A non beta cell tumor located in the abdomen of a
patient with hypoglycaemia secreting high levels of
big insulin-like growth factor (IGF)-II and IGF-
binding protein-6.
Wouter van de Hoef
Intern
Division Biomedical genetics
Biomedical sciences
Bachelor thesis
(received 19 January 2015)
Summary
Non-islet-cell tumor hypoglycaemia
(NICTH) is the result of a non beta
cell tumor overproducing an
intermediate form of IGF-II and
secreting it in the circulation. This
high molecular weight form or big-
IGF-II does not readily form ternary
complexes with acid labile subunit
(ALS) and IGFBP-3. Instead IGFs are
predominantly bound to binary
complexes with any of the six IGF
binding proteins enhancing.
Moreover big-IGF-II displaces IGFs
from complexing with any of the
IGFBPs, raising the free fraction of
IGF in blood.
In this report we analyze pre-
postoperative plasma of a 60-year
old man diagnosed with
hypoglycaemia. He had a solitary
fibrous tumor removed that
produced high amounts of big-IGF-
II. Determination of IGF
components in preoperative serum
showed next to low ALS, IGFBP-3
and IGF-1, high concentrations of
big-IGF-II, IGFBP-2 and IGFBP6,
whereas total IGF-II was normal.
These values greatly normalized
after surgery. We also measured and
calculated the gradient from tumor
to plasma before surgery to identify
whether IGF components are
atypical due to the neoplasm or
merely because of systemic effects.
The positive gradients of big-IGF-II
and IGFBP-6 from tumor to plasma
suggest the neoplasm synthesizes
high levels of both these factors,
contrary to IGFBP-2 which is
probably upregulated by other
tissues. A specific
radioimmunoassay (RIA) for
comparing IGFBP-2, -3 and -6 in
control skin fibroblasts and tumor
fluid supports this statement.
IGFBP-6 was enriched in tumor
tissue whereas IGFBP-2 was slightly
reduced. These combined
observations highlight the role of
IGFBP-6 in tumor hypoglycaemia.
INTRODUCTION
Glucose is of paramount importance as
a fuel for the human body. This is why
the body has many counter regulatory
mechanisms to maintain an adequate
supply of blood sugar as some tissues
like the brain, erythrocytes and the
renal medulla depend almost
completely on glycolysis to function
properly under physiologic
circumstances (Cryer et al, 2009).
Low blood sugar or hypoglycemia arises
whenever counter regulatory systems
are unable to restore the fall in blood
sugar (>70 mg/dL or >3.9 mM) to
physiological levels (Brutsaert, Carey &
Zonszein, 2014).
Hypoglycemia can be caused by a non
beta cell tumor. These are rare
neoplasms that produce large amounts
of aberrant processed insulin-like
growth factor-II (pro-IGF-IIE[68-88] or
big-IGF-II). In healthy individuals
circulating IGFs (IGF-I, IGF-II and IGF-
II variants) are mainly transported in
complexes with binding proteins. These
IGFBPs sequester IGFs in binary
complexes (IGF + one of the six IGFBPs)
or in ternary complexes (IGF + IGFBP-3
or -5 and an acid labile subunit) to
prevent excessive cell survival signals
and insulin-like actions (de Groot et al.,
2007).
In NICTH the IGF physiology is
MATERIALS AND METHODS
Tissue preparations
An aliquot of tumor tissue (0.20 grams
wet weight) was derived from a patient
who had a large a solitary fibrous
retroperitoneal mass, without
unfavorable features of necrosis or high
mitotic activity. After surgery, the
imbalanced. Ternary complex
formation is hampered and instead
IGFs are more frequently transported in
binary formations. Big-IGF-II also
displaces IGFs from complexing with
any of the IGFBPs, halting more free
IGF in blood (Frystyk et al., 1998). Both
these phenomena increase IGF tissue
bio-availability . The high production of
big-IGF-II by a non beta cell tumor
causes enhanced insulin receptor
signaling, resulting in non-islet cell
tumor hypoglycemia (NICTH).
GH helps to restore the blood sugar
balance during hypoglycemia and
stimulates ternary complex formation.
Yet, an increase in IGF signaling
downregulates pituitary GH secretion
and further acerbates the hypoglycemic
episodes in patients (de Groot et al.,
2007).
A year ago, Alkemade et al., reported a
60-year old man with a solitary fibrous
tumor located in the abdomen and
retroperitoneum ( 3,1 dm3; 1,76 kg).
The presenting symptoms, a fasting test
and analysis of plasma with high big-
IGF-II concentrations confirmed
NICTH. Surgical resurrection of the
tumor resulted in complete recovery
and healthy insulin, glucose and big-
IGF-II levels. In this report we followed-
up on this patient and quantified,
IGFBPs and IGFs in tumor tissue but
also in pre- and postoperative plasma.
tumor biopt was stored at -80 Co until
further preparations and analysis.
Four series of cultured skin fibroblasts
(i.e. originally derived from 4 different
healthy subjects), which routinely
serves as control cells for diagnostic
purposes, were obtained from the
laboratory of Metabolic Diseases,
University Medical Centre Utrecht.
Fibroblasts have been cultured in
nutrient mixture F-12 Ham, 25 mM
HEPES medium (Sigma-Aldrich St. Louis,
MO, USA) containing 10 % (v/v) foetal
calf serum, 2 mM glutamine, and 1 %
(v/v) penicillin/streptomycin (10000 IU/
10000 g/ml) in three 175 m2 falcon flasks
until confluence. The cells were
harvested, centrifuged (10 min 252x g, 4
C) and the pellet washed 3 times with
PBS. The pellet of each culture was
resuspended in a cocktail (containing 1.8
ml homogenisation buffer (0.2 M
potassium phosphate buffer pH 7.4) and
0.2 ml of a protease inhibitor (Roche
Diagnostics GmbH, Mannheim,
Germany)), and the various suspensions
of fibroblasts were pooled in a final
volume of 2 ml buffer.
Tumor tissue (0.1 g wet weight/ml) and
the pooled fibroblast suspension were
homogenized in homogenization buffer
on ice using a motor driven Potter
Elvehjem tissue grinder (Schuett
homogen plus, Schuett Biotec, Gttingen,
Germany), 5 strokes, 2000 rpm.
Homogenates were centrifuged and
pellets formed at 4oC for 5 min at 8000
rpm. Supernatants were divided into
small aliquots and stored at -80oC until
further analysis.
50 microliter of each sample was used for
whole protein analysis according to
Lowrys method (Lowry et al., 1951)
Determining IGFBP and IGF levels
in plasma and tissue.
Acid labile subunit (ALS) plasma levels
were quantitatively analyzed using an
ELISA kit (Mediagnost, Reutlingen,
Germany).
IGF-IIE[68-88], IGFBP-2, IGFBP-3, and
IGFBP-6 were determined by specific
RIAs, as described previously in detail for
measurements in plasma (van Doorn et
al., 2002; de Kort et al., 2010; Rikken et
al., 1998; van Doorn et al., 1999,
respectively). Multiple dilutions of the
samples were made in the appropriate
assay buffer for measuring homogenates
of tumor and fibroblasts. In each RIA,
the concentrations measured in serial
dilutions of homogenates of either tumor
tissue or fibroblasts paralleled the
respective standard curves (i.e. rhpro-
IGF-IIE[68-104], rbIGFBP-2, purified
hIGFBP-3, and rhIGFBP-6, respectively).
Moreover, additions of differing known
amounts of either rhpro-IGF-IIE[68-104]
or IGFBP-2, -3 or -6 to the samples
showed recoveries between 87 and 116 %
in the respective RIA measurements.
These data strongly suggest the
specificity of the various determinations
in the tissue specimen investigated. IGF-I
was measured by the use of a non-
extraction immunometric technique on
an Immulite 1000 Analyzer (Siemens
Medical Solutions Diagnostics, Los
Angeles, USA). The lower detection limit
was 12 ng/mL or 1.6 nmol/L and inter-
assay variation was <7.5% at levels
ranging from 46-420 ng/mL (6.0- 55.0
nmol/L) (n=31).
IGF-II was determined by RIA after
acidification and chromatography on
Sep-Pak C18 cartridges. In order to assess
the recoveries of endogenous
IGF-II during the whole procedure, in a
separate series of Sep-Pak C18 extractions
[125I]hIGF-II was added to each
homogenate. For the homogenates of
tumor tissue and fibroblasts the recovery
amounted 42.2 % and 66.4 %,
respectively.
Levels of ALS, IGFBPs and IGFs in
plasma were expressed both in nmol/L
and SDS (i.e. adjusting for age and
gender) (Yu et al., 1999). Concentrations
of IGFBPs and IGFs in tissue
homogenates were expressed as mean
SD, either per g of wet weight and/or mg
protein, as indicated.
RESULTS
and IGFBP-6, whereas IGF-I, ALS and
Levels of the diverse IGF system IGFBP-3 (grey are to a great extent
proteins in patient serum downregulated. All these values
resumed to a more normal state after
surgery. Concentrations of IGFBP-2 and
Measurements of several proteins
-6 do decrease after the resection, yet
involved in the IGF physiology in
these values remain very high during a
patient serum are depicted in table 1.
follow-up control more than 7 months
Preoperative plasma show particularly
later. Total IGF-II serum concentrations
high levels of IGF-IIE[68-88], IGFBP-2
seem normal, but a significant larger
part, compared with healthy controls, is
in the high molecular weight form.

Table 1. Concentration of IGFs, ALS, and IGFBPs determined using different

methods in patients pre- and postoperative serum.
The levels of various components of the IGF system in plasma before and two moments after surgery
(AS). These concentrations were measured by a non-extraction immunoassay (NEIA),
radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The standard score (SDS) is
adjusted for age and gender and are given in parenthesis.
Preoperative 1 month AS 7,3 months AS Immunometric
nM (SDS) nM (SDS) nM (SDS) method
IGF-I 2.28 (- 6.30) 13.14 (- 0.02) 14.04 ( 0.22) NEIA

total IGF-II 61.58 ( 0.57) 59.17 ( 0.34) 45.65 (- 1.10) RIA

IGF-IIE[68-88] 41.61 ( 8.97) 5.05 (- 1.11) 4.42 (- 1.63) RIA

ALS 37.17 (-3.36) 126.02 (-1.71) 133.93 (-1.54) ELISA

IGFBP-2 29.4 ( 3.49) 21.66 ( 3.05) 14.30 ( 2.46) RIA

IGFBP-3 21.61 (-4.58) 46.00 (-1.34) 40.33 (-1.87) RIA

IGFBP-6 14.79 ( 4.15) 9.42 ( 1.36) 12.24 ( 2.86) RIA

Analysis of IGFs and IGFBPs in levels of this IGF in tumor tissue was
tumor compared with plasma not even detectable (ND). IGF-IIE[68-
88] and IGFBP-6 levels inside the
before surgery
tumor are even higher than the
abundance already seen in the patients
Preoperative IGF system proteins
blood before resection. Total IGF-II and
already tend to have extreme values.
IGFBP-3 are both substantially
Analysis of tumor material show in
increased. IGFBP-2 is the only
some cases even more peculiar values,
measured binding protein to be more
as seen in table 2. IGF-I levels in
concentrated in plasma than in tumor
preoperative plasma of the patient are
fluid.
very low (see table 1), however the
Concentration of multiple IGFBPs
in tumor and control tissue
The different IGFBP RIAs have been
used in order to assess changes of
IGFBP levels in tumor tissue by
comparing them with healthy skin
fibroblast, as shown in figure 1. IGFBP-
2 concentrations are 1.6-fold decreased
in neoplasm tissue, from 0,179 0,061
pmol/mg protein (mean SD) in
control fibroblasts to 0.112 0.021
pmol/mg protein in tumor tissue.
IGFBP-3 is even more depressed to
approximately 6.5-times in tumor tissue
as opposed to controls. IGFBP-3 levels
are 6.430 1,82 pmol/mg protein (mean
SD) in fibroblasts but only reach
0.990 0.149 pmol/mg protein in the
non beta cell tumor. IGFBP-6 is the

Table 2. Concentrations of multiple IGFs and IGFBPs in tumor tissue and

comparison with preoperative plasma.
The concentrations of IGFs and IGFBPs were determined in aliquots of tumor tissue homogenate, at
different dilutions, by specific immunometric methods as described in the materials and methods. Data
represent the mean SD, for the number of determinations indicated. ND means not detectable.
number of wet weight ratio of tumor tissue (nmol/g)/
determinations tumor (nmol/g) preoperative plasma (nM)
IGF-I 2 ND ND

total IGF-II 14 220 34 3.57 0.55

IGF-IIE[68-88] 6 445 69 10.69 1.66

IGFBP-2 10 9 2 0.31 0.06

IGFBP-3 14 80 12 3.70 0.56

IGFBP-6 6 139 13 9.81 0.94

Fig. 1. The concentrations of

IGFBP-2, -3 and -6 in both non
beta cell tumor tissue (black
bars) and control tissue:
cultured skin fibroblasts
(striped bars). This figure shows
the mean values of the IGFBPs
SD. The content of the binding
proteins were determined by
specific RIAs. Lowrys method
was applied to quantify protein
only
mass in IGFBP increased in
both tissues.
tumor tissue. The
concentration difference is almost
threefold, from 0.60 0.059 pmol/mg
protein (mean SD) in control
fibroblasts to 1.720 0,165 pmol/mg
protein in tumor tissue.
DISCUSSION
NICTH tumors are mostly derived from
tissues of epithelial or mesenchymal
origin as in this case. Characteristics of
the syndrome are not only low glucose
levels, also production of free fatty acids
and ketone bodies is suppressed. This
makes it very hard for the body to keep
functioning properly. These findings
indicate excessive insulin activity but
contrarily insulin in these patients
serum is very low. Overexpressing the
IGF2 gene and secretion of the high
molecular weight form of IGF-II in the
circulation does increase IGF signalling
but does not solely account for the
hypoglycaemia(de Groot et al., 2007;
Bodnar, et al, 2013). Surely, Big-IGF-II
is greatly increased preoperatively, but
total IGF-II is often within the normal
range, also seen in this case. Therefore
other mechanisms are involved in the
hypoglycaemic attacks in patients.
A shift in IGFBP distribution is
detectable in this patient, as is the case
in all NICTH patients. IGFBP-3 (albeit
indirectly ) and ALS plasma levels are
depressed due to low GH production of
the anterior pituitary (Ono et al., 1996).
Circulatory IGFBP-2 and occasionally
IGFBP-6 are reported to be upregulated
in NICTH (Bond, Meka & Baxter, 2000;
Baxter, 2014). The result of this altered
IGFBP distribution is a reduction of
ternary complexes and as a
consequence, the formation of binary
complexes along with a larger free IGF
fraction (Frystyk et al., 1998) An excess
in bio-active IGF activates IGF-I
receptors and insulin receptors. This
causes hypoglycemia all the while
stimulating tumor growth, survival and
malignancy (Livingstone, 2013;
Riedemann & Macaulay, 2006). All
serum IGF physiology concentrations
we have assessed in patient serum are
compatible with earlier findings in
other NICTH patients.
IGFBP-2 concentration was strongly
elevated in preoperative serum,
decreases after surgery but remains
high after removal of the tumor.
Comparison of this binding protein in
tumor tissue with both preoperative
plasma and fibroblasts shows an inverse
gradient of IGFBP-2 from tumor to the
blood. This implies IGFBP-2 is not the
prime product of the tumor but is
primarily synthesized by other tissues.
The most striking feature in this case
are the high IGFBP-6 values in
preoperative serum, that are very
possibly are the result of non beta cell
tumor production. Evidence for this
claim is the positive gradient in table 2
for IGFBP-6. Even though IGFBP-6
content is already staggering, this
binding protein is even more enriched
in tumor fluid as seen in figure 1. The
same phenomena is observed for IGF-
IIE[68-88] in serum and tumor tissue.
The abundance of this pro-protein has
been demonstrated to be caused by
overexpressing the IGF2 gene by non
beta cell tumors.
So far, reports determining the
expression or presence of IGFBPs
directly in non beta cell tumor tissue
are relatively scarce, although a few
have been reported in the medical
literature (van Doorn et al., 1999;
Rikhof et al., 2009; Holt et al., 1998).
The results of these reports indicate
IGFBP-2 is not abundantly produced by
the tumor, but a mere physiological
response of the body to increased IGF-2
signaling. This mechanism however
remains uncharacterized. IGFBP-6 on
the other hand can be overproduced
abundantly by a non beta cell tumor
(van Doorn et al., 1999) and can
therefore act as a reservoir for IGF in
the paracellular space. Due to the rarity
of direct measurement of IGFBP-6 in
this type of tumors, this has to be
further investigated. Some proteases
produced by tumors can degrade IGFs
from binary complexes (including IGF-
II and IGFBP-6) and are able to increase
IGF bio-availability in close proximity of
the tumor (Miyamoto et al., 2007).
Further research could investigate the
tumor suppressive or tumor stimulating
IGF- independent effects of the IGFBPs
in NICTH. Another topic might be to
find the source of IGFBP-6. Not only
tumor but also surrounding tissues
and/or other tissues could contribute to
high IGFBP-6 concentrations in serum
of patients. Immunohistochemistry
could be used to detect this binding
protein in tissue enclosing the tumor.
Unfortunately we were not able to
perform these experiments with healthy
mesenchymal tissue from the patient
that could act as control tissue. That is
why cultured skin fibroblasts was the
best solution for comparison with
healthy tissue, as tumor and fibroblasts
are both from mesenchymal origin. The
first IGF-II determination by RIA after
acidification and chromatography on
Sep-Pak C18 cartridges was
unsuccessful. The second time
succeeded. New homogenates of
cultured skin fibroblasts showed a
slight variance in the concentration of
IGF system components but no
deviating values.
In conclusion we present a patient with
a large non beta cell tumor located in
the abdomen and retroperitoneum
producing high quantities of big-IGF-II
and IGFBP-6. The extreme values of IGF
and IGFBPs in preoperative serum
greatly normalized after surgical
resection. Future research can hopefully
elucidate the IGF-dependant and -
independent effects of this IGFBP in
NICTH.
Acknowledgments
I want to thank Jaap van Doorn for
accepting me as his laboratory assistant,
for his advice writing these articles and
for carefully reading these manuscripts.
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