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First published online 1 November 2006
doi: 10.1242/dev.02659
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Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.
* Author for correspondence (e-mail: clive.wilson{at}anat.ox.ac.uk)
Accepted 25 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key words: Drosophila, Insulin, PTEN, Obesity, Oogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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; Goberdhan and Wilson,
2003a; Holm,
2003). Disruption of this homeostatic mechanism can have
significant physiological consequences. For example, reduced insulin
signalling underlies the defects in glucose metabolism observed in diabetes,
whereas changes in lipid metabolism associated with obesity are an important
causative factor in establishing cellular insulin resistance in patients with
Type 2 diabetes (Haslam and James,
2005).
Genetic analysis of the insulin/insulin-like growth factor signalling (IIS)
cascade in Drosophila has highlighted the importance of this pathway
in development, revealing a central role in controlling protein synthesis and
cell growth (Goberdhan and Wilson,
2003a; Hafen,
2004). This evolutionarily conserved process is mediated by
cell-surface activation of the downstream effector kinase Akt (also known as
Akt1 - Flybase). Akt is activated by increased levels of phosphatidylinositol
3,4,5-trisphosphate [PtdIns(3,4,5)-P3], a phospholipid
signalling molecule produced by IIS-regulated class I PI3-kinases
(Downward, 1998).
Hyperactivation of IIS has been implicated in the generation of many different
human tumours, which are frequently defective for the tumour-suppressor
protein PTEN, a PtdIns(3,4,5)-P3 phosphatase that directly
antagonizes the effects of PI3-kinases
(Goberdhan and Wilson,
2003b).
IIS also affects sugar metabolism
(Rulifson et al., 2002) and
lipid storage during fly development, thus modulating starvation sensitivity
(Böhni et al., 1999;
Oldham et al., 2002;
Teleman et al., 2005).
However, the lack of a good in vivo system in which to study cellular changes
in lipid accumulation has made it difficult to dissect the genetic mechanisms
involved. Interestingly, studies in mice have indicated that, in addition to a
global effect on growth, increased IIS can strongly stimulate lipid storage in
a narrower range of cell types (Magun et
al., 1996; Horie et al.,
2004), demonstrating that the effects of IIS on lipid metabolism
are highly cell type-specific.
In order to genetically dissect IIS-dependent lipid-storage mechanisms, we analysed the effects of increasing downstream IIS signalling in the nutrient-storing nurse cells of the Drosophila ovary. We find that nurse cells lacking Pten exhibit a remarkable cell type-specific enlarged lipid-droplet phenotype. Mutant cells have highly elevated levels of activated Akt in their cytoplasm. We show that this pool of Akt is essential for lipid accumulation and controls the expression of an evolutionarily conserved lipid-storage protein, LSD2/perilipin. Our data therefore reveal a novel mechanism by which triglyceride storage can be controlled independently of other IIS-dependent events in specific tissues, potentially explaining how these processes might become uncoupled in certain disease states.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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).
Males hemizygous for a hsp70-flp122
X-chromosome insertion, which produces leaky expression of flp even
in the absence of heat shock (Britton et
al., 2002), and heterozygous for Pten1 FRT40A
(Goberdhan et al., 1999) were
crossed to females homozygous for
P[w+;Ubi-GFPnlsS65T] FRT40A (a gift from Bruce
Edgar, Fred Hutchinson Cancer Research Center, Seattle, USA), which
preferentially expresses GFP in the nucleus. Newly eclosed
yw1118
hsp70-flp122/w1118;Pten1
FRT40A/P[w+;Ubi-GFP] FRT40A females were heat shocked in a
water bath for 1 hour at 37.5°C and aged for up to 3 days before
dissection.
Males hemizygous for the same hsp70-flp122
chromosome and heterozygous for UAS-Dp110 were crossed to females
homozygous for the actin flp-out stock Act>CD2>Gal4,
UAS-GFPnlsS65T (Neufeld et
al., 1998). Newly eclosed F1 females, heterozygous for
hsp70-flp and transheterozygous for the flp-out and
UAS-Dp110 chromosomes, were heat shocked as described above. In all
egg chambers examined, every nurse cell appeared to overexpress the GFP and
Drosophila (D)p110 transgenes, presumably because
of leaky hsp70-flp122 expression.
Staining of ovaries
Dissected ovaries from 3-day old females were fixed for 30 minutes in 4%
paraformaldehyde in PBS. F-actin was stained with 2.5 µg/ml
TRITC-phalloidin (Sigma). Rabbit anti-phospho-Ser-505-Akt antibody (Cell
Signalling Technologies) was used at 1:500 and rabbit anti-LSD2
(Welte et al., 2005) at 1:500
in PBS, 0.1% Tween-20. The secondary antibody was a Cy5-coupled anti-rabbit
antibody raised in donkey (Jackson Labs; 1:800). A 10 mg/ml solution of Nile
Red (Sigma) in acetone was diluted 1:2500 or 1:1250 in PBST for staining. To
visualize DNA, egg chambers were incubated for 2 hours at room temperature
with 0.5 mg/ml RNAase A in PBST, then stained with 1 µg/ml propidium
iodide. Images were collected on a Leica TCS SP Confocal System and processed
with Adobe Photoshop CS2 version 9.
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RESULTS AND DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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).
Cells in 90% (n=50) of clones mutant for the IIS antagonist
Pten (Goberdhan et al.,
1999; Huang et al.,
1999) showed cell-autonomous formation of highly enlarged large
lipid droplets (Fig. 1D-I) and
a corresponding reduction in the number of small perinuclear droplets. This
phenotype was never observed in groups of nurse cells in wild-type egg
chambers (n=50; P<0.001 by chisquared test), and has not
previously been reported in Pten mutant cells in other tissues.
Indeed, analysis of mutant clones in the developing eye imaginal disc revealed
no obvious changes in lipid droplet accumulation (see Fig. S1 in the
supplementary material).
Cell-surface activation of Akt in response to elevated
PtdIns(3,4,5)-P3 levels involves phosphorylation of both
Thr-342 and Ser-505 (equivalent to Ser-473 in mammalian Akt)
(Downward, 1998;
Pinal et al., 2006). The
latter can be assessed using an antibody that cross-reacts specifically with
Ser-505-phosphorylated Akt (P-Akt;
Fig. 2; see
Fig. 3J for a schematic of the
signalling cascade). Interestingly, unlike Drosophila photoreceptor
cells in which P-Akt is localized at the cell surface
(Pinal et al., 2006), we
observed modest accumulation of P-Akt throughout the cytoplasm of
wild-type nurse cells (Fig.
2A,B). Cytoplasmic P-Akt levels were greatly increased in
Pten mutant cells (Fig.
2C-L). Nuclear P-Akt was not similarly increased
(Fig. 2C,D). A change in
cytoplasmic P-Akt is observed despite the fact that the germ-line
nurse cells and oocyte within an egg chamber form a syncytium via
intercellular ring canals, suggesting that IIS signalling complexes (and GFP)
are unable to diffuse through these connections. As found in other cell types
(Goberdhan et al., 1999;
Pinal et al., 2006), there
were no gross abnormalities in the actin cytoskeleton of Pten mutant
cells (Fig. 2G,K) or
indications of premature apoptosis (see nuclear staining in Fig. S2 in the
supplementary material), indicating a specific defect in lipid storage.
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). Viable
females of this genotype showed normal storage of lipids in nurse cells
(Fig. 1C), demonstrating that
Akt is an essential mediator of the effects of PTENs on lipid storage.
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) in
nurse cells. Overexpression of Dp110 does not fully phenocopy the effects of
Pten mutation in other tissues
(Gao et al., 2000), in part,
perhaps, because these molecules can control different subcellular pools of
activated Akt. Indeed, levels of cell-surface P-Akt were selectively
increased by Dp110 overexpression (Fig.
2M-P), but there was no obvious change in bulk cytoplasmic
P-Akt. Many of these overexpressing cells appeared to contain a
normal distribution of small lipid droplets. However, in 16% of overexpressing
egg chambers (n=100), elongated cell-surface Nile Red-positive
structures, which are not observed in normal egg chambers (n=100,
P<0.001), were seen in nurse cells at the periphery of the egg
chamber (Fig. 1J-O).
In vertebrates. triglyceride storage is regulated by a number of
evolutionarily conserved lipases and droplet-binding proteins, including
perilipin. Perilipin can promote lipid-droplet formation, and its expression
and activity are upregulated by insulin
(Holm, 2003;
Prusty et al., 2002;
Akimoto et al., 2005). In
wild-type Drosophila nurse cells the perilipin homologue LSD2, which
normally modulates lipid storage, is distributed throughout the cytoplasm, but
excluded from lipid droplets (Teixera et al., 2003)
(Fig. 3A,B). Levels of LSD2
were greatly increased in Pten mutant cells
(Fig. 3E-I). As expected, the
protein was excluded from all large lipid droplets and, in some cases, there
was increased accumulation at the periphery of these structures.
PI3-kinaseoverexpressing nurse cells do not express LSD2 at elevated levels
(Fig. 3C,D), but this protein
is excluded from the surface-localized Nile Red-stained structures formed in
these cells.
Subcellular localization of IIS components facilitates independent regulation of multiple processes
We have shown that the accumulation of large lipid droplets in bulk
cytoplasm requires activation of Akt throughout this compartment, as is
observed in Pten-mutant nurse cells. Previous functional studies of
Pten in Drosophila have primarily highlighted a crucial role
for this molecule in controlling cell growth in the wing, eye and other
developing adult tissues (Goberdhan et
al., 1999; Huang et al.,
1999; Oldham et al.,
2002). The effects on lipid-droplet formation appear more cell
type-specific and, indeed, our analysis of the developing eye imaginal disc
indicates that the large droplet phenotype observed in nurse cells is not
reproduced in photoreceptors, in which, at least at later stages of
development, activated Akt is localized to the cell surface
(Pinal et al., 2006).
Plasma membrane-associated P-Akt mediates many normal functions of
IIS, including growth (Goberdhan and
Wilson, 2003a). Furthermore, surface activation in restricted
plasmamembrane domains potentially permits independent regulation of other
localized cellular properties such as polarity and migration
(Sulis and Parsons, 2003;
Pinal et al., 2006). However,
significant levels of P-Akt also dissociate into the cytoplasm and
nucleus in some cell types (Downward,
1998; Ayala et al.,
2004; Kumar and Hung,
2005). Nuclear P-Akt has been proposed to have several
cell-biological roles
(Déléris et al.,
2006), but the specific functions of the cytoplasmic pool have not
previously been genetically analysed. Although we have focused our study on
the effects of downstream IIS signalling components, insulin receptor
signalling normally modulates Akt activity throughout the entire fly
(Goberdhan et al., 2003a), so we anticipate that upstream IIS components will
also play a modulatory role in the lipid-storage process. Indeed, these latter
components do affect ovarian development
(Drummond-Barbosa and Spradling,
2001; Brogiolo et al.,
2001). In fact, we believe our study has uncovered a conserved
function of IIS in lipid-storage cells, as insulin can also increase perilipin
expression in mammalian adipocytes and sebocytes
(Prusty et al., 2002;
Akimoto et al., 2005).
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),
whereas decreasing IIS elevates lipid levels both in flies and mice
(Böhni et al., 1999;
Burks et al., 2000). We have
not been able to show biochemically whether triglycerides are increased rather
than merely redistributed in Pten mutant ovaries, because only a
small minority of the nurse cells is mutant. However, in some late-stage
clones, in which cytoplasmic volumes are decreasing, lipid levels often appear
elevated in mutant cells (e.g. Fig. S2 in the supplementary material).
Insulin-stimulated increases in lipid-droplet number and size have been
observed in mammals (Schwertfeger et al.,
2003; Magun et al.,
1996), and, interestingly, when tumours are induced in mouse liver
by raising IIS, lipid-storage mechanisms are also activated
(Horie et al., 2004). We
propose that these cell type-specific responses are due to selective
accumulation of an IIS-modulated, cytoplasmic activated Akt pool that must, to
some extent, be controlled independently of cell-surface P-Akt.
Indeed, we have recently identified a Drosophila phosphatase that
regulates levels of cytoplasmic P-Akt in nurse cells and have shown
that mutations in this gene produce a very similar enlarged lipid-droplet
phenotype, confirming this hypothesis (our unpublished observations).
How could elevated cytoplasmic P-Akt induce such a dramatic
lipid-droplet phenotype in nurse cells? In mammals, Akt can promote the
transcription of genes involved in lipid biosynthesis and storage pathways
(Eberle et al., 2004). Our
data indicate LSD2/perilipin is one of these targets. IIS also
post-translationally upregulates the activity of mammalian perilipin
(Holm, 2003). Interestingly,
ovaries mutant for Lsd2 show altered lipid accumulation, but droplets
are still formed (Teixeira et al.,
2003). Therefore, LSD2 is almost certainly one, but not the only,
target for IIS in the control of lipid-droplet accumulation in nurse cells. In
this context, it is interesting to note that, in addition to its proposed role
in coating lipid droplets, LSD2 has recently been shown to regulate
microtubule-dependent trafficking of these organelles
(Welte et al., 2005). As
cytoplasmic P-Akt could still be associated with intracellular
membranes or the droplet surface, it may be well positioned to modulate this
transport process.
Obesity is a well-established predisposing factor in the acquisition of
cellular insulin resistance and Type 2 diabetes
(Haslam and James, 2005).
Increased levels of circulating free fatty acids (FFAs) associated with
obesity appear to be important in this link
(Kovacs and Stumvoll, 2005).
However, it is unclear whether other mechanisms are also involved or how
reduced insulin sensitivity ultimately impacts on lipid storage. Molecules
downstream of Akt are known to regulate cell-surface IIS through at least two
negative-feedback loops (Fig.
3J) involving downstream S6 kinase and the transcription factor
FOXO (Harrington et al., 2005;
Goberdhan et al., 2005;
Puig and Tjian, 2005). Our
work therefore raises the possibility that any predisposition towards
increased cytoplasmic P-Akt could specifically promote lipid storage
and also selectively suppress insulin-dependent events at the cell surface
(Fig. 3J). It will be
interesting to investigate further the molecules involved in controlling this
P-Akt pool and whether the feedback mechanisms have any role to play
in linking obesity and insulin resistance in Type 2 diabetes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4731/DC1
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ACKNOWLEDGMENTS |
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