Allatostatin-C reversibly blocks the transport of citrate out of the mitochondria and inhibits juvenile hormone synthesis in mosquitoes

https://doi.org/10.1016/j.ibmb.2014.12.003Get rights and content

Highlights

  • Allatostatin-C (AST-C) is a strong and fast reversible inhibitor of JH synthesis in mosquitoes.

  • AST-C has no effect on the activity of JH biosynthetic enzymes.

  • The target of AST-C is located before the entry of acetyl-CoA in the pathway.

  • AST-C blocks the CIC carrier that transports citrate from the mitochondria to the cytosol.

Abstract

Aedes aegypti allatostatin-C (AeaAST-C or PISCF-AST) is a strong and fast reversible inhibitor of juvenile hormone III (JH III) synthesis by the corpora allata (CA) of mosquitoes; however, its mechanism of action remains poorly understood. AeaAST-C showed no inhibitory activity in the presence of any of the intermediate precursors of JH III indicating that the AeaAST-C target is located before the entry of acetyl-CoA in the pathway. Stimulation experiments using different sources of carbon (glucose, pyruvate, acetate and citrate) suggest that AST-C acts after pyruvate is transformed to citrate in the mitochondria. In vitro inhibition of the citrate mitochondrial carrier (CIC) mimicked the effect of AeaAST-C, and was overridden by addition of citrate or acetate. Our results provide compelling evidence that AeaAST-C inhibits JH III synthesis by blocking the CIC carrier that transports citrate from the mitochondria to the cytosol, obstructing the production of cytoplasmic acetyl-CoA that sustains JH III synthesis in the CA of mosquitoes.

Introduction

Allatostatins (AST) are pleiotropic neuropeptides which act as reversible inhibitors of juvenile hormone (JH) synthesis in insects (Stay and Tobe, 2007). JHs are synthesized in the corpora allata (CA), a pair of endocrine glands connected to the brain, and play critical roles as regulators of development and reproduction (Goodman and Cusson, 2012). Three types of ASTs have been described in insects: cockroach ASTs (FGLamides or AST-A), cricket ASTs (W(X)6 amides or AST-B) and Manduca ASTs (PISCFs or AST-C) (Gilbert et al., 2000, Bendena et al., 1999). The mechanism of action of ASTs in the regulation of JH synthesis remains poorly understood (Weaver and Audsley, 2009, Stay and Tobe, 2007); AST-C receptors have been described in two dipteran species, Drosophila melanogaster (DAR-1 and DAR-2) (Kreienkamp et al., 2002) and Aedes aegypti (AeaAST-Cr-A and AeaAST-Cr-B) (Mayoral et al., 2010); they are G-protein-coupled receptors (GPCRs) operating through the cAMP and phosphatidylinositol signaling pathways (Tuteja, 2009).

We have previously described that the biosynthetic activity of the CA in vitro was reversibly inhibited by mosquito brain extracts, as well as by Anopheles gambiae PISCF-AST (Li et al., 2004). A. aegypti AST-C (AeaAST-C) was isolated and characterized by MS/MS analysis (Li et al., 2006). AeaAST-C is a 15 amino acid amidated peptide synthesized by neurosecretory cells in the protocerebral lobes of the brain (Hernandez-Martinez et al., 2005). Maximum level of the peptide in the brain of female mosquitoes is detected at 3 days after adult eclosion, correlating with an increased sensitivity of the CA to inhibition by AeaAST-C (Li et al., 2006). Lastly, the AeaAST-C GPCR receptor expression in CA of mosquitoes was reported (Mayoral et al., 2010). In vitro studies have previously shown that AST inhibition is developmentally dependent (Yagi et al., 2005, Tobe et al., 2000, Weaver et al., 1998, Lorenz and Hoffmann, 1995); the sensitivity to inhibition by AeaAST-C in the in vitro assay also fluctuates, revealing that the mosquito CA is most sensitive during periods of low synthetic activity (Li et al., 2006).

JH III synthesis is very dynamic during the gonotrophic cycle of female mosquitoes; and its regulation involves a complex interplay of changes of precursor pools and enzyme levels (Rivera-Perez et al., 2014, Nouzova et al., 2011). JH III is synthesized through the mevalonate pathway (MVAP), which consists of multiple enzymatic steps through which acetyl-CoA is gradually transformed into the 5-carbon compound isopentenyl-pyrophosphate (IPP), and later onto the 15-carbon farnesyl-pyrophosphate (FPP) (Belles and Piulachs, 2005). In the CA of mosquitoes, FPP is sequentially transformed to farnesol (FOL), farnesal (FAL), farnesoic acid (FA), methyl farnesoate (MF) and JH III (Nouzova et al., 2011). Experimental increases in the magnitude of any individual precursor pool generally raise rate of JH III synthesis, suggesting that enzyme concentrations in the CA are often in excess (Rivera-Perez et al., 2014, Nouzova et al., 2011, Feyereisen et al., 1984).

In the present study, we explored the mechanism of action of AeaAST-C in the CA of female A. aegypti mosquitoes. AST receptors transcripts were detected in both CA and corpora cardiaca (CC). In vitro treatment of the adult CA with AeaAST-C resulted in a fast, strong and reversible inhibition of JH III biosynthesis; which was overridden by stimulation with early or late JH III intermediate precursors. The mitochondrial membrane citrate transport protein (CIC) was established as a potential target for AeaAST-C by assessing the contribution of different carbon metabolites as sources of 2C building blocks for the production of JH III. Pharmacological inhibition of CIC mimicked the effect of AeaAST-C, resulting in abolishment of JH III biosynthesis. This inhibitory effect could be superseded by addition of acetate or citrate, reinforcing the idea that the CIC carrier is a main target for AeaAST-C. Altogether our studies provided compelling evidence that AeaAST-C inhibits JH III synthesis by inactivation of the CIC transporter that mobilizes citrate from the mitochondria to the cytosol, obstructing the production of cytoplasmic acetyl-CoA that sustains JH III synthesis in the CA of mosquitoes.

Section snippets

Insects

A. aegypti of the Rockefeller strain were reared at 28 °C and 80% humidity as previously described (Nouzova et al., 2011). Adult mosquitos were offered a cotton pad soaked in a 20% sucrose solution.

Chemicals

Custom made peptide A. aegypti AST-C (AeaAST-C) (QIRYRQCYFNPISCF) was provided by Biopetide Co. (San Diego, CA), purified by reverse phase liquid chromatography and assessed to be 98% pure by analytical mass spectrometry and amino acid analysis. Stock of aqueous solutions of AeaAST-C were prepared at

AeaAST-C has a reversible inhibitory effect on JH III synthesis

Incubation of Br-CA-CC complexes dissected from 3-day old sugar-fed females with 10−9 M AeaAST-C resulted in over 80% reduction in JH III synthesis (Fig. 1). To test if the effect of AeaAST-C is reversible, Br-CA-CC complexes were first incubated with or without AeaAST-C for a 2 h period. During the first 2 h incubation, JH III released from CA was significantly inhibited in the presence of the synthetic peptide (Fig. 2A). Afterwards, AeaAST-C-treated and control Br-CA-CC complexes were rinsed

AeaAST-C is a fast and strong reversible inhibitor of JH III synthesis in adult female mosquitoes

It took almost 40 years from the first demonstration that neuropeptides influence CA activity (Scharrer, 1952) to the identification and functional characterization of the first FGLamide (Woodhead et al., 1989) and PISCF ASTs (Kramer et al., 1991). These two types of allatostatins are associated with unique GPCR receptor families that have vertebrate orthologs; the FGLamide receptors are related to the vertebrate galanin receptors (Birgül et al., 1999), and PISCF receptors show similarity to

Acknowledgments

This work was supported by NIH Grant No AI 45545 to F.G.N.

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Both authors contributed equally to this work.

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