Regulation of amino acid and nucleotide metabolism by crustacean hyperglycemic hormone in the muscle and hepatopancreas of the crayfishProcambarus clarkii

Abstract

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-silenced and saline-treated control 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 were summarized and discussed.

The Crustacean Hyperglycemic Hormone Superfamily: Progress Made in the Past Decade

“…The stimulatory effects of CHH on the release of amylase from the hepatopancreas ( 162 ) and hemolymph levels of triacylglycerols and phospholipids ( 163 ) have been reported but not further characterized. Recently, the metabolic roles of CHH in the crayfish P. clarkii were more comprehensively characterized using an RNAi approach (double-stranded RNA) followed by profiling the hepatopancreas and muscle metabolomes ( 77 , 78 ). The combined data indicated that CHH has more diverse effects than previously realized, and the two target tissues are differentially regulated.…”

“…The combined data indicated that CHH has more diverse effects than previously realized, and the two target tissues are differentially regulated. The main effects of CHH are stimulation of glycolysis and lipolysis in the hepatopancreas, and higher rate of utilization of carbohydrates (glucose and other sugars, including fructose, galactose, sucrose, and lactose) via glycolysis and TCA cycle (resulting in higher levels of ATP), stimulation of the pentose phosphate pathway (PPP) flux (leading to increased levels of nucleotide biosynthesis), and elevation of amino acid biosynthesis in the muscle ( 77 , 78 ). Stimulation of the “Nicotinate and nicotinamide metabolism”, which concerns the metabolism of two nicotinamide coenzymes (NAD + and NADP + ), is central to the metabolic effects of CHH in the muscle.…”

“…Stimulation of the “Nicotinate and nicotinamide metabolism”, which concerns the metabolism of two nicotinamide coenzymes (NAD + and NADP + ), is central to the metabolic effects of CHH in the muscle. Thus, the higher levels of NAD + (and higher NAD + /NADH ratio) and NADP + , respectively, drive these fluxes through glycolysis, the TCA cycle, and the PPP ( 77 , 78 ). The tissue-specificity of CHH regulation is consistent with the results showing the transcript expression of carbohydrate metabolism-related enzyme genes were differentially regulated by CHH in the same two tissues of M. japonicus ( 159 ).…”

“…Physiologically adaptive roles of CHHs have been put to test using animal models that undergo life-history stages that presumably require regulation by CHH ( 75 , 76 ). Recently, the metabolic effects of CHH on the muscle and hepatopancreas of the crayfish Procambarus clarkii were comprehensively characterized using a metabolomic analysis, revealing that the effects are more wide-ranging than previously realized and that the two tissues are differentially regulated by CHH ( 77 , 78 ). The metabolic effects of CHH have also been implicated in the pathogenesis of diseases in infected crustaceans ( 77 – 81 ).…”

“…Recently, the metabolic effects of CHH on the muscle and hepatopancreas of the crayfish Procambarus clarkii were comprehensively characterized using a metabolomic analysis, revealing that the effects are more wide-ranging than previously realized and that the two tissues are differentially regulated by CHH ( 77 , 78 ). The metabolic effects of CHH have also been implicated in the pathogenesis of diseases in infected crustaceans ( 77 – 81 ). On the other hand, several studies suggested that CHH modulates the immune functions ( 82 – 84 ), although the proposed immunomodulatory effects of CHH have not been thoroughly assessed in pathologically relevant conditions.…”