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1 December 2013 Effects of 20-Hydroxyecdysone on the Reversible Mitochondrial Transhydrogenase in the Tobacco Hornworm, Manduca sexta
Kurt P. Vandock, Emily C. Perregaux, Brianna M. Consiglio
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Abstract

The reversible, mitochondrial membrane-associated transhydrogenase from the midgut of Manduca sexta (L.) (Lepidoptera: Sphingidae) catalyzes hydride-ion transfer between NADP(H) and NAD(H). The effects of ecdysone and 20-hydroxyecdysone were evaluated and compared to both the NADH-NADP and NADPH-NAD transhydrogenations. In the direction of NADPH-formation, the developmentally significant transhydrogenations occur as non-energy- or energy-linked reactions. The energy-linked activity couples with either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg -dependent ATPase. Upon the addition of ecdysone alone, all energy-linked reactions in the direction of NADPH formation exhibited a notable increase in activity level over the control reaction. The addition of 20-hydroxyecdysone yielded no significant increase in the activity of any of the transhydrogenations. Synergistic addition of both ecdysone and 20-hydroxyecdysone resulted in no significant effect on transhydrogenase activity. The results of this study make evident a relationship between the presence of ecdysone and 20-hydroxyecdysone on the overall activity of M. sexta midgut mitochondrial transhydrogenations. The potential mediation of the energy-linked mitochondrial transhydrogenations involved with NADPH synthesis through the developmental relationship of ecdysone and 20-hydroxyecdysone is considered.

Introduction

The enzyme transhydrogenase (E.C. 1.6.1.1) has been characterized in humans, mammals, insects, bacteria, and parasites (White et al. 2000; Pestov and Shakhparonov 2009; Yousuf et al. 2010). In parasites (e.g., Hymenolepis diminuta), transhydrogenase plays a crucial role in metabolism and serves as an additional site for anaerobic phosphorylation (Park and Fioravanti 2006; Mercer-Haines and Fioravanti 2008; Fioravanti and Vandock 2010). In the tobacco hornworm, Manduca sexta (L.) (Lepidoptera: Sphingidae), the mitochondrial transhydrogenase has been characterized as a membrane-associated, phospholipid-dependent, and energy-linked enzyme that contributes to NADPH synthesis and larval development (Vandock et al. 2011). With regard to M sexta, the mitochondrial transhydrogenase has been shown to facilitate the reversible reaction noted here (Vandock et al. 2008):

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The complete adult life cycle of M. sexta consists of five larval stadia lasting approximately 4–6 days each (Kingsolver 2006). At the end of the fifth larval stadium, the insect molts, forms a pupa, and completes its holometabolous metamorphosis. Achievement of post-embryonic development is dependent upon the presence of the molting hormone, ecdysone (Gilbert et al. 2002). This ecdysteroid targets midgut, fatbody, and Malphigian tubules (Smith 1985; Weirich 1997; Gilbert et al. 2002). The P450-dependent ecdysone 20-monooxygenase (E.C. 1.14.99.22; E-20M) catalyzes the conversion of ecdysone (E) to the active form of the molting hormone 20-hydroxyecdysone (20-HE) (Smith et al. 1979; Feyereisen 2005; Lafont et al. 2005). The transitionfrom larval tissues into pupal tissues is characterized by an increase in the level of E-20M activity and a peak in hemolymph ecdysteroid titer (Lafont et al. 2005). During the fifth larval stadium, midgut E-20M activity increases 50-fold between day four and five, corresponding to the increased expression of the shade gene, which encodes E-20M (Mitchell et al. 1999; Gilbert and Rewitz 2009).

Preliminary developmental studies suggest that the role of M. sexta transhydrogenase in the energy-linked formation of NADPH may serve as a source of this reducing equivalent which in turn is required for the completion of post-embryonic development (Vandock et al. 2010):

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The potential for transhydrogenase to act as a mediator during endocrine-controlled postembryonic development in M sexta is evident. In light of this, studies of known flavonoids that act as inhibitors of both E-20M and transhydrogenase have suggested that altering transhydrogenase activity may provide the means for specific control of insect development (Vandock et al. 2012). The following data demonstrates that both the presence of the ecdysteroids E and 20-HE have a direct effect on the energy-linked activities of M. sexta mitochondrial transhydrogenations.

Materials and Methods

Manduca sexta rearing

Fifth instar M. sexta were selected and raised on an artificial diet in a standard growth incubator. The environment was controlled under non-diapausing conditions at a photo-period of 16:8 L:D with interruption only for care and feeding. Temperature was controlled at 26° C, and relative humidity maintained at ∼60% (Bell and Joachim 1976).

Mitochondrial isolation procedure

Mitochondria from 5th instar M sexta (Day 4) were extracted by a procedure similar to that described by Vandock et al. (2010). Insects were immobilized on ice prior to isolation of the midguts. Midguts were obtained through careful dissection. Subsequently, the midguts were washed and reserved in a mitochondrial medium (9.0 mL/g tissue) consisting of 250 mM sucrose, 15 mM EDTA, and 10 mM Tris-HCl (pH 7.5). Midguts were homogenized in the mitochondrial medium using a Hielscher Ultrasonic Immersion blender ( www.hielscher.com). Cellular debris was separated via differential centrifugation at 480 × g for 10 min. The resulting supernatant was centrifuged for 30 min at 9050 × g to form the mitochondrial pellet. The pellet yielded from the second spin was resuspended in the mitochondrial medium and washed at 11025 × g for