Adenosine monophosphate-activated protein kinase (AMPK) is the main energy sensor in mammals, but limited information is available regarding its role as an energy sensor in nutrient-restricted fish particularly in period of overwinter starvation. The present study aimed to investigate the role of AMPK in the grass carp Ctenopharyngodon idellus through characterization of AMPK full-length cDNAs and the measurement of transcriptional activity in response to overwinter starvation. AMPK is a heterotrimeric serine/threonine kinase that consists of a catalytic alpha (α) subunit complexed with two regulatory subunits, beta (β) and gamma (γ). In our study, we identified nine isoforms of the AMPK family in grass carp and obtained their complete coding sequences (CDS). In the grass carp, the α subunit is encoded by two isoforms (α1 and α2). The β and γ subunits are encoded by three (β1a, β1b, β2) and four isoforms (γ1, γ2a, γ2b, γ3), respectively. AMPK isoforms in grass carp possess structural features similar to mammalian AMPK and exhibit a high degree of homology with other fish and vertebrate AMPK sequences. The mRNA of nine grass carp AMPK isoforms were found to be expressed in a wide range of tissues in vivo, but the abundance of each AMPK mRNA demonstrated a tissue-dependent expression pattern, indicating that they might be key complexes playing the role of energy metabolism sensors during overwinter starvation conditions. Compared to expression levels in control fish (week 0), the expression of various AMPK isoforms significantly increased in the hepatopancreas of fish exposed to 1 week or more of overwinter starvation conditions as follows: week 1 (AMPK α1 and AMPK α2), week8 (AMPK β1b and AMPK γ2b), week 12 (AMPK β2 and AMPK γ1), and week 16 (AMPK β1a, AMPK γ2a, and AMPK γ3). Additionally, compared to expression levels in control fish (week 0), the expression of various AMPK isoforms significantly increased in the adipose tissue of fish exposed to 1 week or more of overwinter starvation conditions as follows: week 1(AMPK β1a and AMPK β1b), week 4 (AMPK α1, AMPK α2, AMPK γ1, AMPK γ2b and AMPK γ3), and week 8 (AMPK β2 and AMPK γ2a). Further in vitro analysis revealed that the mRNA levels of AMPK isoforms in hepatocytes (AMPK α1, AMPK α2, AMPK β1a, AMPK β1b, AMPK β2, AMPK γ2b and AMPK γ3) and adipocytes (AMPK γ2a, AMPK γ2b and AMPK γ3) changed significantly with in the first 24 h of exposure to the overwinter starvation conditions. These findings confirm that nine AMPK subunits are present in grass carp and that all encode proteins with conserved functional domains. The nine AMPK subunits are all regulated at the transcriptional levels to manage excess energy expenditure during overwinter starvation stress.
Keywords: AMP-activated protein kinase; Ctenopharyngodon idellus; Gene expression; Molecular cloning; Overwinter starvation.
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