Review

The insulin-like growth factor (IGF) system and

non-islet-cell tumor hypoglycemia: mechanism,
management, and the effects of IGF binding
proteins
Wouter van de Hoef
Intern
Division Biomedical genetics
Biomedical sciences
Bachelor thesis

(received 19 January 2015)

Abstract

The IGF system is composed of the protein ligands IGF-I and IGF-II, inducing their mitogenic
and metabolic effects by interacting with the IGF-I receptor (IGF-IR), the insulin receptor (IR)
and hybrid receptors of the former. The IGF-II receptor does not possess tyrosine kinase
activity but acts by means of taking up and degrading extracellular IGF-II.
Other ways for limiting IGF-II bio-availability are genomic imprinting, and circulatory IGF
sequestering by binding any of the six IGFBPs. These complexes can still cross the vasculature,
therefore most IGFs are transported in ternary complexes with an acid labile subunit and
IGFBP-3 or -5 that are unable to do so. Deregulation in any of these IGF inhibiting mechanisms
can stimulate tumorigenesis. Non beta cell tumors are rare tumors and known for
overproducing large volumes of aberrant processed IGF-II, also called big-IGF-II. This IGF-II
variant distorts ternary complex formation and displaces IGFs from binary complexes. The
consequences are a shift from ternary to binary complex formation and a larger free bio-active
fraction of IGFs. This in turn enhances signaling through IGF-IR, downregulating growth
hormone (GH), and stimulates IRs, causing hypoglycemia in a process called non-islet-cell
tumor hypoglycemia or (NICTH). This rare syndrome is diagnosed in patients with a
concurrent illness by excluding other pathologies. Next to the presenting symptoms of
hypoglycemia, a correct diagnosis is made by measuring insulin, IGF-I and big-GF-II.
Patients can fully recover if the tumor is completely removed. IGFBP ratios in NICTH are
different from healthy individuals. These changes can be systemic and/or due to the tumor.
The exact role of all IGFBPs in NICTH remain poorly understood. IGF-independent effects of
these binding proteins could give new insights in how these proteins interact with these
neoplasms and elucidate the hypoglycemic events in patients.

1
1. Introduction
Insulin-like growth factor-2 (IGF-II) is
one of the two protein ligands of the
IGF system. The hormone has
mitogenic and anti-apoptotic effects on
various cell types, including healthy and
tumor cells. It also has an important
role in pre-adolescent and in utero
growth, promoting growth as autocrine
and paracrine regulators. This article
however will only address IGF-II action
in postnatal life.
2. The IGF-II system
Multiple mechanisms have evolved to
suppress inappropriate IGF-II signaling
after birth. These consist of genomic
imprinting, sequestration by IGF
binding proteins and removal of
circulatory IGF-II by the IGF-II receptor
(Khandwala, McCutcheon, Flyvbjerg, &
Friend, 2000).
The actions of the IGFs are mainly
mediated through the interaction with
different plasma membrane receptors,
namely the IGF-I receptor (IGF-IR), the
insulin receptor (IR) and hybrid
receptors of the former two, see figure 1.
Both IGF-I and IGF-II are synthesized
by many tissues, predominantly the
liver and excreted into the circulation,
see figure 2. Association of the peptide
hormones to IGF binding proteins
(IGFBPs) lowers their bioavailability via
sequestration. (de Groot et al., 2007;
Fang, Hwa, & Rosenfeld, 2004;
Livingstone, 2013).
A. IGF2 gene and IGF-II
The IGF2 gene is located on
chromosome 11. It comprises
approximately 30.000 base pairs, nine
exons and four promotors. The first six
exons can be transcribed into various
RNAs from the different promoters, but
do not code for protein. Exons 7, 8 and
9 code for a protein of 20 kDa called
pre-pro-IGF2. This 180-amino acid
protein consists of 5 domains (A-E) and
include a signal peptide of 67 amino
acids, the mature protein and 89
residues designated as the E-domain.
First the signal peptide is removed after
which pro-IGF-II is formed. In the next
step a sugar group is added via O-linked
glycosylation of the E-domain. The
carboxy terminal or E-domain of pro-
IGF-II is enzymatically cleaved by
prohormone convertase 4 (PC4). This
yields the final 67-amino acid mature
IGF-II that makes up around 80 to 90
procent of the total IGF-II in the blood.
Not all IGF-II is processed this way and
multiple forms, together called big-
IGF-II, are intermediates formed by
incomplete PC4 processing with parts
of the E-domain still attached to the
mature protein. The most relevant
variant of big-IGF-II for this review
article is IGF-IIE[1-88].
Insulin and IGF-II are homologous,
sharing almost 50 percent identical
amino acids. This is mainly due to the
conservation of the hydrophobic core of
the proteins that interact with the
insulin receptor and the IGF-I receptor
(Livingstone, 2013; O'Dell & Day, 1998).
B. Imprinting of the IGF2 gene
Normally the expression of autosomal
genes inherited from each parent is
biallelic. In the past 30 years it was
recognized that not all genes are
expressed from both alleles equally.
Genes preferentially expressed from
only one allele are called imprinted
genes. They all play important roles in
embryogenesis, development,
2
metabolism, brain physiology and
behavior (Nordin, Bergman, Halje,
Engstrm, & Ward, 2014).
An example of an imprinted gene is
IGF2. The gene copy of the mother is
silenced during life by methylation of
the CpG-rich cis-elements, better
known as the differentially methylated
regions. Human IGF2 is therefore
maternally imprinted and prevents
inordinate IGF2 expression. The gene
copy of the father is transcribed into
IGF-II mRNA and translated into
various IGF-II peptides (Giannoukakis,
Deal, Paquette, Good-yer, & the IGF-I receptor (IGF-IR), isoform A
Polychronakos, 1993; Livingstone, 2013;
Harris & Westwood, 2012).
Transcription of the IGF2 gene takes
place from four promoters in the
human liver, designated P1 - P4. The
gene is maternally imprinted in the
liver of a human embryo, whereas in
the adult liver both alleles are
expressed. Transcripts from P2 P4 are
always derived from only one allele.
Transcription from P1 during adulthood
results in biallelic expression.
Genomic imprinting was always
thought to be a process in which a
complete allele or an entire part of a
chromosome is made transcriptionally
inactive. IGF2 imprinting is an example
in which only a specific defined region
within a gene is silenced (Vu &
Hoffman, 1994).
Directly downstream the IGF-II gene
resides H19, a gene solely expressed
from the maternal allele. The two genes
are very closely connected, sharing the
same 3 enhancer. H19 is transcribed to
a non-coding mRNA that is steadily
expressed in utero. The role of this RNA
had not been elucidated until recently.
It appears to interact with microRNAs,
short sequences of nucleotides that are
able to interfere with translation and
RNA destruction. It has been
demonstrated H19 inhibits genes that
enhance myoblast development and
stimulates genes promoting growth of
the placenta, for instance the gene
coding for the IGF-I receptor
(Ratajczak, 2012; Nordin et al., 2014).
C. IGF-II signaling, receptors
and downstream targets
The mitogenic and growth promoting
effects of IGF-II are mediated through
of the insulin receptor (IR-A) and a
hybrid receptor composed of the two
receptors mentioned above, see figure 1.
Two types of the insulin receptor (IR)
exist, namely IR-A and IR-B. Isoform B
is involved in taking up glucose from
the circulation. It is mainly expressed in
muscle, liver and adipose tissue. IR-A is
an alternatively spliced variant of the
insulin receptor that binds IGF-II with
an affinity of 15% compared to insulin.
The receptor is expressed throughout
life, especially in foetal tissue and some
tumor cells. The physiological role of
IR-A in adults still has to be elucidated
(Riedemann & Macaulay, 2006).
The IGF-IR and the IR are strikingly
similar. An example of their
equivalency are the comparable size of
their genes and the construct of the
exons. Both are synthesized as pre-
proreceptors and consist of a single
chain of amino acids with a signal
peptide. This end piece is enzymatically
cleaved during translation. The pre-
proreceptors are further processed in
several steps. These involve rounds of
glycosylation, folding and dimerisation
aided by chaperones. Once transported
into the Golgi, the receptors are cleaved
3
by proteases to yield the mature
receptors consisting of two and two -
subunits. The -subunits are located
extracellularly wheras the -subunits
have multiple domains. These are
located extracellularly, transmembrane
and intracellularly. The intracellular -
subunit inside the cell also contains the
tyrosine kinase domain.
Heterodimeric hybrid receptors will
form if a cell expresses the genes for
both the IGF1R and the IR, as depicted
in figure 1 (Riedemann & Macaulay,
2006).
Signaling via IGF-II mainly occurs by
interaction with the IGF-IR. Upon
binding of the ligand a signal
transduction route is set in motion.
First, a receptor tyrosine kinase is made
active and autophosphorylates the -
subunit of the receptor on several
tyrosine residues: namely 1131, 1135 and
1136 in the kinase domain. This is
followed by a phosphorylation step of
the juxtamembrane tyrosines and
carboxy-terminal serines. Insulin
receptor substrates (IRS) are recruited
to the site. Phosphoinositide 3-kinase
(PI3-kinase) can interact with IRS-1 via
its regulatory element, followed by an
increase of phosphatidylinositol 3,4,5-
triphosphate (PIP3). This in turn
mobilizes and activates
phosphoinositide-dependent kinase-1
(PDK) and AKT/protein kinase B. This
activation step inhibits pro-apoptotic
signals by inactivating various proteins
along the cell death inducing signal
transduction pathway (Livingstone,
2013; Riedemann & Macaulay, 2006).
These substrates include:
Bad: a member of the Bcl-2
family and a pro-apoptotic factor
(Ouyang et al, 2012)
Caspase 9: an initiator caspase
that is able to cleave effector
caspases, the factors involved in
the biochemical and
morphological changes inside an
apoptotic cell (Wrstle,
Laussmann, & Rehm, 2012;
Ouyang et al, 2012)
FoxO, The O subgroup of the
Forkhead protein family of
transcription factors. They
influence the transcription of
various genes and induce DNA-
repair mechanisms as well as
genes involved in cell death, cell
cycle arrest and metabolism
(Webb, & Brunet, 2014).
On top of that, activated AKT triggers
various kinds of proteins promoting cell
survival. These include: (Riedemann &
Macaulay, 2006).
NF-kB: a family of transcription
factors involved in cell survival,
proliferation and migration
(Dolcet et al., 2005).
Bcl-xL and Bcl-2: are among the
proteins that can inhibit the
caspase-9-induced
mitochondrial pathway of cell
death. Both belong to the same
protein family as bad (Ouyang et
al, 2012).
D. The IGF-II receptor
IGF-II also has its own receptor, called
the cation-independent mannose-6-
phosphate/insulin-like growth factor-2
receptor, in short the IGF-IIR. It
consists of a large extracellular part
with multiple domains of which two
bind IGF-II and three others bind
Mannose-6-phosphate (M6P). The
exact binding interactions between the
receptor and its ligands is not exactly
known. The receptor has a small
transmembrane unit and a long tail
residing in the cytoplasm (Bergman,
Halje, Nordin, & Engstrm, 2012).
4
 Fig 1. The ligands and receptors of the IGF and insulin system
The ligands roughly bind with an affinity for the receptors as stated in the figure on the left.
Insulin receptors preferentially bind insulin over any of the IGFs. The hybrid receptors and the
IGF-1R especially bind IGF-1 and IGF-2 over insulin. Signaling through the insulin receptor
isoforms A and B have predominantly metabolic actions, whereas the IGF-1R and the hybrids have
more effects on growth than on metabolism as depicted by the arrows.. The receptors are
homologues and dimerize randomly if multiple receptor types are expressed. They all share a -
domain with tyrosine kinase activity initiating downstream signaling upon ligand binding. The
extracellular -domain is primarily involved in ligand binding. The IGF-2 receptor is different from
the other transmembrane proteins. It consists of only a single subunit and does not possess any
tyrosine kinase activity. The receptor is involved in taking up as well as degrading extracellular
IGF-II and is one of the mechanisms limiting IGF-II signaling (Dynkevich et al. 2013).

IGF-IIR has an higher affinity for IGF-II
than does IGF-IR, as depicted above.
Although the receptor lacks tyrosine
kinase activity and an autophos-
phorylation site, it can still signal by
linking to G-proteins (Harris &
Westwood, 2012). Binding of IGF-II to
this cell surface protein results in
endocytosis and breakdown of the
ligand by degradation in the lysozome.
In this way, the concentration of free
IGF-II is tightly controlled by clearing it
from the circulation.
IGF-IIR not only functions as a receptor
on the cell surface but also has an
important intracellular function. It
facilitates the transport of newly
synthesized peptides marked with the
sugar Mannose-6-phosphate (M6P).
These tagged lysosomal enzymes,
growth factors and cytokines are then
moved from the trans-Golgi network to
late endosomes via clathrin-coated
vesicles. The M6P receptor can be
recycled, back to the cell membrane or
the Golgi network, while the peptides
end up in the lysozome.

5

E. IGFBPs, ALS and complex
formation
IGFs in the circulation are bound to one
of the six IGF binding proteins
(IGFBPs), designated IGFBP-1 through
IGFBP-6. Binding IGFs expands the
half-lives of these growth factors, as
depicted in figure 2. All IGFBPs have an
higher affinity for IGFs (kd 10-10M)
than the IGF-I receptor (kd ; 10-8 10-
9
M). This means the binding proteins
are not just simple carriers of IGFs, but
also have an import function in
modulating the bio-availability of these
peptide hormones (Hwa, Oh &
Rosenfeld, 1999a).
IGFBP-3 transfers three quarters of IGF-
I and IGF-II in ternary complexes. The
third component of this complex is an
acid labile subunit (ALS), a glycoprotein
rich in leucine with a molecular mass of
85 kDa. IGFBP-5 is ten times less
abundant in the circulatory system than
IGFBP-3, but can form the same ternary
complexes. In adult individuals 90% of
IGFBP-3 and 55 % of IGFBP-5 is present
in the circulation in these ternary
complexes (Firth & Baxter, 2002).
The concentration of the IGFs and
IGFBP-3 in serum is equivalent,
approximately 100 nmol/L. Serum
values of IGFBP-3 appear to be
controlled post-transcriptionally when
forming complexes and is very
dependent on ALS concentration. ALS
and IGF-I are both dependent on the
amount of circulating growth hormone
(GH). This gives the impression that
IGFBP-3 is also susceptible to GH. This
is not the case, as it is only indirectly
sensitive to GH signaling due to its two
GH-dependent accessories (Baxter,
2014).
IGFs are also present in the circulation
in their free form and in binary
complexes. In these forms IGFBPs are

Figure 2. free and bound IGFs in the circulatory system

The liver, kidney, tumors and many other tissues synthesize IGFs. The peptide hormones are
excreted into the circulation if not used as a ligand for auto- or paracrine signaling. All IGFBPs
form binary complexes with IGF, increasing the half-life more than tenfold compared to the
fraction circulating freely. Binary complexes containing IGFBP-3 and -5 can combine with the
glycoprotein ALS to form ternary complexes. These complexes are not able to pass the
endothelium and are very stable, extending the half-life of the IGF up to a day (Baxter, 2014).

6
able to quickly pass the endothelium
into the tissue, whereas ternary
complexes are restricted from passing
the endothelial walls. Figure 2 describes
IGF dynamics in blood (Firth & Baxter,
2002).
The six IGFBPs encompass a family of
cysteine-rich proteins (the pre-peptides
have 16-20 cysteines) and have a very
similar primary amino acid sequence.
They also share uniformity in their
structural design. This includes: a
preserved N-terminal domain (IGFBP-1
trough IGFBP-5 have 12 cysteines; IGFB-
6 only has 10), a non-preserved
intermediate segment and a preserved
C-terminal domain comprising 6
cysteines.
A striking feature is that all IGFBPs
have an even number of cysteines. This
implies that within each domain the
cysteines form covalent disulphide
bonds without any cysteine reaching
out to a free cysteine located in another
domain. The intermediate protein
segment may have an important role in
the formation of the notch for binding
IGF. Moreover, the diversity of this
segment illustrates the variety of IGFBP
function. These include functions that
are independent of IGF (Hwa, Oh &
Rosenfeld, 1999b).
3. IGF-II and cancer
IGF-II is a very important factor to
stimulate growth, cell survival and
transformation and is essential during
embryogenisis. This means the level of
this IGF should be carefully controlled
after birth as excessive signaling
through IGF-IR and IR-A could
promote tumorigenesis. An abundance
in IGF-II signaling is mostly the
consequence of one (or more) of the
following mechanisms (Yu & Rohan,
2000)
Deregulation in the IGF2/H19 cluster
IGF1-R upregulation
Loss of functioning IGF-IIR
High IR-A:IR-B ratio
High big-IGF-II: mature IGF-II ratio
A. IGF2/H19 and cancer
In adults, IGF-II is mainly transcribed
from P1, the first of the four present
promoters. Transcription from P3 and
P4 occurs mainly in utero but
overexpression has also been linked to
various cancers. This upregulation can
be caused by a loss of transcriptional
suppressors, changing promoter sites
and loss of imprinting (Yu and Rohan,
2000). Deregulation in the IGF2/H19
cluster owing to loss of imprinting has
been linked to tumors originating from
the gastrointestinal tract (Ratajczak,
2012). In some tumors loss of
imprinting is connected with low H19
expression (Khandwala et al., 2000).
The largest amount of IGF-II expression
was found in solitary fibrous tumors.
B. IGF1-R and cancer
The IGF1-R is very often overexpressed
in tumor cells, for instance in colon,
renal and pancreatic cancers. This is
almost always related to loss of tumor
suppressor activity, like p53. IGF1-R
signaling is an essential component in a
developing tumor. The signal
transduction route is necessary for
transformation, proliferation and cell
survival.
Stimulating the IGF1R is a powerful
mechanism to block apoptosis. In fact,
increasing IGF1R signaling is
proportional to the resistance of cell
death induced by multiple pro-
7
apoptotic factors, like osmotic shock,
oxygen deprivation and anti-cancer
medicines.
This cell survival mechanism is mainly
mediated through the (PI3K) pathway
(Livingstone, 2013; Riedemann &
Macaulay, 2006). The environment in
the core of big progressing tumors is
very low in oxygen and deprived of
nutrients. Protection from cell death in
these cells is increased by activating
IGF1-R. Moreover, IGF and insulin
activity have been found to increase the
hypoxic response by inducing AKT and
MAP kinase, stimulating vascular
endothelial growth factor.
Abundantly expressed IGF-IR is also
correlated with later stages of cancer,
namely invasion and metastasis.
Phosporylated IRS-1 interacts with -
catenine stimulating E-cadherin to
dissociate from the actin cytoskeleton.
Losing functional E-cadherin
disconnects epithelial cells from one
another and enhances the invasiveness
of cancerous cells. By suppressing
signaling through IGF-IR, it is possible
to inhibit metastasis in vivo. All in all,
Upregulated IGF-IR is an important
factor in tumor development and
progression to malignancy. (Riedemann
& Macaulay, 2006).
C. IGF-IIR and cancer
IGF-IIR is responsible for the uptake
and degradation of circulating IGF-II.
Loss of function could therefore play a
role in Neoplasm formation. Mutated
forms of the receptor are not able to
process the amount of circulating IGF-
II, resulting in abnormal levels of the
circulating hormone. Furthermore,
cancer cells lacking the ability to break
down IGF-II have shown to have a
greater growth potential. Loss of gene
expression has been described in
malignancies of the liver, prostate,
adrenals and the endometrium (Harris
& Westwood, 2012; Yu & Rohan, 2000).
D. IR-A and cancer
Like IGF-IR signaling, binding of IGF-II
to IR-A induces mitogenesis and is
another way in which abundant IGF-II
signaling could lead to cancer. It has
been demonstrated that binding of IGF-
II to IR-A activates different
downstream pathways in comparison
with insulin stimulating this receptor.
High insulin levels in blood and
enhanced signaling through IR-A might
be a factor explaining the high
incidence of cancer in type 2 diabetics.
As the human body ages, a shift is
observable involving more IR-A and less
IR-B expression and this increases the
chance of neoplasm formation. Isoform
A of the receptor is particularly
enriched in cancers that become more
and more undifferentiated. In some
cancer cells IGF-II can induce all its
tumor stimulating effects even in the
absence of IGF-IR. IRS1 is an important
signal transduction protein in IGF1-R
and IR-A signaling and is less prone to
internal degradation by IGF-II signaling
compared to insulin. This way the
mediator can sustaining its mitogenic
signals in healthy and cancer cells.
(Livingstone, 2013).
E. Big-IGF-II and cancer
Some tumors produce excessive
amounts of IGF-II to stimulate growth
in an autocrine and paracrine way
(Livingstone, 2013). These neoplastic
cells not only express a lot of IGF-II
mRNA but also cannot process all the
newly formed pro-IGF-II. This means
that in comparison to normal cells a
greater part of pro-protein leaks into
8
the circulation (Davda & Seddon, 2007).
These high molecular weight forms of
IGF-II have not undergone all post-
translation processing and some still
contain the 21 amino acids of the E-
domain (E(6888)). The bio-availibility
of IGF-II is futher increased as big-IGF-
II does not form ternary complexes
with ALS and IGFBP-3. High serum
big-IGF-II concentrations, like mature
IGF-II promote mitogenic and insulin-
like effects. The mitogenic actions can
stimulate tumor progression, whereas
the insulin-like effects can cause
hypoglycemia (van Doorn et al., 2002).
4. Hypoglycemia
Hypoglycemia is a serious medical
condition in which patients suffer from
low blood sugar. It is difficult to give an
exact definition but true hypoglycemia
is the state in which the body activates
hormonal countermeasures to maintain
adequate circulatory glucose levels
(Cryer et al., 2009).
A. Blood sugar balance
Glucose is an essential fuel for the brain
under normal physiological
circumstances. This is because the brain
is incapable of synthesizing glucose on
its own and cannot use alternatives like
amino acids and fatty acids
energetically. The only endogenous
energy reservoir for this tissue is a small
amount of glycogen that can only
sustain brain function for a couple of
minutes. Besides the brain, erythrocytes
and the renal medulla are also
dependent on glycolysis. This is why at
the blood sugar level is always so
intensively monitored and adjusted to
the needs of the body. Low plasma
glucose triggers a hormonal response in
the human body, namely by
downregulating insulin followed by
upregulation of glucagon. This
hormonal shift stimulates the
breakdown of glycogen, predominantly
in the liver. In turn, this elevates
plasma glucose and restores the balance
in healthy subjects under normal
physiological circumstances (Cryer et
al., 2009).
B. Hypoglycemia
Counter mechanisms against
hypoglycemia kick in when glucose
levels drop below 70 mg/dL (3.9 mM)
and consist of secreting epinephrine
and glucagon into the circulation.
When these regulatory mechanisms are
insufficient in resolving the
hypoglycemic state, additional
hormonal mechanisms are activated:
the release of GH and cortisol
(Brutsaert, Carey & Zonszein, 2014).
Glucose levels in the blood that fall
below 55 mg/dL (3.1 mM) trigger the
activation of the sympathetic nervous
system and the adrenal medulla. This
happens in two ways and gives rise to
the autonomic symptoms:
The first mechanism is the adrenergic
effect: the release of catecholamines,
adrenaline and noradrenaline,
synthesized by chromaffine cells in the
adrenal medulla, into the circulation.
This effect leads to adrenergic
symptoms and include: irregular
heartbeat, quivering, and anxiety.
The second is the cholinergic effect,
mediated by acetylcholine secreted by
activated postganglionic sympathic
neuron. The cholinergic effects are
responsible for sweating and food
craving. (Martens & Tits, 2014).
An hypoglycemic patient at this point
will feel the need to eat protecting
against a further drop in blood glucose.
If however blood sugar concentration
9
declines to approximately 50 mg/dL
(2.8 mM) patients can get
neuroglycopenic symptoms because of
inadequate glucose supply to the brain.
Patients will feel dizzy, confused and or
tired. In the worst case the symptoms
can result in amnesia, seizures and even
coma (Brutsaert et al., 2014; Martens &
Tits, 2014).
C. Spontaneous hypoglycemia
An important risk factor for
hypoglycemia is the intake of insulin or
glucose lowering medicine. Therefore it
is a condition that mostly affects
patients with diabetes mellitus. Other
notable drugs that can induce
hypoglycemia are alcohol, quinine, beta
blockers, quinolones and cibenzoline.
However, spontaneous hypoglycemia
can appear in a non-diabetic or non
drug taking patient. The human body
has many defense mechanism to cope
with an hypoglycemic event. This
means spontaneous hypoglycemia is a
serious medical problem that should be
carefully investigated. Diagnosis of
hypoglycemia is made definitive by
using Whipples criteria or triad.
A clinician can determine if a patient
has hypoglycemia on the basis of the
following criteria.
1. The before mentioned
(autonomic and neuroglyco-
penic) symptoms are in
concordance with hypoglycemia
2. A low blood sugar level. A
venous blood sample with a
plasma glucose level below 55
mg/dL (3.1 mM) measured using
an appropriate laboratory
method.
3. Resolution of the hypoglycemic
signs and symptoms when
plasma glucose level is raised.
D. Clinical status
A patient diagnosed with hypoglycemia
according to Whipples triad has to
have its clinical status classified. Key is
to identify whether a patient seems well
or has a concurrent disease. The
clinician should always have in mind
that various kinds of drugs can interfere
with glucose metabolism. Even non-
diabetic drugs can be the underlying
cause of drug-induced hypoglycemia.
The patient history, physical
examination and laboratory tests are
needed to diagnose the specific type of
hypoglycemia, see figure 3 (Martens &
Tits, 2014).
E. Seemingly well
This category is specified by patients
presenting with spontaneous
hypoglycemia without any apparent
hormone deficiencies, tumors or any
other serious illnesses. In these
seemingly healthy patients blood
analysis should be carried out after a
spontaneous or induced hypoglycemia.
The following parameters should be
covered using an adequate laboratory
method: glucose, insulin, C-peptide,
pro-insulin, and -hydroxybutyrate, all
measured in plasma .
Furthermore, a clinician should take
into account autoantibodies and oral
glucose lowering medicine. In this way,
he can diagnose a patient according to
the parameter values. The
hypoglycemia can be either endogenous
(from inside the body) or exogenous
(from outside the body). An example of
exogenous hypoglycemia is a diabetic
injecting himself with too much insulin.
He will have an elevated plasma insulin
level but no corresponding rise in C-
peptide and pro-insulin. This way the
clinician can discriminate between the
10
two types. (Martens & Tits, 2014;
Iglesias & Dez, 2014).
Exogenous hyperinsulinism
This type of hyperinsulinism is
characterized by intake of insulin or an
hypoglycemic medicine leading to
hypoglycemia. An example is diabetic
hypoglycemia in which a diabetic
injects himself accidentally with too
much insulin. Also, a suicide attempt in
which insulin or other hypoglycemic
agents are severely abused. Rarely due
to psychiatric disorders in which the
patient injects himself (Mnchausen
syndrome) or is a victim of someone
else injecting (Mnchausen syndrome
by proxy) in order to gain attention and
or sympathy.
Endogenous hyperinsulinism
Endogenous hyperinsulinism is a rare
condition in which the amount of
circulating insulin fails to decrease,
even though plasma glucose level drops
to hypoglycemic proportions.
Spontaneous hypoglycemia is
confirmed by Whipples triad but
elaborate test are needed for
investigating the source of
hypoglycemia for further treatment. In
follow-up laboratory tests levels of
insulin, C-peptide and pro-insulin are
measured in blood sample of a fasting
patient. Hypoglycemic medicines used
by the patient and autoantibodies
detectable in plasma must be
considered. Insulin auto antibodies
present in serum indicate an insuloma
(Martens & Tits, 2014).
Insulinomas are pancreatic -cells
insulin secreting tumors. They are
mostly small, benign and tumor mass is
evenly distributed throughout the
pancreas. It is a very rare tumor with
only four cases per million per year
(Iglesias & Dez 2014). The best
treatment is to surgically remove the
tumor. When done successfully, disease
symptoms vanish almost completely.
Autoantibodies present in blood plasma
can be a sign of autoimmune
hypoglycemia. Antibodies raised against
the insulin receptor (type B insulin
resistance) stimulate insulin production
in a similar fashion as thyroid hormone
antibodies bind and activate the TSH
receptor leading to Graves disease.
Immunoglobulins recognizing insulin
can lead to insulin autoimmune
syndrome or Hirata disease and also
induces hypoglycemia. In this
pathology antibodies bind insulin but
are released at a later timepoint
interfering with normal glucose
homeostasis (Iglesias & Dez 2014). The
primary way of treatment is the use of
immune suppressing medicine like
corticosteroids. Patients will have to eat
multiple small meals, also at night to
prevent hypoglycemic attacks.
Noninsulinoma pancreatogenous
hypoglycemia syndrome (NIPHS)
Patients suffering from NIPHS suffer
from grave attacks of neuroglycopenia,
especially a few hours after a meal. A
biopt of the pancreas shows
nesidioblastosis, hyperplasia of -cells
in combination with hypertrophy and
proliferation of islet cells in the ducts of
the pancreas. This pathology results in
a prolonged and exaggerated insulin
production after a meal. Discriminating
between an insuloma not visible on a
scan and NIPHS is possible using
SPACI. Calcium stimulates insulin
secretion and is injected separately in
the three arteries supplying the
pancreas to measure the insulin
11
response in the draining right hepatic
vein. Overall increased insulin response
illustrates nesidioblastosis, whereas a
single artery response points to an
insuloma (Martens & Tits, 2014; Iglesias
& Dez 2014).
Multiple ways of treating NIPHS are
available but therapy is still very
personalized. A diet low in sugar
combined with multiple small meals is
advised in mild cases. In patients with
severe neuroglycopenic symptoms who
do not benefit enough from taking
medicine (diazoxide, acarbose or
octreotide), a partial pancreatectomy is
a last resort to relieve symptoms (Cryer
et al, 2009).
Concurrent illness
These patients are classified as a group
in which the hypoglycemia is due to
their primary disease. This means that
most of them are at danger when
attacks of hypoglycemia occur.
Concurrent illnesses can be caused by
critical illnesses, hormone deficiencies
and non-beta cell tumors
Critical illnesses
Hypoglycemia can be the result of
malfunctioning organs like the liver,
kidney or heart. Starvation and sepsis
are also critical situations in which the
body cannot maintain adequate glucose
homeostasis. Toxic and ischemic
hepatitis make the liver unable to
activate gluconeogenesis to raise
plasma glucose in case of an
hypoglycemic event. In renal
insufficiency the clearance of insulin is
impaired. Circulating cytokines during
sepsis (this includes malaria) induce
hypoglycemia by elevating sugar
metabolism.
Hormone deficiencies
Growth hormone and cortisol are very
important hormones maintaining
steady glucose levels when blood sugar
levels drop. Deficiencies in these two
glucose regulators owing to
hypopituitarism and primary adrenal
insufficiency (Addison's disease) result
in hypoglycemia primarily after a long
fasting period. Cortisol is important in
stimulating hepatic gluconeogenesis.
Additional symptoms are an helpful
tool in the diagnosis of GH or cortisol
deficiency. Hormones can be replaced
in order to treat these endocrine
diseases. The last form of hypoglycemia
in very ill patients is caused by non beta
cell tumors. These are rare neoplasms
originating from extrapancreatic tissues
and are among the tumors causing
episodes of hypoglycemia in patients.
These type of tumors will be further
discussed in the next chapter
Non-islet-cell tumor-induced
hypoglycemia.
In the figure below the steps for
diagnosing hypoglycemia in a patient
are summarized.
12
Figure 3. A step by step plan for a clinician to discover the underlying disease or cause of a
patient with hypoglycemia
A patient is hypoglycemic if he fulfills Whipples criteria. The clinician has to make an evaluation
whether a patient is severely ill or is seemingly well, but most also rule out any hypoglycemic agent
used by the patient. A patient with an healthy impression will have his blood analyzed in the
laboratory. Determination of plasma glucose, insulin, C-peptide, pro-insulin, beta-hydroxybutyrate,
insulin antibodies or any hypoglycemic agent could help to find the cause of hypoglycemia. A patient
with a concurrent illness needs immediate care as hypoglycemic episodes in these patients can be
detrimental. These seriously ill people most often have a severe disease, hormone deficiency or an
non beta cell tumor explaining the hypoglycemic attacks. The words written in red depict the
preferential diagnostic means to come to a diagnosis.
Abbreviated words in this figure: HH means hyperinsulinemic hypoglycemia, Ab stands for antibody,
SPACI means selective pancreatic arterial calcium injective and NIPHS means non-insulinoma
pancreatogenous hypoglycemia syndrome (Martens & Tits, 2014).

13
5. Non-islet-cell tumor-
induced hypoglycemia
NICTH or written in full: non-islet cell
tumor-induced hypoglycemia is a very
rare pathology. It is one of the
conditions whereby tumor activity, in
this case the secretion of a specific
protein is predominantly responsible
for hypoglycemic attacks in patients.
A. Tumors and hypoglycemia
Tumor-induced hypoglycemia (TIH)
can be the consequence of different
underlying mechanisms and lead to
hypoglycemic events in patients in
various ways. Insulomas are known to
directly cause hypoglycemia by
overproducing insulin.
Big tumors are a burden for their high
energy consumption (Iglesias & Dez
2014). Malignant neoplasms can
infiltrate and destroy liver, kidney and
adrenal tissue, making it difficult to
maintain glucose homeostasis. The final
way for TIH to develop is the
overproduction of tumor derived
proteins that distort glucose
metabolism. Among these substances
are various cytokines, catecholamines,
IGF-I, glucagon-like peptide 1 and
incomplete processed forms of IGF-II.
Overexpression of the last-mentioned
peptide by non beta cell tumors gives
rise to hypoglycemia in a process called
NICTH (de Groot et al., 2007).
B. NICTH
NICTH is a very rare condition with
approximately 300 cases reported in the
English medical literature over the past
25 years. So far, patients do not seem to
be predisposed by genetics or gender in
developing a non beta cell tumor. This
statement is backed up by the facts that
so far no multiple cases have been
reported within families and no
mutations have been found to be
associated with promoting NICTH. On
average, a patient is well above his
fifties at diagnosis, even though a
handful of reports involve children
(Bodnar, Acevedo & Pietropaolo, 2013).
The syndrome is thought to be four
times less common than an insuloma,
although many patients with NICTH go
undiagnosed (de Groot et al., 2007).
Many different kind of tumors are able
to produce big-IGF-II. NICTH cases are
often reported in tumors originating
from the mesenchyme or epithelium.
Mesenchymal tumors are
predominantly originating from the
pleura, retroperitoneum, abdomen and
pelvic area, slow growing and well
differentiated, sometimes weighing
multiple kilograms before being
diagnosed. A vast amount of non beta
cell tumors develop in the trunk, with
only a handful of reports in tumors
originating from haematopoietic or
neuroendocrine tissue. Both benign
(33%) and malignant tumors (67%) are
associated with NICTH. (de Groot et al.,
2007; Bodnar, et al, 2013). The most
frequently observed big-IGF-II
producing neoplasms are the following:
14
 Mesenchymal origin
I. Solitary fibrous tumor
II. Mesothelioma
III. Hemangiopericytoma
IV. Gastrointestinal stromal tumor
Epithelial origin
I. Hepatocellular carcinoma
II. Adenocarcinoma
As described previously, the IGFs are
mitogens transported in the circulation
mainly bound to ternary complexes
with ALS and either IGFBP-3 or IGFBP-
5. This expands the half-life of the
growth factors by sequestration,
making them unable to bind insulin or
IGF receptors .
Some tumors overexpress the IGF2 gene
resulting in exorbitant amounts of the
precursor of IGF-II, also called big-
IGF-II. The tumor cells are not able to
process the large quantity of this newly
produced IGF-II and a part leaks into
the circulation without the necessary
post-translational steps (Davda &
Seddon, 2007). These steps are
described in the chapter IGF2 gene
and IGF-II.
Big- IGF-II is hindered from forming
ternary complexes with IGFBP-3 and
ALS and is mainly transported in binary
complexes with one of the six IGFBPs or
circulating freely. In healthy people
around 80% of IGF-II is transported in
ternary complexes, the remainder
almost exclusively in binary complexes.
In NICTH the opposite is seen: 80% of
total IGF is bound to binary complexes,
the remaining amount in ternary
complexes or circulating in its free
form. The abundance of IGF-II
signaling seen in NICTH results, via
negative feedback, in diminished GH
and insulin secretion respectively by the
anterior pituitary gland and pancreatic
-cells (de Groot et al., 2007). This
causes problems in multiple ways.
Suppressing GH makes it even harder
to deal with hypoglycemia. GH action is
even more needed to defend against
15
 Figure 4. The actions of increased IGF-2 signaling on various tissues
A tumor synthesizes and secretes an abundance of IGF-2 or big-IGF-2 into the circulation. The
diverse effects as depicted in the figure are caused by signaling through IGF-1R and IR and affect
many different target tissues. The great amount of IGF-2 decreases glyconeogenesis as well as
ketogenesis in the liver and lypolysis along with free fatty acids secretion by adipose tissue. Glucose
consumption by insulin sensitive tissue like the muscle is increased. On top of this, many hormones
like GH, insulin and glucagon are downregulated. Lastly, the synthesis of IGF binding components
in the liver is decreased, primarily due to low GH. All these effects combined deprive the bodies
essential energy supply. Tumor IGF-II production is a vicious cycle resulting in more bio-active
systemic IGF, more IGF signaling and lower energy resources (Dynkevich et al. 2013).

low blood sugar but is rendered merely
useless due to negative feedback.
Furthermore, decreased GH signaling
suppresses the level of circulating IGF-I,
ALS and indirectly IGFBP-3. The result
of the decline in the latter two proteins
is a reduced formation of ternary
complexes, instead more big- IGF-II
and IGF-II are bound as binary
complexes or IGFs are circulating in
their free form. This means the IGFs
can readily leave the blood, into the
tissue and activate IGF and insulin
receptors throughout the body,
especially in the liver and muscle. The
end result is the uptake of glucose by
insulin sensitive tissue, mainly the
muscle and reduced hepatic glucose
production creating hypoglycemia.
Moreover, IGF-II signaling inhibits
ketogenesis in the liver making it even
harder for the body to cope with an
hypoglycemic event. In diagnostic
terms this means serum -
hydroxybutyrate is very low. On top of
that, lipolysis is shut down partially due
to IGF by means of less free fatty acid
release by adipocytes.
In short: a patient with NICTH
experiences a vicious cycle in which

16
big-IGF-II production lowers ternary
complex formation, abolishes GH, in
turn leading to an even greater fraction
of bioactive IGF-II, all the while
suppressing, hepatic glucose
production, glyconeogenesis,
ketogenesis and lipolysis, these actions
of IGF-2 are depicted in figure 4.
Hypoglycemia is the consequence
whenever counter regulatory
mechanisms are unable to make up for
the increased insulin-like activity.
Symptoms
The symptoms of a patient with NICTH
can be categorized in hypoglycemia and
other signs that are sometimes
present in patients owing to the insulin-
like and mitogenic actions of big-IGF-
II .
Hypoglycemia
- NICTH is typically a fasting
hypoglycemia presenting when a
patient does not eat for several
hours. Symptoms arise in between
meals and in the morning after a nights
sleep. Patients can experience
neuroglycopenic symptoms (described
in chapter Hypoglycemia), like
confusion and inactivity, before
becoming comatose (de Groot et al.,
2007).
Other signs of NICTH
- Hypokalemia
- Acromegaloid skin changes
- Goitre
- Elevated IGFBP-2 and BP-6
(Livingstone, 2013).
Diagnosis
The initial diagnosis in an
hypoglycemic patient is established
using Whipples triad, see chapter
hypoglycemia. At first the physician
needs to exclude any hypoglycemic
medicine, critical illness, organ failure,
but also hormone deficiencies and
lastly, any form of endogenous
hyperinsulinism that would otherwise
clarify the patients illness, as shown in
figure 3. NICTH should be considered
in a patient in which the cause of
hypoglycemia is unknown and
symptoms are compatible with the
syndrome (Bodnar et al., 2013).
NICTH is characterized by suppressed
insulin, C-peptide and GH combined
with low serum IGF-I, IGFBP-3 and -
hydroxybutyrate. big- IGF-II levels are
high, although the amount of IGF-II
and its high molecular weight form
(total IGF-II) is hardly affected. The
ratio of total IGF-II/IGF-I can increase
to more than tenfold (de Groot et al.,
2007). In 2008, Fukuda et al., showed
that total IGF-II is increased in 42% of
patients with big-IGF-II producing
NICTH whereas in all 31 patients IGF-I
levels are markedly depressed. His data
demonstrates that apart from the
presence of big-IGF-II, low serum IGF-
I and an enhanced total IGF-II/IGF-I
ratio are great tools for diagnosis.
Once the diagnostic work-up points to
NICTH, the next step is to identify the
source. A scan of the trunk can help to
determine the presence of a tumor,
because the pelvis, abdomen or thorax
is the primary location for a non beta
cell tumor to reside (Bodnar et al.,
2013).
Treatment
The ideal therapy following NICTH
diagnosis is to completely remove the
tumor producing the IGF-II abundance.
Complete removal of the tumor can
restore the balance and puts an end to
the hypoglycemic attacks. Nonetheless,
17
a surgical procedure or chemotherapy Genetic analyses of tumor biopts rarely
is not always feasible, for example in an show a mutation in any of the IGFBPs.
end stage cancer patient with multiple The exact role of these binding proteins
metastases. The aim then should be to in cancer formation is therefore harder
use additional medical therapies to to determine, in comparison with genes
assuage the hypoglycemic events. A that are in fact regularly mutated.
short-term remedy is the acute Nonetheless, epigenetic mechanisms
injection of glucose/dextrose or like hypermethylation in promoter
glucagon. Long-term treatment include regions are able to prevent the
the administration of glucocortico- expression of IGFBP genes and have
steroids, high doses of recombinant been found in several human cancers.
human GH, somatostatin analogues or In addition, IGFBPs are involved in
a combination of these medicines chemo resistance. Cancer cells that
(Livingstone, 2013). signal through hyperphosphorylated
IGF1R constitutively activate PI3K and
6. IGF-independent
effects of IGFBPs in
cancer
The IGFBPs are an old family of
proteins that are shared by all
vertebrates and appeared way
before their emergence. Lancelets
and todays vertebrates share a
common ancestor more than 500
million years ago. The lancelet
IGFBP possesses all important
domains found in all vertebrate
IGFBPs. This IGFBP is localized in
the nucleus as a transcription
factor, but has no affinity for
vertebrate IGFs. A striking
discovery is that a single mutation
in the lancelet IGFBP gene, Figure 5. Multiple actions and interactions decide the affinity
changing a phenylalanine into a of IGFBP-1 for IGF-1
leucine, is able to make the (i) IGFBP-1 interacts with transmembrane 51-integrin
lancelet IGFBP a useful receptors for carrying out its IGF-independent effects.
vertebrate IGF binding protein. (ii) IGFBP-1 in serum can be phosphorylated at different
positions. This phosphorylated isoform has a much
This implies IGF-independent higher affinity for IGF-1. Phosphorylation and
actions of the vertebrate dephosphorylation are mechanisms by which the IGF-I
IGFBPs are very old whereas its affinity in the circulation is regulated although this so
function as modulator of IGFs far unknown.
originated later in evolution (iii) IGFBP-1 can polymerize hereby decreasing its binding
affinity for IGF-I
(Zhou, Xiang, Zhang, & Duan, (iv) The binding protein can also interact with 2-
2013). macroglobulin even though it can no longer bind IGFs
(Wheatcroft, & Kearney, 2009).

18
IRS-1 and express only little IGFBP-3 Catecholamine, glucagon and corstisol
and -4, addition of recombinant IGFBP- promote IGFBP-1 synthesis, whereas
3 was able to halt tumor growth (Guix insulin halts it.
et al., 2008).
IGFBP-1 without binding an IGF is able
The IGF-dependent actions of the to activate a transmembrane receptor.
binding proteins are predominantly This interaction is mediated by an
tumor suppressive, by sequestering the Arginine-Glycine-Aspartic acid (RGD)
two insulin-like growth factors. As sequence located in the IGFBP-1 C-
mentioned in the chapter on the terminal domain by linking them with
binding proteins of the IGFs, the integrins on the outer cell membrane.
IGFBPs have a variety of functions next Hereby inducing migration through
to being a transporter in the circulation. 51 integrin signaling.
The 51 integrin pathway is also
IGFBP-1 involved in insulin resistance, but
whether IGFBP-1 is a factor
IGFBP-1 is synthesized by the liver and contributing has to be further
brought In the circulation with many investigated. (Wheatcroft & Kearney,
phosphate groups attached to the 2009).
binding protein. This phosphorylated
configuration compared to its
dephosphorylated counterpart has a
stronger binding
affinity for IGF-I
(Brandt, Grnler,
Brismar, & Wang,
2014), more than
sixfold. Multiple ways
have evolved to fine-
tune the IGFBP-1/IGF
dependent binding.
Processes like
dephosphorylation,
multimarization and
2-macroglobulin
(2M) association of
IGFBP-1 can decrease
the affinity of the
binding protein for Figure 6. The linear structure of IGFBP-2, before and after
IGF, enabling it to proteolysis
carry out its growth Like all IGFBPs, the protein contains three domains. The N-terminal
promoting effects, as domain is rich in cysteines. The linker domain and binds IGFs with high
affinity by forming a pocket with the IGF binding domain located in the
depicted in figure 5. C-terminal domain. As shown in this figure, cleaving a specific motif
IGFBP-1 expression within the linker domain creates two protein fragments with reduced IGF
is mainly adjusted by binding capacity. IGFBP-2 has two heparin binding domains for
the nutritional association with integrins located in the extracellular matrix (ECM) of
status of the body. various cells, creating a reservoir of matricine bound binary complexes
(Russo et al., 2014)

19

IGFBP-2
IGFBP-2 is apart from IGFBP-3 the most
common binding protein for the growth
factors in the circulation and its actions
are mainly to counter excessive IGF
signaling. The binding proteins has a
higher binding affinity for IGF-II, albeit
only twofold (Firth & Baxter, 2002). The
mature protein is 31 kDa and contains
multiple binding domains. IGFB-2
corresponds to IGFBP-1 in that they
both share cysteine rich IGF binding
domains and a RGD sequence in their
C-terminal for associating with various
integrins. The structure of IGFBP-2 is
pictured in figure 6. Furthermore, they
both form complexes with 2M to
prevent degradation, even though it
cannot bind IGF in this way, increasing
IGF bio-availability. IGFBP-2 with
bound IGF can associate with proteins
of the ECM via its heparin binding
motif. This pool of bound IGF is
believed to be a mitogenic reserve in
the extracellular space and can be
abused by cancer cells. By this
mechanism complexes of binding
protein with IGF in the pericellular
space are dissociate by proteolysis as
the remaining fragments of IGFBP-2
have no or reduced affinity for the
peptide hormones. Aggressive tumors
secrete IGFBP proteases to stimulate
their growth and become more
resistant to cell death.
IGFBP-2 also has IGF-independent
effects, besides binding IGFs and
interacting with extracellular proteins
in the pericellular space. It is also
transported actively into the nucleus
and acts as an activator of transcription
(Russo et al., 2014). Azar & Azar et al.
demonstrated IGBP-2 can stimulate
angiogenesis in neuroblastoma cells by
promoting vascular endothelial growth
factor (VEGF) gene expression. This in
turn stimulated the formation of new
small blood vessels. In a follow-up
study it appeared IGFBP-2 is
transported into the nuclear space by
forming a complex with importin-,
necessary steps to induce its
angiogenetic effects via VEGF in cancer
cells. (Azar et al., 2013).
IGFBP-3
The most abundant binding protein in
the circulation for the IGFs is IGFBP-3.
One of the two proteins that forms
ternary complexes with ALS and IGF.
IGFBP-3, independent of IGF can be a
pro-apototic factor and is able to
decrease growth stimuli in healthy and
cancer cells that have been affected by
various stressful situations. However,
in some instances IGFBP-3 induces cell
growth and acts anti-apoptotic. This
striking feature might be explained by
the many interactions this binding
protein has with multiple regulators,
receptors and different cell types in
various cellular
circumstances. IGFBP-3 turned out to
be a very versatile protein with many
functions, of which cell biologists are
starting to unravel its role inside and
outside the cell (Johnson & Firth, 2014).
IGFBP-3 interacts with many
transmembrane and several nuclear
receptors which act predominantly
growth inhibiting. In the
endoplasmatic reticulum the binding
protein can make the heat shock
protein GRP78 lose its affinity for
caspase-7, an initiator of cell death
(Johnson & Firth, 2014).
Ingermann, Yang, Han, et al.,
discovered a cell membrane receptor
that selectively recognizes IGFBP-3. The
authors used in vitro breast cancer cells
and demonstrated that the IGFBP-3
20
receptor upon ligand binding is able to
activate caspase-8 leading to apoptosis
in these malignant cells. Another way
for IGFBP-3 for mediating apoptosis
this time in prostate cancer cells is by
binding retinoid X receptor- (RXR-),
a protein located in the nucleus. Cells
in which (RXR) was knocked out were
cell survival in environments deprived
of glucose and oxygen by means of
mediating autophagic effects. On top of
this IGFBP-3 in the nucleus was found
to interact with EGFR and DNA-PK,
factors for non-homologous end-joining
repair mechanisms upon stress like
radiation (Baxter, 2013). This double

Figure 7. IGFBP-3 has both oncogenic and tumor suppressive effects

The versatile binding protein is intracellularly primarily located in the endoplasmtaic reticulum (ER)
and can be transported out of the cell. In the extracellular space it can interact with many receptors,
like the sphingosine rheostat, cermides, fibronectins, integrins, TRV and IGF-1R (A) In many ways
IGFBP-3 has been found to be cell preservative. The binding protein is involved in autophagy
processes by association with GRP78 in the ER. IGFBP-3 is also located in the nucleus and interacts
with DNA-PK and EGFR. In diverse stressful cellular circumstances these proteins are important for
non-homologous end-joining repair mechanisms. Proteolysis of IGFBP-3/IGF complexes liberates bio-
active IGF and stimulates growth via IGF-1R signaling (B) on the other hand IGFBP-3 associates with
various caspases to halt cellular growth and promote apoptosis. Examples are IGFBP-3 receptor
activation resulting in caspase-8-induced apoptosis. The binding protein can make the heat shock
protein GRP78 lessen its affinity for caspase-7 in the ER with the same result. Apoptosis is also
archieved by interacting with retinoid X receptor- (RXR) in the nucleus. Finally, IGFBP-3 is growth
limiting by aiding the translocation of RXR and Nur77 from the nucleus to mitochondria (Johnson &
Firth, 2014).

unable to become apoptotic by IGFBP-3 face of IGFBP-3 is depicted in the figure

effects (Liu et al., 2000). above.
The binding protein is involved in
autophagy by interacting with the heat
shock protein GRP78. In solid tumors
IGFBP3 has shown to be important for

21
IGFBP-4
IGFBP-4, like many other IGFBPs is
synthesized by liver tissue and excreted
in the blood, although many tumors
also express this binding protein.
Bound IGF is biologically inactive, but
pregnancy-associated plasma protein-A
(PAPP-A), an IGFBP-4 protease is able
to cleave BP-4 resulting in fragments of
binding protein and free bioactive IGF.
Production of IGFBP-4 and its proteases
creates a local reservoir for IGFs that
stimulate tumor growth. Blocking
either IGFB-4 or PAPP-A has proven to
halt tumor progression. Therapy with
recombinant IGFBP-4 resistant to
proteolysis may be a new way to treat
people with breast cancer (Ryan et al.,
2009). Fibroblasts are the main source
of PAPP-A synthesis. This protease
preferentially cleaves IGFBP-4 bound to
IGF-II. The structural cleavage domain
of the binding protein is this way more
susceptible to proteolysis.
Under normal physiological
circumstances IGFBP-4 binds the excess
of IGF-II in the circulatory system or
the paracellular space. But special
events like cancer or pregnancy can
increase the demand for IGF signaling.
Exorbitant amounts of IGF-II stimulate
the breakdown of IGFBP-4 via PAPA-A
and further increase IGF-II bio-
availability creating a positive feedback
loop (Qin et al., 2000).
IGFBP-4 has an important function in
preventing cancer formation. It limits
cell growth, proliferation and stimulates
apoptosis. The binding protein seems to
play important role in counteracting
angiogenesis in neoplasms (Contois et
al, 2012).
Hermani et al. investigated the IGF-
independent effects of IGFBP-4 and
IGFBP-5 in MC7 breast cancer cells and
found they limited estradiol-induced
cell proliferation. This effect was
unaltered in cancer cells that did not
express IGF-IR.
IGFBP-5
The fifth member of the IGFBP family is
IGFBP-5, a 252 amino acid protein with
a molecular mass of 28.5 kDa. Together
with IGFBP-3 the only protein to form
ternary complexes. It contains a motif
for binding heparin in its C-terminal
domain and a smaller variant of this
binding motif in its central domain,
needed for interacting with ECM
proteins, especially glycosaminoglycans.
IGFBP-5 in the extracellular space
functions chiefly to modulate IGF bio-
availability in many connective tissues
like stroma, bone and cartilage. As in
many cancers the actions of IGFBP on
tumorigenesis are sometimes hard to
determine. A good example is IGFBP-5
and breast cancer. IGFBP-5 actions
depend on many factors: type of cell
line, presence of hormones, proteases,
ligands, transcriptional regulators and
posttranslational modifiers but also its
location inside the cell. This makes it
very hard to compare experimental
results and to elucidate its true
oncogenic and tumor supressive effects
(Beattie, Allan, Lochrie & Flint, 2006;
Akkiprik et al., 2008).
However, an in vivo study indicates
IGFBP-5 may be an excellent target for
anti-cancer treatment due to its strong
reduction of angiogenesis on tumor
vasculature (Rho et al., 2008).
22
IGFBP-6

The sixth and final member

of the IGFBP family is
IGFBP-6. The binding
protein is unique in that it
has a 50-times higher
binding affinity for IGF-II
compared to IGF-I (Bach,
2005). In contrast to all
others that preferentially
bind IGF-I or both IGFs with
almost the same affinity.
The mature protein contains
in between 213-216 amino
acids and is O-glycosolated
in its L-domain. This IGF
binding protein is present
throughout the body, not
only in serum for binding
IGFs, but significantly more
in tissues like: smooth muscle Figure 8. A suggested mechanism for IGFBP-6 to stimulate
cells, ganglion cells and migration in rhabdomyosarcoma cells
retinal cells (Bach, 2014). Like IGFBP-6 associates with a yet uncharacterized transmembrane
all other binding proteins of receptor (1). Also, it connects with transmembrane prohibitin-2
(PHB2) aiding in its phosphorylation processes. These effects
IGF IGFBP-6 has three result in migration through IGFBP-6 with a paramount role for
domains: PHB2. The binding protein is a modulator of MAPK-induced
N-domain for binding migration (3) or stimulates this process directly without the
IGFs, supported by the C- downstream MAPK pathway (4) (Fu et al,2013).
domain for high affinity
binding affinity for IGFs were able to stimulate
L-domain for posttranslational migration. In a follow-up study Fu et al.
modifications, stabilizing the used Rh30 RMS cells that just like the
protein RD cells employed by Fu, Thompson &
C-domain for binding IGFs and IGF- Bach express IGF-II. They propose a
independent effects, like mechanism in which IGFBP-6 binds a
interactions with various extra- and receptor resulting in migration through
intracellular proteins (Bach, Fu, & chemotaxis. The results from both
Yang, 2013). studies indicate IGFBP-6 can induce
cancer cell migration and promote
Fu, Thompson & Bach investigated the tumor malignancy through at least 2
IGF-independent effects in a RD different pathways in RMS cell lines.
rhabdomyosarcoma (RMS) cell line and
concluded IGFBP-6 is involved in The downstream pathway of IGFB-6
migration of these cells through p38 binding to one or more transmembrane
MAPK. The C-domain of both the intact proteins leading to migration remain to
protein and a mutant who has lost its elucidated. It appears the C-domain of

23
IGFBP-6 binds prohibitin-2 (PHB2) and
is essential for IGFBP-6-inducef RMS
cell migration, although binding of the
two is independent for migration via
MAPK signaling. In figure 8 is this
pictured schematically (Fu, Yang, &
Bach, 2013).
These combined observations of IGFBP-
6 stimulating tumor malignancy are in
deep contrast with earlier findings. In
this particular study it was concluded
that IGFBP-6 limited cell growth and
stimulated cell death in both RD and
Rh30 RMS cells, both in vitro and in
vivo, although these effects are mainly
mediated by IGFBP-6 sequestrating
IGF-II and hereby inhibiting growth
factor stimulation (Gallicchio et al.,
2001).
IGFB-6 can have IGF-independent
tumor suppressive effects. It halts
angiogenesis in vivo and in vitro. Both
the natural IGFBP-6 and a mutated
form that binds IGF-II with 10.000 fold
less affinity, also used by Fu, Thompson
& Bach, was able to decrease
angiogenesis induced by VEGF in
vascular endothelium (Zhang et al.,
2012).
IGFBPs and NICTH
IGF-II is present in the circulation in
many different forms differing in how
they are processed post-
transcriptionally by PC4. Pro-IGF-II
includes an E-domain, whereas IGF-
IIE[1-88] retains only 21 of the 89 amino
acids and mature IGF-II lacks it
altogether, see IGF2 gene and IGF-II.
IGF-II is overexpressed in some extra-
pancreatic tumors resulting in a rise of
high molecular forms of IGF-II,
especially pro-IGF-IIE[1-88]. This pro-
IGF bio-active stimulates tumor growth,
angiogenesis and metastasis
predominantly via IGF1R signalling. On
top of this, they cause insulin-like
effects by activating insulin receptors.
NICTH develops in patients whenever
hormonal and other physiogical
countermechanisms fail, as described in
the chapter Non-islet-cell tumor-
induced hypoglycemia.
The physiologic response to an increase
in IGF signaling is normally resolved by
negative feedback. The tumor however
is probably insensitive to these input
signals and will keep on expressing the
IGF2 gene. Although the result of this
overexpression is a lot of incomplete
processed peptide, it stimulates tumor
growth in both an autocrine and
paracrine way. This is beneficial for the
tumor and its tendency therefore will
be to keep overproducing IGF-II.
In NICTH the GH-IGF-I axis is
markedly downregulated by negative
feedback mechanisms of the IGFs on
the anterior pituitary.
24
An important feature
is the way in which
the IGF binding
proteins are not
resolving the
abundance of big-
IGF-II by sequestering
them, but are actually
exacerbating the
insulin-like effects.
The concentration of
IGFBP-3 is depressed
whereas all other
IGFBPs are
upregulated or within
the normal range.
However, this is not
enough to stop the
overwhelming
insulin-like actions
and maintain healthy
glucose homeostasis.
IGFBP-5 could form
ternary complexes
and help to resolve
the excess. Bond,
Meka & Baxter state
that the glycosolated
pro-protein is not
sterically hindering
ALS to form ternary
Figure 9. A possible model explaining the excess of IGF-II signaling
complexes together
leading to hypoglycemia in NICTH
with IGFBP-5 as is the
Non beta cell tumors excrete high amounts of big-IGF-II in the circulation.
case with IGFBP-3.
Ternary complex formation of the high molecular weight form of IGF-II with
This is in sharp
ALS and IGFBP-3 or -5 is hampered. Instead, more binary complexes (IGFs
contrast with recent
with any of the six IGFBPs) and free fractions of IGFs circulate in the blood.
findings. It appears These IGFs can pass the endothelium and induce their insulin-like and growth
ternary complex promoting effects in the body resulting in hypoglycemia (via IRs) and tumor
formation with growth (via IRs and IGF1R). Moreover, pituitary GH production, lipolysis and
IGFBP-5, big-IGF-II hepatic gluconeogensis is inhibited and glucose uptake by the muscle and
and ALS is also adipose tissue is increased, limiting the bodys ability to resolve the
weakened (Greenall et hypoglycemic attack. Low GH and high big-IGF-II cause a shift in circulatory
al., 2012). IGFBP composition. ALS, IGFBP-3 and BP-5 are downregulated further limiting
On top of this, IGFBP- ternary complex formation and exacerbating the hypoglycemic episodes (De
5, like ALS and Groot et al, 2007).
indirectly IGFBP-3 are
GH-dependent (Ono

25
et al., 1996). These combined
mechanisms explain why IGFBP-5
cannot restore ternary complex
formation in NICTH as a replacement
of IGFBP-3.
The formation of binary complexes of
pro-IGF-II with any of the IGFBPs is not
impaired (Bond, Meka & Baxter, 2000).
This means in time as the tumor is
progressing, more and more binary
complexes arise instead of ternary
complexes. the IGFBPs are capable of
transporting the IGFs from the
circulation to target tissues including
the tumor. The half life of IGF-II in
binary complexes is shorter than IGF
bound to ternary complexes. IGF-II will
more readily bind to one of its receptors
in target tissues, including the IGF-IIR.
However big-IGF-II has a lower affinity
for this receptor than does IGF-II
(Greenall et al., 2012). The mechanism
by which IGF-IIR might be a factor
contributing to NICTH could be an
interesting topic for research.
Not only the tendency to form binary
complexes is increased, also the levels
of free circulating IGFs rise as shown in
figure 9. Frystyk et al. reported multiple
NICTH patients with high unbound
fractions of IGF-I, IGF-II and pro-IGF-II,
although the free fraction IGF-II was
the most discernible. These results can
be explained by the large amount of
pro-IGF-II displacing mature IGF-II and
IGF-I from any of the binding proteins.
Big-IGF-II binds all IGFBPs with
almost the same affinity as does IGF-II
(Bond, Meka & Baxter, 2000), shifting
the equilibrium and increasing the ratio
of unbound fraction/IGFBP bound
fraction. In patients with progressive
disease the GH/IGF-I axis is
downregulated. The total amount of
circulating IGF-I will therefore be
decreased. However, when enough
big-IGF-II is brought into the
circulation even the affinity of IGFBPs
for IGF-I can decrease, clarifying the
results found by Frystyk et al. IGF-I has
a very potent effect as inhibitor of GH
release on the level of both the
hypothalamus and the pituitary
(Romero et al., 2012). A high free
fraction can worsen disease as it further
limits GH production. This model of
disease is shown in figure 9.
On top of this, not all IGFBPs, apart
from IGFBP-3 are equally upregulated
to bind the excess IGFs. Especially
IGFBP-2 and sometimes IGFBP-6
concentrations are elevated (Bond,
Meka & Baxter, 2000). Occasionally,
case reports mention increased plasma
IGFBP-6 in patients diagnosed with
NICTH (Baxter et al., 1995; Hoekman et
al., 1999; Nanayakkara et al., 2002), but
in some instances the levels are
reported to be normal (Singh et al.,
2006; Escobar et al, 2007; van Veggel et
al, 2014). The same phenomena is
observed when blood samples of patient
groups are scanned for IGFBP-6 levels .
Van doorn, Ringeling et al. analysed
plasma samples and used
radioimmunoassay to determine
IGFBP-6 concentrations in healthy
controls and patients with various
diseases. Among these patients were
six people diagnosed with NICTH and
four of them had high IGFBP-6 plasma
concentrations. The authors suggest
IGFBP-6 can only rise in very special
pathologies like NICTH to act as a
modulator of the excess IGF-II variants.
26
Rikhof et al measured IGF-I, mature
IGF-II, IGF-IIE[6888], IGFBP-2, and
IGFBP-3 in 24 patients diagnosed with a
gastrointestinal stromal tumor (GIST)
that could not be surgically resolved.
These patients were prescribed
a possible drug resistant role of IGFBP-
2. The authors compared levels of the
binding protein in both cyst fluid and
normal tissue and considered that not
only tumor but also other tissues could
be contributors to high IGFBP-2 levels

Figure 10. The enzymatic activity of metalloproteinase7 on the extracellular matrix

bound IGFBP-2-IGF-II binary complexes and the resulting IGF-II signaling in human
colon cancer cells.
Serum starved human colon cancer (HT29) cells secrete MMP-7 (a metalloproteinase), IGFBP-2,
heparan sulfate proteoglycan (HSPG) and express IGF-1R on their cell membrane. HSPG can
combine with the heparin binding domain (HBD)of IGFBP-2 and retain BP-2/IGF-II binary
complexes in the extracellular space. MMP-7 is able to cleave IGFBP-2 in the extracellular
matrix (ECM), increasing local IGF-2 bio-availability. This free IGF-2 can now stimulate growth
and survival in these cancer cells via phosphorylating and hereby activating IGF1R (Miyamoto et
al, 2007).

imatinib, a tyrosine-kinase inhibitor, in serum. Three out of these 24 patients

used as an anticancer drug. Serum
samples were obtained before and
during therapy. In most patients IGFBP-
2 concentrations were high. Even
though serum concentrations of this
binding protein were not correlated
with tumor growth or degree of
recovery, patients not responding to
imatinib had increased levels of IGFBP-
2 in plasma compared to treatment
responders. These results would suggest
experienced or developed severe
hypoglycemic attacks during or after
therapy. One of three patients
presented with a significantly higher
IGFBP-6 plasma concentration.
Obviously the circulatory IGFBP
composition is shifting in the
pathogenesis of NICTH. Big-IGF-II is
preferentially bound to IGFBP-3
compared to IGFBP-2 in healthy

27
controls with a normal ratio of the IGF-
II variants. In NICTH, the opposite is
observed. Increased levels of the high
molecular weight protein preferentially
associate with IGFBP-2 rather than
IGFBP-3. This would imply the total
amount of IGF-II forming ternary
complexes in progressive disease is
further reduced as big-IGF-II
concentrations are going up, many
times at the expense of mature IGF-II.
The high molecular weight form is ten
times more associated with binary
complexes compared with healthy
individuals (Qiu et al., 2010).
Binary complexes of (Big-)IGF-
II/IGFBP-2 can interact with proteins of
the ECM through the heparin binding
motif of IGFBP-2. This pool of bound
IGF is believed to be a mitogenic
reserve in the extracellular space and
can be abused by cancer cells.
Miyamoto et al studied the interactions
of metalloproteinase7 on ECM bound
binary complexes and the resulting IGF-
II signaling in human colon cancer
cells. Metalloproteinase7 or MMP-7 is
an IGFBP-2 degrading agent solely
secreted by cancer cells. The enzyme
breaks down the binding protein and
liberates bio-active IGF-II in the local
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