Biochem. J. (2010) 425, 13–26 (Printed in Great Britain)
13
doi:10.1042/BJ20091181
REVIEW ARTICLE
Molecular mechanisms of metabolic regulation by insulin in Drosophila
Aurelio A. TELEMAN1
German Cancer Research Center (Deutsches Krebsforschungszentrum), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
The insulin signalling pathway is highly conserved from mammals to Drosophila. Insulin signalling in the fly, as in mammals,
regulates a number of physiological functions, including carbohydrate and lipid metabolism, tissue growth and longevity. In
the present review, I discuss the molecular mechanisms by which
insulin signalling regulates metabolism in Drosophila, comparing
and contrasting with the mammalian system. I discuss both the
intracellular signalling network, as well as the communication
between organs in the fly.
INTRODUCTION
INSULIN MEDIATES MUCH OF THE NUTRIENT-DEPENDENT
SIGNALLING IN DROSOPHILA
In the past decade, Drosophila has been one of the important
model systems for studying the insulin and IGF (insulin-like
growth factor)/TOR (target of rapamycin) signalling pathway
(Figure 1). For instance, some components of the pathway such
as Rheb, Tsc1 (tuberous sclerosis complex 1) and Tsc2 (tuberous
sclerosis complex 2) were first placed into the TOR pathway in the
fly [1–5]. Such work has been supported by the powerful genetic
tools available in Drosophila, allowing researchers to quickly
generate loss-of-function animals to query gene function, and
epistasis experiments to query gene relationships, both of which
are fundamental for elucidating signalling networks.
More recently, Drosophila has also become a model system
for studying how insulin signalling regulates organismal and
cellular metabolism (Table 1). Although the metabolic regulation
of Drosophila is interesting in itself, work in this field has also
been sparked by an increased interest in organismal metabolism
due to the emerging world-wide epidemic of Type 2 diabetes
and obesity. At first, one might be sceptical that the metabolic
regulation of flies and humans have anything in common, yet a
surprising amount of conservation and parallelism between the
two systems has emerged. As discussed below, the conservation
occurs not only at the level of the molecular components of the
insulin signalling pathway, but also at the level of the physiological
outputs of the pathway. This is probably because multicellular
organisms needed to regulate their response to varying nutrient
conditions early in evolutionary history. Indeed Drosophila, like
mammals, regulate their circulating sugar levels and store excess
energy in the form of glycogen and lipids [6]. These energy stores
are then mobilized when energy is needed [7]. In the light of
this, in this review I will highlight both the similarities and the
differences between the fly and mammalian systems.
Key words: Akt, Drosophila metabolism, insulin signalling, target
of rapamycin (TOR), total body glucose, total body lipid.
Two sets of observations support the conclusion that most of the
nutrient sensing in Drosophila takes place via the insulin pathway.
First, mutations in components of the insulin pathway phenocopy
the effects of nutrient deprivation. For instance, mutation of
the kinase TOR phenocopies amino acid deprivation, leading
to a block in tissue growth, reduced nucleolar size and cell
cycle arrest despite the presence of nutrients [8]. As another
example, reduction in S6K [RPS6 (ribosomal protein S6)-p70protein kinase] activity in the Drosophila brain leads to similar
changes in feeding behaviour to those observed upon fasting,
including an increased rate of food intake and reduced aversion to
unappealing food [9]. Secondly, artificially elevating signalling
through the insulin pathway is sufficient to bypass the need
for nutrients for many cell-biological processes. This means
that signalling through the pathway is ‘epistatic’ to nutrient
availability. For instance, activation of PI3K (phosphoinositide
3-kinase) signalling bypasses the usual nutrient requirement of
many larval cell types for growth and DNA replication [10].
Thus PI3K activation can lead to cell growth and proliferation
despite a lack of nutrient availability, causing death of the animal
[10].
It is worth mentioning that in Drosophila, organismal growth is
restricted to the larval stages of development, which can therefore
be considered similar to the childhood and teenage years in
humans. In contrast, after metamorphosis, the adult fly no longer
increases in size. This means that genetic manipulations that alter
insulin signalling in the fly during larval stages result in both
growth and metabolic phenotypes, whereas genetic manipulations
that alter insulin signalling in the adult only result in metabolic
consequences.
Abbreviations used: 4E-BP, 4E-binding protein; AKH, adipokinetic hormone, AMPK, AMP-activated protein kinase; Atg1, autophagy-specific gene 1; CC,
corpus cardiacum; CREB, cAMP-response-element-binding protein; dALS, Drosophila acid-labile subunit; dLip4, Drosophila lipase 4; eIF4B, eukaryotic
initiation factor 4B; eIF4E, eukaryotic initiation factor 4E; ERK, extracellular-signal-regulated kinase; FOXO, forkhead box O; GAP, GTPase-activating protein; GEF, guanine-nucleotide-exchange factor; GFP, green florescent protein; GLUT4, glucose transport protein 4; GRB2, growth-factor-receptor-bound
protein 2; GSK-3β, glycogen synthase kinase 3β; IGF, insulin-like growth factor; ILP, insulin-like peptide; Imp-L2, imaginal morphogenesis protein-late 2;
InR, insulin-like receptor; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; MAP4K3, MAPK kinase kinase kinase-3; MAPK, mitogen-activated
protein kinase; Melt, Melted; NLaz, neural lazarillo; NS3, nucleostemin 3; NSC, neurosecretory cell; Path, pathetic; PDK1, phosphoinositide-dependent
kinase 1; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PP2A, protein phosphatase 2A; PRAS40, proline-rich Akt
substrate of 40 kDa; PTEN, phosphatase and tensin homologue deleted on chromosome 10; RNAi, RNA interference; S6K, RPS6 (ribosomal protein
S6)-p70-protein kinase; Sgg, shaggy; SIK2, salt-inducible kinase 2; Slif, slimfast; sNPF, short neuropeptide F; NPFR1, sNPF receptor; SREBP, sterolregulatory-element-binding protein; Step, steppke; TIF-IA, transcriptional intermediary factor-IA; Tobi, target of brain insulin; TOP, terminal oligopyrimidine
tract TOR, target of rapamycin; TOR-C1, TOR complex 1; TOR-C2, TOR complex 2; Tsc1, tuberous sclerosis complex 1; Tsc2, tuberous sclerosis complex 2; Wdb, widerborst.
1
email [email protected]
c The Authors Journal compilation c 2010 Biochemical Society
14
Figure 1
A. A. Teleman
The ‘core’ intracellular insulin signalling pathway in Drosophila
Functional relationships between components are indicated. Arrows indicate activation, but not
necessarily indicate direct physical interactions, whereas bar-ended lines indicate inhibitory
interactions. Broken lines indicate indirect interactions or interactions requiring further study.
Red arrows indicate transcriptional regulation. Details of each interaction are described in the
main text, and outlined in Table 1.
Metabolic regulation is inherently a complex system, designed
to maintain homoeostasis in a robust way, yet to be responsive to
varying inputs. As a consequence, it is rich in feedback loops
and interconnections, both within a single cell and between
organs. In the following sections I will first describe the
intracellular insulin/IGF signalling network in flies, together
with the metabolic phenotypes that have been reported for
components of the pathway. I will then address the roles and
interconnections of various organs involved in insulin signalling
in the fly. Finally, I will point out some differences between the
Drosophila and mammalian insulin systems.
COMPONENTS OF THE INSULIN PATHWAY AND METABOLIC
PHENOTYPES
ILPs (Insulin-like peptides)
Signalling through the insulin/IGF pathway commences upon
binding of ligand to the insulin receptor, in Drosophila
InR (insulin-like receptor) (Figure 1). Drosophila has seven ILPs,
termed ILP1–7, which are homologues of human insulin and
IGFs. Mammalian insulin and Drosophila ILPs are homologues
c The Authors Journal compilation c 2010 Biochemical Society
at the protein level [11], as well as being functionally equivalent;
mammalian insulin can activate the Drosophila InR [12,13] and
Drosophila extract has insulin bioactivity in mice [14]. Although
it was initially surprising that Drosophila has seven ILPs, as the
complexity of Drosophila insulin signalling tends to be lower
than that of its mammalian counterpart, it is now apparent that
mammals also have a large number of ILPs as at least nine have
been identified in rodents {insulin, Igf1, Igf2, relaxin, Insl3, Ins4,
Ins5 and Ins6 (for a review see [15])}. The seven Drosophila
ILPs are similar at the amino acid level with the exception of
ILP6, which has some structural differences that might distinguish
it functionally from the others [16]. However, each ILP has a
different expression pattern and unique regulation, suggesting that
the functions of the various ILPs are not overlapping. ILPs 1, 2, 3
and 5 are expressed in seven median NSCs (neurosecretory cells)
of the Drosophila brain [11,17,18], ILPs 4, 5 and 6 are expressed in
the midgut, ILP2 is expressed in the imaginal discs and the salivary
gland [11,17] and ILP7 is expressed in the ventral nerve cord of
the brain [11]. In the larva, expression of ILPs 2, 3 and 5 reduce
upon fasting [17,19] in a manner analogous to that of human
insulin, whereas, surprisingly, expression of ILPs 6 and 7 increase
[19].
Overexpression of any of the seven ILPs during larval
development leads to increased body size [17] indicating that
all seven ILPs can activate the insulin receptor. Larval ablation
of the NSCs that produce ILPs 2, 3 and 5 yields adults
that are developmentally delayed, reduced in body size and
have elevated circulating sugars, total body glycogen and lipid
levels [18,20,21]. Ablation of the same cells in adults leads to
increased trehalosaemia without the growth phenotypes [21].
Selective knockdown of ILP2 expression in the NSCs by
RNAi (RNA interference) yields adults with elevated total
body trehalose levels, but none of the other defects, possibly
due to compensatory changes in ILP 3 and 5 expression
[22].
These physiological effects of ablating the NSCs cells, reducing
insulin levels systemically and hence causing elevated circulating
sugars and lipid accumulation, are analogous to the effects seen
in diabetic patients or in mice when there is generalized insulin
resistance, such as in insulin receptor knockouts (for a review see
[15]). Furthermore, the attenuated growth observed in animals
with reduced insulin signalling (either via ablation of the NSCs
or other manipulations as described below) parallel the reduction
in body size seen in mice mutant for insulin and IGF receptors
[23].
Although it is probable that the various ILPs have distinct
functions, this has not yet been carefully studied and the role
of each individual ILP in fly physiology is only starting to be
elucidated. For example, overexpression of ILPs 2 and 4 in adult
NSCs induce behavioural changes that are not induced by ILP3.
When larvae are starved of nutrients, they undertake a set of
hunger-induced behaviours, including an enhanced feeding rate.
This is caused by reduced insulin production from the NSCs.
Expression of ILPs 2 and 4 are able to suppress this hungerdriven feeding activity, whereas expression of ILP3 does not
[9]. The above example also illustrates that in Drosophila, ILPs
form part of a homoeostatic feedback loop. Nutrient conditions
regulate expression of several ILPs, and expression of ILPs in
turn regulate feeding behaviour. The generation of knockout flies
for each of the individual ILPs should shed light on the function
of each ILP.
Three secreted inhibitors of ILP function have recently been
described, Imp-L2 (imaginal morphogenesis protein-late 2, also
known as Ecdysone-inducible gene L2) [24], dALS (Drosophila
acid-labile subunit, also known as convoluted) [25] and NLaz
Metabolic regulation by insulin in Drosophila
Table 1
15
Summary of insulin genes with demonstrated metabolic phenotypes in Drosophila
The metabolic phenotypes are given for the loss-of-funtion condition unless otherwise stated.
Protein
CG number(s)
Function
Inputs
Metabolic phenotypes
Experimental model
Source(s)
CG14173; CG8167;
CG14167; CG6736;
CG33273; CG14049;
CG13317
activates insulin receptor
nutrient availability; stress via
p53, JNK and FOXO; ERK
signalling
larvae and adults
[9,18,20,21,22]
InR
CG18402
transcriptionally regulated by
FOXO
adult
[30]
Chico
CG5686
Lnk
CG17367
Melt
CG8624
TOR-C1
CG5092
Tobi
CG11909
transduces insulin signal
intracellularly; activates
PI3K
recruits downstream signalling
components to InR
recruits downstream signalling
components to InR
recruits Akt targets (FOXO and
Tsc1/2) to Akt
promotes cellular translation
and growth and nutrient
uptake; inhibits autophagy
glucosidase
elevated circulating sugars,
glycogen and lipids;
overexpression in adult
NCSs leads to altered
feeding behaviour
elevated total body sugars and
lipids
sNPF
CG13968
neuropeptide; activates ERK
signaling and ILP secretion
in NSCs
CG15009
binds and antagonizes ILP2
dALS
CG8561
binds and modulates ILP2
activity
NLaz
CG33126
lipocalin
PTEN
CG5671
Wdb
CG5643
FOXO
CG3143
phosphatase counteracting
PI3K action
phosphatase
dephosphorylating Akt
transcription factor activating
stress-response and
catabolic genes
4E-BP
CG8846
TORC
CG6064
Positive factors
ILP1–7
Negative factors
Imp-L2
translation inhibitor via
binding of eIF4E
CREB transcriptional
co-activator
inhibited by S6K
phosphorylation
not known
elevated lipids
adult
[31]
elevated lipids
adults
[32]
none known
lean
adults
[66]
activated by insulin signaling
via Akt; activated by amino
acid availability
induced transcriptionally by
insulin
not known
decreased lipid stores in
adipose tissue; reduced
circulating sugar levels
reduced glycogen upon
overexpression
reduced circulating
carbohydrates upon
overexpression
larvae
[67,94, 98,100,
102,110,119]
adult
[147]
larvae
[143]
larvae
[24]
larvae
[25]
adults
[26]
adults
[39]
oocyte nurse cells
[54]
adults; lavae
[57,58,62,64]
adults
[68]
adults
[93]
transcriptionally induced upon increased mortality in adverse
adverse nutrient conditions
nutrient conditions
transcriptionally downdecreased haemolymph
regulated upon starvation
trehalose; increased
haemolymph lipids upon
fat-body-specific
knockdown
activated transcriptionally
reduced trehalose, glycogen
by JNK
and triacylglycerol levels
not known
reduced total body glycogen
and lipids
not known
enlarged lipid droplets
in oocytes
inhibited by Akt
required for wasting in
phosphorylation
response to infection; on
overexpression arrest in
feeding and growth
and accumulation
of lipid aggregates
inhibited by TOR-C2
elevated rate of lipid use
phosphorylation
upon starvation
inhibited by insulin signalling reduced glycogen and
and activated upon
lipid stores
starvation
(neural lazarillo) [26]. These will be described in detail in the
section on insulin signalling in tissues.
Insulin receptor and IRS (insulin receptor substrate)
Drosophila has one insulin receptor, InR, which is similar in
sequence to the mammalian insulin receptor, except that it
contains 400 additional amino acids at the C-terminus. This
extension contains three YXXM motifs similar to those found
in mammalian IRS1, allowing Drosophila InR to bind PI3K in
the absence of an IRS [27,28]. Whereas complete removal of
InR in Drosophila throughout development causes early larval
lethality [12,29], a more mild reduction in InR levels, specifically
in the adult, leads to live adults with increased total body free
sugar and lipid levels [30].
Upon ligand binding, InR autophosphorylates [28], recruiting
the IRSs Chico and Lnk [31,32]. Although a large number of
adapter proteins have been described as binding to the mammalian
insulin/IGF receptors {IRS1, IRS2, IRS3, IRS4, SHC, CBL,
APS, SH2B, GAB1, GAB2, DOCK1 (dedicator of cytokinesis or
downstream of Crk-180 homologue 1), DOCK2 and CEACAM1
(carcinoembryonic antigen-related cell-adhesion molecule 1); for
a review see [33]}, only Chico (the fly homologue of IRS1),
dreadlocks (which is involved in axon guidance [34]) and
Lnk [the fly homologue of SH2B (SH2B adaptor protein 1)]
have been described as binding to the Drosophila InR. As
Drosophila has homologues for some of these other adapter
proteins, future work might shed light on whether they are also
involved in transducing signals downstream of the fly insulin
receptor. Both Chico and Lnk mutant adults are strongly reduced
in size and viability, and have elevated total body lipid levels
c The Authors Journal compilation c 2010 Biochemical Society
16
A. A. Teleman
[31,32], whereas simultaneously mutation of Chico and Lnk
is lethal, suggesting these two proteins transduce the majority
of the signal downstream of InR. The metabolic phenotypes
observed in InR, Chico and Lnk mutants, which are essentially
insulin-resistant in the whole body, are consistent with the
metabolic phenotypes observed upon ablation of the NSCs
as described above, which also causes a reduction in insulin
signalling.
In mammals, a number of connections have been described
between insulin and Ras signalling. In mammals, activation of the
insulin receptor induces interaction of the GRB2 (growth-factorreceptor-bound protein 2)–SOS (Son of sevenless) complex with
IRS1 thus activating Ras and subsequently ERK (extracellularsignal-regulated kinase) [35,36]. Furthermore, Ras-GTP binds
to and activates the catalytic subunit of PI3K [37]. These
interconnections are starting to be studied in the fly, yielding
insights into the physiological relevance of these interactions.
Although insulin treatment of Drosophila cells leads to activation
of ERK [38], mutating the consensus binding site for the Ras
pathway adaptor Drk (downstream of receptor kinase)/GRB2 in
the Chico protein does not interfere with growth [39]. As Ras
is required for viability, this suggests that either Ras is activated
in a Chico-independent manner in Drosophila, perhaps via the
C-terminal extension of the Drosophila insulin receptor, or that
the effect of insulin signalling on Ras is modulatory and not
required for insulin-mediated growth. A study has shown that
mutating the Ras-binding domain in the PI3K Drosophila p110
protein (also known as PI3K92E), the catalytic subunit of PI3K,
in vivo is dispensable for viability but is required for maximal
PI3K signalling, leading to a phenotype of small flies with
dramatically lowered egg production [40]. This lowered egg
production might reflect a defect in lipid metabolism given lipids
are required in large amounts in eggs. As PI3K is required
for viability, these results suggest the impact of Ras on insulin
signalling is modulatory in function.
PI3K, PTEN (phosphatase and tensin homologue deleted on
chromosome 10), PDK1 (phosphoinositide-dependent kinase 1)
and Akt/PKB (protein kinase B)
Upon phosphorylation of InR and Chico, PI3K (p110) [41,42],
together with its adapter subunit p60 (also known as PI3K21B)
[43], is recruited to the cell membrane and activated. This
leads to the generation and accumulation of PtdIns(3,4,5)P3
at the cell membrane. The kinase activity of PI3K is opposed
by the phosphatase activity of the tumour suppressor PTEN. Step
(Steppke), a member of the cytohesin GEF (guanine-nucleotideexchange factor) family, has now been found to act upstream of
PI3K to regulate growth and metabolism [44,45]. Step mutant
flies are small, and blocking the function of the Step homologues
in mice causes elevated gene transcription of insulin-repressed
gluconeogenic genes, as well as inhibition of glycogen and fatty
acid synthesis [44,45]. The exact mechanism by which Step
affects PI3K function remains to be elucidated.
Accumulation of PtdIns(3,4,5)P3 recruits the two kinases
PDK1 [also known as PK61C (protein kinase 61C)] and Akt
to the plasma membrane, via their lipid-binding PH (pleckstrin
homology) domains, and leads to their phosphorylation and
activation (for a review see [46]). These proteins and interactions
are highly conserved between flies and mammals. The most
dramatic phenotypes observed in mutants for these components
are growth-related. Overexpression of PI3K, Akt or PDK1 leads
to tissue overgrowth, whereas loss-of-function leads to tissue
undergrowth and lethality [41,42,47,48]. Because of this, it
c The Authors Journal compilation c 2010 Biochemical Society
is difficult to reveal any ‘metabolic’ phenotypes. In contrast,
mutation of the counteracting phosphatase PTEN has the opposite
size effects and inactivating mutations in PTEN are not lethal
[49–52]. In PTEN mutants, which can be considered insulin
‘hyperactive’ in the entire body, it would be expected that we
would see the opposite phenotype from that in diabetic patients.
Indeed both total body glycogen and lipid levels are reduced
in PTEN mutant adult flies [39] (although, as will be discussed
below, in one specific cell type in Drosophila, the nurse cells,
PTEN mutation leads to the opposite effect, namely formation of
enlarged lipid droplets [53]).
Akt activity is regulated by two additional inputs. First, recent
work has shown that Akt is dephosphorylated and inactivated by
the phosphatase PP2A (protein phosphatase 2A) via its PP2A-B
subunit, Wdb (widerborst) [54]. Mutation of Wdb leads to
activation of Akt and to the same lipid-droplet phenotype in nurse
cells as seen in PTEN mutants [53,54]. It would be interesting to
know whether, at the whole organism level, Wdb mutants have
the same phenotypes of reduced body glycogen and lipid levels
as are observed in PTEN mutants. Secondly, Akt is phosphorylated and activated by TOR-C2 (TOR complex 2) both in
Drosophila and in mammals [55]. This regulation appears to be
modulatory, as removal of TOR-C2 activity by mutation of the
essential component Rictor (rapamycin-insensitive companion
of Tor) only resulted in mild growth impairment and no
observable metabolic effects ([55]; V. Hietakangas, personal
communication).
Akt targets and FOXO (forkhead box O) proteins
In mammals, Akt phosphorylates a large number of proteins
involved in metabolic control, including GSK-3β [glycogen
synthase kinase 3β; the Drosophila orthologue is known as
Sgg (shaggy)], TBC1D4 (TBC1 domain family, member 4), the
FOXO transcription factors, Tsc2, PRAS40 (proline-rich Akt
substrate of 40 kDa; the Drosophila orthologue is known as Lobe),
6-phosphofructo-2-kinase and ATP-citrate lyase [46,56]. A
number of these interactions have also been studied in
Drosophila.
One important target of Akt in Drosophila is the single
homologue of the FOXO transcription factors, dFOXO [57,58].
In Drosophila, as in mammals, phosphorylation of FOXO
by Akt leads to its retention in the cytoplasm, inhibiting its
nuclear transcriptional activity [57,59]. As one of the principle
transcription factors in the insulin signalling pathway, FOXO has
a profound impact on animal metabolism and a large number
of functional studies have been performed in the fly [57–66].
These studies all converge at the concept that part of the anabolic
effect of insulin results from blocking FOXO activity, which
otherwise promotes the conservation of energy or, in extreme
cases, even catabolism. Overexpression of dFOXO in larvae
phenocopies starvation, leading to growth arrest and causing
larvae to wander away from food [58]. In contrast targeted
overexpression of dFOXO in fly tissues reduces their size by
reducing cell number [57]. Analogously, growth suppression can
be seen when endogenous FOXO is activated by reducing Akt
activation [60,61]. Along these lines, one study showed that
flies infected with Mycobacterium marinum undergo a process
similar to wasting; they progressively lose metabolic stores, in
the form of fat and glycogen, and become hyperglycaemic. This
process is mediated in part via dFOXO, as dFOXO mutants
were found to exhibit less wasting [62]. Finally, a recent report
showed that dFOXO is activated upon amino acid starvation of
the animal and that this activation is required for the animals
Metabolic regulation by insulin in Drosophila
to survive the adverse conditions [63]. Surprisingly, dFOXO
functions both autonomously and non-autonomously in cells;
expression of dFOXO in adult head fat body causes reduces
ILP2 secretion from the NSCs via an unknown mechanism [64]
and causes the same metabolic consequences as in flies with
reduced insulin production, including increased longevity [65].
This will be discussed in more detail in the section on tissue
specificity below. A further Drosophila scaffolding protein, called
Melt (melted), has been found to modulate the ability of Akt
to inhibit FOXO in vivo, possibly by encouraging the physical
interaction between Akt and FOXO [66]. Melt mutants display
elevated FOXO activity and reduced total body lipid levels, a
phenotype which could be rescued by removal of FOXO [66].
FOXO exerts its effects by modulating transcription of a
very large number of target genes. One canonical target of
dFOXO is the translational repressor 4E-BP (4E-binding protein,
in Drosophila also known as Thor). Upon activation, 4EBP binds eIF4E (eukaryotic initiation factor 4E) and blocks
recruitment of the ribosome to the 5 end of mRNAs. As a
result, cellular translation rates are dampened [67]. Although
this has been suggested to regulate tissue growth, no growth
defects are observed in 4E-BP-null flies [68], nor in 4E-BP1or 4E-BP2-null mice [69, 70]. Instead, both 4E-BP-null flies and
4E-BP1-null mice have elevated metabolic rates [68,69], which is
consistent with the idea that protein synthesis is an energetically
expensive process and that its regulation impacts overall organismal energy balance. Studies have shown that FOXO regulates
close to 2000 genes, half of which are regulated in a tissue-specific
manner [19,71]. Among these are many components of the translational apparatus, as well as mitochondrial components, leading to
an overall reduction in translation and in mitochondrial biogenesis
upon dFOXO activation. Furthermore, dFOXO was found to
regulate expression of Myc (the Drosophila orthologue is also
known as diminutive), which in turn will also affect mitochondrial
biogenesis [19,72]. Finally, dFOXO also regulates expression of
an acid lipase (dLip4; Drosophila lipase 4) [73], and so is involved
in lipid homoeostasis. In summary, FOXO regulates expression
of a large number of genes that have an impact upon cellular
metabolism. Furthermore, a large number of metabolism-related
targets for FOXO1 have been described in mammals (for a review
see [74]), which have not yet been studied in depth in the fly.
A second important phosphorylation target of Akt is the tumour
suppressor Tsc2. This phosphorylation presents a conundrum.
Tsc2 inhibits TOR-C1 (TOR complex 1) activity both in flies and
in mammals via the small GTPase Rheb. Tsc2 acts as a GAP
(GTPase-activating protein) for Rheb, thereby inhibiting it and,
in turn, inhibiting TOR (Figure 1). In both systems, inhibition of
Tsc2 therefore leads to TOR hyperactivation and consequently
strong effects on tissue growth and metabolism [5,75–77].
Phosphorylation of Tsc2 by Akt can inhibit Tsc2 function, at
least when Tsc2 is overexpressed in mammalian cell culture, in
Drosophila cell culture or in vivo in the fly [78–81]. However, the
in vivo functional significance of this phosphorylation event is not
clear, as flies lacking Akt phosphorylation sites on Tsc2, or flies
simultaneously lacking Akt phosphorylation sites on Tsc1 and
Tsc2, are viable and normal in size, with only very mild metabolic
defects [82,83]. Therefore, although this phosphorylation does
occur in vivo, the functional consequences of this regulation
are not yet clear. Further work, perhaps with ‘knockin’ mice,
should shed light on whether this reflects a difference between
mammals and flies, or whether phenotypes are being masked by
redundant regulatory mechanisms such as the possible mechanism
mediated via PRAS40/Lobe. In an analogous manner to Tsc2,
mammalian PRAS40 was identified as a TOR-C1 inhibitor that
is phosphorylated and inhibited by Akt [84,85]. However, the
17
functional role of PRAS40 is not completely clear, as it has
also been reported that PRAS40 is actually a TOR-C1 target,
required for signalling downstream of the complex [86–88].
PRAS40 and Tsc2 therefore could represent two redundant pathways by which Akt activates TOR. Knockdown of the Drosophila
orthologue Lobe in tissue culture does activate phosphorylation
of TOR-C1 targets, suggesting that Lobe/PRAS40 function might
be conserved in flies [85]. However, Lobe mutants display
phenotypes suggestive of Notch pathways function, rather than
TOR-C1 function [89], so further work will be required to shed
light on the possible role of Lobe in TOR-C1 signalling.
In mammals, Akt phosphorylates and inactivates GSK-3β,
leading to the dephosphorylation and activation of glycogen
synthase and hence to an acceleration of glycogen synthesis [90].
The Drosophila orthologue of GSK-3β, Sgg, was also shown
to be phosphorylated downstream of PI3K signalling in vivo
[91]. Future work will shed light on whether this regulation has
metabolic consequences.
Another phosphorylation target of mammalian Akt is the
kinase SIK2 (salt-inducible kinase 2; the Drosophila orthologue
is CG4290) [92]. Upon feeding, Akt phosphorylates SIK2,
leading to phosphorylation and degradation of the transcriptional
co-activator TORC (in mammals also known as CRTC2
[CREB (cAMP-response-element-binding protein)-regulated
transcription co-activator 2]. As a consequence, TORC no longer
functions as a CREB co-activator and inducer of genes involved in
catabolism and gluconeogenesis. Recently, this signalling cascade
was found to be conserved in Drosophila. The Drosophila TORC
homologue is phosphorylated by Drosophila SIK2 in an Aktdependent manner [93]. TORC mutant adult flies are sensitive to
starvation and oxidative stress [93]. Surprisingly, TORC mutant
flies have reduced glycogen and lipid stores [93], rather than the
elevated levels of nutrient stores that might be expected from
mutation of a catabolic gene.
The Tsc1/2, Rheb and TOR signalling cassette
A central regulator of cellular metabolism is the anabolic
kinase TOR. TOR exists in one of two complexes, termed TOR-C1
and TOR-C2, with some components that are shared and some
that are distinct between the two complexes (for a review see
[94]). TOR-C1 is traditionally thought of as a regulator of cell
size and cell growth, as manipulation of TOR-C1 activity affects
cell size in eukaryotes from yeast to humans. However, TOR also
regulates processes that at first seem quite disparate, including
carbohydrate metabolism, lipid metabolism and autophagy. One
way to unify these functions is to consider TOR-C1 principally
as a regulator of cellular metabolism, controlling a cell’s decision
on whether to use energy and nutrients or whether to conserve
them. Growth can then be considered a metabolic process. To
grow, a cell needs biochemical building blocks, including amino
acids, proteins, lipids and carbohydrates, which in turn need
to by synthesized via metabolic pathways. Furthermore, protein
synthesis is a highly energetically expensive process, using 35–
80 % of a cell’s energy [95,96]. Therefore cell size and growth
are readouts or consequences of a cell-metabolic decision. When
TOR-C1 activity is high, this leads to accumulation of glycogen,
lipids and cell mass, whereas low TOR-C1 activity in a cell leads
to the catabolism of carbohydrate and lipid stores, turnover of
excess mass and, in extreme cases, autophagy. For this reason, I
also mention growth phenotypes in the present review, as a readout
of cellular metabolism.
In Drosophila, TOR-C1 function is conserved relative to
mammals. Genetic manipulation of TOR activity results in
c The Authors Journal compilation c 2010 Biochemical Society
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A. A. Teleman
significant changes in tissue and animal size. Loss-of-function
reduces tissue size, by reducing cell size and cell number, whereas
gain-of-function leads to the opposite effects [8,97]. Loss of TOR
also leads to lipid vesicle aggregation in the larval fat body [8]
and mild impairment of TOR-C1 activity in the whole larva also
results in decreased lipid stores in adipose tissue and reduced
circulating sugar levels [98]. This is consistent with the phenotype
of mice lacking one of the TOR-C1 targets, S6K1, which are
hypersensitive to insulin and are protected against obesity [99].
Strong reduction in TOR-C1 activity in the Drosophila adipose
tissue induces autophagy [100] (for a review see [101]) and
TOR-C1 activation is sufficient to suppress starvation-induced
autophagy [102].
Mechanistically, one of the main cellular processes that TORC1 regulates is translation. This occurs at a number of levels. TORC1 regulates ribosome biogenesis by controlling the production
of both components of the ribosome, ribosomal proteins and
rRNA. In mammals and in the fly, TOR-C1 activity promotes
the function of the transcription factor TIF-IA (transcriptional
intermediary factor-IA), which is required for RNA polymerase Imediated expression of rRNA [103,104]. Although the regulation
is conserved, the mechanistic details might be different in
the two systems, as phosphorylation of TIF-IA by mTOR
influences TIF-IA subcellular localization in mammalian cells
[104], whereas this does not seem to be the case in
Drosophila (A. A. Teleman, unpublished work). Furthermore,
in mammals, TOR-C1 also regulates activity of another rRNA
transcription factor, UBF (upstream binding transcription factor)
[105]; however, Drosophila contains no obvious homologue.
In Drosophila, TOR-C1 also regulates transcription of genes
involved in ribosome synthesis and assembly [106]. This was
recently shown to occur via regulation of the transcription factor
Myc [19]. Inhibition of TOR-C1 leads to a rapid reduction
in Myc protein levels, and consequently in expression of genes
involved in ribosome biogenesis. Consistent with this result, Myc
activity is also required for TOR-C1 to drive tissue growth [19,72].
Close connections between TOR-C1 and Myc appear to also exist
in mammals [107]. In addition to regulating ribosome biogenesis,
in mammals TOR-C1 also regulates expression of tRNAs by
RNA polymerase III. Mammalian TOR-C1 phosphorylates the
transcription factor Maf1, causing it to relocalize to the nucleolus
where it activates expression of tRNA genes [108]. Whether this
is also the case in Drosophila has not yet been investigated.
Finally, TOR-C1 regulates translation initiation and elongation.
Both in mammals and in flies TOR-C1 phosphorylates the
inhibitor of translation initiation 4E-BP [109] (for reviews see
[67,94,110]). This leads to the dissociation of 4E-BP from
the initiation factor eIF4E, allowing recruitment of the ribosome
to the cap complex at the 5 end of cellular mRNAs, and
an increase in overall cellular translation rates. TOR-C1 also
phosphorylates and activates S6K, which phosphorylates the
40S ribosomal protein S6 [109] (for reviews see [67,94,110]).
S6K requires phosphorylation at two sites for full activation
and the second site is phosphorylated by PDK1. Activation of
S6K was initially thought to regulate translation of mRNAs
containing 5 TOPs (terminal oligopyrimidine tracts), which
include many mRNAs encoding components of the translation
apparatus, although this has now been shown not to be the case,
as mice lacking S6K have normal translation of 5 TOP mRNAs
[111]. Therefore it is likely that TOR-C1 regulates translation of
5 TOP mRNAs in an S6K-independent manner, which remains
to be explored. Nonetheless, mammalian S6K phosphorylates
two other regulators of translation initiation and elongation,
eIF4B (eukaryotic initiation factor 4B) and eEF2K (eukaryotic
elongation factor-2 kinase) [112,113].
c The Authors Journal compilation c 2010 Biochemical Society
Regulation of translation by these TOR-C1 effectors has a
strong impact on organismal size and metabolism. TIF-IA mutants
arrest as first instar larvae and fail to grow [103]. Drosophila
deficient in S6K exhibit a strong delay in development and a severe
reduction in body size [114]. Although the S6K mutant phenotype
is less severe than that of dTOR mutants, S6K appears to be a key
mediator of TOR-C1 activity, as overexpression of S6K in vivo
can rescue dTOR the viability of mutant animals [8] and removal
of one copy of S6K can rescue the lethality of Tsc1/2 loss-offunction [115]. As mentioned above, mutants for the other TOR
target, 4E-BP, do not display growth abnormalities, but rather
have elevated metabolic rates, leading to reduced organismal lipid
levels, consistent with translation being an energetically expensive
process [68].
In addition to translation, TOR-C1 regulates a number of other
cellular processes. TOR-C1 inhibits autophagy in the Drosophila
fat body, and perhaps in other tissues as well [100,102]. Although
the molecular mechanism by which this occurs is not completely
clear, it is known to be S6K-independent [102] and that Atg1
(autophagy-specific gene 1), an important regulator of autophagy,
is involved. TOR-C1 physically interacts with Atg1 and promotes
its phosphorylation, although more work will be required to
understand the implications of this regulation [116]. There
are probably other molecular mechanisms by which TOR-C1
regulates autophagy and these remain to be explored. In addition,
Atg1 was shown to inhibit S6K phosphorylation [117] suggesting
a regulatory feedback loop between TOR activity and autophagy.
Autophagy is important for lipid mobilization as inhibition of
autophagy leads to increased lipid storage, indicating this is
probably one means by which TOR-C1 regulates metabolism
[118].
TOR signalling also stimulates bulk endocytic uptake and
targeted uptake of nutrients, although again the mechanism is
not yet completely elucidated [119]. This may occur in part via
inhibition of the endocytic degradation of nutrient transporters
such as Slif (slimfast) [119]. Additionally, calderon [also known
as Orct2 (organic cation transporter 2)], an organic cation
transporter similar to transporters believed to function in the
uptake of sugars and other organic metabolites, was shown to
be transcriptionally induced by S6K activity [120]. Calderon is
required cell autonomously and downstream of S6K for normal
growth and proliferation of larval tissues [120].
Finally, a recent study in mammalian cells showed that TORC1 activity is required for Akt-induced activation of de novo lipid
synthesis [121]. Inhibition of mTOR-C1 by rapamycin prevented
Akt-dependent accumulation of unsaturated and saturated fatty
acids as well as phosphatidylcholine and phosphatidylglycerol
[121]. This probably occurs via regulation of SREBP [sterolregulatory-element-binding protein, also known in Drosophlia
as HLH106 (helix-loop-helix 106)] by TOR-C1. SREBP is a
transcription factor that regulates expression of genes involved in
sterol biosynthesis. The nuclear accumulation of mouse SREBP1
in response to Akt activation was prevented by rapamycin [121].
This interaction is probably conserved in flies as the study showed
that silencing of Drosophila SREBP blocked the induction of cell
growth caused by PI3K [121].
It is clear that the regulation of TOR-C1 is complex and
not completely understood (for reviews see [67,110,122,123]).
Briefly, TOR-C1 is activated in response to growth factors,
especially insulin, and nutrient availability. Activation of TORC1 in response to insulin signalling occurs via Akt, but the exact
mechanism is still unclear and, as described above, this activation
might occur via two redundant pathways: by phosphorylation and inhibition of the Tsc1/2 complex or by phosphorylation
and inhibition of PRAS40/Lobe. The Tsc1/2 complex acts as a
Metabolic regulation by insulin in Drosophila
GAP, and inhibitor, for the small GTPase Rheb, which in turn
activates TOR. Rheb was reported to be activated by the GEF
TCTP (translationally controlled tumour protein) [124]; however,
a later study called this into question [125]. In mammalian cells
TOR-C1 is also regulated by cellular energy status via AMPK
(AMP-activated protein kinase). High intracellular AMP levels
activate AMPK, which directly phosphorylates Tsc2, leading to
Tsc2 activation and TOR-C1 inhibition. Whether activation of
AMPK also leads to TOR-C1 inhibition in the fly remains to
be directly tested. TOR-C1 is also regulated by the availability
of amino acids in mammals and in Drosophila. Three factors
have been reported to mediate the amino acid availability signal
to TOR-C1, Vps34 (also known in Drosophila as PI3K59F),
the Rag GTPases and MAP4K3 [MAPK (mitogen-activated
protein kinase) kinase kinase kinase-3; the Drosophila orthologue
is CG7097]. In mammals, activity of the class III PI3K Vps34 is
regulated by amino acid availability via a calcium-dependent
mechanism [126]. This leads to accumulation of PtdIns3P in cells,
which is thought to cause the recruitment of proteins recognizing
PtdIns3P to early endosomes, forming an intracellular signalling
platform that leads to TOR-C1 activation [127,128]. This feature
of the pathway may be specific for vertebrates, as flies mutant for
Vps34 have been reported not to show TOR-C1 signalling defects
[129]. Recently, two groups discovered that Rag GTPases mediate
amino acid signalling to TOR-C1, both in mammals and in flies
[130,131]. The emerging picture is that amino acids change the
GDP/GTP loading of the Rag GTPases, thereby stimulating
the binding of Rag heterodimeric complexes to TOR-C1. This
in turn causes TOR-C1 to change its intracellular localization,
perhaps relocalizing it to vesicles containing the activator Rheb.
Finally, Lamb and co-workers identified human MAP4K3 in cell
culture as a kinase that is activated by amino acid sufficiency, and
in turn it is necessary for S6K activation in response to amino
acids [132]. This mechanism also appears to be conserved from
flies to humans; flies mutant for MAP4K3 have reduced TOR-C1
activity levels and display TOR-C1 hypomophorphic phenotypes
(B. Bryk, K. Hahn, S. M. Cohen and A. A. Teleman, unpublished
work). The relationships between Vps34, the Rag GTPases and
MAP4K3 remain to be investigated. In addition, novel regulators
of TOR-C1 have been identified, whose mechanisms of action are
not yet clear. TOR-C1 activity was found to be down-regulated
upon exposure of cells to hypoxic conditions [133]. This downregulation was mediated by Ptp61F (protein tyrosine phosphatase
61F), although the mechanism remains to be elucidated [133].
TOR-C1 activity is also modulated by the amino acid transporter
Path (pathetic). Path mutants are reduced in size and yield genetic
interactions consistent with reduced TOR-C1 activity [134].
When expressed in Xenopus oocytes, Path allows extracellular
amino acids to activate TOR-C1. Surprisingly, however, Path has
very low transport capacity for amino acids, suggesting Path may
be acting as an amino acid sensor which regulates TOR-C1 via an
unknown intracellular mechanism [134].
Outputs of the insulin signalling pathway
As described above, the ‘upstream’ signalling events in the insulin
signalling pathway are now fairly well understood. In brief, they
lead to activation of a number of kinases such as Akt and TOR
via a relay of phosphorylation events. In comparison, however,
at least in Drosophila, the outputs of the pathway are not as
clearly defined. Activation of the kinases in the insulin cascade,
in one way or another, has an impact on cellular metabolism by
regulating the enzymes that act as effectors, controlling lipid and
carbohydrate biosynthesis and breakdown. One well-understood
example is the regulation of GSK-3β by Akt. Activation of Akt
19
leads to phosphorylation and inactivation of GSK-3β, leading
to the dephosphorylation and activation of glycogen synthase
[90]. However, there are probably a large number of links
between the insulin pathway and metabolic enzymes that are
not yet known. Work in the future on this ‘downstream’ part
of the insulin signalling pathway will be fundamental for us to
understand how metabolic pathways are regulated by insulin.
This will require the coming together of multiple fields of
biological research, including cell signalling, biochemistry and
metabolomics. Furthermore, such research will probably require
the study of phenotypes that are more subtle and quantitative in
nature, compared with the phenotypes the field has studied so
far. This is because the few ‘master regulator’ kinases of the
insulin pathway, such as Akt and TOR, control both directly
and indirectly a large number of effector enzymes. Therefore
mutation of these master regulatory kinases leads to obvious and
easily studied phenotypes. In contrast, single mutations of each
effector enzymes will only lead to a subset of effects. Nonetheless,
an understanding of the molecular connections between insulin
signalling components and cellular metabolic enzymes will be
necessary for us to have a complete picture of how insulin
regulates metabolism.
Intracellular feedback loops
The molecular mechanisms regulating animal metabolism need to
simultaneously meet two constraints. First, the system needs
to be exquisitely sensitive and reactive to nutrient conditions of
the animal, in order to respond quickly to environmental changes.
This is best illustrated by insulin levels and insulin intracellular
signalling in humans; the reaction to changing blood glucose
levels occurs within minutes to maintain the glucose concentration
in human blood between 80 and 110 mg/dl [135]. Secondly, the
system needs to be robust and to function properly in a wide range
of individuals with differing genetic makeups. This ‘engineering’
problem of making a machine that is both flexible and inflexible is
probably partly solved by utilizing the large number of feedback
loops present in the system. In the case of insulin signalling, there
are at least three feedback loops worth mentioning.
First, activation of the insulin signalling pathway leads to activation of PDK1 and TOR-C1, both of which phosphorylate and activate S6K. S6K, in mammalian cells, then phosphorylates IRS1,
inhibiting its function and down-regulating signalling through
the pathway [110]. This mechanism is probably conserved in
Drosophila, as removal of S6K in Drosophila cell culture leads
to increased Akt phosphorylation [136]. Thus this is one negative
feedback loop which attenuates signalling through the pathway.
Furthermore, reducing S6K phosphorylation specifically should
lead to increased signalling through Akt and TOR, and therefore
increased phosphorylation of the remaining TOR targets.
A second feedback loop occurs when activation of the insulin
signalling pathway leads to activation of Akt and inhibition
of the FOXO transcription factor. As the insulin receptor
is a transcriptional target of FOXO, signalling through the
insulin receptor leads to its own transcriptional down-regulation,
attenuating signalling. Conversely, low insulin signalling leads
to increased transcription of insulin receptor levels, sensitizing
the system to renewed insulin signalling. This occurs both in
mammalian cells and in Drosophila [137].
One further mechanism is the antagonistic relationship between
activation of TOR-C1 and activation of TOR-C2. This can be
seen in Drosophila cells by monitoring phosphorylation of S6K
at Thr398 (a readout for TOR-C1 activity) and phosphorylation of
Akt at Ser505 (a readout for TOR-C2 activity) when Rheb, Tsc1
c The Authors Journal compilation c 2010 Biochemical Society
20
A. A. Teleman
or Tsc2 are knocked-down [136]. For instance, knockdown of
Rheb in Drosophila S2 cells causes decreased phosphorylation
of S6K and increased phosphorylation of Akt, indicating that
TOR-C1 activity is reduced, whereas TOR-C2 activity is
increased [136]. In mammalian cells, this also appears to be the
case, as mouse embryonic fibroblasts lacking Tsc1 or Tsc2 have
elevated TOR-C1 activity and severely blunted TOR-C2 activity
[138]. As a consequence, a negative feedback loop is created
between Akt and TOR (Figure 1). Activation of Akt leads to
activation of TOR-C1 and inactivation of TOR-C2. This causes a
reduction in the ability of TOR-C2 to enhance Akt activation via
phosphorylation of its hydrophobic motif (Ser505 in Drosophila)
[55].
Although the metabolic and growth consequences of these three
feedback loops have not been carefully explored, they constitute
an integral part of the insulin signalling network and therefore
warrant attention.
INSULIN SIGNALLING IN TISSUE CONTEXT
The insulin signalling pathway described above can be considered
the core pathway, which functions in almost all cells and
tissues of the animal. In addition to this core pathway, there
are tissue-specific upstream regulators and downstream effectors
of the pathway. Furthermore, the metabolic consequences of
manipulating insulin signalling depend strongly on the identity
of the tissue in which the manipulation takes place. Therefore
it is essential to consider insulin signalling in a tissue-specific
context. Work in the mouse has been at the forefront of our
understanding of how various tissues contribute to metabolic
regulation. Nonetheless, recent studies in the fly have also taken
tissue specificity into account. Future work will probably take
tissue specificity increasingly into account, as tissue-specific
manipulations are relatively easy in Drosophila.
Tissue-specific effects of insulin signalling
A wealth of mouse knockout studies have shown that
the physiological effects of reduced insulin signalling differ
enormously depending on the tissue in which the manipulation
takes place (for a review see [139]). For instance, mice completely
lacking the insulin receptor in the whole body, or lacking one
of the two insulin genes, die soon after birth due to highly
elevated circulating glucose levels. Along the same lines, mice
lacking insulin receptor specifically in muscle develop a metabolic
syndrome with increased fat stores and hypertriglyceridemia,
confirming the central role of this tissue in glucose disposal. In
stark contrast, however, mice lacking insulin receptor in white
and brown adipose tissue have a 30 % decrease in whole body
triacylglycerol (triglyceride) content and have normal levels of
circulating lipids, non-esterified fatty acids (free fatty acids) and
glycerol. This is similar to leanness observed [upon adiposespecific removal of TOR-C1 function [via knockout of the Raptor
(regulatory associated protein of mTOR) protein] in mice [140],
indicating that insulin and TOR-C1 signalling help promote
fat accumulation in this tissue. These differing effects reflect
the function of each individual organ in organismal metabolic
control. Furthermore, they highlight an antagonistic relationship,
in which insulin signalling in muscle promotes the reduction of
total body nutrient stores, whereas insulin signalling in adipose
tissue promotes accumulation of nutrient stores.
Although it has not been tested directly, various studies in
Drosophila suggest that the antagonistic relationship described
above also holds true for the fly. Reduction of insulin signalling
c The Authors Journal compilation c 2010 Biochemical Society
in the entire animal, for instance via ablation of the NSCs that
produce ILPs 2, 3 and 5, yields animals that have elevated levels of
circulating sugars and total body glycogen and lipids [18,20,21].
As another example, flies lacking the IRS Chico similarly have
elevated whole body lipid levels [31]. In contrast, reducing insulin
signalling specifically in adipose tissue, as observed both in
Melt and miR-278 (microRNA-278) mutant animals, has the
opposite effect, leading to reduced adiposity [66,141]. The same
is observed with conditions that up-regulate insulin signalling.
Flies mutant for PTEN, an inhibitor of the pathway, have reduced
total body glycogen and lipid levels [39]. In contrast, adiposespecific activation of TOR activity leads to increased total body
lipid levels [66].
In addition to these results, which parallel those in the mouse,
other studies have highlighted that other tissues respond in a
specific manner to insulin signalling in the fly. For example,
although PTEN mutants have reduced total body glycogen and
lipid levels, the nurse cells in the developing oocytes of PTEN
mutants actually have enlarged lipid droplets [53], suggesting that
nurse cells respond differently to the PTEN mutation compared
with other tissues of the animal. Another recent study looked at the
transcriptional response of adipose tissue and muscle to altered
insulin signalling, and found that insulin signalling modulates the
expression of approx. 2000 genes in each tissue [19]. However,
only half of these genes were affected in both tissues, and the
other half were specific for each tissue, reflecting the different
role of each tissue in organismal metabolism.
Communication between tissues
Metabolic regulation by insulin takes place within the context
of an intricate network of communication between various
tissues (Figure 2). For instance, inhibition of insulin signalling
specifically in larval muscles autonomously reduces muscle size
and non-autonomously reduces the size of the entire body,
most likely by regulating feeding behaviour [72]. Although this
is not seen in mice [142], this highlights the importance of
communication between tissues, which will be discussed below.
Brain
One important organ for insulin signalling in Drosophila is the
brain. The brain contains seven NSCs in each hemisphere, which
express ILPs 1, 2, 3 and 5 [11,17,18]. These NSCs have axon
terminals in the larval ring gland and on the aorta, where the
ILPs are secreted into the haemolymph. Reduced ILP2 expression
in NSCs correlates with reduced insulin signalling in peripheral
tissues, observed by reduced phosphorylation of Akt or increased
nuclear localization of FOXO [64,143–146], which is consistent
with a model in which ILPs secreted from NSCs activate insulin
signalling in most tissues of the animal. Secretion of ILPs
from these cells has profound effects on other tissues of the
animal, affecting tissue growth, lipid metabolism, carbohydrate
metabolism and animal longevity. Knockdown of ILP2 expression
in the NSCs by RNAi yields animals with elevated total body
trehalose levels [22]. Ablation of the NSCs causes developmental
delay, growth retardation, elevated carbohydrate levels in larval
haemolymph and an extension of lifespan [18,20]. Recently, an α1,4-glucosidase named Tobi (target of brain insulin) was identified
as a peripheral transcriptional target of ILP signalling emanating
from the brain [147]. Ablation of NSCs leads to a 17-fold downregulation of Tobi expression in the gut. Consistent with its
glucosidase function, Tobi overexpression causes a reduction in
total body glycogen levels in adult flies [147].
Metabolic regulation by insulin in Drosophila
Figure 2
21
Communication between Drosophila tissues in the insulin signalling pathway
NSCs are one tissue that produces ILPs, which are secreted to influence insulin signalling in all other tissues of the animal. ILP transcription is a central node of regulation, modulated by a number of
inputs including FOXO and S6K activity, as well as sNPF signalling from nearby neurons. Furthermore, FOXO activation in the fat body of the head regulates ILP expression in NSCs by an unknown
mechanism and serotonin (5-hydroxytryptamine) signalling from nearby neurons regulates secretion of ILP2. ILP function is inhibited by a number of secreted factors including dALS, Imp-L2 and
NLaz. Several feedback loops exist in the animal. For instance, insulin signalling in the prothoracic gland promotes ecdysone production, which in turn inhibits insulin signalling in other tissues
such as the peripheral fat body. Insulin signalling in gut promotes expression of the α-1,4-glucosidase Tobi. Further details are described in the main text. a.a., amino acids; EcR, ecdysone receptor.
A large number of factors have been reported to regulate ILP
expression by NSCs, suggesting this is a central node of metabolic
regulation in Drosophila. Neurons adjacent to the NSCs express
sNPF (short neuropeptide F), the fly orthologue of mammalian
neuropeptide Y [143]. In an elegant study, Lee et al. [143]
showed that activation of sNPF signalling, via NPFR1 (sNPF
receptor), in NSCs leads to activation of ERK signalling, inducing
expression of ILPs 1, 2 and 3. This connection was confirmed
in cell culture using CNS (central nervous system)-derived
neural BG2-c6 cells, and is therefore direct. Thus increased
sNPF production by sNPFnergic neurons resulted in increased
insulin signalling in peripheral tissues like fat body, leading to
a reduction in larval haemolymph carbohydrates. In contrast,
reduced sNPF signalling caused an elevation in circulating sugars
and increased median lifespan by 16–21 %. Another set of
neurons adjacent to the NSCs, serotonergic neurons, were also
shown to regulate ILP activity [145]. This study focused on
the function of NS3 (nucleostemin 3), a nucleostemin family
GTPase. NS3 mutants have elevated serotonin signalling in
serotonergic neurons, leading to inhibition of ILP2 secretion,
but not expression, from NSCs by an unknown mechanism.
This causes NS3 mutant animals to have low insulin signalling in peripheral tissues, to grow slowly and to reach an
adult weight of only ∼ 60 % that of control animals [145,148].
Adjacent to the adult brain in the Drosophila head is fat tissue.
Activity of the transcription factor FOXO in this adult head fat
body somehow regulates ILP2 expression in NSCs [64]. Increased
activity of FOXO in head fat leads to reduced ILP2 expression in
NSCs, leading to reduced insulin signalling in peripheral tissues,
such as peripheral fat, leading to increased animal lifespan. The
mechanism by which FOXO activity in head fat causes reduced
expression of ILP2 in NSCs remains to be investigated.
In addition to these inputs from nearby cells, several factors
within the NSCs themselves have been found to regulate ILP
expression. NSCs have high JNK (c-Jun N-terminal kinase)
activity levels [146]. As in other tissues, this JNK activity was
shown to activate FOXO in a cell autonomous manner in NSCs,
causing a reduction in ILP2 expression and consequently a
reduction in growth and an extension of lifespan [146]. Likewise,
increased S6K activity in NSCs has been proposed as a mechanism
to increase ILP expression [9], although further work will be
necessary to test this directly. In both cases, the mechanism by
which S6K and FOXO activity in NSCs regulates ILP expression
remains unclear. A recent study showed that PKA (protein kinase
A) activity in NSCs also affects insulin signalling in peripheral
tissues, and regulates production of ecdysone in the prothoracic
gland [149]. This presumably occurs via modified ILP expression
in the NSCs caused by the altered PKA activity, although this was
not tested directly. Finally, overexpression of Drosophila p53 in
NSCs causes nuclear accumulation of FOXO in NSCs. Consistent
with the studies mentioned above, this leads to reduced ILP2
expression, reduced insulin signalling in peripheral tissues and
c The Authors Journal compilation c 2010 Biochemical Society
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A. A. Teleman
increased lifespan [144]. Future work will shed light on whether
p53 loss-of-function in NSCs has the opposite effect to that seen
with overexpression.
to increased ecdysone signalling which counteracts ILP function
[149].
Fat body
Ring gland
The ring gland in Drosophila is a central endocrine organ which
produces ecdysteroids and juvenile hormones. It is composed of
three parts, the prothoracic gland, the corpus allatum and the CC
(corpus cardiacum). A number of connections involving insulin
signalling have been described between the ring gland and other
organs of the fly.
The CC secretes several factors that counteract insulin
signalling. A recent study showed that CC cells produce ImpL2, which binds ILP2 inhibiting its function and reducing growth
non-autonomously [24]. This function is particularly necessary
when animals are in adverse nutritional conditions. Under these
conditions, Imp-L2 mutant animals are not able to reduce their
insulin signalling in peripheral tissues such as fat body, leading
to increased mortality [24]. In addition, CC cells, together
with fat body cells, produce the Drosophila homologue of the
vertebrate IGF-binding protein acid-labile subunit, dALS. dALS
binds dILPs in a tertiary complex with IMP-L2, antagonizing ILP
function, hence controlling animal growth as well as carbohydrate
and fat metabolism [25]. Finally, CC cells produce AKH
(adipokinetic hormone), a functional homologue of glucagon
[150]. CC cells express ATP-sensitive potassium channels, the
targets of sulfonylureas, such as glyburide and tolbutamide, used
to stimulate glucagon secretion in diabetic patients. Exposure of
Drosophila to tolbutamide led to a 40 % increase in haemolymph
glucose in a manner dependent on CC cells. This indicates that
CC cells regulate glucose homoeostasis using glucose-sensing
and response mechanisms similar to islet cells [150].
The second component of the ring gland, the prothoracic
gland, is the site of synthesis of ecdysone. Ecdysone counteracts
insulin signalling in a number of tissues including the fat body.
Increased ecdysone levels lead to reduced PI3K activity in the
fat body, assayed via the PtdIns(3,4,5)P3 level, as well as by
increased nuclear FOXO [151,152]. The mechanistic details of
how ecdysone signalling counteracts insulin signalling are not
known. Nonetheless, the physiological results of this inhibition
are clear. During metamorphosis, increased ecdysone titres
lead to the induction of autophagy in the fat body, which is
normally suppressed by TOR signalling [100,102]. Experimental
manipulations that lead to increased ecdysone during larval
life inhibit the growth-promoting function of insulin, leading
to reduced growth of the entire animal [151,152]. Surprisingly,
ecdysone is part of a feedback loop involving insulin signalling.
Synthesis of ecdysone is positively regulated by PI3K and
TOR activity in the prothoracic gland [151–153]. This has
several physiological consequences. First, as TOR can sense
nutrients directly, independently of insulin signalling, this couples
the availability of nutrients to developmental timing, which is
regulated by ecdysone. When TOR activity is reduced in the
prothoracic gland, the ecdysone peak that marks the end of larval
development and the beginning of metamorphosis is abrogated,
extending the duration of growth. Thus the developmental delay
induced by food deprivation works in part via TOR signalling in
the prothoracic gland [153]. A second consequence is that ILP2
produced by the NSCs is less ‘powerful’ than ILP2 produced in
other tissues, due to two counteracting functions; ILP2 produced
by NSCs acts directly on peripheral tissues to regulate their
growth and metabolism but also increases insulin signalling in
the prothoracic gland, which the NSCs innervate and this leads
c The Authors Journal compilation c 2010 Biochemical Society
In mammals, adipose tissue not only acts as a fat reservoir
but also functions as an endocrine organ. The same holds true
in Drosophila. The fat body appears to act as a sensor of
nutrients, in particular amino acids, and secretes factors that nonautonomously regulate insulin signalling in other tissues. This
was discovered by studying mutants for the cationic amino acid
transporter called Slif [154]. When Slif is knocked-down in fat
body, this causes a non-autonomous reduction in insulin signalling
in other tissues, such as salivary gland cells, and a reduction in
the total body size of the animal. These effects are partially
rescued by co-expressing S6K in adipose tissue, implicating the
TOR pathway in this process. Indeed, down-regulation of TOR
function in adipose tissue also non-autonomously reduces insulin
signalling and growth in other tissues [154]. This suggests that
removal of Slif in fat body causes a reduction in intracellular
amino acids, which is sensed by TOR. The authors of that study
go on to show that knockdown of Slif in adipose tissue strongly
suppresses expression of dALS in adipose tissue, suggesting that
dALS might function as the secreted second messenger [154].
However, subsequent work has shown that dALS functions as
an ILP antagonist, and not as an agonist, as would be required
by this model [25]. Therefore further work will be required to
identify the secreted messenger by which fat body signals to other
tissues.
A third secreted factor, in addition to dALS and IMP-L2,
was recently identified as an inhibitor of insulin signalling
in Drosophila. Stress-responsive JNK signalling, as well as
starvation were found to induce expression of the secreted
lipocalin, NLaz, in fat body [26]. NLaz mutant larvae have
reduced insulin signalling levels, as measured via localization
of a reporter for PI3K activity, GFP–PH [GFP (green florescent
protein) fused to the PH domain of GRP1 (general receptor of
phosphoinositides-1)]. Conversely, overexpression of NLaz in fat
body caused a decrease in the GFP–PH signal. Overexpression of
NLaz in fat body also led to a decrease in GFP–PH signal in nurse
cells of the Drosophila oocyte, demonstrating non-autonomy of
NLaz action [26]. NLaz mutant animals display all the canonical
phenotypes of animals with increased systemic insulin signalling,
such as larger size and reduced stores of glucose, trehalose,
glycogen and triacylglycerols [26]. This is a second mechanism,
in addition to its effects on ILP2 secretion from NSCs, by which
JNK signalling regulates insulin signalling and hence metabolism
in Drosophila.
SOME DIFFERENCES BETWEEN MAMMALS AND FLIES
Although the insulin signalling pathway is strikingly conserved
between flies and mammals, there are also differences between the
two systems, some of which are mentioned above. Some further
differences are also described in this section.
Insulin and Ras
In mammalian cells, Ras activation has been implicated as
an important mediator of the mitogenic effects of insulin
stimulation [36,155]. Results so far in Drosophila have seen
only a modulatory effect of Ras on the physiological effects
of insulin (discussed above) and this might reflect a difference
between Drosophila and mammals. Alternatively, the Drosophila
Metabolic regulation by insulin in Drosophila
results agree with those presented in a study performed
in CHO (Chinese-hamster ovary) cells, where inhibition of
Ras and MAPK activation downstream of insulin signalling
does not have an impact on proliferation (assayed via DNA
synthesis), activation of S6K or glycogen synthase [156],
suggesting that in this system the effect of Ras is also
modulatory.
GLUT4 (glucose transport protein 4) and glucose
In mammals, one of the principal effects of insulin signalling is
to promote cellular glucose update via the GLUT4 transporter
[157]. In response to insulin signalling, GLUT4 translocates
to the cell surface resulting in an increased glucose uptake. In
Drosophila, this appears not to be the case. Insulin stimulation
does not alter [3 H]-labelled 2-deoxyglucose uptake in Drosophila
Kc cells [158], nor does manipulation of PI3K or Akt activity in
S2 cells [159]. However, one difference between Drosophila and
mammals is that the major circulating sugar in Drosophila is
trehalose, not glucose. Knocking-down ILP2 expression in the
NSCs by RNAi yields animals with elevated total body trehalose
levels [22], indicating that insulin signalling is indeed involved
in homoeostasis of circulating trehalose. Therefore it would be
interesting to test whether cellular uptake of trehalose is induced
upon insulin stimulation.
Glucocorticoids
In mammals, signalling through the glucocorticoid receptor is
known to have strong effects on the insulin signalling pathway
[160]. Drosophila has no obvious orthologue of the glucocorticoid
receptor [161], suggesting that one major layer of regulation might
be missing in the fly.
Reduced complexity of the pathway
Although Drosophila contains a homologue for virtually all
components of the insulin signalling pathway, the overall
complexity of the pathway is somewhat reduced in flies compared
with mammals. This is because flies often have a single
homologue that corresponds to multiple mammalian ones. For
instance, humans have two S6K proteins, three 4E-BP proteins
and multiple FOXOs, whereas flies only have a single S6K,
4E-BP and FOXO protein. On the one hand, this means that
removal of gene function in the fly is more likely to give strong
and clear effects, as there is less redundancy compared with
the mammalian system. On the other hand, direct comparison
between the fly protein functions and the mammalian ones may
not be easy, as the proteins in the mammalian system may have
specialized functions.
CONCLUSIONS
Some aspects of mammalian metabolic control, such as the leptin
signalling pathway, do not exist in the fly. These mechanisms can
be considered additional layers of metabolic regulation that were
added during mammalian evolutionary on top of the more basic
ones found in all animals. Clearly these mechanisms cannot be
studied in the fly. Insulin signalling, however, appears to be both
present and highly conserved, both at the molecular level and the
physiological level, probably due to the basic cellular functions it
regulates. Therefore it is fruitful to exploit the powerful genetic
tools available in the Drosophila model organism in order to
23
effectively study the molecular mechanisms by which insulin
regulates metabolism.
FUNDING
The work in my laboratory was supported by a Helmholtz
Young Investigator grant and an European Union FP7 grant under
the MITIN (integration of the system models of mitochondrial
function and insulin signalling and its application in the study of
complex diseases) project.
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