Comparative Biochemistry and Physiology Part B 136 (2003) 217–226
Review
Insect adipokinetic hormones: release and integration of flight
energy metabolism夞
Dick J. Van der Horst*
Department of Biochemical Physiology and Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht 3584 CH,
The Netherlands
Received 21 February 2003; received in revised form 26 May 2003; accepted 26 May 2003
Abstract
Insect flight involves mobilization, transport and utilization of endogenous energy reserves at extremely high rates.
Peptide adipokinetic hormones (AKHs), synthesized and stored in neuroendocrine cells, integrate flight energy
metabolism. The complex multifactorial control mechanism for AKH release in the locust includes both stimulatory and
inhibitory factors. The AKHs are synthesized continuously, resulting in an accumulation of AKH-containing secretory
granules. Additionally, secretory material is stored in large intracisternal granules. Although only a limited part of these
large reserves appears to be readily releasable, this strategy allows the adipokinetic cells to comply with large variations
in secretory demands; changes in secretory activity do not affect the rate of hormone biosynthesis. AKH-induced lipid
release from fat body target cells has revealed a novel concept for lipid transport during exercise. Similar to sustained
locomotion of mammals, insect flight activity is powered by oxidation of free fatty acids derived from endogenous
reserves of triacylglycerol. However, the transport form of the lipid in the circulatory system is diacylglycerol (DAG)
that is delivered to the flight muscles associated with lipoproteins. While DAG is loaded onto the multifunctional insect
lipoprotein, high-density lipophorin (HDLp) and multiple copies of the exchangeable apolipoprotein III (apoLp-III)
associate reversibly with the expanding particle. The resulting low-density lipophorin (LDLp) specifically shuttles DAG
to the working muscles. Following DAG hydrolysis by a lipophorin lipase, apoLp-III dissociates from the particle,
regenerating HDLp that is re-utilized for lipid uptake at the fat body cells, thus functioning as an efficient lipid shuttle
mechanism. Many structural elements of the lipoprotein system of insects appear to be similar to their counterparts in
mammals; however, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly
different.
䊚 2003 Elsevier Science Inc. All rights reserved.
Keywords: Neuropeptides; Secretory granules; Lipoprotein; Lipophorin; Apolipophorin III; Locusta migratoria; Exercise; Lipid shuttle
Abbreviations: AAP, AKH-associated peptide; AKH, adipokinetic hormone; apoLp, apolipophorin; APRP, AKH-precursor-related
peptide; CCAP, crustacean cardio active peptide; DAG, diacylglycerol; DMPC, dimyristoylphosphatidylcholine; FFA, free fatty acids;
HDLp, high-density lipophorin; HSL, hormone-sensitive lipase; ICG, intracisternal granule; iLR, insect lipophorin receptor; JP, joining
peptide; LDLp, low-density lipophorin; Lom-TK, locustatachykinin; LTP, lipid transfer particle; Q-TOF, quadrupole time of flight;
TAG, triacylglycerol
夞 This paper is based on a lecture presented during the symposium on ‘Neuropeptides integrating physiological, processes in
invertebrates: an evolutionary and comparative approach’ at the American Physiological Society Intersociety Meeting 2002 held in
San Diego, California, USA, August 24–28, 2002.
*Tel.: q31-30-2533723; fax: q31-30-2532837.
E-mail address: [email protected] (D.J. Van der Horst).
1096-4959/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S1096-4959(03)00151-9
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D.J. Van der Horst / Comparative Biochemistry and Physiology Part B 136 (2003) 217–226
1. Introduction
Flight activity of insects involves the highest
metabolic rates in nature, rendering flying insects
fascinating, yet relatively simple model animals
for studying metabolic key processes and their
regulation by metabolic neurohormones. In insects
engaging in long-distance flights, for which the
migratory locust, Locusta migratoria, constitutes a
well-accepted model insect, the dramatic increase
in the fuel demand by the flight muscles is sustained for extended periods. The relative simplicity
of the insect system offers ample possibilities to
unravel the molecular mechanisms of the consecutive processes pertinent to energy metabolism,
such as release of neurohormones, mobilization of
stored reserves and transport of substrates, while
the insight obtained may be important also to more
complex vertebrate systems. The set of consecutive
processes that provides an integrated model for
metabolic regulation is summarized in Fig. 1.
Initiation of flight activity induces the release of
peptide neurohormones (adipokinetic hormones,
AKHs) from the intrinsic AKH-producing cells
(adipokinetic cells) in the glandular lobes of the
corpus cardiacum, a neuroendocrine gland located
caudally to the insect brain and physiologically
equivalent to the pituitary of mammals. The fat
body, which combines many of the properties and
functions of vertebrate liver and adipose tissue,
plays a fundamental role in lipid storage, as well
as in the process of lipolysis controlled by the
AKHs. Binding of these hormones to their G
protein-coupled receptors at the fat body target
cells, triggers a number of coordinated signal
transduction processes that ultimately result in the
mobilization of carbohydrate and lipid reserves as
fuels for flight activity. Both substrates are transported via the circulatory system (hemolymph) to
the contracting flight muscles. Carbohydrate (trehalose) in the circulation is replenished from
glycogen reserves while it provides the energy for
the initial period of flight. However, like during
sustained activity in many animal species, the work
involved during the vast distances covered nonstop by flight activity is powered principally by
mobilization of endogenous reserves of triacylglycerol (TAG), the most concentrated form of energy
available to biological tissues. As a result of TAG
mobilization, the concentration of sn-1,2-diacylglycerol (DAG) in the hemolymph increases progressively and gradually constitutes the principal
fuel for flight. The mechanism for hormonal activation of glycogen phosphorylase, the enzyme
Fig. 1. Schematic overview of AKH-controlled substrate mobilization from locust fat body during flight activity. AKHs: adipokinetic
hormones; R: receptor; G: G protein; HDLp: high-density lipophorin; LDLp: low-density lipophorin; apoLp-III: apolipophorin III; FFA:
free fatty acids. (After Van der Horst et al., 2001).
D.J. Van der Horst / Comparative Biochemistry and Physiology Part B 136 (2003) 217–226
219
Fig. 2. Sequence and proteolytic processing of Locusta migratoria AKH prohormones. The AKH sequences (boxed and shaded) are
followed by a processing site (GKR or GRR), the two basic amino acids (KR or RR) of which are removed while G is used for
amidation of the C-terminal amino acid of the AKH molecules. The resulting parts of the AKH prohormones are the AKH-associated
peptides (AAP I, II and III); identical residues in AAP I and II are shaded. Both AAP I and II are further processed to smaller joining
peptides (JP) (boxed; broken lines). The cysteine residues forming disulfide bridges between AKH-I andyor -II prohormone molecules
and between two AKH-III molecules prior to proteolytic processing are shown in white (Based on Bogerd et al., 1995; Baggerman et
al., 2002).
determining the rate of glycogen breakdown and
trehalose biosynthesis from the resulting glucosyl
units, has been well established. In contrast, little
is known of the mechanism by which the pivotal
enzyme, TAG lipase, catalyzes AKH-controlled
production of the DAG, on which long-distance
flight depends.
The action of AKHs on lipid mobilization has
uncovered an alternative mechanism for lipid
transport during exercise, which utilizes multifunctional lipoprotein and high-density lipophorin, to
load the DAG at the fat body cells and to transport
the lipid in the hemolymph. At the flight muscles,
DAG-derived fatty acids are oxidized for energy
generation; however, the lipophorin is regenerated
and recycled to the fat body for another round of
lipid uptake, thus acting as an efficient lipid
shuttle.
The success of insects such as the migratory
locust in long-distance flights is attributable to the
system of multiple neuropeptide AKHs integrating
flight energy metabolism, but equally to the efficiency of the processes involved in the transfer of
energy substrates, particularly lipids, to the flight
muscles. Therefore, this short review will focus on
recent advances in both the strategy of adipokinetic
cells in hormone storage and release, and the
molecular basis of the reversible lipophorin conversions in the insect blood.
2. Strategy of the adipokinetic cells
In view of their involvement in the regulation
and integration of extremely intense metabolic
processes, the AKH-producing cells (adipokinetic
cells) of the locust corpus cardiacum, constitute a
highly appropriate model system for studying neuropeptide biosynthesis and processing, as well as
for analyzing the coherence between biosynthesis,
storage and release of these neurohormones. Insect
AKHs are short peptides consisting of 8–11 amino
acid residues. To date the structures of over 35
different AKHs are known from representatives of
most insect orders, in spite of considerable variation in their structures they are clearly related
(reviewed in Van der Horst et al., 2001; Oudejans
and Van der Horst, 2003). All AKHs are Nterminally blocked by a pyroglutamate (pGlu)
¨
residue and all but one (Kollisch
et al., 2000) are
C-terminally amidated. The AKHs, synthesized in
the locust adipokinetic cells, consist of a decapeptide AKH-I and two octapeptides (AKH-II and
-III); the transport of these blocked peptides in the
circulation occurs independent of a carrier (Oudejans et al., 1996). AKH-I is by far the most
abundant peptide; the ratio of AKH-I:AKHII:AKH-III in the corpus cardiacum is approximately 14:2:1 (Oudejans et al., 1993). These
peptides are derived from preprohormones translated from separate mRNAs and subsequently
enzymatic processed. Co-translational cleavage of
the signal sequences generates the AKH-I, -II and
-III prohormones, consisting of a single copy of
AKH, a GKR or GRR processing site and an
AKH-associated peptide (AAP). The AKH-I and
-II prohormones are structurally very similar
whereas that of AKH-III is remarkably different
(Bogerd et al., 1995; see Fig. 2). Prior to further
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processing, the AKH-I and -II prohormones dimerize at random by oxidation of their (single) cysteine residues in the AAP, giving rise to two
homodimers and one heterodimer. Proteolytic processing of these dimeric products at their processing
sites, involving removal of the two basic amino
acid residues and amidation using Gly as the donor,
yields the bioactive hormones as well as three
(two homodimeric and one heterodimeric) AKHprecursor-related peptides (APRPs) with as yet
unknown functions (reviewed in Van der Horst et
al., 2001; Diederen et al., 2002; Oudejans and Van
der Horst, 2003). Recent data from capillary liquid
chromatography-tandem mass spectrometry analysis, indicate that these APRPs are further processed
to form smaller peptides, designed AKH joining
peptide 1 (AKH-JP I) and 2 (AKH-JP II), respectively, (Baggerman et al., 2002; see Fig. 2). The
biosynthesis of AKH-III from its prohormone has
only very recently been disclosed (Huybrechts et
al., 2002). By use of sophisticated techniques
including capillary HPLC and nanoflow electrospray ionization Q-TOF mass spectrometry, another (fourth) APRP was identified, a homodimer
resulting from the crosslinking of two AKH-III
prohormone molecules by two disulfide bridges
(in a parallel andyor antiparallel fashion) and
subsequent proteolytic cleavage of the AKH-III
molecules. This finding indicates that the processing of AKH-III prohormone occurs similar to that
of the AKH-I and -II prohormones. However, in
contrast to the APRPs derived from AKH-I and
-II prohormones, no evidence was found for further
processing of the APRP generated along with
AKH-III production.
In situ hybridization showed that the mRNA
signals encoding the three different AKH preprohormones are co-localized in the cell bodies of the
glandular lobes of the corpus cardiacum (Bogerd
et al., 1995). Following their synthesis in the rough
endoplasmic reticulum, the AKH prohormones are
transported to the Golgi complex and packaged
into secretory granules at the trans-Golgi network,
whereas proteolytic processing of the prohormones
to bioactive AKHs is presumed to take place in
the secretory granules (reviewed in Van der Horst
et al., 2001; Diederen et al., 2002; Oudejans and
Van der Horst, 2003). The intracellular location of
the AKHs was probed by using antibodies specific
for the corresponding associated peptides (AAP I,
II and III), whose amino acid sequences of which
differ to a larger degree from each other than those
of the AKHs. All three (dimeric) AAPs are colocalized in the same secretory granules, which
implies that also the three AKHs are co-localized
in these granules and are released during flight
(Harthoorn et al., 1999). Since the membranes of
exocytosed secretory granules fuse with the plasma
membrane, the total content of the granules is
released into the hemolymph. Consequently, in
addition to the bioactive AKHs, also the APRPs
and possibly, other (rest-) products are released.
Whether the AKH-JPs are released is not yet clear
(Baggerman et al., 2002; Huybrechts et al., 2002);
data on AKH-JP I and II indicate that these
peptides neither stimulate lipid release from the
fat body nor activate fat body glycogen phosphorylase, which are both key functions of the AKHs
(Baggerman et al., 2002).
The AKH cells continuously synthesize AKHs,
resulting in a steady increase in the amounts of
the three hormones in the corpus cardiacum with
age. Concurrently, also the number of the AKHcontaining secretory granules (diameter approx.
300 nm) increases. In addition, particularly in older
adults, intracisternal granules (ICGs) are produced.
ICGs are present in both exocrine and endocrine
cells and originate from premature condensation
of peptidergic products within cisternae of the
rough endoplasmic reticulum. In the locust adipokinetic cells, these granules that may attain diameters up to 5 mm and even more, appear to function
as a store for AKH prohormones, at least for
AKH-I and -II as shown immunocytochemically
with specific anti-AAPs (Harthoorn et al., 1999,
2000). The prohormone for AKH-III is absent,
pointing to differences in physiological function
between AKH-III and the other two AKHs.
The secretory activity of the adipokinetic cells,
which has been investigated in vitro, primarily for
AKH-I, is subject to many regulatory substances,
including neurogenic locustatachykinins (LomTKs) and humoral crustacean cardioactive peptide
(CCAP) as initiating factors, trehalose as an inhibitor and several positive and negative modulators
(reviewed in Vullings et al., 1999; Van der Horst
et al., 1999). Recent data on the release of AKH
from the corpora cardiaca in vitro show that
regulatory substances affect the release of all three
AKHs proportionally (Harthoorn et al., 2001). The
only natural stimulus for the release of the AKHs
is flight activity and the relative contributions of
D.J. Van der Horst / Comparative Biochemistry and Physiology Part B 136 (2003) 217–226
all known substances effective in the process of
release of these neurohormones remain to be established in vivo.
Although the amount of AKHs released during
flight represents only a few percent of the huge
stores harbored in the adipokinetic cells, we have
investigated the question of whether such a secretory output would induce a stimulation of the rate
of AKH biosynthesis. However, the mRNA levels
of all three AKH preprohormones appeared to be
not affected by flight activity, while the rate of
synthesis of AKH prohormones and AKHs was
not affected either (Harthoorn et al., 2001). Apparently, a coupling between release and biosynthesis
of AKHs is absent (Fig. 3a).
However, in studies, where young secretory
granules were labeled, we have shown that these
newly formed secretory granules are preferentially
released (reviewed in Van der Horst et al., 2001;
Diederen et al., 2002; Oudejans and Van der Horst,
2003). Following the biosynthesis of new AKH
prohormones, their packaging into secretory granules and their processing to bioactive AKHs, which
takes less than 1 h, granules containing newly
synthesized AKHs appeared to be available for
release during a restricted period (approx. 8 h),
before they are supposed to enter a pool of older
secretory granules that appear to be unable to
release their content upon secretory stimulation.
This indicates that only a relatively small, readily
releasable pool of new secretory granules exists.
Inhibition of the AKH biosynthesis in vitro by
Brefeldin A, a specific blocker of the transport of
newly synthesized secretory proteins from the
endoplasmic reticulum to the Golgi complex,
resulted in a considerable decrease in the release
of AKHs induced by CCAP and highlighted once
more that the regulated secretion of AKHs is
completely dependent on the existence of a readily
releasable pool of newly formed secretion granules
(Harthoorn et al., 2002). Therefore, we conclude
that the strategy of the adipokinetic cells to cope
with variations in secretory output of AKHs, apparently, is to rely on continuous biosynthesis of
AKHs, which produces a readily releasable pool
that is sufficiently large and constantly replenished
(Fig. 3b).
An important question remaining unanswered
is, what might be the rationale for the storage of
such large quantities of hormones that are not
accessible for secretory release. In addition, the
possible function of the prohormones for AKH-I
221
and -II in the ICGs, in providing an additional
source of AKH prohormones when called upon,
remains to be established.
3. Effect of adipokinetic hormone on lipid
mobilization
Binding of the AKHs to their plasma membrane
receptor(s) at the fat body cells is the primary step
to the induction of signal transduction events that
ultimately lead to the activation of target key
enzymes and the mobilization of lipids as a fuel
for flight. The AKHs constitute extensively studied
neurohormones and their actions have been shown
to occur via G protein-coupled receptors (reviewed
in Vroemen et al., 1998; Van Marrewijk and Van
der Horst, 1998), the general properties of which
are remarkably well conserved during evolution
(reviewed in Vanden Broeck, 2001). Yet, the
identification of these receptors has not been successful. However, very recently, the first insect
AKH receptors have been identified at the molecular level, namely those of the fruitfly Drosophila
melanogaster and the silkworm Bombyx mori
(Staubli et al., 2002). They appear to be structurally related to mammalian GnRH receptors. These
data provide possibilities to clone AKH receptors
from other insects; it is envisaged that insects such
as the locust, that produce two or more different
types of AKH, have two or more different AKH
receptors.
The signal transduction mechanism of the three
locust AKHs has been studied extensively and
involves stimulation of cAMP production, which
is dependent on the presence of extracellular
Ca2q. Additionally, the AKHs enhance the production of inositol phosphates, including inositol
1,4,5-trisphosphate (IP3 ), which may mediate the
mobilization of Ca2q from intracellular stores. This
depletion of Ca2q from intracellular stores stimulates the influx of extracellular Ca2q, indicative of
the operation of a capacitative (store-operated)
calcium entry mechanism. The interactions
between the AKH signaling pathways ultimately
result in the mobilization of stored reserves as
fuels for flight (reviewed in Vroemen et al., 1998;
Van Marrewijk and Van der Horst, 1998; Van der
Horst et al., 2001; Van Marrewijk, 2003). The
concentration of DAG in the hemolymph is
increased progressively at the expense of stored
TAG reserves in the fat body, which implies
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D.J. Van der Horst / Comparative Biochemistry and Physiology Part B 136 (2003) 217–226
Fig. 3. Schematic representation of the strategy of the adipokinetic cells in the release and biosynthesis of AKHs during flight activity.
(a) Absence of coupling between the secretory output of AKHs and the rate of AKH biosynthesis. (b) Despite the abundant stores of
AKHs in the adipokinetic cells, only a small pool of newly formed AKHs is readily releasable; the older granules constitute an
unreleasable pool. (Based on Harthoorn et al., 2001, 2002).
hormonal activation of the key enzyme, fat body
TAG lipase. In a bioassay, all three AKHs are able
to stimulate lipid mobilization, although their relative potencies differ, as inferred from the doseresponse curves (Fig. 4). This recalls the concept
of a hormonally redundant system, involving multiple regulatory molecules with overlapping actions
(reviewed in Goldsworthy et al., 1997; Vroemen
et al., 1998). Results obtained with combinations
of two or three AKHs, which are likely to occur
together in locust hemolymph in vivo, revealed
that the maximal responses for the lipid-mobilizing
effects were much lower than the theoretically
calculated responses, based on the dose-response
curves for the individual hormones. In the lower
(probably physiological) range, combinations of
the AKHs were more effective than the theoretical
values calculated from the responses elicited by
the individual hormones (reviewed in Van Marrewijk and Van der Horst, 1998).
The mechanism by which TAG lipase catalyzes
AKH-controlled production of the DAG on which
long-distance flight depends, is only poorly understood, mainly due to technical problems in isolating or activating the lipase. In vertebrates,
hormone-sensitive lipase (HSL) controls mobilization of TAG stores in adipose tissue and although
contrary to insects, free fatty acids (FFA) are
released into the blood for uptake and oxidation
in muscle, there is a clear functional similarity
between vertebrate adipose tissue HSL and insect
Fig. 4. Dose-response curves for the lipid mobilizing effects of
AKH-I, -II and -III in L. migratoria. Adult male locusts were
injected with different doses of AKH or in the controls with
saline and after 120 min the lipid content in hemolymph was
determined. Responses represent increases in hemolymph lipid
in AKH-injected locusts, expressed as gyl. For clarity, data
points and standard error bars were omitted. ED50: effective
dose eliciting 50% of the maximum response. (After Van Marrewijk and Van der Horst, 1998).
D.J. Van der Horst / Comparative Biochemistry and Physiology Part B 136 (2003) 217–226
223
fat body TAG lipase (reviewed in Ryan and Van
der Horst, 2000; Van der Horst et al., 2001).
4. Adipokinetic hormone-induced lipophorin
conversions
For insect species that recruit fat body TAG
depots to power their flight muscles during the
vast distances covered non-stop by migratory
flight, an efficient mechanism for lipid transfer is
a premier issue. The action of AKHs on lipid
release has revealed a novel concept for lipid
transport in the circulation of animal organisms
during exercise. Insect hemolymph generally contains abundant amounts of a single multifunctional
lipoprotein particle and high-density lipophorin
(HDLp), performing the tasks of transporting dietary and endogenously produced lipids to peripheral tissues during all developmental stages
(reviewed in Ryan and Van der Horst, 2000; Van
der Horst et al., 2001). A characteristic feature of
HDLp is its ability to function as a reusable vehicle
for a variety of lipids by the selective loading and
unloading of lipid components at target tissues.
The transport of the AKH-enhanced release of
DAG into the hemolymph requires the transformation of lipophorin, which is capable of alternating between a relatively lipid-poor form (HDLp)
and a lipid-enriched form (low-density lipophorin,
LDLp). In these reversible conversions, the
exchangeable apolipoprotein, apoLp-III, which
exhibits a dual capacity to exist in both lipidbound and lipid-free states, plays an essential role
in stabilizing the lipid-enriched particle. A schematic overview of the process is depicted in Fig.
5 (cf. also Fig. 1).
HDLp (density approx. 1.12 gyml) generally
comprises two non-exchangeable apolipoproteins,
apolipophorin I (apoLp-I; mol. mass approx. 240
kDa) and apolipophorin II (apoLp-II; mol. mass
approx. 80 kDa). Studies on locust apolipophorin
biosynthesis in the fat body revealed that apoLp-I
and -II are derived from a common precursor
through posttranslational cleavage (Weers et al.,
1993; Bogerd et al., 2000). In addition, to phospholipids, DAG constitutes a major lipid component, whereas smaller amounts of hydrocarbons
and sterols are present. The loading of HDLp in
the circulation with additional DAG, mobilized by
the fat body cells in response to AKH, requires a
transfer of lipid between cells and HDLp. Lipid
Fig. 5. Molecular basis of the lipophorin lipid shuttle: schematic overview of AKH-controlled reversible alternation of
lipophorin in a relatively lipid-poor (HDLp) and a lipid-rich
(LDLp) state, and apoLp-III in a lipid-free and lipid-bound
state. DAG: diacylglycerol; apoLp-I, -II, and -III: apolipophorin I, II and III.
transfer particle (LTP), a high molecular weight
complex of three apoproteins and lipid, synthesized
in the fat body and secreted into the hemolymph,
plays an essential role in this process; the precise
mechanism of LTP-mediated lipid transfer, however, is not yet completely understood (reviewed
in Ryan and Van der Horst, 2000; Arrese et al.,
2001).
Insect apoLp-III (approx. 18–20 kDa) is one of
the best examples of the reversible existence of
exchangeable apolipoproteins in lipid-free and
lipoprotein-associated states and serves as a model for
studies of apolipoproteins with lipoprotein surfaces
(reviewed in Van der Horst et al., 1993; Soulages
and Wells, 1994; Ryan, 1994; Ryan and Van der
Horst, 2000; Van der Horst et al., 2001). L.
migratoria apoLp-III represents the only fulllength apolipoprotein of which the thee-dimensional structure has been determined by X-ray
crystallography (Breiter et al., 1991). The protein
is a five-helix bundle; its helices are organized
such that their hydrophobic faces are oriented
toward the center of the bundle while their hydrophilic faces are exposed to the aqueous
environment.
Recent advances on the structural properties of
HDLp and apoLp-III demonstrate a remarkable
similarity to their counterparts in the mammalian
system: the apolipoprotein precursor of HDLp to
the non-exchangeable apoB100 in human (very)
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low-density lipoprotein ((V)LDL) (Babin et al.,
1999; Van der Horst et al., 2002) and apoLp-III
to the 22 kDa N-terminal domain of human apoE
(Wilson et al., 1991; Narayanaswami and Ryan,
2000; Ryan and Van der Horst, 2000). This similarity suggests that the information emerging from
the insect system, additionally to its intrinsic value,
is directly applicable to analogous proteins in
mammals. However, the physiological functioning
of the insect lipoprotein as a shuttle mechanism
operating in energy transport during flight activity
is notably different, since in mammals’ lipoproteins play no role as a carrier of mobilized lipids
during exercise.
A conspicuous phenomenon pertinent to the
lipid shuttle mechanism is the association of the
exchangeable apoLp-III with the expanding surface
of the lipophorin particle as a function of the
loading with additional DAG, which is partitioned
between the hydrophobic core and the surface
phospholipid monolayer of the particle. The molecular basis of this interaction of apoLp-III with the
lipoprotein surface is of great interest and value
for our understanding of exchangeable apolipoprotein function. The molecular architecture of apoLpIII explains the water solubility of the protein in
the absence of lipid and additionally allows for
postulating that conformational changes of the
protein accompany its association with lipid. It has
been proposed that apoLp-III undergoes a pronounced lipid-triggered opening of the helix bundle about putative hinge loops, resulting in
exposure of its hydrophobic interior and interaction
of the hydrophobic face of the helices with the
lipid surface. A plausible model, based particularly
on the interaction of apoLp-III with model phospholipid discoidal bilayer particles consisting of
dimyristoylphosphatidylcholine (DMPC), postulated that the conformational change in the bundle
of five a-helices (H1–H5) would be such that H1,
H2 and H5 move away from H3 and H4 (reviewed
in Narayanaswami and Ryan, 2000; Ryan and Van
der Horst, 2000; Van der Horst et al., 2001, 2002).
Recently, however, additional models for the lipidassociated conformation of the apolipoprotein have
been proposed, involving repositioning of H1 away
from H5 upon encountering a lipid surface and
repositioning of H2 and H3, resulting in a fully
extended helical conformation of apoLp-III around
the periphery of the discoidal DMPC bilayer particles; neighboring molecules are aligned antipar-
allel with respect to each other, possibly offset by
one helical segment (Garda et al., 2002; Sahoo et
al., 2002). However, the different models proposed
may result from the (artificial) hydrophobic surfaces exposed in these apoLp-III-lipid binding
assays. It will be of high physiological importance
to address the precise mechanism of the major
conformational change of apoLp-III, as well as
both helical orientation and geometry of these
exchangeable apolipoproteins, in native lipoprotein
particles. For this purpose, insect lipophorin
(LDLp) would seem to be extremely suitable as it
contains only one type of exchangeable apolipoprotein (apoLp-III), while its non-exchangeable
apolipoprotein (apoLp-I and -II) is fully
characterized.
5. Conclusion and perspectives
A considerable number of basic metabolic processes and their regulation in insects and vertebrates are similar. In view of their specific
requirements during flight activity, however,
insects apply different mechanisms for neurohormonal regulation of substrate mobilization and use
other carriers for substrate transport. In fact, many
physiological capacities of insects such as sensitive
vision, rapid information processing in the nervous
system and efficient oxygen provision to muscles,
can be interpreted as adaptations for flight (Candy
et al., 1997). The flight-related strategy of the
insect corpus cardiacum in the biosynthesis, storage and release of the AKHs, and the effects of
these neuropeptides on the regulation and integration of carbohydrate and lipid mobilization and
transport during flight activity has offered a broad
and profound research model for integrative physiology and biochemistry. In addition, this model
provides a context within which we can interpret
the functions of the multitude of genes and their
protein products emerging from the continuously
increasing pace of gene and genome sequencing
(Arrese et al., 2001). This may involve genome
projects of model insects such as Drosophila and
Anopheles, but also be extended to recent initiatives such as the silkworm genome database, the
honeybee (Apis mellifera) gene index and the A.
mellifera genome sequencing proposed by the US
National Human Genome Institute.
The mechanism whereby neuropeptides modulate and integrate a series of metabolic processes
D.J. Van der Horst / Comparative Biochemistry and Physiology Part B 136 (2003) 217–226
during flight, including the release of DAG into
the circulation through activation of fat body
lipase, represents an important research goal.
Transport of the released DAG requires the AKHstimulated transformation of pre-existing lipophorin particles, which are capable of alternating
between a relatively lipid-poor (HDLp) and a
lipid-enriched (LDLp) form. The appearance of
DAG in the lipophorin phospholipid surface monolayer triggers the binding of several lipid-free
apoLp-III molecules. The conformational switch
of the apoLp-III helix bundle, resulting in an
extended helix organization around the periphery
of discoidal phospholipid complexes, remains to
be examined in the native lipophorin. The resulting
LDLp is transported in the circulation to the flight
muscles; as DAG is hydrolyzed by the flight
muscle lipophorin lipase, apoLp-III dissociates and
returns to its lipid-free state, regenerating HDLp.
This completes a shuttle cycle, which represents
the essential mechanism for long-distance flight in
insects under the action of AKHs. Interestingly, in
solitary-phase locusts, which are unable to perform
long-distance flights, injection of AKH did not
result in LDLp formation (Chino, 1997). This
effect appeared not to be due to the absence of
HDLp or apoLp-III in the hemolymph, but to the
extremely low TAG content in the fat body (0.7%
or less of that in gregarious locust fat body), which
is quite insufficient to maintain the lipophorin
shuttle mechanism.
On many aspects concerning lipophorin biosynthesis, structure, interconversion of subspecies and
evolutionary relationships, considerable information has been gained in the last few years, but
much additional data is required for a full understanding of this unique system and to fully apply
insight into corresponding processes in higher
organisms.
An endocytic receptor for HDLp, the insect
lipophorin receptor (iLR), identified as a novel
member of the low-density lipoprotein (LDL)
receptor family (Dantuma et al., 1999), does not
seem to be involved in the lipophorin shuttle
mechanism operative during flight activity, but
rather in lipid uptake for storage. Three-dimensional models of extra cellular domains of iLR,
based on elucidated atomic structures of the human
LDL receptor, bear a striking resemblance to the
latter (Van der Horst et al., 2002). In contrast to
LDL, however, which is degraded in lysosomes
after dissociating from its LDL receptor, HDLp
225
endocytosed by iLR, stably transfected in a mammalian (CHO) cell line, was recently shown to
follow a transferring-like recycling pathway and is
re-secreted (Van Hoof et al., 2002). Apart from
the intriguing mechanism underlying the recycling
of the insect lipoprotein particle, this data represent
another example of the similarity between structural elements of the lipoprotein system in insects
and mammals, and highlight once more that the
functioning of these structures in similar processes
is different (Van der Horst et al., 2002).
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