Regulation of amino acid and nucleotide metabolism by crustacean hyperglycemic hormone in the muscle and hepatopancreas of the crayfish Procambarus clarkia

To comprehensively characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), metabolites in two CHH target tissues of the crayfish Procambarus clarkii, whose levels were significantly different between CHH knockdown and control (saline-treated) animals, were analyzed using bioinformatics tools provided by an on-line analysis suite (MetaboAnalyst). Analysis with Metabolic Pathway Analysis (MetPA) indicated that in the muscle Glyoxylate and dicarboxylate metabolism, Nicotinate and nicotinamide metabolism, Alanine, aspartate and glutamate metabolism, Pyruvate metabolism, and Nitrogen metabolism were significantly affected by silencing of CHH gene expression at 24 hours post injection (hpi), while only Nicotinate and nicotinamide metabolism remained significantly affected at 48 hpi. In the hepatopancreas, silencing of CHH gene expression significantly impacted, at 24 hpi, Pyruvate metabolism and Glycolysis or gluconeogenesis, and at 48 hpi, Glycine, serine and threonine metabolism. Moreover, analysis using Metabolite Set Enrichment Analysis (MSEA) showed that many metabolite sets were significantly affected in the muscle at 24hpi, including Ammonia recycling, Nicotinate and nicotinamide metabolism, Pyruvate metabolism, Purine metabolism, Warburg effect, Citric acid cycle, and metabolism of several amino acids, and at 48 hpi only Nicotinate and nicotinamide metabolism, Glycine and serine metabolism, and Ammonia recycling remained significantly affected. In the hepatopancreas, MSEA analysis showed that Fatty acid biosynthesis was significantly impacted at 24 hpi. Finally, in the muscle, levels of several amino acids decreased significantly, while those of 5 other amino acids or related compounds significantly increased in response to CHH gene silencing. Levels of metabolites related to nucleotide metabolism significantly decreased across the board at both time points. In the hepatopancreas, the effects were comparatively minor with only levels of thymine and urea being significantly decreased at 24 hpi. The combined results showed that the metabolic effects of silencing CHH gene expression were far more diverse than suggested by previous studies that emphasized on carbohydrate and energy metabolism. Based on the results, metabolic roles of CHH on the muscle and hepatopancreas are suggested: CHH promotes carbohydrate utilization in the hepatopancreas via stimulating glycolysis and lipolysis, while its stimulatory effect on nicotinate and nicotinamide metabolism plays a central role in coordinating metabolic activity in the muscle with diverse and wide-ranging consequences, including enhancing the fluxes of glycolysis, TCA cycle, and pentose phosphate pathway, leading to increased ATP supply and elevated protein and nucleic acid turnovers.

Tissue metabolites whose levels were significantly different between the SAI and 117 CHH DSI groups were subsequently analyzed using resources provided by a

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MetPA combines statistical enrichment analysis with pathway topological 124 characteristics to identify the most relevant pathways under the study condition [36].
125 Briefly, the metabolites whose tissue levels were significantly different between SAI 126 and CHH DSI groups were uploaded. After data processing and compound name 127 mapping, pathway analysis was performed using the fruit fly (Drosophila 128 melanogaster) library from the KEGG database [38]. The hypergeometric test was 129 chosen to be pathway analysis algorithm for the over representation analysis and the 130 relative betweenness centrality for pathway topology analysis.

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The Metabolite Set Enrichment Analysis (MSEA) is a tool to test if there are 132 some biologically meaningful groups (e.g., pathways) of metabolites that are 133 significantly enriched. After data processing and compound name mapping,  180 and nucleotide metabolism will be described in details.

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In the muscle at 24 hpi, levels of several amino acids, including alanine, arginine, 182 aspartate, glutamate, glutamine, proline and serine decreased significantly, while 183 those of 5 amino acids or related compounds, asparagine, anserine, histidine, 184 histamine, and -alanine, significantly increased (Fig 1). At 48hpi, the changes were 185 similar to those at 24 hpi, with aspartate no longer significantly decreased and 186 anserine no longer significantly increased (Fig 1).

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In response to silencing of CHH gene expression in the muscle at 24 hpi, 191 thymidine, uracil, urea and uridine (Fig. 2). Similar trends of changes were observed 192 at 48 hpi (Fig. 2).

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In the hepatopancreas, with regard to nucleotide metabolism only levels of 194 thymine and urea were significantly decreased at 24 hpi and no significant change 195 was found at 48 hpi.

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In this study, comprehensive analysis of the metabolite whose levels were  . 3). Thus, a negatively impacted PPP would, as a result of 231 low NADP + levels, decrease nucleotide biosynthesis, which was clearly demonstrated 232 by significantly lower levels of nucleotides across the board (Fig. 2, 3).

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Another pathway that was significantly affected in the muscle by CHH gene 234 silencing at 24 hpi is Pyruvate metabolism (  (Tables 1 and 3, Supplementary Tables1 248 and 3). On the other hand, levels of several amino acids and related metabolites in 249 the muscle were increased after silencing CHH gene expression; these included those 250 of asparagine, anserine, histidine, histamine, and -alanine (Fig. 1) . Thus, an increase in 255 asparagine levels was likely a spillover from a TCA cycle thus inhibited (Fig. 3). In 256 addition, increases in -alanine, histidine, and anserine levels ( Fig. 1) are worth 257 mentioning. Carnosine (not detected) is a dipeptide, highly concentrated in the 258 muscle and brain, consists of -alanine and histidine, and could be converted to 259 anserine via methylation of the histidine residue (Fig. 3)  325 in the 2 target tissues is proposed (Fig. 4).      and free fatty acids are released into the hemolymph and taken up by the muscle 527 where they are further metabolized via glycolysis and TCA cycle, respectively, for 528 ATP production. In the muscle, central to the effects of CHH is a stimulated 529 Nicotinate and nicotinamide metabolism, which provides two nicotinamide 530 coenzymes (NAD + and NADP + ) that drive glycolysis and TCA cycle, and the 531 pentose phosphate pathway, respectively (Li et al., 2017; the present study), 532 resulting in more ATP supply and higher protein and nucleic acid turnover. 533 Additionally, CHH may provide protective effects to the muscle by increasing 534 carnosine levels.