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Abstract: Hexokinase (HK) catalyzes the phosphorylation of glucose, the first rate-limiting step in glycolysis. HKs are a conserved family of tissue-specific isozymes, from which very little is known in marine crustaceans. This study describes the cloning and characterization of the full-length cDNA sequence for HK from the shrimp Litopenaeus vannamei, the theoretical tridimensional protein structure and response to short-term hypoxia. The full-length cDNA is 1872bp long with an open reading frame of 1452bp coding for a protein of 484 amino acids and predicted molecular mass of 53kDa. Highly conserved amino acid residues are present in the glucose, glucose-6-phosphate, ATP and Mg+2 binding sites. Phylogenetic analysis shows that the shrimp HK is closer to invertebrates than to vertebrate HKs. Hypoxia induced HK expression in gills with the concomitant increase in the specific enzyme activity. In muscle, hypoxia decreased HK mRNAs but increased the enzyme activity. Silencing of the hypoxia inducible factor 1 (HIF-1) affected the expression of HK differentially. In gills, silencing of α or β subunits blocked the induction of HK expression and the enzymatic activity, but not in muscle. This suggests the existence of tissue-specific HK isozymes and post-transcriptional and post-translational regulation of HK. [Copyright &y& Elsevier]

Abstract: Many molecular and physiological responses to hypoxia are controlled by the transcription factor called hypoxia inducible factor 1 (HIF-1). Metabolic responses are particularly important for marine crustaceans that suffer hypoxia in their aquatic environment, including shrimp. HIF-1 is a heterodimer recently described in shrimp and composed by α and β subunits. In the present study, we investigated the effect of silencing HIF-1 using dsRNA in the response to hypoxia in the shrimp Litopenaeus vannamei in vivo. Intramuscular injection of dsRNA for α and β separately, decreased the concentration of the corresponding transcripts in gills from shrimp maintained in normoxia, while no effect was detected in muscle. In contrasts, during hypoxia, silencing of HIF-1α or HIF-1β, did affect the corresponding transcripts. Moreover, silencing of the α and β subunits of HIF-1 affected the concentrations of glucose and lactate in hemolymph and gills from shrimp exposed to hypoxic conditions, giving insights of the action of HIF-1 in these responses. [ABSTRACT FROM AUTHOR]

Abstract: Hypoxia inducible factor 1 (HIF-1) is a key transcription factor that regulates a variety of molecular responses to hypoxia. Some marine crustaceans experience changes of oxygen tension in their aquatic environment, but knowledge about the function and expression of HIF-1 is very limited. HIF-1 is a heterodimer composed by α and β subunits. We report the complete cDNA sequences of HIF-1α and HIF-1β from the white shrimp Litopenaeus vannamei. HIF-1α (LvHIF-1α) is 3672bp and codes for 1050 amino acids, while HIF-1β is 2135bp (LvHIF-1β) and 608 amino acids. Both, the α and β subunits have the helix-loop-helix (bHLH) and PAS domains. HIF-1α also has the oxygen dependent degradation (ODD) and the C-terminal transactivation domain (C-TAD), important for regulation in normoxia. Phylogenetic analyses of the proteins indicate separation of invertebrates from vertebrates. Large differences of HIF-1α and HIF-1β transcripts abundance were detected in gills, hepatopancreas and muscle under normoxia (6mg/L dissolved oxygen, DO) and hypoxia (2.5 and 1.5mg/L DO). HIF-1α was more abundant in gills and HIF-1β in hepatopancreas. Large changes in response to hypoxia were detected for HIF-1α in gills, while HIF-1β remained fairly constant. Glucose and lactate in hemolymph increased rapidly in hypoxia in all cases and up to 4.7 and 5.0-fold, respectively, in response to 1.5mg/L DO for 1h. [Copyright &y& Elsevier]