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

[eng] 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.[spa] 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

Structural and Functional Evolution of Glucose Transporter 4 (GLUT4): A Look at GLUT4 in Fish

The insulin-responsive glucose transporter GLUT4 was first described in 1988 as a result of studies on the regulation of glucose metabolism by insulin [1]. Soon after the discovery of GLUT4, several groups cloned GLUT4 in the human [2], rat [3,4] and mouse [5]. Since its discovery, GLUT4 has received, together with GLUT1, more experimental attention than any other single membrane transport protein. Structurally, GLUT4 follows the predicted model for class I glucose transporters. GLUT4 has a high affinity for glucose, with a Km of approximately 5 mM [6], and also transports mannose, galactose, dehydroascorbic acid and glucosamine [7-10]. In mammals, GLUT4 is mainly expressed in cardiac and skeletal muscle, brown and white adipose tissue, and brain [6,11,12]. GLUT4 plays a pivotal role in whole body glucose homeostasis, mediating the uptake of glucose regulated by insulin [13,14]. GLUT4 is responsible for the reduction in the postprandial rise in plasma glucose levels [6]. Insulin acts by stimulating the translocation of specific GLUT4-containing vesicles from intracellular stores to the plasma membrane (PM) resulting in an immediate increase in glucose transport [6,15]. The disruption of GLUT4 expression has been extensively associated with pathologies of impaired glucose uptake and insulin resistance such as type 2 diabetes and obesity [13,16-18]..

Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion

Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Sistemas de Informação e Inovação: Um estudo bibliométrico

ABSTRACT Information Systems (IS) and innovation are two issues currently discussed. The purpose of this articleis to verify the completeness of the literature with regard to the treatment of these two topics together. With this purpose, a bibliometric research was conducted using bases in ISI Web of Science, Scopus and Scielo. 134 articles were analyzed and generated some results, such as: predominance of publications from Brazil and the United States of America; higher recurrence of the Enterprise Resource Planning citation (ERP) in comparison to other tools; and predominance of practical researches, which apply concepts from the literature in real situations.RESUMO Sistemas de Informação (SI) e Inovação são dois temas discutidos atualmente. O propósito deste artigo consiste em verificar a abrangência da literatura no que diz respeito ao tratamento desses dois temas em conjunto. Para isso, foi realizada uma pesquisa bibliométrica utilizando-se nas bases ISI Web of Science, Scopus e Scielo. Foram analisados 134 artigos e gerados alguns resultados, tais como: predominância de publicações do Brasil e Estados Unidos; maior recorrência na citação do Enterprise Resource Planning (ERP) em comparação a outras ferramentas; e predominância de pesquisas de cunho prático, aplicando conceitos da literatura em situações reais

Cavin-2 is degraded by the proteosome and Cavin-1 redistributes to the cytosol following cholesterol depletion.

<p>3T3-L1 adipocytes were treated with or without MβCD (20 mM, 90 minutes) and subjected to either (<b>A</b>) immunostaining with the indicated antibodies (red) and for nuclei with DAPI (4′,6-diamidino-2-phenylindole, blue) and analyzed by confocal microscopy as described in Methods or (<b>B</b>), subjected to subcellular separation into membrane and cytosolic fractions. Following centrifugation, an equal proportion of each fraction (ca. 6X more cytosol protein than membrane) were analyzed by SDS-PAGE and Western blotting. Glyceraldehyde phosphate dehydrogenase (GAPDH) is a loading control for the cytosolic fraction. (<b>C</b>) Cultured at cells were treated with or without methylβ-cyclodextrin in combination with the proteasome inhibitor MG-132 (10 µM), the lysosomal inhibitor chloroquine (40 µM), or the inhibitors alone for 90 minutes and cell extracts were prepared in lysis buffer and analyzed by SDS-PAGE and Western blotting for the proteins indicated. Shown are representative experiments.</p

Cholesterol repletion restores cavin-2 levels, which allows return of cavin-1 to the plasma membrane.

<p>3T3-L1 adipocytes were treated without or without methyl-β-cyclodextrin (20 mM, 90 minutes), and the medium was changed to that containing either 10% FBS, 10% FBS depleted of lipoproteins (LPDS), or, 10% FBS lipoprotein depleted serum containing cholesterol loaded cyclodextrin (25 µM cholesterol). Following incubation for the times indicated (time 0 being after MβCD removal), cell lysates were analyzed by SDS-PAGE and Western blotting (<b>A</b>) or were processed for immunofluorescence with antibodies for cavin-1 and -2 (<b>B</b>) and for nuclei (DAPI) as in prior figures. The percent cavin-2 rim staining was determined by scoring 104+/−3 cells that were also positive for DAPI staining.</p

Cholesterol depletion by simvistatin and cholesterol-binding antibiotics causes loss of cavin-2 in fat cells (A) and fibroblasts (B).

<p>Adipocytes or NIH-3T3 fibroblasts were treated with or without Simvastatin (10 µM, 18 hrs) methyl-β-cyclodextrin (20 mM, 90 minutes), nystatin (76 µM, 4 hours) or filipin (7.6 µM, 4 hours). Whole cell extracts were then prepared in RIPA buffer and analyzed by SDS-PAGE and Western blotting as in prior figures.</p

ShRNA mediated knockdown of cavin-2 causes redistribution of cavin-1 to the cytosol.

<p>3T3-L1 adipocytes stably expressing shRNA directed against enhanced green fluorescent protein (eGFP) or cavin-2 were processed for freeze drying electron microscopy (<b>A</b>) or subjected to immunostaining (<b>B</b>) with the indicated antibodies (red) and DAPI (blue), and analyzed by confocal microscopy and quantitatively analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034516#pone-0034516-g003" target="_blank">Figure 3</a> for 102 cells or (<b>C</b>) subcellular fractionation into membrane and cytosolic fractions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034516#pone-0034516-g002" target="_blank">Figure 2B</a>. Following fractionation, proteins were analyzed by SDS-PAGE and Western blotting, with glyceraldehyde phosphate dehydrogenase (GAPDH) being used as a loading control for the cytosolic fraction. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034516#pone-0034516-g004" target="_blank">Figure 4A</a>, panel B, black arrows point to torus shaped caveolae and white arrows to flattened caveolae and the scale bar is 200 nm. In 4B, the amount of cavin-2 in whole cell lysates is shown. These are representative of 3 such experiments.</p

Cav1 null adipocytes lack caveolae but cavin-2 is membrane associated and redistributes to the cytosol upon cholesterol depletion.

<p>Cav1 null adipocytes were treated with or without MβCD as in previous figures and lysates (<b>A</b>) were prepared and analyzed by Western blot or (<b>B</b>) labeled with anti-cavin-2 and secondary antibody prior to analysis by confocal microscopy.</p