Two hexokinases of the shrimp Penaeus (Litopenaeus) vannamei are differentially expressed during oxygen limited conditions.

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.

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia.

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.