RESUMEN
The white shrimp Penaeus (Litopenaeus) vannamei is the most cultivated shrimp worldwide. Compared to other shrimp species, it has higher resistance to adverse conditions. During hypoxia, the shrimp reduces oxygen consumption and adjusts energy metabolism via anaerobic glycolysis, among other strategies. Hexokinase (HK) is the first enzyme of glycolysis and a key regulation point. In mammals and other vertebrates, there are several tissue-specific HK isoforms with differences in expression and enzyme activity. In contrast, crustacean HKs have been relatively little studied. We studied the P. vannamei HK isoforms during hypoxia and reoxygenation. We cloned two HK1 sequences named HK1-long (1455 bp) and HK1-short (1302 bp), and one HK2 (1344 bp). In normoxia, total HK1 expression is higher in hepatopancreas, while HK2 is higher in gills. Severe hypoxia (1 mg/L of DO) after 12 h exposure and 1 h of reoxygenation increased HK1 expression in both organs, but HK2 expression changed differentially. In hepatopancreas, HK2 expression increased in 6 and 12 h of hypoxia but diminished to normoxia levels after reoxygenation. In gills, HK2 expression decreased after 12 h of hypoxia. HK activity increased in hepatopancreas after 12 h hypoxia, opposite to gills. These results indicate that shrimp HK isoforms respond to hypoxia and reoxygenation in a tissue-specific manner. Intracellular glucose levels did not change in any case, showing the shrimp ability to maintain glucose homeostasis during hypoxia.
Asunto(s)
Penaeidae , Animales , Penaeidae/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Secuencia de Aminoácidos , Hipoxia/metabolismo , Oxígeno/metabolismo , Isoformas de Proteínas/metabolismo , Glucosa/metabolismo , Hepatopáncreas/metabolismo , Mamíferos/metabolismoRESUMEN
Animals suffer hypoxia when their oxygen consumption is larger than the oxygen available. Hypoxia affects the white shrimp Penaeus (Litopenaeus) vannamei, both in their natural habitat and in cultivation farms. Shrimp regulates some enzymes that participate in energy production pathways as a strategy to survive during hypoxia. Glucose-6-phosphatase (G6Pase) is key to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. We previously reported a shrimp G6Pase gene (G6Pase1) and in this work, we report a second isoform that we named G6Pase2. The expression of the two isoforms was evaluated in oxygen limited conditions and during silencing of the transcription factor HIF-1. High G6Pase activity was detected in hepatopancreas followed by muscle and gills under good oxygen and feeding conditions. Gene expression of both isoforms was analyzed in normoxia, hypoxia and reoxygenation in hepatopancreas and gills, and in HIF-1-silenced shrimp. In fed shrimp with normal dissolved oxygen (DO) (5.0 mg L- 1 DO) the expression of G6Pase1 was detected in gills, but not in hepatopancreas or muscle, while G6Pase2 expression was undetectable in all three tissues. In hepatopancreas, G6Pase1 is induced at 3 and 48 h of hypoxia, while G6Pase2 is down-regulated in the same time points but in reoxygenation, both due to the knock-down of HIF-1. In gills, only G6Pase1 was detected, and was induced by the silencing of HIF-1 only after 3 h of reoxygenation. Therefore, the expression of the two isoforms appears to be regulated by HIF-1 at transcriptional level in response to oxygen deprivation and subsequent recovery of oxygen levels.
Asunto(s)
Glucosa-6-Fosfatasa , Penaeidae , Animales , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Penaeidae/genética , Penaeidae/metabolismo , Hipoxia/metabolismo , Oxígeno/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
The shrimp Litopenaeus vannamei is the main farmed crustacean worldwide. This shrimp suffers environmental changes in oxygen availability that affect its energy metabolism. Pyruvate kinase (PK) catalyzes the last reaction of glycolysis and is key for the regulation of glycolysis and gluconeogenesis. There is ample knowledge about mammalian PK, but in crustaceans, the information is very scarce. In this study, we analyzed in silico the structures of the PK gene and protein. Also, the effects of hypoxia on gene expression, enzymatic activity, glucose, and lactate in hepatopancreas and muscle were analyzed. The PK gene is 15,103 bp and contains 11 exons and 10 introns, producing four mRNA variants by alternative splicing and named PK1, PK2, PK3 and PK4, that results in two proteins with longer C-terminus and two with a 12 bp insertion. The promoter contains putative binding sites for transcription factors (TF) that are typically involved in stress responses. The deduced amino acid sequences contain the classic domains, binding sites for allosteric effectors and potential reversible phosphorylation residues. Protein modeling indicates a homotetramer with highly conserved structure. The effect of hypoxia for 6 and 12 h showed tissue-specific patterns, with higher expression, enzyme activity and lactate in muscle, but higher glucose in hepatopancreas. Changes in response to hypoxia were detected at 12 h in expression with induction in muscle and reduction in hepatopancreas, while enzyme activity was maintained, and glucose and lactate decreased. These results show rapid changes in expression and metabolites, while enzyme activity was maintained to cope with short-term hypoxia.
Asunto(s)
Penaeidae , Piruvato Quinasa , Animales , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Hipoxia/genética , Hipoxia/metabolismo , Oxígeno/metabolismo , Glucosa/metabolismo , Lactatos , Penaeidae/metabolismo , Mamíferos/metabolismoRESUMEN
Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.
Asunto(s)
Penaeidae/enzimología , Penaeidae/genética , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Branquias/metabolismo , Hipoxia/enzimología , Hipoxia/genética , Ácido Láctico/metabolismo , Modelos Moleculares , Fosfofructoquinasa-2/química , Filogenia , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Hypoxic zones are spreading worldwide in marine environments affecting many organisms. Shrimp and other marine crustaceans can withstand environmental hypoxia using several strategies, including the regulation of energy producing metabolic pathways. Pyruvate carboxylase (PC) catalyzes the first reaction of gluconeogenesis to produce oxaloacetate from pyruvate. In mammals, PC also participates in lipogenesis, insulin secretion and other processes, but this enzyme has been scarcely studied in marine invertebrates. In this work, we characterized the gene encoding PC in the white shrimp Litopenaeus vannamei, modelled the protein structure and evaluated its gene expression in hepatopancreas during hypoxia, as well as glucose and lactate concentrations. The PC gene codes for a mitochondrial protein and has 21 coding exons and 4 non-coding exons that generate three transcript variants with differences only in the 5'-UTR. Total PC expression is more abundant in hepatopancreas compared to gills or muscle, indicating tissue-specific expression. Under hypoxic conditions of 1.53 mg/L dissolved oxygen, PC expression is maintained in hepatopancreas, indicating its key role even in energy-limited conditions. Finally, both glucose and lactate concentrations were maintained under hypoxia for 24-48 h in hepatopancreas.
Asunto(s)
Penaeidae , Piruvato Carboxilasa , Secuencia de Aminoácidos , Animales , Glucosa/metabolismo , Hepatopáncreas/metabolismo , Hipoxia/metabolismo , Lactatos/metabolismo , Mamíferos/metabolismo , Estructura Molecular , Penaeidae/metabolismo , Piruvato Carboxilasa/genética , Piruvato Carboxilasa/metabolismoRESUMEN
The white shrimp Penaeus (Litopenaeus) vannamei is the most economically important crustacean species cultivated in the Western Hemisphere. This crustacean shifts its metabolism to survive under extreme environmental conditions such as hypoxia, although for a limited time. Glucose-6-phosphatase (G6Pase) is a key enzyme contributing to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. To our knowledge, there are no current detailed studies about cDNA or gene sequences of G6Pase from any crustacean reported. Herein we report the shrimp P. (L.) vannamei cDNA and gene sequences. The gene contains seven exons interrupted by six introns. The deduced amino acid sequence has 35% identity to other homolog proteins, with the catalytic amino acids conserved and phylogenetically close to the corresponding invertebrate homologs. Protein molecular modeling predicted eight transmembrane helices with the catalytic site oriented towards the lumen of the endoplasmic reticulum. G6Pase expression under normoxic conditions was evaluated in hepatopancreas, gills, and muscle and the highest transcript abundance was detected in hepatopancreas. In response to different times of hypoxia, G6Pase mRNA expression did not change in hepatopancreas and became undetectable in muscle; however, in gills, its expression increased after 3 h and 24 h of oxygen limitation, indicating its essential role to maintain glycemic control in these conditions.
Asunto(s)
Clonación Molecular/métodos , Branquias/metabolismo , Gluconeogénesis/genética , Glucosa-6-Fosfatasa/metabolismo , Hepatopáncreas/metabolismo , Animales , Glucosa-6-Fosfatasa/genética , PenaeidaeRESUMEN
The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1ß and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1ß decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions.
Asunto(s)
Apoptosis , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Proteínas de Peces/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Metalotioneína/metabolismo , Oxígeno/metabolismo , Penaeidae/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica , Branquias/metabolismo , Branquias/patología , Hemocitos/metabolismo , Hemocitos/patología , Hipoxia/genética , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Metalotioneína/genética , Penaeidae/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Shrimp are increasingly exposed to warmer temperatures and lower oxygen concentrations in their habitat due to climate change. These conditions may lead to oxidative stress and apoptosis. We studied the effects of high temperature, hypoxia, reoxygenation, and the combination of these factors on lipid peroxidation, protein carbonylation, and caspase-3 activity in gills of white shrimp Litopenaeus vannamei. Silencing of mitochondrial manganese superoxide dismutase (mMnSOD) was used to determine the role of this enzyme in response to the abiotic stressors described above, to avoid oxidative damage and apoptosis. In addition, mMnSOD gene expression and mitochondrial SOD activity were evaluated to determine the efficiency of silencing this enzyme. The results showed that there was no effect of the abiotic stress conditions on the thiobarbituric acid reactive substances (TBARS), but protein carbonylation increased in all the oxidative stress treatments and caspase-3 activity decreased in hypoxia at 28⯰C. On the other hand, mMnSOD-silenced shrimp experienced higher oxidative stress, since TBARS, carbonylated proteins and caspase-3 activity increased in some silenced treatments. Unexpectedly, mitochondrial SOD activity increased in some of the silenced treatments as well. Altogether, these results suggest that mMnSOD has a key role in shrimp for the prevention of oxidative damage development and induction of apoptosis in response to hypoxia, reoxygenation, high temperature, and their interactions, as conditions derived from climate change.
Asunto(s)
Caspasa 3/metabolismo , Crustáceos/fisiología , Técnicas de Silenciamiento del Gen , Calor , Hipoxia/metabolismo , Mitocondrias/enzimología , Estrés Oxidativo/genética , Oxígeno/metabolismo , Superóxido Dismutasa/genética , Animales , Crustáceos/metabolismo , Silenciador del Gen , Superóxido Dismutasa/metabolismoRESUMEN
Low oxygen concentration in water (hypoxia) and high temperature are becoming more frequent due to climate change, forcing animals to endure stress or decease. Hypoxia and high temperature stress can lead to reactive oxygen species (ROS) accumulation and oxidative damage to the organisms. The shrimp Litopenaeus vannamei is the most cultivated crustacean worldwide. The aim of this study was to evaluate the expression and enzymatic activity of glutathione peroxidase (GPx), catalase (CAT) and cytosolic manganese superoxide dismutase (cMnSOD) in gills and hepatopancreas from L. vannamei in response to two combined stressors: hypoxia and reoxygenation at control and high temperature (28 vs 35 °C, respectively). In addition, glutathione and hydrogen peroxide content were analyzed. The changes were mainly tissue-specific. In gills, cMnSOD expression and enzymatic activity increased in response to the interactions between oxygen variation and thermal stress, while GPx and CAT were maintained. More changes occurred in GPx, CAT and MnSOD in hepatopancreas than in gills, mainly due to the effect of the individual stress factors of thermal stress or oxygen variations. On the other hand, the redox state of glutathione indicated that during high temperature, changes in the GSH/GSSG ratio occurred due to the fluctuations of GSSG. Hydrogen peroxide concentration was not affected by thermal stress or oxygen variations in hepatopancreas, whereas in gills, it was not detected. Altogether, these results indicate a complex pattern of antioxidant response to hypoxia, reoxygenation, high temperature and their combinations.
Asunto(s)
Antioxidantes/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Hipoxia/metabolismo , Oxígeno/metabolismo , Penaeidae/fisiología , Animales , Antioxidantes/química , Catalasa/metabolismo , Branquias/fisiología , Glutatión Peroxidasa/metabolismo , Hepatopáncreas/metabolismo , Homeostasis , Calor , Estrés Fisiológico , Superóxido Dismutasa/metabolismo , TemperaturaRESUMEN
Climate warming has been increasing ocean water temperature and decreasing oxygen concentrations, exposing aquatic organisms to environmental stress conditions. The shrimp Litopenaeus vannamei manages to survive these harsh environmental conditions by enhancing their antioxidant defenses, among other strategies. In this study, we report the mitochondrial manganese superoxide dismutase (mMnSOD) nucleotide and deduced amino acid sequences and its gene expression in L. vannamei tissues. The deduced protein has 220 amino acids with a signal peptide of 20 amino acids. Expression of mMnSOD was analyzed in hepatopancreas, gills and muscle, where gills had highest expression in normoxic conditions. In addition, shrimp were subjected to high temperature, hypoxia and reoxygenation to analyze the effect on the expression of mMnSOD and SOD activity in mitochondria. High temperature and hypoxia showed a synergistic effect in the up-regulation on expression of mMnSOD in gills and hepatopancreas. Moreover, induction in SOD activity was found in the mitochondrial fraction from gills of normoxia at high temperature, probably due to an overproduction of reactive oxygen species caused by an elevated metabolic rate due to the stress temperature. These results suggest that the combined stress conditions of hypoxia and high temperature trigger molecularly the antioxidant response in L. vannamei in a higher degree than only one stressor.
Asunto(s)
Proteínas de Artrópodos , Mitocondrias/metabolismo , Oxígeno , Penaeidae , Superóxido Dismutasa , Temperatura , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , Penaeidae/genética , Penaeidae/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Hypoxia is a frequent source of stress in the estuarine habitat of the white shrimp Litopenaeus vannamei. During hypoxia, L. vannamei gill cells rely more heavily on anaerobic glycolysis to obtain ATP. This is mediated by transcriptional up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The hypoxia inducible factor 1 (HIF-1) is an important transcriptional activator of several glycolytic enzymes during hypoxia in diverse animals, including crustaceans. In this work, we cloned and sequenced a fragment corresponding to the 5' flank of the GAPDH gene and identified a putative HIF-1 binding site, as well as sites for other transcription factors involved in the hypoxia signaling pathway. To investigate the role of HIF-1 in GAPDH regulation, we simultaneously injected double-stranded RNA (dsRNA) into shrimp to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia followed by reoxygenation, and then measured gill HIF-1α, HIF-1ß expression, GAPDH expression and activity, and glucose and lactate concentrations at 0, 3, 24 and 48â¯h. During normoxia, HIF-1 silencing induced up-regulation of GAPDH transcripts and activity, suggesting that expression is down-regulated via HIF-1 under these conditions. In contrast, HIF-1 silencing during hypoxia abolished the increases in GAPDH expression and activity, glucose and lactate concentrations. Finally, HIF-1 silencing during hypoxia-reoxygenation prevented the increase in GAPDH expression, however, those changes were not reflected in GAPDH activity and lactate accumulation. Altogether, these results indicate that GAPDH and glycolysis are transcriptionally regulated by HIF-1 in gills of white shrimp.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Factor 1 Inducible por Hipoxia/genética , Penaeidae/genética , Secuencia de Aminoácidos/genética , Animales , Regulación de la Expresión Génica , Branquias/metabolismo , Glucólisis/genética , Hipoxia/genética , Consumo de Oxígeno/genética , Penaeidae/fisiologíaRESUMEN
Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pKa of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants Km for NADH was 23.4 ± 1.8 µM, and for pyruvate was 203 ± 25 µM, while Vmax was 7.45 µmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia.
Asunto(s)
Proteínas de Artrópodos , Expresión Génica , L-Lactato Deshidrogenasa , Penaeidae/genética , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , L-Lactato Deshidrogenasa/biosíntesis , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/aislamiento & purificación , Penaeidae/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
HIF-1 is a transcription factor that controls a widespread range of genes in metazoan organisms in response to hypoxia and is composed of α and ß subunits. In shrimp, phosphofructokinase (PFK) and fructose bisphosphatase (FBP) are up-regulated in hypoxia. We hypothesized that HIF-1 is involved in the regulation of PFK and FBP genes in shrimp hepatopancreas under hypoxia. Long double stranded RNA (dsRNA) intramuscular injection was utilized to silence simultaneously both HIF-1 subunits, and then, we measured the relative expression of PFK and FBP, as well as their corresponding enzymatic activities in hypoxic shrimp hepatopancreas. The results indicated that HIF-1 participates in the up-regulation of PFK transcripts under short-term hypoxia since the induction caused by hypoxia (~1.6 and ~4.2-fold after 3 and 48h, respectively) is significantly reduced in the dsRNA animals treated. Moreover, PFK activity was significantly ~2.8-fold augmented after 3h in hypoxia alongside to an ~1.9-fold increment in lactate. However, when animals were dsRNA treated, both were significantly reduced. On the other hand, FBP transcripts were ~5.3-fold up-regulated in long-term hypoxic conditions (48h). HIF-1 is involved in this process since FBP transcripts were not induced by hypoxia when HIF-1 was silenced. Conversely, the FBP activity was not affected by hypoxia, which suggests its possible regulation at post-translational level. Taken together, these results position HIF-1 as a prime transcription factor in coordinating glucose metabolism through the PFK and FBP genes among others, in shrimp under low oxygen environments.
Asunto(s)
Fructosa-Bifosfatasa/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Penaeidae/fisiología , Fosfofructoquinasas/metabolismo , Animales , Fructosa-Bifosfatasa/genética , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hepatopáncreas/metabolismo , Hipoxia , Factor 1 Inducible por Hipoxia/genética , Lactatos/metabolismo , Fosfofructoquinasas/genéticaRESUMEN
During hypoxia the shrimp Litopenaeus vannamei accelerates anaerobic glycolysis to obtain energy; therefore, a correct supply of glucose to the cells is needed. Facilitated glucose transport across the cells is mediated by a group of membrane embedded integral proteins called GLUT; being GLUT1 the most ubiquitous form. In this work, we report the first cDNA nucleotide and deduced amino acid sequences of a glucose transporter 1 from L. vannamei. A 1619 bp sequence was obtained by RT-PCR and RACE approaches. The 5´ UTR is 161 bp and the poly A tail is exactly after the stop codon in the mRNA. The ORF is 1485 bp and codes for 485 amino acids. The deduced protein sequence has high identity to GLUT1 proteins from several species and contains all the main features of glucose transporter proteins, including twelve transmembrane domains, the conserved motives and amino acids involved in transport activity, ligands binding and membrane anchor. Therefore, we decided to name this sequence, glucose transporter 1 of L. vannamei (LvGLUT1). A partial gene sequence of 8.87 Kbp was also obtained; it contains the complete coding sequence divided in 10 exons. LvGlut1 expression was detected in hemocytes, hepatopancreas, intestine gills, muscle and pleopods. The higher relative expression was found in gills and the lower in hemocytes. This indicates that LvGlut1 is ubiquitously expressed but its levels are tissue-specific and upon short-term hypoxia, the GLUT1 transcripts increase 3.7-fold in hepatopancreas and gills. To our knowledge, this is the first evidence of expression of GLUT1 in crustaceans.
Asunto(s)
Transportador de Glucosa de Tipo 1/genética , Penaeidae/metabolismo , Regulación hacia Arriba , Secuencia de Aminoácidos , Animales , Hipoxia de la Célula , Clonación Molecular , Secuencia Conservada , Transportador de Glucosa de Tipo 1/metabolismo , Datos de Secuencia Molecular , Penaeidae/genética , FilogeniaRESUMEN
Polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PHE), are common pollutants found in coastal areas where shrimp farming is developed. Even though PAHs can have adverse effects on physiology, shrimp can detoxify and metabolize toxic compounds and neutralize the reactive oxygen species (ROS) produced during this process. This requires the activation of multiple antioxidant enzymes, including peroxiredoxin 6 (Prx6). Prx6 uses glutathione (GSH) to reduce phospholipid hydroperoxides, a function shared with GSH peroxidase 4 (GPx4). Prx6 has been scarcely studied in crustaceans exposed to pollutants. Herein, we report a novel Prx6 from the shrimp Penaeus vannamei that is abundantly expressed in gills and hepatopancreas. To elucidate the involvement of Prx6 in response to PAHs, we analyzed its expression in the hepatopancreas of shrimp sub-lethally exposed to PHE (3.3 µg/L) and acetone (control) for 24, 48, 72, and 96 h, along with GPx4 expression, GSH-dependent peroxidase activity, and lipid peroxidation (indicated by TBARS). We found that GPx4 expression is not affected by PHE, but Prx6 expression and peroxidase activity decreased during the trial. This might contribute to the rise of TBARS found at 48 h of exposure. However, maintaining GPx4 expression could aid to minimize lipid damage during longer periods of exposure to PHE.
RESUMEN
Due to global warming, world water bodies have higher temperatures and lower oxygen concentrations that affect aquatic species including the white shrimp Litopenaeus vannamei. This species withstands these conditions, but the information of the physiological responses that allow them to survive are scarce. We analyzed the effects of high temperature, hypoxia, reoxygenation, and the combination of these factors on the relative expression of selected genes: HSF1, Hsp70, p53, TIGAR, HIF-1α, and VEGF1-3 in gills of L. vannamei. Additionally, glucose, lactate, NADP, and NADPH were determined. HSF1 was up-regulated in the high temperature and oxygen stress conditions, but Hsp70 was up-regulated only in reoxygenation at both temperatures. HIF-1α was also up-regulated by reoxygenation in both temperatures. Meanwhile, the VEGF genes were not altered by the stress conditions, since none of them changed expression drastically. p53 relative expression remained stable at the tested stress conditions, which prompts to the maintenance of antioxidant defenses. TIGAR expression was induced in normoxia and hypoxia at high temperature, which induced NADPH content helping to scavenge reactive oxygen species (ROS). Additionally, high temperature caused higher glucose and lactate content in normoxia and hypoxia, indicating carbohydrate mobilization and a switch to anaerobic metabolism. The results showed that HSF1, the anaerobic metabolism and the pentose phosphate pathway (PPP) are crucial for the shrimp response to these abiotic stress conditions and contribute to their survival.
Asunto(s)
Penaeidae , Vía de Pentosa Fosfato , Animales , Temperatura , Secuencia de Aminoácidos , Anaerobiosis , NADP/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Hipoxia , Oxígeno/metabolismo , Respuesta al Choque Térmico , Glucosa/metabolismo , Penaeidae/genéticaRESUMEN
Glutathione peroxidases (GPxs) are important antioxidant enzymes that act at distinct levels of the antioxidant defense. In vertebrates, there are several glutathione peroxidase (GPx) isoforms with different cellular and tissue distribution, but little is known about their interrelationships. The shrimp Litopenaeus vannamei is the main crustacean cultivated worldwide. It is affected by environmental stressors, including hypoxia and reoxygenation that cause reactive oxygen species accumulation. Thus, the antioxidant response modulation is key for shrimp resilience. Recently, several GPx isoforms genes were identified in the L. vannamei genome sequence, but their functions are just beginning to be studied. As in vertebrates, shrimp GPx isoforms can present differences in their antioxidant responses. Also, there could be interrelationships among the isoforms that may influence their responses. We evaluated shrimp GPx2 and GPx4 expressions during hypoxia, reoxygenation, and GPx4 knock-down using RNAi for silencing, as well as the enzymatic activity of total GPx and GPx4. Also, glutathione content in hepatopancreas was evaluated. GPx2 and GPx4 presented similar expression patterns during hypoxia and reoxygenation. Their expressions decreased during hypoxia and were reestablished in reoxygenation at 6 h in non-silenced shrimp. GPx2 expression was down-regulated by GPx4 knock-down, suggesting that GPx4 affects GPx2 expression. Total GPx activity changed in hypoxia and reoxygenation at 6 h but not at 12 h, while GPx4 activity was not affected by any stressor. The GSH/GSSG ratio in hepatopancreas indicated that at early hours, the redox status remains well-modulated but at 12 h it is impaired by hypoxia and reoxygenation.
Asunto(s)
Antioxidantes , Oxígeno , Animales , Antioxidantes/metabolismo , Oxígeno/metabolismo , Hipoxia/genética , Hipoxia/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Glutatión , Isoformas de ProteínasRESUMEN
The invertebrate lysozyme (i-lyz or destabilase) is present in shrimp. This protein may have a function as a peptidoglycan-breaking enzyme and as a peptidase. Shrimp is commonly infected with Vibrio sp., a Gram-negative bacteria, and it is known that the c-lyz (similar to chicken lysozyme) is active against these bacteria. To further understand the regulation of lysozymes, we determined the gene sequence and modeled the protein structure of i-lyz. In addition, the expression of i-lyz and c-lyz in response to lipopolysaccharide (LPS) was studied. The shrimp i-lyz gene is interrupted by two introns with canonical splice junctions. The expression of the shrimp i-lyz was transiently down-regulated after LPS injection followed by induction after 6 h in hepatopancreas. In contrast, c-lyz was up-regulated in hepatopancreas 4 h post-injection and slightly down-regulated in gills. The L. vannamei i-lyz does not contain the catalytic residues for muramidase (glycohydrolase) neither isopeptidase activities; however, it is known that the antibacterial activity does not solely rely on the enzymatic activity of the protein. The study of invertebrate lysozyme will increase our understanding of the regulatory process of the defense mechanisms.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Modelos Moleculares , Muramidasa/química , Muramidasa/genética , Penaeidae/enzimología , Penaeidae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Hypoxia (<2 mg O2/L) is one of the main environmental stressors that affects aquatic organisms, including the white shrimp (Litopenaeus vannamei). During hypoxia, reactive oxygen species (ROS) accumulation induces oxidative stress and damage to biomolecules. Redox state and ROS overproduction are modulated by the antioxidant system that is composed of several antioxidant enzymes, proteins, and other small compounds. Glutathione peroxidase 4 (GPx4) has emerged as an important antioxidant enzyme with cytoprotective roles. In vertebrates, antioxidant and pro-oxidant stress responses are regulated by several factors, including the p53 protein. However, little is known about GPx4 responses in crustaceans and the regulation by p53. Herein we analyzed and characterized the L. vannamei GPx4 and evaluated the responses to hypoxia and p53 knock-down. We found a unique GPx4 gene that produces five transcript variants (TVs) and only two protein isoforms with distinct cellular localization. GPx4 expression in hepatopancreas during hypoxia and p53 knock-down changed during short and long-term hypoxia, suggesting that GPx4 may be a sensitive indicator of antioxidant imbalance during stress. Knock-down of p53 induced a reduction in GPx4 expression, indicating that p53 modulates GPx4 responses during stress. This agrees with our findings of putative consensus sequences for p53 in the GPx4 gene promoter by in silico analysis. Also, the antioxidant response was effective in preventing major protein damage during hypoxia since no changes were detected in carbonylated proteins content in hepatopancreas during hypoxia. Conversely, p53 knock-down produced significant changes in carbonylated proteins.
Asunto(s)
Hepatopáncreas , Penaeidae , Animales , Antioxidantes/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Hepatopáncreas/metabolismo , Hipoxia/genética , Hipoxia/metabolismo , Penaeidae/genética , Penaeidae/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Some marine crustaceans like the white shrimp Litopenaeus vannamei are tolerant to environmental hypoxia. Under oxygen deprivation, shrimp tissues obtain energy by enhancing anaerobic glycolysis. In mammals, hypoxia increases the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown a "moonlighting" role in cells. However, the effect of hypoxia on the GAPDH expression has not been studied in crustaceans. In the present work, we obtained a 2744 bp gene sequence with a 999 bp ORF split by a single intron. The deduced protein is 332 amino acids and corresponds to the L. vannamei GAPDH (LvGAPDH), which is highly similar in sequence and structure to other animal GAPDHs. During hypoxia, LvGAPDH expression is significantly induced in gills but not in hepatopancreas, suggesting that it may play a role in the molecular and cellular response of shrimp to hypoxia.