Post on 31-Aug-2019
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POSITIVE CROSS-TALK BETWEEN ECDYSONE AND INSULIN SIGNALING IN
INSECT GROWTH
A dissertation presented
by
Srikanth Subramanian
to
The Department of Biology
In partial fulfillment of requirements for the degree of
Doctor of Philosophy
in the field of
Biology
Northeastern University
Boston, Massachusetts
January 2012
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2012
Srikanth Subramanian
ALL RIGHTS RESERVED
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POSITIVE CROSS-TALK BETWEEN ECDYSONE AND INSULIN SIGNALING IN
INSECT GROWTH
A dissertation presented
by
Srikanth Subramanian
ABSTRACT OF DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Biology
in the Graduate School of Arts and Sciences of
Northeastern University, January, 2012
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ABSTRACT
Wing imaginal discs are masses of undifferentiated cells that give rise to the
forewings and hindwings of adult insects. In this dissertation, I examined the roles of
insulin and ecdysone, a steroid molting hormone, in the regulation of imaginal wing disc
growth in the tobacco hornworm, Manduca sexta. Previous studies on a different
lepidopteran insect had suggested a requisite role for both hormones in disc growth
providing a means to coordinate nutritional regulation of growth with metamorphic
development. I studied the short-term and long-term stimulation of signaling events in
the insulin pathway, and in ecdysone receptor content, in order to determine loci at which
insulin and ecdysone might interact. My results clearly revealed a requirement for both
insulin and ecdysone in Manduca disc growth. This result countered earlier research on
growth in Drosophila, which is ecdysone independent, and was more closely in keeping
with steroid regulation of insulin response in vertebrates. The results raised questions
regarding the underlying mechanisms by which disc growth is stimulated. I showed that
ecdysone and insulin were required together to increase cell number, using an EdU click-
chemistry detection assay. I further examined whether specific signaling proteins were
synergistically enhanced by ecdysone and insulin and whether the effects of ecdysone on
growth were dependent upon insulin signaling. I found that RNAi-mediated knockdown
of the insulin receptor blocked hormone-stimulated growth, as well as blocking increases
in the ecdysone receptor, insulin receptor, and insulin receptor signaling pathway.
This research was the first demonstration of knockdown of the insulin receptor in M.
sexta. The results pinpointed critical cellular targets in the insulin pathway, specifically
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Akt, involved in cross-talk with ecdysone. Inhibitors of PI3-kinase and TOR, i.e.
upstream and downstream regulators of Akt, blocked growth and ecdysone receptor
content in a manner similar to insulin receptor knockdown. The results suggest that
downstream effects of Akt, such as reduced nuclear import of FOXO, may play crucial
roles in steroid-regulated growth. This work helps to improve our understanding of the
cellular mechanisms between insulin signaling and steroids that underlie post-embryonic
animal development.
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This thesis is dedicated to Pops, Mom and Eileen,
whose never-ending love and support
made this dissertation possible.
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TABLE OF CONTENTS
Abstract 4
Dedication 6
List of Figures 8
Abbreviations 9
Acknowledgements 12
Introduction 15
Chapter 1: The control of growth and differentiation of wing imaginal discs
in Manduca sexta. 24
Chapter 2: Effects of ecdysone and insulin on cell division 38
Chapter 3: Cellular interactions between ecdysone and insulin in the regulation
of disc growth 48
References 72
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LIST OF FIGURES
Figure 1. Ecdysteroid levels during the fifth instar of M. sexta.
Figure 2. A model of the insulin signaling pathway.
Figure 3. Growth and differentiation of the forewing imaginal disc.
Figure 4. Wing imaginal disc growth during first three days after entering the
wander stage.
Figure 5. Growth of wing imaginal discs in vitro.
Figure 6. Phosphoproteins response of cultured wing discs.
Figure 7. Phosphoprotein Akt and total protein response of cultured wing discs after
48 hour incubation.
Figure 8. Click reaction between EdU and azide modified dye.
Figure 9. Detection of cell proliferation with EdU in wing discs after 48 hour
incubation.
Figure 10. Number of proliferating cells (incorporating EdU) in wing discs after 48
hour incubation.
Figure 11. Effects of siRNA, LY294002, and rapamycin on growth in wing discs.
Figure 12. Detection of transcript in wing discs after 48 hour treatment.
Figure 13. Activation of phosphoproteins in wing discs after 48 hour incubation.
Figure 14. Total Akt in wing discs after 48 hour incubation.
Figure 15. Effects of rapamycin on p4EBP and EcR in wing discs after 48 hour
incubation.
Figure 16. Ecdysone receptor proteins in wing discs after 48 hour incubation.
Figure 17. Suggested mechanism through which ecdysone and insulin synergistically
interact to modulate EcR expression and activity.
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ABBREVIATIONS
4EBP initiation factor 4E binding protein
Akt2 human homolog of v-akt oncogene
BSA bovine serum albumin
Cdk cyclin-dependent kinase
cDNA complementary deoxyribonucleic acid DILP Drosophila insulin-like peptide
DIR Drosophila insulin receptor
DNA deoxyribonucleic acid
dsRNA double stranded ribonucleic acid E2 17- -estradiol Ec ecdysone EcR ecdysone receptor EcR-A ecdysone receptor isoform A EcR-B1 ecdysone receptor isoform B1 EdU 5-ethynyl-2’-deoxyuridine ERK extracellular signal-regulated kinase FOXO forkhead box-containing protein GFPsiRNA small-interfering RNA directed against green fluorescent protein Grb2 growth factor receptor-bound protein 2 GSK glycogen synthase kinase
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HRP horseradish peroxidase IDGF imaginal disc growth factor IGF insulin-like growth factor Ins insulin IR insulin receptor IRS insulin receptor substrate IRsiRNA small-interfering RNA directed against the
bombyxin/insulin receptor JH juvenile hormone MAPK mitogen-activated protein kinase MEK mitogen-activated ERK-activating kinase mRNA messenger ribonucleic acid mTOR mammalian target of rapamycin
p phosphorylated p70S6K 70 kDa S6 kinase PBS phosphate buffered saline PCR polymerase chain reaction PDK phosphoinositide-dependent protein kinases PI3K phosphatidylinositol-3-kinase
PIP3 phosphatidylinositol-(3,4,5)-triphosphate
PKB/Akt protein kinase B
PPAR- peroxisome-proliferator-activated-receptor PTEN phosphatidylinositol-3,4,5-triphosphate 3-phosphatase
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PTTH prothoracicotropic hormone qPCR quantitative real time PCR
Raf Raf serine/threonine kinase
Ras Ras GTP-binding protein
RNA ribonucleic acid
RNAi ribonucleic acid interference
ROS reactive oxygen species
Rp49 ribosomal protein 49 RT room temperature RXR retinoid X receptor S6 ribosomal protein S6 siRNA small-interfering RNA SOS son of sevenless STAT signal transducers and activators of transcription TOR target of rapamycin TSC tuberous sclerosis complex TZDs thiazolidinediones USP ultraspiracle V fifth instar
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ACKNOWLEDGEMENTS
I would like to take a moment, as my time at Northeastern University comes to an
end, to thank the people who have supported me in various ways and have helped make
my success possible. First and foremost, I thank my advisor, Dr. Wendy Smith. She has
been a great mentor and even greater friend to me. Her enthusiasm and sharp sense of
humor made the lab a wonderful place to work. She offered guidance when needed but
she also allowed me the independence to really take possession of my project. She
helped me grow as a person and as a scientist. I have learned so much from her and I am
grateful to be part of her lab. I hope to make her proud in the future. I also thank the
members of my doctoral committee Dr. Gail Begley, Dr. Erin Cram, Dr. Dick Deth and
Dr. Rebecca Rosengaus for their time and expert advice. I owe Dr. Rosengaus extra
thanks for helping me with statistics.
I thank the current and past members of the Smith lab, with whom I have had the
pleasure to work. First, I thank Dr. Amy (Walsh) D’Amico, a.k.a. “Coach” for
welcoming me into the lab, steering me clear of pitfalls, making me laugh constantly, and
for being a dear friend. I also thank Dr. Lou D’Amico for his constant encouragement
and sound advice. I thank Leon DeLalio for his willingness to listen and for sharing his
excellent taste in food, movies, and his lamp with me. There are also many
undergraduates that have been part of the lab who have helped me over the past few
years. I thank Abby Bootes and Erin Greguske for taking over management
responsibilities in the lab. I thank Jackie Olender for helping me troubleshoot real time
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PCR experiments and I thank Jade McPherson for her patience and hard work with the
EdU experiments. I also thank Bethany Kirpalani, Rhamy Zeid, Sauveur Jeanty, Dana
Galacchi, and Erin Vinoski for their friendship and dedication to the lab.
I thank the O’Malley lab for help with the confocal microscope. I thank Dr. Don
O’Malley for taking the time to teach me how to use the microscope properly. I also
thank Dr. Kristen Severi, Dr. Rebecca Westphal, and Sucharita Saha for being gracious
hosts during my lengthy visits to their lab.
The Davis lab has always had an open door policy with me and I thank them for
letting me take advantage of that. I thank Dr. Davis for his friendship and sense of
humor. I thank Dr. Andy Cary for sharing his technical expertise and offering
encouragement in his own unique way. Finally, I thank Dan Wreschnig for always
taking time to talk with me about science, teaching, literature, and life in general.
I thank the office staff in the Biology Department, especially Adrian Gilbert and
Aaron Roth, who have always found a way to make things work. I thank Frauke Argyros
and Patti Hampf for doing everything they could to make the teaching labs run smoothly.
I also thank Patti for letting me invade her office on long teaching days.
I thank the biology graduate students (including the Richards-Hall-Night-crew
and the Biohazards broomball team) for making this whole graduate experience as fun as
possible. I also have to thank my BHS friends (BA, Sarah, Larae, Karen, Jenn and Rene)
who have known me since we were kids and have been encouraging me ever since.
Finally, and most importantly, I need to thank my family. Their unconditional
love helped make this dissertation possible. Pops and Mom have been a constant source
of emotional support. I also thank Mani Mama, Bhagya Auntie, Karthik and Kavitha. I
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thank the Loughmans for welcoming me into their family. I’m saving the best for last; I
thank my wife, Eileen. There are no words that can fully encompass how much her love
and support (and patience) mean to me. I love you all.
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Introduction
Insect growth/development
The growth of a cell or tissue involves complex interactions between genes,
metabolism, nutrition, and extracellular signals. In order to understand these processes,
we need to use a model that can be studied physiologically and on a molecular level. The
tobacco hornworm, Manduca sexta has been a useful model to study the endocrine
regulation of growth, particularly post-embryonic growth that directs the transition from
juvenile to adult, because these holometablolous insects develop synchronously and have
clear markers for staging larval development (Nijhout and Williams, 1974; Gilbert et al.,
1980). The duration of growth in M.sexta, as in all insects, is controlled by changing
levels of hormones (Gilbert et al., 2002). The group of hormones that regulate insect
development are ecdysteroids, juvenile hormone (JH) and prothoracicotropic hormone
(PTTH) (Nijhout, 1994; Gilbert et al., 2002). During the fifth larval stage (instar), the
largest growth phase, PTTH secretion and ecdysone are inhibited by JH, a terpenoid
hormone with no vertebrate homolog (Nijhout and Williams, 1974; Roundtree and
Bollenbacher 1986; Gilbert, 2012). Larvae feed during the first part of the instar until
they reach a critical weight of about 6g (Nijhout and Williams, 1974; D’Amico et al.,
2001). JH levels then decrease, allowing the photoperiodically gated release of PTTH, a
neuropeptide, which causes the prothoracic glands to release ecdysone, a steroid (Figure
1) (Nijhout and Williams, 1974; Rountree and Bollenbacher, 1986). Markers for the
release of ecdysone, which is converted to the active ecdysteroid 20-hydroxyecdysone,
include cessation of feeding, purging of the gut, exposure of the dorsal vessel and
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wandering (increased motility) associated with searching for a suitable place to
pupate(Nijhout, 1994; Riddiford et al., 2003). Two days later, a second ecdysteroid peak
causes the animals to pupate (Nijhout, 1994, Riddiford et al., 2003). Body growth is
restricted to the larval instars, especially the fifth instar, so adult size is fixed at the larval-
to-pupal transition.
Figure 1. Ecdysteroid levels during the fifth instar of M. sexta. Abbreviations: wandering (W), ecdysis (E) (Bollenbacher et al., 1981; from Smith, 1995).
19 8 7 6 5 4 3 2 1 0 0
30
Day of Fifth
Ecd
yste
roid
s
W E
0 1 2 3 4 5 6 7 8 9 0 1 2
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Insulin signaling pathway
In addition to the conventional hormones known to regulate insect growth, it is
becoming clear that insulin is also a key player in integrating metabolism and response to
nutrition. The insulin signaling pathway is conserved across species, and insulin-like
signaling molecules have been found in a variety of invertebrates including nematodes,
molluscs and insects (Claeys et al. 2002). Bombyxin, from the silkmoth Bombyx mori,
was the first insect insulin-like molecule to be discovered. A heterodimer of insulin-like
A- and B-chains, it is clearly a member of the insulin superfamily (Ishizaki and Suzuki,
1994; Claeys et al. 2002). Over 40 bombyxin genes have been identified, yet their
function(s) remain unclear (Adachi et al., 1989; Nagata et al., 1995; Kondo et al., 1996).
The insulin receptor (IR) is one of many growth factor receptors that regulate
cellular processes via the activation of receptor tyrosine kinases in the plasma membrane
(Myers et al. 2003; Claeys et al. 2002). The IR is comprised of - and -subunits joined
by disulfide bridges to form an -heterodimer (Myers et al. 2003). Ligand binding
specificity depends on cysteine-rich regions in -subunits located towards the
extracellular face of the plasma membrane. -subunits on the cytoplasmic face of the
membrane contain tyrosine kinase activity and undergo tyrosine phosphorylation by
adjacent subunits as a result of ligand binding (Claeys et al. 2002). These features are
also present in the insulin-like growth factor (IGF) receptor (Fernandez et al. 1995;
Myers et al. 2003).
The insulin receptor uses a group of scaffolding molecules called insulin receptor
substrates (IRS), to initiate cytosolic signaling (White, 1998; Lannigan, 2003; Claeys et
al. 2002). As shown in Figure 2, IRS binds to the phosphotyrosine in activated IR and
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itself becomes phosphorylated (White, 1998). IRS can then activate many growth related
pathways (Claeys et al. 2002; Lannigan, 2003). For example, the activation of Grb2/Sos
(son of sevenless) activates the mitogen-activated ERK-activating kinase/extracellular-
signal-regulated kinase (MEK/ERK) pathway via the proteins Ras and Raf (Lannigan,
2003). A second pathway leads to the activated IRS-phosphatidylinositol-3-OH kinase
(IRS-PI3K) complex, which generates phosphatidylinositol-(3,4,5)-triphosphate (PIP3)
and recruits phosphoinositide-dependent protein kinases (PDK), protein kinase B
(PKB/Akt), and other downstream kinases which affect cell growth and survival (Claeys
et al. 2002; Lannigan et al. 2003). Typically, Akt phosphorylation blocks the activity of
proteins such as glycogen synthase kinase (GSK), forkhead box-containing protein
(FOXO), and tuberous sclerosis complex (TSC) that stop growth in their non-
phosphorylated state (Figure 2) (Burgering and Medema, 2003; Krymskaya, 2003). For
example, non-phosphorylated TSC indirectly inhibits the growth regulating kinase, target
of rapamycin (TOR) (Krymskaya, 2003). TOR is a nutrition sensor that can regulate
translation by phosphorylating initiation factor 4E binding protein (4E-BP) and
p70S6kinase (Hay and Sonenberg, 2004).
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Figure 2. A model of the insulin signaling pathway. See text for details. Abbreviations: InR, insulin receptor; IRS, insulin receptor substrate; Grb2/SOS, adapter and guanine-nucleotide-exchange factor for Ras; Ras, Ras GTP-binding protein; Raf, Raf serine/threonine kinase; MEK, MAPK/ERK kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol-(3,4,5)-triphosphate; PDK, phosphoinositdie-dependent protein kinase; Akt protein kinase B; GSK, glycogen synthase kinase; FOXO, forkhead box-containing protein; TSC, tuberous sclerosis complex; TOR, target of rapamycin. 4EBP, binding protein for ribosomal initiation factor 4E; S6, ribosomal protein S6; p70S6K, 70 kDa S6 kinase.
Ecdysone and ecdysone receptor
Ecdysone is a steroid molting hormone which also stimulates metamorphosis-
related development. 20-hydroxyecdysone is the mature form of the hormone derived
from enzymatic modification of cholesterol. Ecdysone synthesis occurs in glands known
as the prothoracic glands, and is controlled by the brain via the neurosecretory hormone
prothoracicotropic hormone (PTTH). A PTTH-stimulated rise in ecdysone triggers each
larval molt. During the fifth instar, a rise in ecdysone induces the cessation of feeding
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and entry into the prepupal wandering stage (Bollenbacher et al., 1981; Baker et al.,
1987; Smith, 1995, and see Fig. 1).
The ecdysone receptor is part of the superfamily of nuclear receptors that are
ligand-dependent transcription factors (Riddiford et al., 2000; Billas and Moras, 2005).
This superfamily plays an important role in regulating the expression of an array of genes
during development and reproduction (Riddiford et al., 2000). Steroid receptors are
characterized by a C-terminal ligand-binding domain, a DNA-binding region, and
transcriptional activation domains (Jindra et al. 1996, 1997). The ecdysone receptor
(EcR) heterodimerizes with a nuclear receptor known as ultraspiracle (USP) (Yao et al.,
1993; Billas and Moras, 2005). USP is an ortholog of the vertebrate retinoid X receptor
(RXR) (Yao et al, 1993; Riddiford et al., 2001). There are two main ecdysone receptor
isoforms, EcR-A and EcR-B1, which differ in the N-terminal domain (Truman, 1996;
Jindra et al., 1996, 1997). EcR sub-types have been found to play specific roles in some
metamorphic events, particularly in the nervous system (Truman, 1996; Scauer et al.,
2011; Schwedes et al., 2011).
Insulin signaling and ecdysone in insects
A growing body of evidence indicates that metamorphosis involves an interplay
between ecdysteroid and insulin-stimulated developmental events. The regulation of
insect growth via the insulin signaling pathway is best understood in imaginal discs,
particularly in Drosophila. Imaginal discs are simple epithelia made up of a single layer
of cells that rapidly grow into complex structures in the adult (Johnston and Gallant,
2002). There is only one Drosophila insulin receptor (DIR) but it has been shown to be
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highly expressed in larval imaginal discs (Garofalo and Rosen, 1988). Overexpression or
loss-of-function of DIR increases or decreases body size, respectively (Brogiolo et al.,
2001). The DIR loss-of-function mimics the effects of starvation/nutrient-deficiency
(Brogiolo et al., 2001). Drosophila insulins or insulin-like peptides referred to as DILPs,
work together with a group of growth factors (secreted glycoproteins) called imaginal
disc growth factors (IDGFs) to control tissue and body growth primarily by regulating
cell size (Kawamura et al.,1999; Bryant, 2001; Varela et al., 2002; Goberdhan and
Wilson, 2003; Arquier et al., 2008; Zhu et al., 2008). At least part of this growth effect is
mediated by two small groups of neurons in the Drosophila brain which secrete DILPs
into the circulatory system at levels that are modulated by nutrition (Goberdhan and
Wilson, 2003; Arquier et al., 2008). Though DILPs and IDGFs are required for disc
growth in Drosophila, ecdysone does not seem to be required. In Drosophila fat body,
ecdysone appears to inhibit insulin/insulin-like growth factor signaling (Colombani et al.
2005, Francis et al., 2010).
In contrast, imaginal disc growth in Lepidoptera requires insulin and ecdysone.
Wing discs of Lepidoptera grow continuously and exponentially during the later larval
and prepupal stages (Kremen and Nijhout, 1998; Miner et al., 2000; Nijhout and Grunert,
2002; Nijhout et al., 2007). In feeding larvae, starvation and JH expression have been
shown to inhibit disc growth (Miner et al. 2000). However, in non-feeding larvae, i.e.,
once the animals have begun metamorphosis, discs continue to grow. In other words,
control of imaginal disc growth changes from being dependent on feeding to being
independent of feeding (Nardi et al., 1985; Nijhout et al., 2007). In the buckeye butterfly,
Precis coenia, imaginal discs from feeding animals stop growing (Nijhout and Grunert,
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2002). Disc growth in vitro is promoted by supplementing media with ecdysone and with
hemolymph containing the insulin-like growth factor, bombyxin (Nijhout and Grunert,
2002). Neither alone stimulates growth. Hence, unlike Drosophila, both ecdysone and
insulin-like hormones are required to stimulate growth. At the time I began my research,
little else was understood about the nature of the interaction between ecdysone and
insulin in regulating disc development. In the experiments described in Chapter 1, I
examined the roles of insulin and ecdysone in the regulation of wing imaginal disc
growth in Manduca sexta and explored the short-term and long-term stimulation of
insulin signaling events. Precedence for steroid regulation of insulin signaling can be
found in other animals as described in the introduction of Chapter 1.
Similar to Precis, I hypothesized that in Manduca, ecdysone was working synergistically
with insulin to enhance the insulin pathway and promote growth of imaginal discs. In
short-term (30 min) incubations, insulin activated growth factor signaling elements such
as Akt, while ecdysone did not. But after longer (2 day) incubations, ecdysone
stimulated identifiable members of the insulin pathway. Together, insulin and ecdysone
also stimulated disc growth. These observations were further explored in Chapters 2
and 3.
In experiments described in Chapter 2, I determined whether ecdysone was
responsible for cellular division, as is typical of steroid hormones (Revelli et al., 1998;
Fox et al., 2009). There was the possibility that insulin and ecdysone acted
synergistically, with neither having sole responsibility for an increase in cell number. By
using a novel DNA synthesis detection assay, I determined the effects of each hormone
specifically upon cell division (Chapter 2). Both insulin and ecdysone were seen to
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increase cell division, with ecdysone having a greater effect, in keeping with traditional
effects of steroid hormones.
In experiments described in Chapter 3, I further explored the interaction between
ecdysone and insulin in modulating growth. I hypothesized that insulin and ecdysone
would have mutually positive effects, particularly with regard to receptor expression.
Precedence in vertebrates is discussed in greater detail in Chapter 3. Using RNAi,
inhibitors, and antibodies directed against specific elements of the insulin pathway, I
further identified synergistic interactions between ecdysone and insulin, including but not
limited to receptor content, in the regulation of disc growth (Chapter 3).
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CHAPTER ONE
The control of growth and differentiation of wing imaginal discs
in Manduca sexta
(Published as Nijhout HF, Smith WA, Schachar I, Subramanian S, Tobler A, Grunert
LW. The control of growth and differentiation of the wing imaginal disks of Manduca
sexta. Dev Biol. 2007 Feb 15;302(2):569-76).
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INTRODUCTION
Initial studies with lepidopteran imaginal discs indicated that conditions required
for normal growth in Precis coenia differed from those in Drosophila (Nijhout and
Grunert, 2002). In Precis, during the feeding phase, growth of the discs depended on
nutrition (Nijhout and Grunert, 2002). When larvae were starved, disc growth ceased
within 4-6 hours (Miner et al., 2000; Nijhout and Grunert, 2002). Similarly, if larvae
were fed a low-nutrient diet, larval growth rate and growth rate of the discs were also
slow. In other words, wing disc growth was modulated to remain proportional to the
growth of the rest of the body.
When discs taken from feeding and growing larvae were placed in nutrient-rich
culture medium they failed to grow. However, discs were made to grow at a normal rate
by adding bombyxin and a steroid molting hormone 20-hydroxyecdysone to the culture
medium (Nijhout and Grunert, 2002). Precis disc growth thus required a classic insect
developmental trigger (ecdysone) as well as a hormone regulated by nutrition
(bombyxin), providing an appealing explanation for coordination of nutrition and
development in the regulation of body and organ size. In Precis, the bulk of disc growth
occurs during feeding. This may be an exception in developing insects rather than the
rule. For example, in most lepidopterans discs grow continuously and exponentially
throughout the late larval and prepupal periods (Miner et al., 2000).
In Manduca, imaginal discs continue to grow on their normal exponential trajectory, even
after larvae have stopped feeding (Williams, 1980; Nardi et al., 1985; Nijhout et al.,
2007). Given that growth continues in non-feeding in Manduca, the question arose as to
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whether insulin (and/or ecdysone) continued to play a positive role. To answer this
question, we studied the post-feeding wandering stage growth of the imaginal wing discs
of Manduca sexta. We showed that, as in Precis, disc growth in Manduca requires
ecdysone and insulin in vitro.
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MATERIALS AND METHODS
Animals and tissue culture
Manduca sexta larvae were reared on a standard artificial laboratory diet (Bell and
Joachim, 1976) at 25°C under a long-day 16:8 light:dark cycle. Day five, fifth instar
(wandering) animals were anesthetized for 10 min in water then surface-sterilized by
immersion for 2–3 min in a 1/750 solution of benzalkonium chloride (Matheson Coleman
and Bell). Discs were dissected in sterile saline in a sterile hood. Disks were cultured in
24-well plates (Costar 3524) in 300 l Grace's medium (Gibco, Invitrogen) supplemented
with 10% fetal calf serum (Gibco) and 10% antibiotic–antimicotic (Gibco), under a 95%
O2/5% CO2 atmosphere. Following incubations, disc length was measured under a
dissecting microscope using an ocular micrometer. Wing discs were then homogenized
in 2x SDS sample buffer, incubated for 3-5 minutes at 90°C, and stored at -20° C.
Western blots were subsequently performed (see below).
Hormones
Insulin (NovoRapid) was diluted in Grace’s medium to a final concentration of
30μM. 20-Hydroxyecdysone (Sigma) was dissolved in Grace’s medium to a final
concentration of 0.1 μg/ml. These concentrations were determined to provide maximal
growth based on preliminary tests of a range of doses for each hormone (0.1 μg/ml to 0.5
μg/ml for ecdysone, and 10 μM to 30μM for insulin, data not shown).
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Western blot analysis
Wing discs were dissected in pairs, placed in 40 μL of 2X SDS sample buffer, and
incubated at 90°C for three to five minutes. The samples were then run on a 10-20%
SDS-PAGE gradient gel (Bio-Rad) to separate the proteins. They were transferred from
gels to nitrocellulose membranes at 4°C. Membranes were then blocked in 3% BSA or
5% milk for one hour, rinsed quickly (15 minutes total) and placed on primary antibody
overnight. Primary antibodies included antiphospho-insulin receptor, antiphospho-Akt,
total Akt, and anti-Actin (Cell Signaling). Blots were rinsed and placed on secondary
anti-rabbit antibody with attached HRP (Cell Signaling) for 75 minutes. Blots were
treated with Western blotting chemiluminescence reagents (Pierce ECL Western blotting
substrate, Thermo Scientific) and were exposed on blue- sensitive autoradiographic film
(Marsh Bio Products) and developed (Kodak GBX fixer and developer). Blots were
scanned and analyzed using ImageJ (Abramoff et al., 2004). Protein bands were
normalized to actin as a loading control where appropriate.
Statistical Analysis
Protein expression in the wing imaginal discs was subjected to statistical analysis
using one way ANOVA, with a Dunnett’s post hoc test to compare specific treatments
against a reference. In all cases, p<0.05 was used to determine significance. All
statistical analyses were performed using SPSS v.19 (licensed to Northeastern University,
2011).
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RESULTS
Growth and differentiation
The morphology of the growing Manduca wing disc is shown in Fig. 3. During
the feeding stage (white box, Fig. 3) wing disc size stayed in proportion to body size. On
day 5 (V5) of the fifth instar, larvae enter the wandering stage and stop feeding. At this
point discs continued to grow and began to differentiate (Fig. 3). Disc differentiation
during the wandering stage involved development of the lacunae (spaces) that would
form the wing veins and the subsequent expansion of the tracheal system into those same
spaces. Wing vein differentiation or tracheal migration began 12 hours after the start of
wandering and the tracheal system developed fully at 60 hours (Fig. 3). In vivo, trachea
started to develop while the ecdysteroid levels were still low. In this regard, imaginal
discs of Manduca significantly differed from those of Precis. In Precis, tracheal
migration and wing vein differentiation occurred during the feeding stage of the last
larval instar (Miner et al., 2000). In Manduca, tracheal migration began before the peak
of ecdysone that initiates the pupal molt. At the same time growth rate increased
significantly in subsequent days after wandering (Fig. 4). It is clear that disc growth
increased as the larvae entered the non-feeding wandering stage. The size of the discs
increased from roughly 1 mm to several mm over a 3-day span (Fig. 3). The question
arose as to whether, as in Precis, ecdysone and insulin were involved in this change.
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Figure 3. Growth and differentiation of the forewing imaginal disc. The larval disc growth during the feeding stage is represented by the enclosed gray box. The developmental “age” of the feeding larva is indicated below each disc by the weight of the larva (in grams). During the wandering stage, developmental “age” is indicated as hours after the initiation of wandering. Numbers below wandering stage discs indicate scoring system used to quantify tracheal differentiation (from Nijhout et al., 2007).
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Figure 4. Wing imaginal disc growth during first three days after entering the wandering stage (from Nijhout et al., 2007).
Control of imaginal disc growth in vitro
We studied the direct effects of ecdysone and insulin on growth of wing imaginal
discs of Manduca sexta in tissue culture. Insulin was used rather than bombyxin as
Manduca bombyxin was not available in purified form. Insulin has been shown to affect
developing tissues in Manduca in a manner indistinguishable from bombyxin (Koyama
et al., 2008). As shown in Fig. 5, discs cultured with the addition of both ecdysone and
insulin doubled in size. Neither ecdysone alone nor insulin alone had a significant effect
on growth (Fig. 5).
32
Figure 5. Growth of wing imaginal discs in vitro. All discs were cultured for 48 hours alone in Grace’s medium (Control), with 20-hydroxyecdysone (Ec), with insulin (Ins) or with 20-hydroxyecdysone and insulin (Ec+Ins). Significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05) (disc pictures from Nijhout et al., 2007).
Short-term and long-term stimulation of insulin signaling events
Given a clear synergistic effect of ecdysone and insulin on growth, we next
investigated whether ecdysone and insulin had identifiable effects on growth-related
signaling pathways. Insulin-like hormones stimulate tyrosine phosphorylation of a highly
conserved amino acid sequence (YETDYY) of the insulin receptor (Nijhout et al., 2007).
Discs were exposed to ecdysone and/or insulin for 30 minutes, to capture short-lived
phosphorylation events. As seen in Figs. 6A and B, insulin alone stimulated the
a a a
b
33
phosphorylation of an 85kD protein, corresponding in size to the subunit of the
mammalian insulin receptor. A clone of the B. mori insulin receptor predicted a protein
of similar size (Swevers and Iatrou, 2003) which was also recently confirmed by the
sequencing of the M. sexta insulin receptor (Koyama et al., 2008). Anti-actin antibody,
which detected two actin fragments, one at 45kD and a smaller 30 kD fragment, was used
to control for loading of lanes on the Western blot (Fig. 6A). Samples were normalized
by dividing each respective band intensity by the intensity of actin.
An additional signaling kinase typically activated by insulin-like growth factors,
Akt/protein kinase B, was also studied using antibody directed against a highly conserved
phosphorylation domain (Fig. 6A and C). After 30-min incubation, insulin stimulated the
phosphorylation of Akt (65kD) in wing discs. Results from this experiment indicate that
mammalian insulin significantly stimulated (p<0.05) typical phosphoproteins in the
insulin signaling pathway. Conversely, ecdysone alone did not stimulate phosphorylation
of the insulin receptor or Akt within the 30 min incubation period (Fig. 6). When
combined, ecdysone and insulin significantly stimulated (p<0.05) phosphorylation of
both insulin receptor and Akt, though to no greater degree than insulin alone (Fig. 6B and
C).
34
Figure 6. Phosphoproteins response of cultured wing discs. A. 30-minute challenge in medium containing ecdysone (Ec), insulin (Ins), both (Ec+Ins), or no hormones (Control, C) (n=8). Molecular mass is shown in kilodaltons (kD) at right. B. Quantification of pIR. C. Quantification of pAkt Western blots were done using ImageJ (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).
b
b
b
b
a a
a a
B
C
Ec C Ins C Ec+Ins C
pIR
pAkt
Actin
- 85kD
- 62 kD
- 40 kD
- 30 kD
A
35
The same phosphoproteins were examined after long-term incubation. After 48
hours, insulin, or insulin combined with ecdysone (Ec+Ins), significantly increased pAkt
(p<0.05) (Fig. 7). 48 hour incubation in ecdysone alone also led to a significant increase
in pAkt (p<0.05) (Fig. 7B). The observed increases in pAkt after 48 hours were due, at
least in part, to increases in total Akt. As seen in Fig. 7C, there was a significant increase
in total Akt when discs were incubated with both ecdysone and insulin (Ec+Ins) (p<0.05)
(Fig. 7C).
Figure 7. Phosphoprotein Akt and total protein response of cultured wing discs after 48 hour incubation. Challenge in medium containing ecdysone (Ec), insulin (Ins), both (Ec+Ins). A. Representative Western blots. Molecular mass shown in kilodaltons (kD) at right. B. Quantification of pAkt. C. Quantification of total Akt. Western blots were done using Image J (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05)
a a a
a
36
DISCUSSION
The primary focus of the experiments described in this Chapter was to determine
whether growing discs in post-feeding insects, representing a typical period of
exponential disc growth, required both steroid hormones and insulin. Insulin-like growth
hormones had been implicated as the primary regulators of late larval insect growth,
using Drosophila as a model. Mutations in insulin signaling for genes such as IR, Chico
(an insulin receptor substrate), PI3K, TSC1/2, TOR, and FOXO, all caused changes in
Drosophila cell size or cell proliferation and affected overall body size (Brogiolo et al.,
2001; Britton et al., 2002; Kim et al., 2002; Garami et al., 2003; Hariharan and Bilder,
2006). In Drosophila, ecdysteroids reduced insulin signaling and inhibited growth
(Colombani et al., 2005). More recently, in lepidopteran insects such as Precis,
ecdysteroids were shown to work with insulin-like hormones to stimulate wing disc
growth. In Precis, disc growth occurs only in feeding larvae. A more typical pattern of
lepidopteran growth, however, is continuous through feeding and non-feeding (prepupal)
stages. We thus set out to determine if growth in the more typically developing tobacco
hornworm was driven by insulin alone, as in Drosophila, or by both insulin-like and
steroid hormones, as in Precis. Our results clearly implicate both hormones in Manduca
disc growth, despite the fact that growth occurs largely post feeding.
In the present studies, growth was shown to occur via the insulin signaling
pathway as indicated by phosphorylation of the Manduca insulin receptor and
downstream signaling kinase Akt (Fig. 6 and Fig. 7A). Short-term experiments indicated
that insulin was able to significantly activate the pathway by itself, but that ecdysone was
37
not (Fig. 6). After 48 hours, ecdysone significantly increased both total and
phosphorylated Akt on its own, as did insulin, although neither alone stimulated growth.
The maximal activation of Akt and significant growth in the wing discs was achieved by
incubating with both ecdysone and insulin (Fig. 7B and 7C). These results point strongly
to synergistic effects of ecdysone and insulin in regulating growth. Discs do not increase
in size when exposed to insulin alone despite clear demonstration that insulin can
enhance the phosphorylation of an endogenous insulin receptor as well as levels of
phospho- and total Akt. The dual-hormone requirement for Manduca disc growth means
that changes in somatic growth could come about by varying ecdysone levels, insulin
levels, or both.
The experiments described in this chapter delineated the parameters by which we
could reliably stimulate disc growth in Manduca sexta. The results raise questions
regarding the underlying mechanisms by which growth is stimulated. We chose to
investigate this in two ways. First we examined whether ecdysone and insulin were
together required to increase cell number. Cell division, a typical effect of steroids, may
necessitate additional input by insulin-like hormones in developing insects. Second, we
examined whether specific signaling proteins were enhanced by ecdysone and insulin and
whether the effects of ecdysone on growth were dependent upon insulin signaling. The
results of these approaches are developed in Chapters 2 and 3.
38
CHAPTER TWO
Effects of ecdysone and insulin on cell division
39
INTRODUCTION
During development, insulin signaling molecules function as growth factors
influencing size as well as differentiation of specialized cells. Insulin-like growth factors
(IGFs) have been known to affect cellular proliferation by stimulating cell cycle
progression (Dupont et al., 2000). Other growth factors such as fibroblast growth factor
and vascular endothelial growth factor, have been characterized as more potent mitogens,
but IGFs often synergize with these growth factors to produce an enhanced mitogenic
response (Kurenova et al., 2009; Shimotake et al., 2010; Guillemot et al., 2011; Wesche
Et al., 2011).
Steroids mediate their activity through binding to specific intranuclear receptor
proteins. These receptors may also be functionally linked to signaling pathways, at the
plasma membrane, for example activating tyrosine kinases or G protein coupled receptors
(Revelli et al., 1998). Steroids such as 17-beta-estradiol (E2), via binding to cytoplasmic
or membrane-associated receptors, have been seen to rapidly activate intracellular
signaling cascades such as ERK, PI3K and STATs (Fox et al., 2009).
Given this information, we wondered if ecdysone worked as a typical steroid
hormone and stimulated cellular division on its own, or if the requirement for insulin for
growth involved a co-requisite role in cell division. We used a novel detection assay,
EdU (5-ethynyl-2’-deoxyuridine), to directly measure de novo DNA synthesis (S-phase
of the cell cycle) using click chemistry (Salic and Mitchison, 2008; Click-iT EdU
imaging kit manual, Invitrogen, 2009). Click chemistry is a method of covalently
coupling an azide with an alkyne (Fig. 8). Detection of EdU employed the copper (I)
40
catalyzed click reaction with an azide modified fluorescent dye to form a stable triazole
ring. Because of the small size of the alkyne labeled nucleotide, no harsh denaturation
steps were needed to gain access to the DNA (Salic and Mitchison, 2008). In other
words, an alkyne modified nucleotide, EdU, once incorporated into DNA, could be easily
detected with an azide-modified fluorescent dye. These reagents were used to distinguish
the relative roles of insulin and ecdysone in cell division.
Figure 8. Click reaction between EdU and azide modified dye. EdU (5-ethynyl-2’-deoxyuridine, a thymidine analog, carries a terminal alkyne group. The terminal alkyne group reacts with azide in the presence of catalytic amounts of copper.
41
MATERIALS AND METHODS
Animals, tissue culture, and hormones
Protocols for animal rearing, disc culture, and hormones employed for
experiments are as described in Chapter 1.
EdU labeling, fixing of and detection
EdU (Click-It EdU Imaging Kits-Invitrogen) was added to the Grace’s culture
medium at the start of incubation at a concentration of 10 M. After 48 hour incubation,
discs were washed two to three times in 3.7% formaldehyde in PBS and then incubated at
RT for 15 minutes. The discs were then washed twice in 3% BSA in PBS and
permeabilized and fixed using a standard formaldehyde fixation protocol (Invitrogen)
overnight. Discs were once again washed in 3% BSA in PBS and 0.5mL of Click-iT
reaction cocktail (43 μL 10X reaction buffer; 38 μL distilled water; 20 μL copper
sulphate; 1.2 μL Alexa Fluor 594 azide; 50 reaction buffer additive) was added to each
well. Samples were incubated for 30 minutes in the dark before being washed again with
3% BSA in PBS (Click-iT EdU imaging kit manual, Invitrogen). Discs were washed in
75% ethanol (x2), 95% ethanol (x2) and 100% ethanol (x2). Then discs were placed in
xylene for 60 seconds and moved to fresh xylene for 8 minutes. Discs were mounted on
slides (Fisherbrand Superfrost/Plus) with Permount and covered with cover slip. Slides
were stored at 4°C.
42
Imaging and analysis
Images were acquired using a BioRad MRC600 confocal imaging system
(Hercules, Ca, USA) with a Zeiss inverted microscope stand (wavelength 594 nm;
magnification of 40x, neutral density =5, F6 =1.0). Confocal microscopy equipment was
provided courtesy of the O’Malley Lab, Northeastern University. A grid was overlayed
on top of the slide to consistently map locations on the discs and subsequently removed
before pictures were taken.
Statistical analysis
Number of cells incorportated in EdU in the wing imaginal discs was subjected to
statistical analysis using one way ANOVA, with a Dunnett’s post hoc test to compare
specific treatments against a reference. In all cases, p<0.05 was used to determine
significance. All statistical analysis was performed using SPSS v.19 (licensed to
Northeastern University, 2011).
43
RESULTS
Cell proliferation
In conjunction with the stimulation of growth, ecdysone and insulin (Ec+Ins)
produced the greatest number of proliferating cells (Figs. 9 and 10). The center of the
discs were easy to distinguish under the microscope; but the edges were not. To solve this
problem, a grid was placed on top of the slides (at 40x magnification), and we
consistently chose the same 2-3 grids in the lower left area (roughly between the 6 and 8
o’clock positions) of the disc for the edge. Following incubation with ecdysone and
insulin, incorporation of EdU (DNA replication) increased significantly in both the center
and along the edge of the disc, although replication appeared more prevalent along the
outer edge of the disc (Fig. 9). Control samples showed little incorporation of EdU (Fig.
9). Discs treated with ecdysone alone showed significant cell division along the edges
(p<0.05), but not towards the center of the discs (Figs. 9 and 10). Conversely, discs
treated with insulin alone showed significant cell proliferation near the center of the disc
(p<0.05), but not along the edge. Overall, these results suggest that ecdysone and insulin
increase cell division, but ecdysone has a greater effect. Insulin does, however, augment
cell division when combined with ecdysone.
44
Figure 9. Detection of cell proliferation with EdU in wing discs after 48 hour incubation. Discs were treated with Grace’s medium (Control), ecdysone (Ec), insulin (Ins), or ecdysone and insulin (Ec+Ins) (n=8 for each treatment). Images are at 40x magnification taken at the center or at the outer edge of the disc using a confocal imaging system.
45
Figure 10. Number of proliferating cells (incorporating EdU) in wing discs after 48 hour incubation. Images were taken of discs treated with Grace’s medium (Control), ecdysone (Ec), insulin (Ins), or ecdysone and insulin (Ec+Ins) (n=8 for each treatment). A. The number of proliferating cells was counted in the center of each disc. B. The number of proliferating cells was counted at the outer edge of each disc. Significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).
b
b
b
b
a a
a a
A
B
46
DISCUSSION
In this chapter, we distinguished the influence of each hormone, ecdysone and
insulin, on cell division. As expected, the steroid hormone ecdysone successfully
stimulated cell division. Insulin by itself was also able to stimulate cell division in wing
discs. Potter et al. (2001) showed in Drosophila wing discs that insulin signaling was
responsible for increasing cell number as well as cell size. They overexpressed tuberous
sclerosis complex (TSC) homologues 1 and 2, which led to a decrease in cell number
(Potter et al., 2001). TSC is a downstream target that is normally inhibited in the insulin
pathway. Inactivating positive signaling components of the insulin pathway including
Drosophila insulin receptor (dinr), Drosophila PI3K (Dp110), and Drosophila Akt (dAkt)
all led to decreases in cell number and cell size (Leevers et al., 1996; Verdu et al., 1999;
Potter et al., 2001).
In Manduca, insulin significantly enhanced cell division by itself, but more
strongly in conjunction with ecdysone. This synergistic effect by a steroid and insulin on
cell division has also been shown in human breast cancer cells (Dupont et al., 2000;
Castoria et al., 2001; Hamelers and Steenbergh, 2003). Dupont et al. (2000) showed that
MCF-7 cells were stimulated to proliferate by both insulin-like growth factor I (IGF-I)
and the steroid estradiol (E2), individually. IGF induced proliferation by activating the
PI3K pathway. E2, via activation of the estrogen receptor, enhanced the expression of
growth related genes, including cyclin D1 as well as the PI3K pathway (Dupont et al.,
2000; Simoncini et al., 2000). It was further shown that a combination of these mitogens
resulted in a synergistic increase in cell division (Hamelers and Steenbergh, 2003).
47
Finally, if one of the receptors, either estrogen receptor or insulin receptor, was blocked,
then the proliferative response was suppressed (Dupont et al., 2000). This suppression
suggests that both hormones are needed to promote breast cancer cell proliferation.
In wing discs, ecdysone and insulin are both required for maximal cell division.
Ecdysone and insulin can also promote cell division on their own, suggesting that each
hormone can affect cyclins, proteins that control the progression of cells through the cell
cycle by activating cyclin-dependent kinase (Cdk) enzymes (see Uhlmann and Lopez-
Aviles, 2011). In MCF-7 breast cancer cells, insulin-like growth factor 1 receptor and
estrogen receptor were co-expressed and resulted in enhanced growth (Dupont and le
Roith, 2001). Estradiol induced expression of insulin receptor substrate (IRS-1) which
led to Akt activation. Estradiol also potentiated the effect of IGF-1 on the expression of
cyclin D1 and cyclin E, and phosphorylation of the retinoblastoma protein, a tumor
supressor (Dupont et al, 2000; Dupont and LeRoith, 2001). There are many cyclin
proteins, and they could have different sensitivities to ecdysone or insulin. These
hormones may also differentially regulate the expression of cyclin dependent kinase
inhibitors. This variability could explain why insulin promotes cell proliferation in the
center of the discs and ecdysone promotes cell proliferation towards the edge. When
ecdysone and insulin are both present, both sets of co-factors could be stimulated,
yielding greater overall proliferation and uniform tissue growth. Cell proliferation results
confirmed that the ecdysone and insulin signaling pathways were intertwined. Further
delineation of the relationships among signaling pathways involved in hormonally-
stimulated disc growth are described in Chapter 3.
48
CHAPTER THREE
Cellular interactions between ecdysone and insulin in the
regulation of disc growth
49
INTRODUCTION
As outlined in Chapter 1, lepidopteran growth occurs as an interplay between
insulin and the steroid hormone ecdysone. How is growth positively regulated by insulin
and steroids? Steroid hormones act through nuclear transcription factors to regulate gene
expression. In vertebrates, steroids such as progesterone, testosterone, and aldosterone
have all been shown to upregulate the insulin pathway (Berrie, 2001, Wu et al., 2010).
This relationship is best characterized for the interactions between estrogens and insulin-
like growth factors (IGFs). There is growing evidence to suggest that estrogen- and IGF-
mediated signaling pathways are linked. For example, in the brain, estrogen receptors
and IGF-I receptors are frequently expressed in the same cells. Estradiol and IGF-I
cooperate to regulate neuronal development, and both are activated in response to neural
tissue injury (Cardona-Gomez et al. 2003; Garcia-Segura et al., 2007). While the exact
molecular mechanisms involved in these interactions are still not well understood,
estrogen has been found to increase the expression of IGF-I receptors and IRS-I (Lee et
al. 2007). The estrogen receptor subtype known as estrogen receptor interacts with
PI3K, enhancing the activation of Akt and the phosphorylation of glycogen synthase
kinase-3 (GSK3) (Garcia-Segura et al. 2006). In addition, estradiol treatment results in
an increase in the phosphorylation of ERK (Garcia-Segura et al. 2006). These findings
suggest that estrogen effects in the brain may be mediated in part by the activation of the
signaling pathways of the IGF-I receptor.
A positive relationship also exists between estradiol and elements of the insulin
signaling cascade in cancer cell lines. The ligand-bound estrogen receptor was reported to
50
directly bind and activate the IGF type I receptor in human breast tumor cells (Hamelers
and Steenbergh, 2003). Castoria et al. (2001) reported that estradiol in MCF-7 breast
carcinoma cells activated the ERK pathway, PI3K, and Akt. They further showed that
inhibition of PI3K with the inhibitor LY294002 abolished the estradiol stimulation of Akt
and inhibited cell cycle progression (Castoria et al., 2001; Hamelers and Steenbergh,
2003).
Like the estrogen receptor, the peroxisome-proliferator-activated-receptor-
(PPAR- ) is a member of the nuclear hormone receptor family. Activation of PPAR-
enhances the regulation of glucose and lipid metabolism through regulation of insulin
signal transduction (Pandey et al., 2007). In some tissues, PPAR- activation leads to the
activation of insulin signaling pathways (Seto-Young et al., 2007). Thiazolidinediones
(TZDs), acting as PPAR- agonists, improve downstream insulin transduction in muscle
and stimulate PI3K and MAPK in blood vessels and liver cells (Belifiore et al., 2009),
and enhance insulin-signaling pathways and glucose uptake in type 2 diabetics (Pandey et
al., 2007; Choi et al., 2011). Seto-Young et al. (2007) have shown similar effects of
TZDs on insulin signaling in ovaries. This further supports the trend of positive cross-
talk between insulin signaling and the nuclear receptor family.
Testosterone, a steroid hormone, can also rapidly activate the insulin signaling
pathway. In cultured cardiomyocytes and in myoblasts, testosterone increased the
phosphorylation of ERK, Akt, TOR , and the downstream targets of TOR, 40S ribosomal
protein S6 kinase 1 (S6K1), and eukaryotic initiation factor 4E-binding protein 1 (4E-
BP1) (Altamirano et al., 2009; Wu et al., 2010). The S6K1 phosphorylation induced by
testosterone led to cardiac hypertrophy, and phosphorylation was blocked by the TOR
51
inhibitor, rapamycin. However, when Akt was inhibited, testosterone still activated TOR
and S6K1 (Altamirano et al., 2009). In a similar fashion, Serra et al. (2011) showed that
IGF-I was not essential for mediating testosterone’s effects on androgen-responsive
skeletal muscle. These findings suggest that although testosterone and insulin can work
together, testosterone does not necessarily require insulin; testosterone can stimulate
TOR by itself.
Just as steroids can enhance insulin signaling, insulin can alter steroid receptors.
For example, IGF-I and IGF-II are two of the most abundant growth factors in the
prostate (Pinches et al., 1991). IGF-I has been shown to activate androgen receptors and
thus stimulate cancer proliferation and growth in the absence of androgens
(Gnanapragasam et al., 2000; Pandini et al., 2005; Wu et al., 2006). Likewise, IRS-1
overexpression has been associated with tumor development in breast cancer.
Specifically, in estrogen sensitive breast cancer cell lines, IRS-1 was found to enhance
estrogen receptor activity, which led to an overgrowth of cells (Cesarone et al., 2006).
The effects of insulin on steroid receptors have received relatively little attention because
steroids are generally potent enough to stimulate growth on their own. A recent paper by
Morelli et al. (2010) provided underlying mechanisms for stimulatory effects of insulin
on steroid sensitivity in breast cancer cells. In estrogen-positive breast cancer cells, Akt2
modulated estrogen receptor transcriptional activity at multiple levels, including the
regulation of estrogen receptor expression, its nuclear retention, and the activation of
Forkhead transcription factor, FoxO3a (Morelli et al., 2010). FoxO3a co-localized and
co-precipitated with estrogen receptor in the nucleus, and further investigation suggested
a repressive effect of FoxO3a on estrogen receptor transcription (Morelli et al., 2010).
52
Hence, precedent exists for positive interactions between steroids and insulin in
vertebrates. Earlier chapters showed that, in insects, ecdysone and insulin stimulate
growth and cell division. In the present chapter, RNA interference (RNAi), kinase
inhibitors, and antibodies to receptors and signaling enzymes are employed to provide a
more detailed identification of interactions between ecdysone and insulin that stimulate
growth. In addition to further investigating the effects of ecdysone on insulin signaling,
the converse effects of insulin on ecdysone receptor content were explored. Together,
results confirm a mutually positive influence of steroids and insulin on growth-related
signaling pathways in growing wing discs.
53
MATERIALS AND METHODS
Animals and tissue culture
Manduca sexta larvae were reared and discs were cultured as decribed in
Chapter 1.
Hormones
Insulin (NovoRapid) was diluted in Grace’s medium to a final concentration of
30μM. 20-Hydroxyecdysone (Sigma) was dissolved in Grace’s medium to a final
concentration of 0.1 μg/ml.
Knockdowns via RNAi
To determine if the observed disc growth was dependent upon the insulin receptor
(InR), RNA interference (RNAi) was used to reduce receptor content. The small-
interfering RNA (siRNA) was designed using the Whitehead siRNA Selection Web
Server (http://jura.wi.mit.edu/bioc/siRNA) (Yuan et al, 2004). Two separate siRNA
sequences,
IR-1 (5’-CCACCACGAACGGUUUAGUtt -3’) and
IR-2 (5’- CGCAGACGUUGUGAACAAUtt -3’), were directed against two different
regions of the insulin/bombyxin receptor gene. The Manduca insulin receptor sequence,
against which primers were designed, was obtained from the partial gene sequence of
Stefan Girgenrath (1999). A complete Manduca insulin receptor sequence was
subsequently published by Koyama et al. (2008) (GenBank accession no FJ169469) and
54
matched to the partial sequence used. A third siRNA, (5’-
GACACGUGCUGAAGUCAAGtt -3’), directed against green fluorescent protein (GFP),
was used as a non-specific sequence to test the effects of nonsense siRNA in culture.
Custom siRNA (40 nmol, standard purity, annealed) was synthesized by Ambion, Inc.
(www.ambion.com). siRNA was also generated using a siRNA synthesis PCR kit
(Invitrogen).
Quantitative real time PCR (qPCR) analysis of gene expression
Manduca bombyxin/insulin receptor (IR) and ribosomal protein 49 (RP49) were
analyzed by qPCR using a real-time sequence detection system (Applied Biosystems
7000). Individual wing discs were used from day five, fifth instar larvae. Total RNA
was collected from samples using SV Total RNA isolation kit (Promega). RNA was
tested spectrophometrically for purity and concentration, and run on a formaldehyde gel
to ensure quality. 100 ng of each RNA sample were reversed transcribed using random
hexamer primers in a Superscript III First Strand Synthesis System (Invitrogen)
according to the manufacturer’s protocol. Primers for qPCR analysis were designed
using Primer Express (Applied Biosystems). Primer sequences were as follows:
IR forward 5’- GGGATTTCGGCATGACCAGAGATATT-3’
IR reverse 5’-TCGTTCGACAGGCCCTGATATGG-3’
rp49forward 5’-GAGGAATTGGCGTAAACCTAGAG-3’
rp49reverse 5’-TGACGGGTCTTCTTGTTGGA-3’
PCR reactions were performed using SYBR green mastermix (Applied Biosystems). IR
55
PCR reactions contained 0.1 μM of each primer. RP49 PCR reactions contained 0.9 μM
of each primer. qPCR was performed in a final volume of 20 μl. All quantitative
reactions were subjected to 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of
95°C for 15s, and 60°C for 1 min. In addition, products were subjected to gel
electrophoresis to check product size for each primer pair. In order to account for
potential contamination, non-reverse-transcribed total RNA (with potential genomic
DNA contamination) and non-template controls were included. No products were
observed in these reactions. Dilution curves generated by serial dilutions of cDNA were
used to calculate amplification efficiencies. Transcript levels of the target genes were
normalized to the Manduca ribosomal gene rp49 after correcting for differences in
amplification efficiency (Pfaffl, 2001).
Western blot analysis
Wing discs were homogenized in 40 μL of 2X SDS sample buffer, and
incubated at 90°C for three to five minutes. The samples were then run on a 10-20%
SDS-PAGE gradient gel (Bio-Rad) to separate the proteins (Laemmli, 1970). They were
transferred from gels to nitrocellulose membranes at 4°C. Membranes were then blocked
in 3% BSA or 5% milk for one hour, rinsed quickly (15 minutes total) and placed on
primary antibody overnight. Primary antibodies included: anti-phospho-4EBP,
antiphospho-Akt, total Akt, anti-phospho-ERK, anti-actin, anti-phospho-GSK (Cell
Signaling), anti-USP (a gift from Prof. Fotis Kafatos, currently at Imperial College
London) and anti-EcR (Developmental Studies Hybridoma Bank, University of Iowa,
Department of Biology). Blots were rinsed and placed on secondary anti-rabbit antibody
56
with attached HRP (Cell Signaling) for 75 minutes. They were treated with Western
blotting chemiluminescence reagents (Pierce ECL Western blotting substrate, Thermo
Scientific). The blots were exposed on blue-sensitive autoradiographic film (Marsh Bio
Products) and developed (Kodak GBX fixer and developer). Blots were scanned and
analyzed using ImageJ (Abramoff et al., 2004). Protein bands were normalized to actin
as a loading control where appropriate.
Statistical analysis
Gene expression in the wing imaginal discs was subjected to statistical analysis
using one way ANOVA, with a Dunnett’s post hoc test to compare specific treatments
against a reference. Kaplan-Meir and Cox-regression tests were used to evaluate %
detection of real-time PCR data. In all cases, p<0.05 was used to determine significance.
All statistical analyses were performed using SPSS v.19 (licensed to Northeastern
University, 2011).
57
RESULTS
Inhibition of disc growth via siRNA against the bombyxin/insulin receptor (InR)
As previously observed in Chapter 1, discs incubated with ecdysone and insulin
(Ec+Ins) grew significantly (Dunnett Multiple Comparison test, p<0.05) (Fig. 11). The
relevance of InR to glandular growth was explored using RNAi-mediated knockdown of
the receptor. In the presence of siRNA directed against the bombyxin/insulin receptor,
ecdysone and insulin (Ec+Ins+IRsiRNA) no longer stimulated growth (Fig. 11).
Knockdown of the receptor was confirmed using real-time PCR (Fig. 12A, discussed in
greater detail on the following pages). Control RNAi, directed against green fluorescent
protein (GfpsiRNA), did not inhibit growth (Fig. 11). This indicated that the effects of
the RNAi were specific and that merely incubating discs with RNAi was not toxic. As
seen previously, discs treated with ecdysone alone (Ec) or insulin alone (Ins) did not
increase significantly in size relative to Grace’s medium alone (Control). The size of
discs incubated in ecdysone alone or insulin alone was not affected by IRsiRNA (Dunnett
Multiple Comparison test, p=0.123, p= 0.074, respectively) (Fig. 11).
58
Figure 11. Effects of siRNA, LY294002, and rapamycin on growth in wing discs. Discs were removed from day 5 fifth stage larvae and cultured for 24 hours in Grace’s medium/10% FBS/10% antibiotic-antimycotic, then transferred to the same medium containing Grace’s medium alone (control), 20-hydroxyecdysone and insulin (Ec+Ins), 20-hydroxyecdysone alone (Ec), or insulin alone (Ins) for 2 days. Discs were also treated with small interfering RNA directed against the bombyxin/insulin receptor (IRsiRNA), LY294002 (LY), an inhibitor of PI3K, or rapamycin, an inhibitor of TOR. Growth was assessed by measurement of disc width. Significant increase in growth is designated by “b” (Dunnett Multiple Comparison test, p<0.05).
Measurement of insulin receptor transcript
Levels of insulin receptor transcript (InR) were measured using real-time PCR
(Fig. 12A, C). Real-time PCR uses SYBR Green to detect the presence of double-
stranded DNA (dsDNA). The amount of fluorescence increases as the amount of double-
stranded DNA increases. As a control, the transcript levels of Rp49, a ribosomal gene,
were also measured (Fig. 12B, D). Fig. 12A and 12C indicate that there was a significant
and faster increase in InR in discs treated with both ecdysone and insulin (Ec+Ins)
(Kaplan-Meir/Mantel-
b b
a a a
a a
a
a
a
a a
a a a
a
a a
a a
59
Figure 12. Detection of transcript in wing discs after 48 hour treatment. RNA was collected from wing discs (n=8) after treatment with ecdysone (Ec), insulin (Ins), ecdysone and insulin (Ec+Ins), or ecdysone and insulin and siRNA against the insulin receptor (Ec+Ins+IRsiRNA). 100ng of RNA from each sample was reverse transcribed with random hexomer primers and cDNA was analyzed using quantitative real time PCR A. The detection of insulin receptor transcript (InR) is indicated at various cycle times depending on treament. B. The detection of Rp49 transcript, a Manduca ribosomal gene was measured as a control. C. Fold changes in mRNA were calculated for InR and D. Rp49 using the Pffafl method. Significant differences are indicated by “b.”
Cox, p=0.00006; Pffafl method, p<0.05). InR dsDNA in the (Ec+Ins)-treated samples
was detected much earlier (cycle 5) than in controls (cycle 36), reflecting an increase in
InR transcript (Fig. 12A). InR dsRNA in (Ec+Ins)-treated samples were detected earlier
than ecdysone-treated (Ec) or insulin-treated (Ins) samples (Kaplan-Meir/Mantel-Cox,
p=0.00008). InR dsRNA in (Ec)- and (Ins)-treated samples were also detected
A B
b
a a a
C D
a a a a
60
significantly earlier than in untreated controls (Kaplan-Meir/Mantel-Cox, p=0.00048),
but differences were not observed between (Ec) and (Ins) treated groups (Kaplan-
Meir/Mantel-Cox, p=0.026) (Fig. 12A). The increase in insulin receptor transcript
stimulated by ecdysone and insulin independently was not enough to cause an increase in
overall disc size. Treatment with ecdysone, insulin and control siRNA
(Ec+Ins+GfpsiRNA) produced results similar to (Ec+Ins) (data not shown). As expected,
none of the treatments altered transcription of the ribosomal protein, Rp49 (Fig. 12B, D).
Treatment was a significant and independent predictor of detection (Wald 55.7, df
=4, p<0.00001). In this case “detection” refers to statistical distinction relative to control.
Relative to controls, ecdysone-treated samples were 27 times more likely to be detected
and insulin-treated samples were 23 times more likely to be detected relative to controls
(Wald 41.3, df=1, p<0.00001; Wald 40.6, df=1, p<0.00001). Together, ecdysone and
insulin were 52 times more likely to be detected than the controls (Wald 56.3, df= 1;
p<0.00001). Primer was an independent predictor (Wald 56, df=1, p<0.00001) after
controlling for the effect of treatment. Primer 1 (InR) was 27 times more likely to be
detected than primer 2 (Rp49) (Wald 56, df=1, p<0.00001).
Examination of insulin signaling pathways
RNAi against the bombyxin/insulin receptor blocked growth, so it was
hypothesized that it would also decrease the activation of downstream signaling proteins.
Western blots were used to measure changes in phosphorylated Akt (pAkt),
phosphorylated ERK (pERK), and phosphorylated 4EBP (p4EBP) (locations in signaling
61
Figure 13. Activation of phosphoproteins in wing discs after 48 hour incubation. Western blots of pAkt, pERK, and p4EBP were all quantified using ImageJ (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).
pathway shown in Fig. 2, results shown in Fig. 13). There was no significant increase in
pERK stimulated by any of the treatments (Fig. 13). Combined treatment with ecdysone
and insulin (Ec+Ins) significantly increased pAkt and p4EBP (Fig. 13, Fig. 16A). RNAi
against the bombyxin/insulin receptor (IRsiRNA) significantly decreased both pAkt and
p4EBP (Fig. 13). Insulin alone (Ins) stimulated a significant increase in pAkt. p4EBP
was not significantly increased by insulin alone in this particular set of experiments but
was in a later set of experiments (Fig. 16). Ecdysone (Ec) was able to stimulate pAkt, but
62
had almost no effect on p4EBP (Fig. 13). This result suggested that Akt is a possible site
of interaction/cross-talk between ecdysone and insulin in the regulation of growth.
The effects of insulin and ecdysone on pAkt are due at least in part to changes in
total Akt (Fig. 14). There was a significant increase in total Akt in response to ecdysone
treatment combined with insulin (Ec+Ins) (Dunnett Multiple Comparison test, p<0.05).
Though not statistically significant, both insulin (Ins) and ecdysone (Ec) showed a trend
in increasing total Akt. IRsiRNA significantly inhibited total Akt expression. As
expected, samples treated with control RNAi directed against GFP (COsiRNA, same as
GFPsiRNA) did not show a change in total Akt expression (Fig. 14).
Figure 14. Total Akt in wing discs after 48 hour incubation. Western blots of total Akt were quantified using ImageJ (Abramoff et al., 2004). Samples were treated with ecdysone (Ec), insulin (Ins), ecdysone and insulin (Ec+Ins), or ecdysone and insulin and siRNA against the insulin receptor (Ec+Ins+IRsiRNA) or LY294002 (LY). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).
b b
a a a a a
a a a
a
a a
63
Effects of PI3 kinase and TOR inhibitors on disc growth
Disc growth was successfully inhibited via knockdown of the insulin receptor
which also led to a decrease in activation of downstream signaling proteins. Given the
importance of PI3K in the insulin signaling pathway, we anticipated the PI3K inhibitor
LY294002 (LY) would have effects similar to siRNA. As expected, LY blocked growth
stimulated by ecdysone and insulin (Ec+Ins) (Fig. 11), as well as accompanying changes
in pAkt, total Akt, and p4EBP (Figs. 13 and 14).
We next examined effects of the TOR inhibitor, rapamycin, on disc growth.
Rapamycin significantly decreased growth in discs treated with ecdysone and insulin
(Ec+Ins+Rapamycin) (Fig. 11). Rapamycin also decreased the size of discs treated with
insulin alone (Ins) or ecdysone alone (Ec) (Fig. 11). As expected, rapamycin
significantly decreased the phosphorylation of 4EBP (Fig. 15A). Non-phosphorylated
4EBP serves as a potent inhibitor of translation (refer to Fig. 2, Introduction). Together,
results shown in Figs. 11 through 15 argue strongly that ecdysone and insulin enhance
growth through the activation of IR, PI3K, pAkt, TOR, and 4EBP.
64
Figure 15. Effects of rapamycin on p4EBP and EcR in wing discs after 48 hour incubation. Western blots of A. p4EBP and B. EcR were quantified using Image J (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05). Effects of insulin on ecdysone receptor protein content
We next examined whether insulin could in turn affect the ecdysone receptor.
Antibodies were available for the Manduca ecdysone receptor (isoform A, EcR-A) and
the transcription factor with which it heterodimerizes, ultraspiracle (USP). The same
Western blots that were previously used to detect insulin pathway proteins were stripped
and re-probed for EcR and USP. As shown in Fig. 16, combined treatment with
ecdysone and insulin (Ec+Ins) led to increased EcR and USP relative to controls.
Ecdysone (Ec) significantly stimulated EcR content on its own, but insulin (Ins) did not
(Fig. 16). USP was only slightly stimulated by ecdysone (Ec), and not at all by insulin
(Ins). All three inhibitors discussed previously, RNAi directed against the
bombyxin/insulin receptor (IRsiRNA), LY294002 (LY), and rapamycin, significantly
decreased disc content of EcR and USP (Fig. 15B, Fig. 16). Together, these results
65
suggest that the insulin receptor, PI3K, and TOR affect EcR, even though insulin does
not enhance cellular content of EcR directly.
Figure 16. Ecdysone receptor proteins in wing discs after 48 hour incubation. Western blots of ecdysone receptor (EcR) and untraspiracle (USP) were quantified using ImageJ (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).
b b
a a a a
a a a a a a a
a a a a a a a a a
b b
b
66
DISCUSSION
In Manduca wing discs, growth in culture occurs only in the presence of both
ecdysone and insulin. In our initial study of Manduca discs (Chapter 1), replicated in the
present chapter, we found that ecdysone stimulates elements of the insulin pathway,
specifically pAkt, but has the greatest effect when combined with insulin. We used
siRNA directed against the bombyxin/insulin receptor (IRsiRNA), to determine if the
synergistic effects of insulin and ecdysone on growth of the wing discs were in fact
occurring through the insulin receptor. The IRsiRNA successfully inhibited growth in
ecdysone- and insulin-treated discs and confirmed that the receptor was a requisite player
in the signaling pathway. A central focus of Akt was also suggested by the inhibitory
effects of upstream and downstream inhibitors (LY294002 and rapamycin, respectively).
Our findings with regard to the enhancement of Akt by members of the nuclear
receptor superfamily are in keeping with what others have found in several human
diseases including cancer. For example, in vertebrate thyroid cancer, the thyroid
hormone receptor interacts with PI3K and activates Akt to increase cell proliferation and
motility (Furuya et al., 2006; Furuya et al., 2007; Furuya et al., 2009). Cheng et al.
(1996) found that Akt was overexpressed estrogen-sensitive ovarian tumor cell lines and
subsequently showed that siRNA silencing of Akt blocked transformation of these cell
lines.
Furthermore, Akt has also been shown to activate steroid receptors. The androgen
receptor plays an important role in early prostate cancer. Studies have shown that
androgen receptor transcriptional activity and expression are regulated by Akt (Paliouras
and Diamandis, 2008). The mechanism by which Akt influences steroid receptor
67
activation is not entirely clear, but a recent study by Morelli et al. (2010) offers a
potential model. Morelli et al. (2010) showed that estrogen receptor and insulin-like
growth factor I receptor (IGF-IR) pathways engage in cross-talk in breast cancer.
FOXO3a in these tissues inhibits estrogen receptor expression. Activation of IGF-I/IGF-
IR and concomitant activation of Akt leads to phosphorylation of FOXO3a, reducing its
nuclear content and enhancing estrogen receptor expression. Morelli et al. (2010)
observed a strong decrease in estrogen receptor expression, at both RNA and protein
levels, and a decrease in growth, in FoxO3a-overexpressing cells (Morelli et al., 2010).
On the flip side, in FoxO3a-silenced cells, Morelli et al. (2010) noted an increase in cell
proliferation. The experiments were repeated in two additional estrogen-receptor-
positive cell lines. Our own results are in keeping with an inhibitory effect of FOXO on
EcR transcription. Insulin may remove this inhibition via its effects on Akt and FOXO,
although in wing discs, insulin does not, alone, increase EcR content.
We have generated a model for the stimulatory effects of insulin and ecdysone, in
keeping with our results (Fig. 17A). Ecdysone stimulates growth by increasing cellular
content of its own receptor, as well as the phosphorylation and activation of Akt. The
stimulatory effects of insulin are also mediated by activation of Akt. Akt phosphorylates
FOXO, preventing its entry into the nucleus. A reduction in nuclear FOXO augments
EcR expression. Akt also activates TOR, enhancing growth through the phosphorylation
and inhibition of 4EBP, and the phosphorylation and activation of 70S6K. However,
activation of Akt alone is insufficient to stimulate growth in the absence of ecdysone,
which enhances cellular content and activity of the activated ecdysone receptor. Thus,
growth is stimulated by the combined effects of both hormones (Fig. 17A).
68
This model also explains what we see in the presence of siRNA, and inhibitors of
the insulin signaling pathway (Fig. 17B). siRNA against the insulin receptor (IRsiRNA),
prevents increases in pAkt and total Akt. LY294002 (LY) and rapamycin also cause a
reduction in Akt phosphorylation and action. Reduced Akt activity allows FOXO into
the nucleus, causing a decrease in EcR transcription. The reduction in EcR transcription
blocks growth-promoting effects of ecdysone and further reduces Akt activity and
growth.
Figure 17. Suggested mechanism through which ecdysone and insulin synergistically interact to modulate EcR expression and activity. A. In the presence of ecdysone and insulin EcR is liganded (1) and enhances the activation of Akt (2), allowing Akt to effectively phosphorylate and thus inhibit FOXO transcription factor (3), and also activate TOR (4), promoting the nuclear exclusion of and retention of FOXO in the cytoplasm so FOXO can no longer inhibit EcR transcription and expression (5). B. Akt inhibition (1) is a result of siRNA directed against the bombyxin/insulin receptor or via LY294002 inhibiting PI3K, leading to the activation and subsequent nuclear recruitment of FOXO (2). Akt inhibition causes a reduction in TOR (or direct inhibition of TOR by rapamycin) allowing non-phosphorylated 4EBP to block protein synthesis (3) while FOXO causes EcR transcriptional repression, reduction of liganded-EcR retention in the nucleus and consequently, less EcR recruitment on the promoter leading to downregulation (4).
69
Our results focus new attention on changes in ecdysone receptor content,
particularly as regulated by Akt/FOXO, in steroid-dependent growth. FOXO proteins are
evolutionarily conserved across species and are involved in diverse cellular and
physiological processes including cell proliferation, cancer, and cellular response to
reactive oxygen species (ROS). Though insect growth has been studied extensively in
Drosophila, that animal model does not seem to share the antagonistic relationship
between insulin and nuclear FOXO seen in other animal models. As mentioned earlier,
reduced insulin signaling in Drosophila reduces body size. Recently, Slack et al. (2011)
showed that removal of FOXO failed to return normal function to flies with decreased
insulin signaling. By contrast, in C. elegans, removing DAF-16, the worm FOXO
transcription factor, suppressed all negative phenotypes in insulin-deficient worms (Slack
et al., 2011). This suggests that there is evolutionary divergence in the mechanisms that
control effects of insulin signaling in Drosophila relative to other organisms.
It may be of interest to see if growth of Manduca wing discs is sensitive to
reactive oxygen species (ROS). FOXO is important for defensive response to cellular
stress, including increased oxidative stress levels (Greer and Brunet, 2005). In mammals,
there are four FOXO family members: FOXO1, FOX3, FOXO4, and FOXO6.
Phosphorylation of FOXO1, FOXO3, and FOXO4 by Akt leads to their retention in the
cytoplasm, thereby inhibiting their transcriptional activities (Brunet et al., 1999; Rafalski
and Brunet, 2011). When FOXO6 is phosphorylated, subcellular localization is not
affected but transcription activity is still inhibited (van der Heide et al., 2005; Rafalski
and Brunet, 2011). In neural stem cells, FOXO3 is involved in the transcriptional
regulation of genes involved in cellular response to hypoxia (low oxygen) (Renault et al,
70
2009; Rafalski and Brunet, 2011). For example, genes known to be upregulated in
hypoxic brains are downregulated in FOXO3-null neural stem cells (Renault et al, 2009).
In the liver, accumulation of lipid metabolites leads to a reduction in Akt activity, and
enhances the entry of FOXO into the nucleus, which then activates transcription of redox
enzymes (Goldstein et al., 2005; Papconstantinou, 2009; Rafalski and Brunet, 2011).
Hypoxia may be a stronger regulator of growth than previously seen. Under hypoxic
conditions, FOXO would be highly active, and EcR transcription would be down-
regulated, leading to less active Akt and a decrease in growth.
Creating a FOXO siRNA would be an interesting future experiment to test our
model. A FOXO knockdown would reduce the amount of FOXO in the nucleus, leading
to an increase in EcR, and more active Akt to remove even more FOXO from the
nucleus, enhancing growth. FOXO knockdown might even promote/sustain growth in
the absence of insulin or ecdysone. FOXO knockdown may also reveal other players
involved in ecdysone-stimulated growth, or in the response of growing tissues to cellular
stress. Likewise, an Akt knockdown would provide useful information relative to our
model for disc growth. We would expect an elevation in nuclear FOXO following Akt
knockdown, regardless of the presence of ecdysone or insulin. A resulting decrease in
EcR transcription would lead to a drastic decline in growth.
To summarize, the results of the present study shed new light on the roles of
ecdysone and insulin in insect disc growth. Insulin appears to permit a requisite increase
in ecdysone receptor content. Only when discs are fully sensitive to ecdysone can
maximal growth occur, through insulin- and ecdysone-stimulated activation of Akt. The
same pathways are likely to regulate post-embryonic development in other organisms,
71
with our results placing a fresh focus on the importance of insulin in steroid-driven
growth.
72
REFERENCES
Abramoff, M.D., Magelhaes, P.J., and Ram, S.J. (2004). Image Processing with ImageJ. Biophotonics International 11, 36-42.
Adachi T, Takiya S, Suzuki Y, Iwami M, Kawakami A, Takahashi SY, Ishizaki H, Nagasawa H, Suzuki A. cDNA structure and expression of bombyxin, an insulin-like brain secretory peptide of the silkmoth Bombyx mori. J Biol Chem. 1989 May5;264(13):7681-5.
Aljamali MN, Bior AD, Sauer JR, Essenberg RC. RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum. Insect Mol Biol. 2003 Jun;12(3):299-305. Altamirano F, Oyarce C, Silva P, Toyos M, Wilson C, Lavandero S, Uhl ©n P,
Estrada M. Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway. J Endocrinol. 2009 Aug;202(2):299-307.
Arquier N, Geminard C, Bourouis M, Jarretou G, Honegger B, Paix A, Leopold P.
Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides. Cell Metab. 2008 Apr;7(4):333-8. Erratum in: Cell Metab. 2008 Nov;8(5):446.
Auzoux-Bordenave S, Hatt PJ, Porcheron P. Anti-proliferative effect of 20- hydroxyecdysone in a lepidopteran cell line. Insect Biochem Mol Biol. 2002 Feb;32(2):217-23. Bansilal S, Farkouh ME, Fuster V. Role of insulin resistance and hyperglycemia in the
development of atherosclerosis. Am J Cardiol. 2007 Feb 19;99(4A):6B-14B. Baker, F.C., Tsai, L.W., Reuter, C.C., and Schooley, D.A. (1987). In vivo fluctuation of
JH, JH acid, and ecdysteroid titer, and JH esterase activity, during development of fifth stadium Manduca sexta. Insect Biochem 17, 989-996.
Belfiore A, Genua M, Malaguarnera R. PPAR-gamma Agonists and Their Effects on IGF-I Receptor Signaling: Implications for Cancer. PPAR Res. 2009;2009:830501.
Bell, R.A., amd Joachim, F.G. Techniques for rearing laboratory colonies of tobacco
hornworms and pink bollworms. Ann. Ent. Soc. Am. 1976. 69; 365-373.
Berrie CP. Phosphoinositide 3-kinase inhibition in cancer treatment. Expert Opin Investig Drugs. 2001 Jun;10(6):1085-98.
73
Billas IM, Moras D. Ligand-binding pocket of the ecdysone receptor. Vitam Horm. 2005;73:101-29. Review.
Bollenbacher, W.E., Smith, S.L., Goodman, W., and Gilbert, L.I. (1981). Ecdysteroid titer during larval-pupal-adult development of the tobacco hornworm, Manduca sexta. Gen Comp Endocrinol 44, 302-306.
Britton JS, Lockwood WK, Li L, Cohen SM, Edgar BA. Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev Cell. 2002 Feb;2(2):239-49. Brogiolo W, Stocker H, Ikeya T, Rintelen F, Fernandez R, Hafen E. An evolutionarily conserved function of the Drosophila insulin receptor and
insulin-like peptides in growth control. Curr Biol. 2001 Feb 20;11(4):213-21. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC,
Blenis J, Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999 Mar 19;96(6):857-68.
Bryant PJ. Growth factors controlling imaginal disc growth in Drosophila. Novartis Found Symp. 2001;237:182-94; discussion 194-202. Burgering BM, Medema RH. Decisions on life and death: FOXO Forkhead transcription
factors are in command when PKB/Akt is off duty. J Leukoc Biol. 2003 Jun;73(6):689-701.
Caldwell PE, Walkiewicz M, Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release. Curr Biol. 2005 Oct 25;15(20):1785-95. Cardona-Gómez GP, Mendez P, DonCarlos LL, Azcoitia I, Garcia-Segura LM. Interactions of estrogen and insulin-like growth factor-I in the brain: molecular mechanisms and functional implications. J Steroid Biochem Mol Biol. 2002 Dec;83(1-5):211-7. Castoria G, Migliaccio A, Bilancio A, Di Domenico M, de Falco A, Lombardi M, Fiorentino R, Varricchio L, Barone MV, Auricchio F. PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells. EMBO J. 2001 Nov 1;20(21):6050-9. Cesarone G, Garofalo C, Abrams MT, Igoucheva O, Alexeev V, Yoon K, Surmacz E,
Wickstrom E. RNAi-mediated silencing of insulin receptor substrate 1 (IRS-1) enhances tamoxifen-induced cell death in MCF-7 breast cancer cells. J Cell Biochem. 2006 May 15;98(2):440-50
74
Champlin DT, Truman JW. Ecdysteroids govern two phases of eye development during metamorphosis of the moth, Manduca sexta. Development. 1998 Jun;125(11):2009-18.
Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA, Watson DK, Testa JR.
Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3636-41.
Choi SS, Cha BY, Iida K, Lee YS, Yonezawa T, Teruya T, Nagai K, Woo JT.
Artepillin C, as a PPARE. ligand, enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells. Biochem Pharmacol. 2011 Apr 1;81(7):925-33.
Claeys I, Simonet G, Poels J, Van Loy T, Vercammen L, De Loof A, Vanden Broeck
J. Insulin-related peptides and their conserved signal transduction pathway. Peptides. 2002 Apr;23(4):807-16.
Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H, Hafen E, Leevers SJ,
Partridge L. Extension of life-span by loss of CHICO, a Drosophila insulin Receptor substrate protein. Science. 2001 Apr 6;292(5514):104-6.
Click-iT EdU imaging kit manual, Invitrogen. 2009 Colombani J, Bianchini L, Layalle S, Pondeville E, Dauphin-Villemant C,
Antoniewski C, Carre C, Noselli S, Leopold P. Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science. 2005 Oct 28;310(5748):667-70.
D'Amico LJ, Davidowitz G, Nijhout HF. The developmental and physiological basis of body size evolution in an insect.Proc Biol Sci. 2001 Aug 7;268(1476):1589 -93. de Keizer PL, Burgering BM, Dansen TB. Forkhead box o as a sensor, mediator,
and regulator of redox signaling. Antioxid Redox Signal. 2011 Mar 15;14(6):1093-106. Epub 2010 Sep 20. Review.
Di Paolo S, Teutonico A, Leogrande D, Capobianco C, Schena PF. Chronic inhibition of
mammalian target of rapamycin signaling downregulates insulin receptor substrates 1 and 2 and AKT activation: A crossroad between cancer and diabetes?
J Am Soc Nephrol. 2006 Aug;17(8):2236-44. Dobashi Y, Watanabe Y, Miwa C, Suzuki S, Koyama S. Mammalian target of
rapamycin: a central node of complex signaling cascades. Int J Clin Exp Pathol. 2011 Jun 20;4(5):476-95. Epub 2011 Jun 14. Review.
75
Dupont J, Le Roith D. Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects. Mol Pathol. 2001 Jun;54(3):149-54.
Dupont J, Karas M, LeRoith D. The potentiation of estrogen on insulin-like
growth factor I action in MCF-7 human breast cancer cells includes cell cycle components. J Biol Chem. 2000 Nov 17;275(46):35893-901.
Dzitoyeva S, Dimitrijevic N, Manev H. Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi). BMC Genomics. 2003 Aug 12;4(1):33. Fernandez R, Tabarini D, Azpiazu N, Frasch M, Schlessinger J. The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signalingpotential. EMBO J. 1995 Jul
17;14(14):3373-84. Fox EM, Andrade J, Shupnik MA. Novel actions of estrogen to promote
proliferation: integration of cytoplasmic and nuclear pathways. Steroids. 2009 Jul;74(7):622-7.
Francis VA, Zorzano A, Teleman AA. dDOR is an EcR coactivator that forms a
feed-forward loop connecting insulin and ecdysone signaling. Curr Biol. 2010 Oct 26;20(20):1799-808.
Fujiwara H, Jindra M, Newitt R, Palli SR, Hiruma K, Riddiford LM. Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings. Insect Biochem Mol Biol. 1995 Jul;25(7):845-56. Furuya F, Hanover JA, Cheng SY. Activation of phosphatidylinositol 3-kinase
signaling by a mutant thyroid hormone beta receptor. Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1780-5.
Furuya F, Lu C, Guigon CJ, Cheng SY. Nongenomic activation of phosphatidylinositol 3-kinase signaling by thyroid hormone receptors. Steroids. 2009 Jul;74(7):628-34.
Furuya F, Ying H, Zhao L, Cheng SY. Novel functions of thyroid hormone
receptor mutants: beyond nucleus-initiated transcription. Steroids. 2007 Feb;72(2):171-9.
Garcia-Segura LM, Diz-Chaves Y, Perez-Martin M, Darnaudéry M. Estradiol, insulin-like growth factor-I and brain aging. Psychoneuroendocrinology. 2007 Aug;32 Suppl 1:S57-61.
76
Gao X, Pan D. TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 2001 Jun 1;15(11):1383-92. Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, Stocker H, Kozma SC,
Hafen E, Bos JL, Thomas G. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell. 2003 Jun;11(6):1457-66.
Garofalo RS, Rosen OM. Tissue localization of Drosophila melanogaster insulin receptor transcripts during development. Mol Cell Biol. 1988 Apr;8(4):1638-47. Gilbert, LI. (2012). Insect Endocrinology. Elsevier Publishing. London, UK. Gilbert LI, Bollenbacher WE, Granger NA. Insect endocrinology: regulation of
endocrine glands, hormone titer, and hormone metabolism. Annu Rev Physiol. 1980;42:493-510. Review.
Gilbert LI, Rybczynski R, Warren JT. Control and biochemical nature of the
ecdysteroidogenic pathway. Annu Rev Entomol. 2002;47:883-916. Review. Girgenrath, S. Aspects of endocrine regulation in the tobacco hormwaorm (Manduca
setxa). Ph.D. dissertation, Northeastern University, 1999 Gnanapragasam VJ, McCahy PJ, Neal DE, Robson CN. Insulin-like growth factor II
and androgen receptor expression in the prostate. BJU Int. 2000 Oct;86(6):731-5. Goberdhan DC, Wilson C. The functions of insulin signaling: size isn't
everything, even in Drosophila. Differentiation. 2003 Sep;71(7):375-97. Review. Goldstein BJ, Mahadev K, Wu X, Zhu L, Motoshima H. Role of insulin-induced
reactive oxygen species in the insulin signaling pathway. Antioxid Redox Signal. 2005 Jul-Aug;7(7-8):1021-31.
Guillemot F, Zimmer C. From cradle to grave: the multiple roles of fibroblast
growth factors in neural development. Neuron. 2011 Aug 25;71(4):574-88. Review.
Hamelers IH, Steenbergh PH. Interactions between estrogen and insulin-like growth
factor signaling pathways in human breast tumor cells. Endocr Relat Cancer. 2003 Jun;10(2):331-45.
Hariharan IK, Bilder D. Regulation of imaginal disc growth by tumor-suppressor
genes in Drosophila. Annu Rev Genet. 2006;40:335-61. Review .
77
Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004 Aug 15;18(16):1926-45. Ishizaki H, Suzuki A. The brain secretory peptides that control moulting and metamorphosis of the silkmoth, Bombyx mori. Int J Dev Biol. 1994 Jun;38(2):301-10. Jindra M, Huang JY, Malone F, Asahina M, Riddiford LM. Identification and mRNA developmental profiles of two ultraspiracle isoforms In the epidermis and wings of Manduca sexta. Insect Mol Biol. 1997 Feb;6(1):41-53. Jindra M, Malone F, Hiruma K, Riddiford LM. Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta. Dev Biol. 1996 Nov 25;180(1):258-72. Jones G, Sharp PA. Ultraspiracle: an invertebrate nuclear receptor for juvenile hormones. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13499-503. Johnston LA, Gallant P. Control of growth and organ size in Drosophila. Bioessays. 2002 Jan;24(1):54-64. Jun T, Gjoerup O, Roberts TM. Tangled webs: evidence of cross-talk between cRaf-1 and Akt. Sci STKE. 1999 Dec 21;1999(13):PE1. Kawamura K, Shibata T, Saget O, Peel D, Bryant PJ. A new family of growth factors Produced by the fat body and active on Drosophila imaginal discs cells. Development. 1999 Jan;126(2):211-9. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst
P, Sabatini DM. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002 Jul 26;110(2):163-75.
King-Jones K, Charles JP, Lam G, Thummel CS. The ecdysone-induced DHR4 orphan nuclear receptor coordinates growth and maturation in Drosophila. Cell. 2005 Jun 3;121(5):773-84. Kondo H, Ino M, Suzuki A, Ishizaki H, Iwami M. Multiple gene copies for
bombyxin, an insulin-related peptide of the silkmoth Bombyx mori: structural signs for gene rearrangement and duplication responsible for generation of multiple molecular forms of bombyxin. J Mol Biol. 1996 Jun 28;259(5):926-37.
Koyama T, Syropyatova MO, Riddiford LM. Insulin/IGF signaling regulates the
change in commitment in imaginal discs and primordia by overriding the effect of juvenile hormone. Dev Biol. 2008 Dec 15;324(2):258-65.
78
Krymskaya VP. Tumour suppressors hamartin and tuberin: intracellular signalling. Cell Signal. 2003 Aug;15(8):729-39. Kurenova EV, Hunt DL, He D, Fu AD, Massoll NA, Golubovskaya VM, Garces CA,
Cance WG. Vascular endothelial growth factor receptor-3 promotes breast cancer cell proliferation, motility and survival in vitro and tumor formation in vivo. Cell Cycle. 2009 Jul 15;8(14):2266-80.
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of
Bacteriophage T4. Nature. 1970. 227; 680-685. Lannigan DA. Estrogen receptor phosphorylation. Steroids. 2003 Jan;68(1):1-9. Lee AV, Jackson JG, Gooch JL, Hilsenbeck SG, Coronado-Heinsohn E, Osborne CK, Yee D. Enhancement of insulin-like growth factor signaling in human breast cancer: estrogen regulation of insulin receptor substrate-1 expression in vitro and in vivo. Mol Endocrinol. 1999 May;13(5):787-96. Leevers, SJ, and Hafen E, 2004. Growth regulation by insulin and TOR signaling in
Drosophila. In: Hall, MN, Raff, M, Thomas, G (Eds.), Cell growth: Control of Cell Size. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 167-192.
Leevers SJ, Weinkove D, MacDougall LK, Hafen E, Waterfield MD. The Drosophila
phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J. 1996 Dec 2;15(23):6584-94.
Leslie NR. The redox regulation of PI 3-kinase-dependent signaling. Antioxid
Redox Signal. 2006 Sep-Oct;8(9-10):1765-74. Review. Melander A. Kinetics-effect relations of insulin-releasing drugs in patients with type 2
diabetes: brief overview. Diabetes. 2004 Dec;53 Suppl 3:S151-5. Review. Mendez P, Azcoitia I, Garcia-Segura LM. Interdependence of oestrogen and insulin-like growth factor-I in the brain: potential for analysing neuroprotective mechanisms. J Endocrinol. 2005 Apr;185(1):11-7. Mieulet V, Lamb RF. Tuberous sclerosis complex: linking cancer to metabolism.
Trends Mol Med. 2010 Jul;16(7):329-35. Miner AL, Rosenberg AJ, Frederik Nijhout H. Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae). J Insect Physiol. 2000 Mar;46(3):251-258.
79
Mirth C, Truman JW, Riddiford LM. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster. Curr Biol. 2005 Oct 25;15(20):1796-807.
Morelli C, Lanzino M, Garofalo C, Maris P, Brunelli E, Casaburi I, Catalano S,
Bruno R, Sisci D, Andos S. Akt2 inhibition enables the forkhead transcription factor FoxO3a to have a repressive role in estrogen receptor alpha transcriptional activity in breast cancer cells. Mol Cell Biol. 2010 Feb;30(3):857-70.
Myers JW, Jones JT, Meyer T, Ferrell JE Jr. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat Biotechnol. 2003
Mar;21(3):324-8. Nagasawa H, Kataoka H, Isogai A, Tamura S, Suzuki A, Ishizaki H, Mizoguchi A,
Fujiwara Y, Suzuki A. Amino-terminal amino Acid sequence of the silkworm prothoracicotropic hormone: homology with insulin. Science. 1984 Dec 14;226(4680):1344-5.
Nagasawa H, Kataoka H, Isogai A, Tamura S, Suzuki A, Mizoguchi A, Fujiwara Y,
Suzuki A, Takahashi SY, Ishizaki H. Amino acid sequence of a prothoracicotropic hormone of the silkworm Bombyx mori. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5840-5843.
Nagata K, Hatanaka H, Kohda D, Kataoka H, Nagasawa H, Isogai A, Ishizaki H,
Suzuki A, Inagaki F. Identification of the receptor-recognition surface of bombyxin-II, an insulin-like peptide of the silkmoth Bombyx mori: critical importance of the B-chain central part. J Mol Biol. 1995 Nov 10;253(5):759-70.
Nardi JB, Hardt TA, Magee-Adams SM, Osterbur DL. Morphogenesis in wing
imaginal discs: its relationship to changes in the extracellular matrix. Tissue Cell. 1985;17(4):473-90.
Nijhout, HF. 1994. Insect Hormones. Princeton University Press, Princeton, NJ. Nijhout HF, Grunert LW. Bombyxin is a growth factor for wing imaginal disks in
Lepidoptera. Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15446-50. Epub 2002 Nov 12.
Nijhout HF, Kremen C. Control of pupal commitment in the imaginal disks of
Precis coenia (Lepidoptera: Nymphalidae). J Insect Physiol. 1998 Mar;44(3-4):287-296.
Nijhout HF, Smith WA, Schachar I, Subramanian S, Tobler A, Grunert LW. The
control of growth and differentiation of the wing imaginal disks of Manduca sexta. Dev Biol. 2007 Feb 15;302(2):569-76.
80
Nijhout HF, Williams CM. Control of moulting and metamorphosis in the tobacco
hornworm, Manduca sexta (L.): cessation of juvenile hormone secretion as a trigger for pupation. J Exp Biol. 1974 Oct;61(2):493-501.
Nijhout HF, Williams CM. Control of moulting and metamorphosis in the tobacco hornworm, Manduca sexta (L.): growth of the last-instar larva and the decision to pupate. J Exp Biol. 1974 Oct;61(2):481-91. Oldham S, Bohni R, Stocker H, Brogiolo W, Hafen E. Genetic control of size in
Drosophila. Philos Trans R Soc Lond B Biol Sci. 2000 Jul 29;355(1399):945-52. Paliouras M, Diamandis EP. An AKT activity threshold regulates
androgen-dependent and androgen-independent PSA expression in prostate cancer cell lines. Biol Chem. 2008 Jun;389(6):773-80.
Pandey NR, Benkirane K, Amiri F, Schiffrin EL. Effects of PPAR-gamma knock-down and hyperglycemia on insulin signaling in vascular smooth muscle cells from hypertensive rats. J Cardiovasc Pharmacol. 2007 Jun;49(6):346-54.
Pandini G, Mineo R, Frasca F, Roberts CT Jr, Marcelli M, Vigneri R, Belfiore
A. Androgens up-regulate the insulin-like growth factor-I receptor in prostate cancer cells. Cancer Res. 2005 Mar 1;65(5):1849-57. Papaconstantinou J. Insulin/IGF-1 and ROS signaling pathway cross-talk in
aging and longevity determination. Mol Cell Endocrinol. 2009 Feb 5;299(1):89- 100.
Pinches C, Peehl DM, Lamson G, Rosenfeld RG. Insulin-like growth factor, IGF receptors and IGF binding proteins in primary cultures of prostate epithelial cells. J Clin Endocrinol Metabol 1991; 73: 401-7.
Potter CJ, Huang H, Xu T. Drosophila Tsc1 functions with Tsc2 to antagonize
insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell. 2001 May 4;105(3):357-68. Preston-Martin, S, Pike, M.C., Ross, R.K., Jones, P.A., and Henderson, B.E. (1990). Increased cell division as a cause of human cancer. Cancer Res. 50, 7415-7421. Rafalski VA, Brunet A. Energy metabolism in adult neural stem cell fate. Prog
Neurobiol. 2011 Feb;93(2):182-203. Renault VM, Rafalski VA, Morgan AA, Salih DA, Brett JO, Webb AE, Villeda SA,
Thekkat PU, Guillerey C, Denko NC, Palmer TD, Butte AJ, Brunet A. FoxO3 regulates neural stem cell homeostasis. Cell Stem Cell. 2009 Nov 6;5(5):527-39.
81
Revelli AM, Massobrio, Tesarik J. Nongenomic actions of steroid hormones in reproductive tissues. Endocr Rev 1998;19:3–17.
Riddiford LM, Cherbas P, Truman JW. Ecdysone receptors and their biological
actions. Vitam Horm. 2000;60:1-73. Review. Riddiford LM, Hiruma K, Zhou X, Nelson CA. Insights into the molecular basis of the
hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol. 2003 Dec;33(12):1327-38.
Riehle MA, Brown MR. Insulin stimulates ecdysteroid production through a conserved
signaling cascade in the mosquito Aedes aegypti. Insect Biochem Mol Biol. 1999 Oct;29(10):855-60.
Rountree DB, Bollenbacher WE. The release of the prothoracicotropic hormone in the
tobacco hornworm, Manduca sexta, is controlled intrinsically by juvenile hormone. J Exp Biol. 1986 Jan;120:41-58.
Rusten TE, Lindmo K, Juhasz G, Sass M, Seglen PO, Brech A, Stenmark H. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev Cell. 2004 Aug;7(2):179-92. Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of
DNA synthesis in vivo. Proc Natl Acad Sci U S A. 2008 Feb 19;105(7). Sarbassov DD, Ali SM, Sabatini DM. Growing roles for the mTOR pathway. Curr
Opin Cell Biol. 2005 Dec;17(6):596-603. Epub 2005 Oct 13. Review. Satake S, Masumura M, Ishizaki H, Nagata K, Kataoka H, Suzuki A, Mizoguchi A. Bombyxin, an insulin-related peptide of insects, reduces the major storage
carbohydrates in the silkworm Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol. 1997 Oct;118(2):349-57.
Scanga SE, Ruel L, Binari RC, Snow B, Stambolic V, Bouchard D, Peters M, Calvieri B,
Mak TW, Woodgett JR, Manoukian AS. The conserved PI3'K/PTEN/Akt signaling pathway regulates both cell size and survival in Drosophila. Oncogene. 2000 Aug 17;19(35):3971-7.
Schauer S, Callender J, Henrich VC, Spindler-Barth M. The N-terminus of
ecdysteroid receptor isoforms and ultraspiracle interacts with different ecdysteroid response elements in a sequence specific manner to modulate transcriptional activity. J Steroid Biochem Mol Biol. 2011 Apr;124(3-5):84-92.
Schwedes C, Tulsiani S, Carney GE. Ecdysone receptor expression and activity
in adult Drosophila melanogaster. J Insect Physiol. 2011 Jul;57(7):899-907.
82
Serra C, Bhasin S, Tangherlini F, Barton ER, Ganno M, Zhang A, Shansky J, Vandenburgh HH, Travison TG, Jasuja R, Morris C. The role of GH and IGF-I in mediating anabolic effects of testosterone on androgen-responsive muscle. Endocrinology. 2011 Jan;152(1):193-206.
Seto-Young D, Avtanski D, Strizhevsky M, Parikh G, Patel P, Kaplun J, Holcomb
K, Rosenwaks Z, Poretsky L. Interactions among peroxisome proliferator activated receptor-gamma, insulin signaling pathways, and steroidogenic acute regulatory protein in human ovarian cells. J Clin Endocrinol Metab. 2007 Jun;92(6):2232-9.
Shimotake J, Derugin N, Wendland M, Vexler ZS, Ferriero DM. Vascular
endothelial growth factor receptor-2 inhibition promotes cell death and limits endothelial cell proliferation in a neonatal rodent model of stroke. Stroke. 2010 Feb;41(2):343-9.
Shingleton AW, Das J, Vinicius L, Stern DL. The temporal requirements for insulin
signaling during development in Drosophila. PLoS Biol. 2005 Sep;3(9):e289. Simoncini TA, Hafezi-Moghadam DP, Brazil K, et al. Interaction of oestrogen receptor
with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000;407:538–41.
Smith WA. Regulation and consequences of cellular changes in the prothoracic glands of
Manduca sexta during the last larval instar: a review. Arch Insect Biochem Physiol. 1995;30(2-3):271-93.
Smith WA, Priester J, Morais J. PTTH-stimulated ecdysone secretion is dependent
upon tyrosine phosphorylation in the prothoracic glands of Manduca sexta. Insect Biochem Mol Biol. 2003 Dec;33(12):1317-25.
Swevers L, Iatrou K. The ecdysone regulatory cascade and ovarian development
in lepidopteran insects: insights from the silkmoth paradigm. Insect Biochem Mol Biol. 2003 Dec;33(12):1285-97. Review.
Szypowska AA, Burgering BM. The peroxide dilemma: opposing and mediating
insulin action. Antioxid Redox Signal. 2011 Jul 1;15(1):219-32 Tatar M, Kopelman A, Epstein D, Tu MP, Yin CM, Garofalo RS. A mutant Drosophila
insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science. 2001 Apr 6;292(5514):107-10.
Truman JW, Hiruma K, Allee JP, Macwhinnie SG, Champlin DT, Riddiford LM.
Juvenile hormone is required to couple imaginal disc formation with nutrition in insects. Science. 2006 Jun 2;312(5778):1385-8.
83
Tu MP, Yin CM, Tatar M. Impaired ovarian ecdysone synthesis of Drosophila melanogaster insulin Receptor mutants. Aging Cell. 2002 Dec;1(2):158-60. Uhlmann F, Bouchoux C, Lopez-Aviles S. A quantitative model for
cyclin-dependent kinase control of the cell cycle: revisited. Philos Trans R Soc Lond B Biol Sci. 2011 Dec 27;366(1584):3572-83.
van der Heide LP, Jacobs FM, Burbach JP, Hoekman MF, Smidt MP. FoxO6
transcriptional activity is regulated by Thr26 and Ser184, independent of nucleo-cytoplasmic shuttling. Biochem J. 2005 Nov 1;391(Pt 3):623-9.
Vanfleteren JR, Braeckman BP. Mechanisms of life span determination in
Caenorhabditis elegans. Neurobiol Aging. 1999 Sep-Oct;20(5):487-502. Vardatsikos G, Sahu A, Srivastava AK. The insulin-like growth factor family:
molecular mechanisms, redox regulation, and clinical implications. Antioxid Redox Signal. 2009 May;11(5):1165-90. Review.
Varela PF, Llera AS, Mariuzza RA, Tormo J. Crystal structure of imaginal disc
growth factor-2. A member of a new family of growth-promoting glycoproteins from Drosophila melanogaster. J Biol Chem. 2002 Apr 12;277(15):13229-36.
Verdu J, Buratovich MA, Wilder EL, Birnbaum MJ. Cell-autonomous regulation of
cell and organ growth in Drosophila by Akt/PKB. Nat Cell Biol. 1999 Dec;1(8):500-6.
Vermehren A, Qazi S, Trimmer BA. The nicotinic alpha subunit MARA1 is necessary for
Cholinergic evoked calcium transients in Manduca neurons. Neurosci Lett. 2001 Nov 9;313(3):113-6.
Wesche J, Haglund K, Haugsten EM. Fibroblast growth factors and their
receptors in cancer. Biochem J. 2011 Jul 15;437(2):199-213. White MF, Yenush L. The IRS-signaling system: a network of docking proteins that
mediate insulin and cytokine action. Curr Top Microbiol Immunol.1998;228:179-208.
Wu Y, Bauman WA, Blitzer RD, Cardozo C. Testosterone-induced hypertrophy of L6
myoblasts is dependent upon Erk and mTOR. Biochem Biophys Res Commun. 2010 Oct 1;400(4):679-83.
Wu JD, Haugk K, Woodke L, Nelson P, Coleman I, Plymate SR. Interaction of IGF
signaling and the androgen receptor in prostate cancer progression. J Cell Biochem. 2006 Oct 1;99(2):392-401. Review
84
Yao TP, Forman BM, Jiang Z, Cherbas L, Chen JD, McKeown M, Cherbas P, Evans RM. Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature. 1993 Dec 2;366(6454):476-9.
Yuan B, Latek R, Hossbach M, Tuschl T, Lewitter F. siRNA Selection Server: an
automated siRNA oligonucleotide prediction server. Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W130-4.
Zhu Q, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S. Characterization of
recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum. Insect Biochem Mol Biol. 2008 Apr;38(4):467-77.