated by plasma membrane structures called caveolae. The formation of these structures is dependent on the caveolin-1 protein. To analyze the role of caveolae in GLUT4 internalization we blocked its formation by knocking down caveolin-1 and observed an increase of RatGLUT4 and BtGLUT4 internalization; however, both GLUT4 isoforms showed less internalization through the clathrin-mediated and cholesterol-dependent pathways in the absence of cavolin-1. Therefore, we suggest that in 3T3-L1 adipocytes caveolin-1 knockdown induces internalization of GLUT4 through alternative pathways. GLUT4 trafficking is regulated by cellular machinery that interacts with GLUT4 protein motifs. To analyze the role of the mammalian N-terminal FQQI8 and C-terminal TELEY502 motifs in GLUT4 trafficking we mutated the corresponding motifs in BtGLUT4 (FQHL8 and TELDY495, respectively) and observed that mutations in the C-terminal had little effect on BtGLUT4 trafficking whereas mutations on the N-terminal (especially FQQL8 mutant) improved BtGLUT4 intracellular retention in the absence of insulin. Furthermore, we verified that FQQL8 mutation increased BtGLUT4 retention in a syntaxin-6-rich compartment, possibly the trans-Golgi network. In addition to studying BtGLUT4 mutants we also analyzed the trafficking of a chimera consisting of a RatGLUT4 backbone with the large cytoplasmic loop of BtGLUT4 (L-GLUT4). We observed that L-GLUT4 possessed higher plasma membrane levels in the absence of insulin and as a result a weaker translocation. Moreover, we observed that this was caused, at least in part, by a reduction in the endocytosis of L-GLUT4 in the absence of insulin. We also analyzed the contribution of the clathrin-mediated and cholesterol-dependent pathways for L-GLUT4 internalization and observed that the loop substitution (L-GLUT4) reduced RatGLUT4 internalization through the cholesterol-dependent route. Moreover, in the absence of insulin and in caveolin-1, L-GLUT4 internalization did not increase as much as that of RatGLUT4. The internalization of L-GLUT4 in the absence of caveolin-1 and insulin occurred through a clathrin-mediated pathway, similarly to BtGLUT4, but it also internalized through a cholesterol-dependent pathway, unlike RatGLUT4 and BtGLUT4. In summary, in this thesis we have contributed to increase the knowledge on GLUT4 trafficking and on the roles of the FQQI8 motif and large cytoplasmic loop in this process, in 3T3-L1 adipocytes.

El transportador de glucosa GLUT4 tiene la capacidad de, en respuesta a insulina, cambiar su localización celular y de esta forma regular el transporte de glucosa. En este trabajo, hemos utilizado una variante de GLUT4 de trucha (BtGLUT4) para estudiar el trafico de GLUT4, así como sus dominios proteicos involucrados en este proceso, en adipocitos 3T3-L1. Hemos observado que en comparación con el GLUT4 de mamíferos (RatGLUT4), el BtGLUT4 tenia una menor capacidad de translocación a la membrana plasmática en respuesta a insulina y que esto se debía a una trafico celular mas lento (exocitosis y endocitosis) y a una peor retención en las vesículas responsables por retener el transportador dentro de la célula en ausencia de insulina. En este trabajo hemos observado que RatGLUT4 ha internalizado por la vía de endocitosis mediada por clatrina y por la vía dependiente de colesterol, mientras que BtGLUT4 solo ha utilizado la primera. Además, hemos inhibido la internalización caveolar, mediante bajada de la expresión de caveolina-1, y hemos observado un aumento de la internalización de RatGLUT4 y BtGLUT4. Con el objetivo de estudiar el papel del dominio FQQI8 (extremo -N) de mamífero en el trafico de GLUT4, hemos mutado la secuencia correspondiente en BtGLUT4 (FQHL8) y hemos observado que mutaciones en este dominio han mejorado la retención intracelular de BtGLUT4 en ausencia de insulina. También hemos estudiado el trafico de una quimera que consiste en la secuencia de RatGLUT4 con el lazo citoplasmático largo de BtGLUT4 (L-GLUT4). Hemos observado que la sustitución del lazo ha aumentado los niveles de RatGLUT4 en superficie en ausencia de insulina y que esto era debido, por lo menos en parte, a una menor endocitosis en ausencia de la hormona. También hemos observado que la sustitución del lazo de RatGLUT4 ha reducido su internalización a través de la vía dependiente de colesterol en ausencia de insulina. Además, en ausencia de caveolina-1 y insulina, la internalización de L-GLUT4 ha aumentado menos que la de RatGLUT4 y ha ocurrido a través de las vías mediada por clatrina y dependiente de colesterol.