Mechanisms regulating GLUT4 transcription in skeletal muscle cells are highly conserved across vertebrates

The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu (Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates

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]..

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing

Zebrafish ankrd1a as a common player in heart regeneration and skeletal muscle repair

In contrast to humans, zebrafish have a remarkable ability to regenerate their hearts after injury, while both humans and zebrafish efficiently repair the wounded skeletal muscle. Common players in these two processes might represent potential targets for the development of efficient therapies to stimulate human heart to regenerate after injury. We identified ankrd1a expression to be upregulated in both regenerating zebrafish hearts and in repairing skeletal muscle. Its mammalian homolog ANKRD1/CARP encodes a stress responsive cardiac ankyrin repeat protein involved in transcriptional regulation, sarcomere assembly and mechanosensing. Using a TgBAC(ankrd1a:EGFP) line, we showed that activation of ankrd1a in cryoinjured heart is restricted to border zone cardiomyocytes, implicating this gene in dedifferentiation and proliferation of regenerating cardiomyocytes. After stab wound injury of skeletal muscle expression of the fluorescent reporter was observed from 3 dpi, when new EGFP-positive muscle cells emerged inside the injury zone. At later time points, EGFP-positive myofibers were visible in the deeper tissue layers, concomitant with active repair of the injured tissue. In cryoinjured skeletal muscle, strong activation of ankrd1a was also observed in myofibers adjacent to the injury, and in those on uninjured side. Detection of the transgene in both newly formed myofibers that invade the wound and in the apparently uninjured tissue surrounding the injury suggests the role of ankrd1a in skeletal muscle tissue repair and adaptive processes in uninjured myofibers surrounding the injury site. Our results implicate ankrd1a in zebrafish muscle regeneration, repair and remodeling, promoting it as an attractive target for translational studies, as a player in muscle healing and as a sensor of stressed muscle.10th Strategic Conference of Zebrafish Investigators, January 6-9, 2024 at the Asilomar Conference Grounds in Pacific Grove, Californi

Robotic injection of zebrafish embryos for high-throughput screening in disease models

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines

Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish

BACKGROUND: Although the responses to many pathogen associated molecular patterns (PAMPs) in cell cultures and extracted organs are well characterized, there is little known of transcriptome responses to PAMPs in whole organisms. To characterize this in detail, we have performed RNAseq analysis of responses of zebrafish embryos to injection of PAMPs in the caudal vein at one hour after exposure. We have compared two ligands that in mammals have been shown to specifically activate the TLR2 and TLR5 receptors: Pam3CSK4 and flagellin, respectively. RESULTS: We identified a group of 80 common genes that respond with high stringency selection to stimulations with both PAMPs, which included several well-known immune marker genes such as il1b and tnfa. Surprisingly, we also identified sets of 48 and 42 genes that specifically respond to either Pam3CSK4 or flagellin, respectively, after a comparative filtering approach. Remarkably, in the Pam3CSK4 specific set, there was a set of transcription factors with more than 2 fold-change, as confirmed by qPCR analyses, including cebpb, fosb, nr4a1 and egr3. We also showed that the regulation of the Pam3CSK4 and flagellin specifically responding sets is inhibited by knockdown of tlr2 or tlr5, respectively. CONCLUSIONS: Our studies show that Pam3CSK4 and flagellin can stimulate the Tlr2 and Tlr5 signaling pathways leading to common and specific responses in the zebrafish embryo system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1740-9) contains supplementary material, which is available to authorized users

Studies on the function and regulation of glucose transporters GLUT2 and GLUT4 in teleost fish / Estudios sobre la función y regulación de los transportadores de glucosa GLUT2 y GLUT4 en peces teleósteos

[eng] The aim of this thesis was to study the function and regulation of two of the major players in the carbohydrate metabolism regulated by insulin, the facilitative glucose transporters GLUT2 and GLUT4, in teleost fish. In order to investigate the role of factors exerting a control on the transcription of the GLUT4 gene, we cloned the GLUT4 promoter in Fugu. The 5 ́-flanking region of the Fugu GLUT4 gene showed similar features to that in mammals. Structurally, comparative analysis between the cloned promoter sequence and that of other fish promoters revealed a high degree of conservation among teleost species. Furthermore, we demonstrated the functionality of the cloned Fugu GLUT4 promoter, and by generating several deletion constructs we were able to determine the minimal promoter. In this study, we have observed that the activity of the Fugu GLUT4 promoter is inhibited by insulin in a dose- and time-dependent manner. Furthermore, all the deletion constructs were repressed by insulin, suggesting the presence of regulatory elements downstream of the TSS. Next, we investigated the effects of PPARγ activation in L6 murine muscle cells transfected with the cloned Fugu GLUT4 promoter. Our results showed that stimulation with PG-J2 significantly stimulated the activity of the Fugu GLUT4 promoter and that this effect was abolished in the Fugu GLUT4 promoter deletions lacking the PPAR/RXR motifs. Finally, we investigated the in vitro effects of experimentally controlled muscle fiber contraction using C2C12 contractile cells expressing the construct containing the Fugu GLUT4 promoter. Using this system to mimic the effects of exercise in vitro in differentiated myotubes we showed an increase in the transcriptional activity of the Fugu GLUT4 promoter. Next studying the function of GLUT2 at early developmental stages we investigated the expression pattern of GLUT2 during embryonic development in zebrafish by ISH, observing transcripts in the liver, pronephric tubules, anterior intestine, endocrine pancreas and neurons surrounding the hindbrain region. To study its function during the early developmental stages in zebrafish we knocked down GLUT2 using antisense morpholinos. Our results showed that embryos lacking GLUT2 display a delay of the whole body development with severe alterations in the midbrain and hindbrain ventricles. Next, by studying the functional alterations triggered by the lack of GLUT2 we observed that morphant embryos displayed an impairment of glucose uptake in the whole body but especially in the head region. Interestingly, a similar pattern was found when assaying cell viability in these embryos, showing a significant increase in apoptotic cell death, mainly located in the cephalic area. Furthermore, blocking the expression of GLUT2 resulted in alterations in the asymmetric distribution of some endoderm-derived organs shown to express this glucose transporter, namely the liver and the endocrine and exocrine pancreas. Additionally, we studied the transcriptional alterations in these embryos by microarray analysis. Finally, we set out to establish an in vitro system using the mammalian β-cell line MIN6 in order to further study the physiological function of zebrafish GLUT2. Our results showed that we were able to knockdown endogenous GLUT2 leading to a loss of glucose-stimulated insulin secretion in MIN6 cells. Furthermore, we successfully established the conditions for the expression of zebrafish GLUT2 in MIN6 and observed a significantly increase in the basal glucose uptake in the pancreatic cells. In addition, preliminary results point to a possible increase in the glucose uptake in cells expressing the rat GLUT2 construct.[spa] El objetivo de esta tesis fue estudiar la función y regulación de dos de los principales implicados en el metabolismo glucídico regulado por la insulina, los transportadores de glucosa GLUT2 y GLUT4, en los peces teleósteos. En vertebrados no mamíferos, GLUT2 ha sido poco caracterizado hasta la fecha. Se ha demostrado en varias especies de teleósteos que GLUT2 se expresa en los principales tejidos sensibles a la insulina, similar a lo que se describe en los mamíferos. Sin embargo, las propiedades funcionales y el papel fisiológico de GLUT2 apenas han sido descritos en peces. En vista de ello, se ha caracterizado GLUT2 en el pez cebra, ya que dicha especie es uno de los modelos más reconocidos para el estudio de la fisiología, el desarrollo y el metabolismo. En cuanto a GLUT4, nuestro grupo ha sido pionero en la investigación de este transportador en peces teleósteos desde que Planas et al. caracterizaron el primer homologo de GLUT4 en vertebrados inferiores. A pesar de que se ha estudiado más este transportador de glucosa que GLUT2, la información sobre los factores que intervienen en la regulación de la transcripción del gen GLUT4 han sido escasamente caracterizados en mamíferos, mientras que no hay datos disponibles en los vertebrados inferiores. Para ello, en este estudio hemos analizado la regulación de un promotor de GLUT4 de teleósteos bajo la acción de estímulos con conocida capacidad para modular la transcripción y la expresión de GLUT4 en los mamíferos, como la insulina, la contracción de fibras musculares y PPARs

Mechanisms regulating GLUT4 transcription in skeletal muscle cells are highly conserved across vertebrates

The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu (Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates

Cell type- and stage-specific gene expression during spermatogenesis in the Senegalese sole testis

26th Conference of European Comparative Endocrinologists, 21-25 August 2012, ZurichPeer Reviewe