Differential effects of silencing crustacean hyperglycemic hormone gene expression on the metabolic profiles of the muscle and hepatopancreas in the crayfish Procambarus clarkii.

Emanuele Buratti,Yi-Chun Tien,Li-Jane Shih,Kuo-Hsun Chiu,Jean-Yves Toullec,Chi-Ying Lee,Shih-Fu Tsai,Wenfeng Li,Chien-Hsun Lee

doi:10.1371/journal.pone.0172557

Abstract

In order to functionally characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), gene expression of CHH in the crayfish (Procambarus clarkii) was knocked down by in vivo injection of CHH double-stranded RNA (dsRNA), followed by metabolomic analysis of 2 CHH target tissues (the muscle and hepatopancreas) using nuclear magnetic resonance spectroscopy. Compared to the levels in untreated and saline-injected (SAI) animals, levels of CHH transcript, but not those of molt-inhibiting hormone (a CHH-family peptide), in the eyestalk ganglia of CHH dsRNA-injected (DSI) animals were significantly decreased at 24, 48, and 72 hour post injection (hpi), with concomitant changes in levels of CHH peptide in the sinus gland (a neurohemal organ) and hemolymph. Green fluorescence protein (GFP) dsRNA failed to affect levels of CHH transcript in the eyestalk ganglia of GFP DSI animals. Number of metabolites whose levels were significantly changed by CHH dsRNA was 149 and 181 in the muscle and 24 and 12 in the hepatopancreas, at 24 and 48 hpi, respectively. Principal component analysis of these metabolites show that metabolic effects of silencing CHH gene expression were more pronounced in the muscle (with the cluster of CHH DSI group clearly being separated from that of SAI group at 24 hpi) than in the hepatopancreas. Moreover, pathway analysis of the metabolites closely related to carbohydrate and energy metabolism indicate that, for CHH DSI animals at 24 hpi, metabolic profile of the muscle was characterized by reduced synthesis of NAD+ and adenine ribonucleotides, diminished levels of ATP, lower rate of utilization of carbohydrates through glycolysis, and a partially rescued TCA cycle, whereas that of the hepatopancreas by unaffected levels of ATP, lower rate of utilization of carbohydrates, and increased levels of ketone bodies. The combined results of metabolic changes in response to silenced CHH gene expression reveal that metabolic functions of CHH on the muscle and hepatopancreas are more diverse than previously thought and are differential between the two tissues.

Highlights

Crustacean eyestalks contain a neuroendocrine tissue, the X-organ/sinus gland complex, which synthesizes and secrets several neurohormones that are implicated in regulating a wide variety of physiological functions, including metabolism, reproduction, growth, ionic balance, and color changes
Animals of the crustacean hyperglycemic hormone (CHH) double-stranded RNA (dsRNA)-injected (DSI) group exhibited a progressive decrease in CHH transcript levels over a period of 72 h post injection
CHH transcript levels in the saline-injected (SAI) animals were relatively stable from 24 to 72 hpi, not significantly different from those in the untreated animals at any time point examined (82.4 ± 26.4%, 88.5 ± 4.7%, and 70.9 ± 21.1% of the untreated value at 24, 48, and 72 hpi, respectively), but were higher than those in the CHH dsRNA-injected group (DSI) animals at each time point (Fig 1A). These results clearly indicate that expression of CHH gene in the eyestalk ganglia was significantly knocked down by CHH dsRNA treatment

Summary

Introduction

Crustacean eyestalks contain a neuroendocrine tissue, the X-organ/sinus gland complex, which synthesizes and secrets several neurohormones that are implicated in regulating a wide variety of physiological functions, including metabolism, reproduction, growth, ionic balance, and color changes. The wide array of biological processes that are under the hormonal control of the eyestalk factors highlights the importance of the XO/SG complex in crustacean physiology [1,2,3] Among these neuropeptides, crustacean hyperglycemic hormone (CHH) is the prototypic member of the CHH family, which includes molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH), and insect ion transport peptide (ITP) [4, 5]. Most early studies employed tissue extracts (eyestalk ganglia or sinus glands) rather than purified CHH, the combined data are consistent with the notion that CHH mobilizes tissue glycogen store through regulating activity and amount of enzymes in glycogen metabolism. Specific CHH binding sites have been characterized in multiple tissues (including the hepatopancreas and muscle) [24,25,26,27]

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