Biochemical and cellular characterization of lipophorin–midgut interaction in the hematophagous Panstrongylus megistus (Hemiptera: Reduviidae)
Introduction
In insects, lipid transport through a circulatory system is accomplished by lipophorin (Lp), the main high-density lipoprotein. It is composed by two non-exchangeable apolipoproteins, apolipophorin I (apoLp-I) and apolipophorin II (apoLp-II). Lipophorin carries a wide variety of lipids, e.g., diacylglycerol, phospholipids, cholesterol and hydrocarbons among others (Soulages and Wells, 1994, Canavoso et al., 2001).
From a physiological view point, lipid loading of lipophorin at the midgut is a critical event during absorption of dietary lipids (Atella et al., 1995, Canavoso and Wells, 2000, Canavoso and Wells, 2001). Thus, lipophorin loads absorbed dietary lipids at the midgut, which in turn will be unloaded in target tissues for storage or utilization. Unlike mammalians, where dietary lipids are co-assembled into chylomicrons, in insects the process of lipid transfer from midgut to lipophorin does not involve the synthesis of a lipoprotein. Rather, lipids are directly transferred to circulating lipophorin, without being internalized or degraded. Thus, lipophorin functions as a reusable shuttle, cycling from sites of absorption or storage to sites of utilization (Ryan and Van der Horst, 2000, Arrese et al., 2001, Canavoso et al., 2001).
In several species, it has been shown that interaction of lipophorin with tissues is mediated by specific binding sites (Tsuchida and Wells, 1990, Dantuma et al., 1996, Grillo et al., 2003, Lee et al., 2003a). In addition, it seems to be restricted to the surface of cells (Van Antwerpen et al., 1988, Bauerfeind and Komnick, 1992), in agreement with its role as reusable lipid shuttle. An alternative mechanism of lipid delivery in insects was proposed almost a decade ago, when an endocytic lipophorin receptor (LpR) was cloned and sequenced from fat bodies of Locusta migratoria (Dantuma et al., 1999). Thereafter, cloning and sequencing of LpRs have been reported for some species such as Aedes aegypti (Cheon et al., 2001); Galleria mellonella (Lee et al., 2003b); Bombyx mori (Gopalapillai et al., 2006) and Blatella germanica (Ciudad et al., 2007), although their physiological relevance during lipid metabolism is still unclear. All of them are members of the low density lipoprotein (LDL) receptor family, but to date, LpR from locust constitutes the only one for which its cellular processing has been characterized (Rodenburg and Van der Horst, 2005).
The hematophagous Panstrongylus megistus is an important vector of Chagas' disease in South America. As most triatomines, this species takes large blood meals, abundant in lipids. During the digestion of dietary lipids the enterocytes experience a noticeable remodeling when they take up lipids from the lumen (Canavoso et al., 2004a). Storage of lipids in the enterocytes is transient, since they will be exported to circulating lipophorin. Recently we demonstrated that both, phosphatidate phosphohydrolase and triacylglycerol lipase are important in regulating intracellular lipid synthesis and mobilization in the midgut of P. megistus (Canavoso et al., 2004a). However, exploration of the mechanisms mediating lipophorin binding at the midgut remains very scarce. At present, our knowledge is limited to Aeshna cyanea (Bauerfeind and Komnick, 1992); Manduca sexta (Gondim and Wells, 2000) and to the hematophagous hemipteran Rhodnius prolixus (Grillo et al., 2003).
In order to better understand the metabolism of dietary lipids in the midgut of hematophagous insects, we have performed a biochemical and cellular characterization of lipophorin–midgut interaction in the vector P. megistus. The physiological relevance of results during the process of lipid digestion and absorption is discussed.
Section snippets
Chemicals
Oregon Green 514 carboxylic acid (OG) and 4',6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, OR, USA), Iodo-gen and D-Salt Excellulose desalting column (Pierce, Rockford, IL, USA), microtiter plates (Delta Lab, Barcelona, Spain), Sephadex G-25 PD-10 columns (Pharmacia, Uppsala, Sweden), Tissue-Tek embedding medium (OCT) (Miles, Elkhart, IN, USA), cell strainers (Becton Dickinson, Franklin Lakes, NJ, USA), enhanced chemiluminescence (ECL) detection kit (PerkinElmer, Waltham, MA,
Interaction of lipophorin with midgut membranes
In this study, the characterization of lipophorin–midgut interaction was analyzed using two types of binding assays. In the solid-phase assays, isolated membranes were immobilized in microplates and then, they were incubated with the ligand. This convenient technique has gained increasing importance due to sensibility, reliability and ease of handling (Fuchs et al., 1995). In addition, quantitative data of binding and the specificity of lipophorin–midgut interaction were assessed using a
Discussion
The hematophagous P. megistus is an important vector of Chagas' disease in South America. At present, the disease affects about 10 million individuals while more than 50 million are at risk of contracting the infection (Schofield et al., 2006).
Like most triatomines, P. megistus takes large blood meals, abundant in lipids. During the digestion of dietary lipids, a process that in this species takes about 20 days, midgut cells transiently store absorbed lipids which will be exported to
Acknowledgements
LEC and ERR dedicated this work in memory of Dr. Michael A. Wells (1938–2006). Authors thank Drs. P. Scaraffía and B. Settembrini for their suggestions and critical reading of the manuscript, the two anonymous reviewers for helpful comments and Dr. O. Romero for the gamma counter facility. Authors also thank R. Stariolo for rearing of insects as well as J. Leyria, J.P. Nicola and S. Aguirre for laboratory assistance. This work was supported by Grants from Consejo Nacional de Investigaciones
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