[CANCER RESEARCH 64, 8093– 8100, November 1, 2004]
Insulin Receptor, Insulin Receptor Substrate-1, Raf-1, and Mek-1 during Hormonal
Hepatocarcinogenesis by Intrahepatic Pancreatic Islet Transplantation in
Diabetic Rats
Matthias Evert,1,2 Jianping Sun,2,3 Sabine Pichler,2 Nadia Slavova,2,4 Regine Schneider-Stock,1 and
Frank Dombrowski1,2
1
Institut für Pathologie der Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany; 2formerly Pathologisches Institut der Rheinischen Friedrich-Wilhelms-Universität
Bonn, Bonn, Germany; 3Surgical Research, Children’s Hospital Los Angeles, Los Angeles, California; and 4Chirurgische Klinik und Poliklinik I, Allgemeine, Gefä␤ und
Thoraxchirurgie, Campus Benjamin Franklin, Freie- und Humboldt-Universität zu Berlin, Berlin, Germany
ABSTRACT
Low-number transplantation of pancreatic islets into the livers of diabetic rats leads to transformation of the downstream liver acini into
clear-cell foci of altered hepatocytes (FAHs). These FAHs correspond to
the glycogen-storing (clear-cell) phenotype of hepatocellular preneoplasias
and develop into hepatocellular adenomas (HCAs) and hepatocellular
carcinomas (HCCs) within 6 to 24 months. In addition, they show metabolic alterations that resemble well-known insulin effects, most likely
constituting the result of the local hyperinsulinemia. Thus, we investigated
FAHs, HCAs, and HCCs for altered expression of insulin receptor, insulin
receptor substrate-1 (IRS-1), Raf-1 and Mek-1. Light and electron microscopic immunohistochemistry revealed a translocation of insulin receptor
from the plasma membrane (normal tissue) into the cytoplasm in clearcell FAHs and an increase in insulin receptor expression in HCAs and
HCCs. FAHs also showed an increase in IRS-1 gene expression, investigated by in situ hybridization and quantitative reverse transcription-PCR.
IRS-1, Raf-1, and Mek-1 proteins were strongly overexpressed in FAHs
and tumors, as compared with the unaltered liver tissue. These overexpressions were closely linked to the clear-cell phenotype of preneoplastic
and neoplastic hepatocytes, because basophilic FAHs (later stages) and
basophilic tumors showed no overexpressions. In this endocrine model of
hepatocarcinogenesis, severe alterations of insulin signaling were induced
by the pathological local action of islet hormones in the livers and may
substantially contribute to the carcinogenic process.
INTRODUCTION
Insulin is a pleiotropic hormone exerting different metabolic and
mitogenic effects on hepatocytes (1– 6). Although insulin signaling is
transducted intracellularly via an extensive signaling network with
multiple alternative pathways, insulin receptor substrate-1 (IRS-1) is
believed to be the first basic cytosolic mediator (7–10). IRS-1 is
activated via phosphorylation of tyrosine residues, mediated by the
␤-subunit of the insulin receptor, which acts as a tyrosine kinase after
extracellular binding of insulin to the ␣-subunit. Phosphorylated
IRS-1 acts as a docking protein and can associate with proteins
containing Src homology 2 (SH2) domains, such as GRB-2. GRB-2
links to IRS-1, and, thus, insulin signaling to the Ras-complex, which
is the most important downstream signaling cascade besides the
phosphoinositol-3-kinase pathway. Ras has been demonstrated to
initiate a stepwise activation of cytosolic kinases, i.e., Raf-1 and,
hence, Mek-1/MAPKK (mitogen-activated kinase kinase), mitogenReceived 6/9/04; revised 8/12/04; accepted 8/23/04.
Grant support: M. Evert was supported by a Start-up-Project of the Magdeburger
Forschungsverbund “Neurowissenschaften” and “Immunologie und Molekulare Medizin
der Entzündung”; F. Dombrowski was supported by the Deutsche Forschungsgemeinschaft (grant numbers Do 622/1-3, 1-4, and 1-5) and by the Schäfersnolte-GedächtnisStiftung (Benningsen-Förder-Preis des Landes Nordrhein-Westfalen).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Frank Dombrowski, Institut für Pathologie, Universität Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Phone: 49-391-67-17869;
Fax: 49-391-67-17952; E-mail: [email protected].
©2004 American Association for Cancer Research.
activated kinase (MAPK) and pp90 S6 kinase, thereby inducing
increased cellular proliferation and anabolic alterations in the carbohydrate and fatty acid metabolism. This pathway is believed to be
essentially involved in the hepatotrophic and hepatocellular mitogenic
effects of insulin (7, 10 –11).
Several studies have shown that insulin signal transduction proteins, in particular insulin receptor and IRS-1, may also contribute to
the development of hepatocellular carcinomas (HCCs) and other
tumors (12–24). However, it has not yet been demonstrated that this
process can be induced at the very beginning, i.e., via insulin itself.
In a model of hormonal hepatocarcinogenesis, long-term local
effects of insulin appear to induce the carcinogenic process (25–27).
After low-number islet transplantation into the livers of streptozotocin-induced diabetic rats, the animals persisted in a mild diabetic state,
and the transplanted islets showed morphologic signs of increased
insulin synthesis and secretion because of the continuous hyperglycemia. As a response to the local hyperinsulinemia, the hepatocytes
downstream of the transplanted islets show an increase in glycogen
and lipid storage, accompanied by corresponding alterations in their
carbohydrate metabolism, as well as a strong increase in proliferative
and apoptotic activity (25, 27). As a result, these foci of altered
hepatocytes (FAHs) bear morphologic similarities to preneoplastic
hepatocellular foci, known from animal models of chemical hepatocarcinogenesis (26, 28), and may develop into hepatocellular adenomas (HCAs) and HCCs within 6 to 24 months (27). We previously
investigated the insulin-like growth factor (IGF) axis in this model
and found that even a few days after transplantation, IGF-I, IGF-IIreceptor, and IGF-binding-protein (IGFBP)-4 were overexpressed,
whereas IGFBP-1 was down-regulated and IGF-II was not detectable
within the FAHs (29 –30). IGF-I-receptor expression, however, remained on the low level of the normal parenchyma. Thus, at this early
stage, not only insulin but possibly also IGF-I-signaling was more
likely transducted via the insulin receptor.
The aim of the present study was to determine whether the hepatocytes of the preneoplastic foci and tumors in this model show
alterations in their intracellular signaling that can be attributed to the
local hyperinsulinemia and that may contribute to the carcinogenic
process. Thus, we examined insulin receptor and IRS-1 for their
function as the basic mediator molecules of insulin action, as well as
the Raf-1/Mek-1/MAPKK pathway as one of the principal downstream cascades involved in insulin action.
MATERIALS AND METHODS
Islet Transplantation Experiment. Diabetes was induced in adult inbred
male Lewis rats (n ⫽ 75; 250 to 300 g) by treatment with a single subcutaneous
dose of streptozotocin (80 mg per kg body weight) and was defined by a
nonfasting blood glucose level higher than 400 mg/dL, manifesting between 1
and 3 days after the administration of streptozotocin. The transplantation
procedure was performed as described previously (25, 27). We chose a low
number of islets (250 – 450 islet grafts per animal) so that mild hyperglycemia
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INSULIN SIGNALING IN RAT HORMONAL HEPATOCARCINOGENESIS
(250 –300 mg/dL) persisted for at least 10 months after islet transplantation.
The animals were killed under anesthesia between 2 days and 24 months after
transplantation. Fourteen nondiabetic rats did not undergo transplantion and
served as a control group regarding the spontaneous development of preneoplastic hepatocellular foci, which is known to occur in the experimental group
in animals that are ⬃2 years old. They were killed simultaneously with the
animals of the experimental group.
Fasting-Refeeding Experiment. In addition, we investigated 10 nondiabetic control rats that had fasted for 24 hours. Six animals were directly killed,
and four animals received food 2 hours before death. This was done to evaluate
physiologic insulin receptor distribution at low (fasting) and high (postprandial) intrahepatic insulin concentrations, simulating the conditions in the transplantation experiment. Insulin concentration in the portal vein was measured
with Linco⬘s rat insulin radioimmunoassay kit (Linco, St. Charles, MO).
Housing and treatment of all animals conformed to the guidelines of the
Society for Laboratory Animals Service (GV-Solas) and the German animal
protection law.
Processing of Tissue. Tissue was processed as described previously (27).
Approximately 2- to 3-mm-thick sections were dehydrated and embedded in
paraffin. Serial sections were cut and stained with hematoxylin and eosin or
cresyl-violet or with periodic acid Schiff reaction (PAS). Additional sections
were analyzed immunohistochemically. FAHs were histologically classified
into clear-cell foci (CCF), mixed-cell foci (MCF), and basophilic-cell foci
(BCF) as described previously (31). CCF showed enlarged cell bodies with
extensive glycogen storage (PAS-positive). The basophilic cytoplasm of BCFs
was PAS-negative because of loss of glycogen, and the nuclear-to-cytoplasmic
ratio was increased. The basophilia is a result of an increase in the number of
ribosomes. FAHs that were composed of glycogen-rich and glycogen-poor
hepatocytes in a close spatial relationship were designated as MCF. Lesions
were classified as HCAs when they were sharply demarcated, compressed the
surrounding liver parenchyma, and showed loss of acinar architecture. HCCs
were diagnosed when the lesion exhibited trabeculae thicker than three cell
layers in at least two separate areas or showed unequivocal signs of malignancy, such as vascular invasion.
Tissue for Western blotting, quantitative reverse transcription-PCR and in
situ hybridization was obtained from the middle lobe of the liver, removed
before perfusion with the fixation cocktail; the tissues were then cut in slices,
were immediately frozen in isopentane (at ⫺120°C), and were stored at
⫺80°C. RNA, DNA, and proteins were isolated with the TRIZOL method
(Life Technologies, Inc., Gaithersburg, MD).
Immunohistochemistry. Seventy-eight CCF, 20 MCF, 3 BCF, 14 HCAs,
and 7 HCCs were morphologically classified and subsequently investigated
immunohistochemically. The ␤ chain of insulin receptor was detected with two
different primary polyclonal rabbit antibodies, A1314 (kindly provided by Dr.
J. W. Unger, Department of Anatomy, University of Munich, Munich, Germany) and SC-711 (Santa Cruz Biotechnology, Heidelberg, Germany), each at
a concentration of 0.5 ␮g/mL (secondary antibody was goat-antirabbit-IgG,
biotinylated, 1.0 ␮g/mL, Dianova, Hamburg, Germany), detected with the
CSA-Kit (Dako, Hamburg, Germany) according to the manufacturer⬘s instructions. Both antibodies resulted in a similar granular staining of the plasma
membrane. A1314 was used for semiquantitative analysis.
Polyclonal rabbit antibodies against IRS-1 (Upstate Biotechnology Inc.,
Lake Placid, New York; dilution: 1:50), Raf-1 (Santa Cruz Biotechnology;
dilution 1:50), and Mek-1 (Santa Cruz Biotechnology; dilution 1:100) were
detected with the LSAB⫹-Kit (Dako).
Microwave treatment (10 min) was used for antigen retrieval. Rabbit IgG
without preliminary immunonization served as a negative control in all cases
(Oncogen Science/Dianova, Hamburg, Germany). The results were evaluated
semiquantitatively by comparing the immunohistochemical reaction within the
lesions and the adjacent unaltered liver tissue. The specificity of all primary
antibodies was tested by Western blotting, except for A1314, which has
already been characterized successfully in a former study (32). Finally, polyclonal anti-insulin, antiglucagon and antisomatostatin antibodies (Dako; dilution 1:200, 4°C incubation for 20 hours) were used as described previously to
prove the existence of the transplanted islets or smaller remnants within the
foci or tumors (27).
Immunoelectron Microscopy of the Insulin Receptor. For electron microscopic evaluation of the ultrastructural localization of insulin receptor, the
tissue was embedded in Lowicryl K4M according to Roth et al. (33), with few
modifications. Briefly, perfusion-fixed liver tissue was cut into pieces of 0.5
mm in length and fixed for 2 hours in a solution containing 3% paraformaldehyde and 0.2% glutaraldehyde in 0.1 mol/L PBS. The tissue was then rinsed
twice for 2 minutes with 0.1 mol/L PBS and put into 50 mmol/L ammonium
chloride solution for 2 ⫻ 30 minutes. After rinsing in 0.1 mol/L PBS (2 ⫻ 5
minutes), the tissue was dehydrated in a graded series of EtOH, beginning with
30% EtOH at 4°C. The last two steps (90% and 100% EtOH) were performed
at ⫺35°C, followed by infiltration with Lowicryl in four steps: Lowicryl/100%
EtOH (1:1) at ⫺35°C overnight, Lowicryl/100% EtOH (2:1) at ⫺35°C for 4 to
6 hours, and finally twice with pure Lowicryl at ⫺35°C overnight. UV
polymerization of the embedded tissue was performed in a nitrogen atmosphere at ⫺35°C for 12 to 16 hours, followed by a 48-hour period at room
temperature.
IR was immunostained with the primary antibody A1314, which was also
used for light microscopy. Staining was performed with a two-step method
with colloidal gold, bound to the secondary antibody (12 nm-gold-donkeyantirabbit-IgG, dilution 1:50; Dianova, Hamburg, Germany), or by interposition of an amplifying step with a streptavidin-biotin system (34 –37). Goatantirabbit-IgG (1 ␮g/mL) and 12 nm-gold-donkey-antigoat-IgG (dilution 1:30,
Dianova) served as secondary and tertiary antibodies, respectively. Sections
were subsequently stained with uranylacetate (10 seconds) and examined on a
Phillips CM 10 electron microscope (Eindhoven, the Netherlands).
In situ Hybridization of IRS-1. Forty-six CCF and 6 hepatocellular tumors were investigated. A 550-bp cDNA fragment [3092–3642 bp of rat
insulin receptor substrate-1 homologue (hIRS-1) DNA; GenBank accession no.
S85963] was used for generating RNA probes. The probe was a kind gift of Dr.
L. Mohr (Molecular Hepatology Laboratory, Massachusetts General Hospital
Cancer Center, Charlestown, MA). After subcloning and plasmid amplification, 35S-labeled UTP labeling of the RNA probes was performed with SP6 or
T7 polymerase. Probes were hydrolyzed to a length of ⬃200 bp. We performed
prehybridization, hybridization, and washing procedures, including the removal of nonspecifically bound probe by RNAse A digestion for both antisense
and sense strand 35S-labeled RNA probes, exposition for 2 to 6 weeks and
autoradiographic detection of activity as described previously (38).
Quantitative Reverse Transcription-PCR of IRS-1. Pooled tissue of
several early clear-cell FAHs of two animals and six hepatocellular tumors was
investigated. Kryostat sections were laser-microdissected for harvesting
mRNA of the FAHs and the unaltered control tissue of the same animal as
described previously (39). After RNA preparation (RNAeasy kit, Qiagen,
Hilden, Germany) and reverse transcription (Reverse transcription system,
Promega, Madison, WI), cloning and PCR amplification were done as described elsewhere (39). Briefly, IRS-1 and ␤-actin (internal control) were
amplified with the LightCycler-FastStart DNA Master Hybridization Probes
kit (Roche, Mannheim, Germany). Sequences of primers, hybridization probes,
length of PCR-products, and annealing temperatures are given in Tables 1 and
2. Relative expression levels of different samples of FAHs were calculated by
quantification of IRS-1 levels, normalized to the endogenously expressed
house keeping gene ␤-actin according to the formula 2(Rt - Et)/2(Rn - En), where
Rt and Et are the threshold cycle numbers for the reference gene ␤-actin and
the IRS-1 gene in FAHs, respectively, and Rn and En fulfill the same purpose
for the unaltered tissue.
RESULTS
Insulin Receptor
Zonal Distribution and Localization in Normal Tissue. Immunohistochemical detection of insulin receptor resulted in a predomi-
Table 1 Sequences of primers, hybridization probes, and lengths of PCR product
rat IRS-1
IRS-1-F
IRS-1-R
hyb IRS-1-X
hyb IRS-1-LC
5⬘-ATC TTC CTT Tgg CgC AgC TA
5⬘-CAg CAC gAA AAA gCg CTT A
5⬘-CCT CCg gAT ACC gAT ggC TTC TC-X
5⬘-LC-ACg TgC gCA Agg Tgg gTT ACC T-p
Note. For rat IRS-1, GenBank accession no. was NM 012969, length was 272 bp, and
annealing temperature was 63°C.
Abbreviations: -F and -R, forward and reverse primer; hyb, hybridization probe; X,
fluorescein; LC, light cycler red 640; p, phosphate.
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INSULIN SIGNALING IN RAT HORMONAL HEPATOCARCINOGENESIS
Table 2 Sequences of primers, hybridization probes, and lengths of PCR product
rat ␤-actin
␤-Actin-F
␤-Actin-R
hyb ␤-Actin-X
hyb ␤-Actin-LC
5⬘-CAC ggC ATT gTA ACC AAC Tgg gAC
5⬘-CAg Tgg TAC gAC CAg Agg CAT ACA gg
5⬘-ACA ggg TgC TCC TCA ggg gCC AC-X
5⬘-LC-CGC AgC TCA TTg TAg AAA gTg Tgg TgC CA-p
Note. For rat ␤-actin, GenBank accession no. was NM 031144; length was 232 bp; and
annealing temperature was 71°C.
Abbreviations: -F and -R, forward and reverse primer; hyb, hybridization probe; X,
fluorescein; LC, light cycler red 640; p, phosphate.
nant reaction of hepatocytes, whereas nonparenchymal cells showed
only weak staining (Fig. 1A). Signal intensity was higher in diabetic
than in healthy control rats. In most animals, there was a zonal
distribution with a predominance of insulin receptor expression in
acinar zone 3 (Fig. 1B). Cytoplasmic staining was weak, whereas
membranous staining was strong, particularly in the sinusoidal part.
Fasting-Refeeding Experiment. Feeding resulted in an approximately 15-fold increase in portal vein blood insulin concentration
(0.1 ng/mL in fasted versus 1.54 ng/mL in re-fed animals). As
expected, this increase in sinusoidal insulin concentration in the
re-fed group was accompanied by translocation of the insulin
receptor from the cell membrane into the cytoplasm, resulting in
diffuse or sometimes granular staining of hepatocytes (Fig. 1C and
D). In contrast to fasted animals, in which the hepatocytes were
glycogen-depleted, re-fed animals showed increased glycogen
storing as expected. Thus, high insulin concentrations in sinusoidal
blood lead to translocation of a large part of the membrane-bound
insulin receptor into cytoplasm, resembling the initial step of
insulin signaling in hepatocytes.
Expression and Localization in FAHs and Hepatocellular Tumors. The morphology of the FAHs was not different from the
former experiments. FAHs developed within the first days after transplantation and had a clear-cell morphology (CCF) in the beginning
because of increased glycogen and storing of fat. The clear-cell
phenotype was also overall predominant (78 of 101 FAHs). The other
Fig. 1. Immunohistochemical expression of the
insulin receptor in nonneoplastic tissue (A–D),
clear-cell FAH (E–F), and hepatocellular neoplasms (G–H). Insulin receptor in normal hepatocytes is nearly exclusively located on the sinusoidal
membrane (A). A zonal distribution with predominant expression in the acinar zone 3 was often
visible (B, portal tract in the upper left part and
central vein in the upper right corner). In animals
that fasted for 24 hours, this membranous pattern of
insulin receptor staining was stable (c). Feeding the
fasted animals resulted in high sinusoidal insulin
concentrations and provoked a translocation of the
receptor into the cytoplasm (D), but membranous
staining is still visible. In E, a transplanted islet is
located in the lower left corner. The hepatocytes of
the surrounding preneoplastic focus, corresponding
to the liver acinus, which drains the hyperinsulinemic blood from the islet graft, show a translocation
of the insulin receptor into the cytoplasm, whereas
the hepatocytes from a neighboring acinus (right
edge and upper right corner) maintain their membranous staining pattern. At higher magnification
(F), the clear-cell morphology and the lack of
membranous insulin receptor staining of the preneoplastic hepatocytes (left part) as well as the
sharp border to hepatocytes of the neighboring nonaltered acinus (right part) can be seen. (G) and (H)
show a HCA (G, upper left part) and a HCC (H, left
part) at the border to the adjacent unaltered parenchyma, respectively. Both tumors were of clear-cell
morphology, because of the maintenance of increased glycogen and lipid storage, and show a
pronounced intracytoplasmic localization of the insulin receptor, when compared not only to the
adjacent unaltered liver tissue but also to preneoplastic foci. ⫻452 (A); ⫻577 (B); ⫻220 (C); ⫻141
(D, E, G); ⫻550 (F); ⫻110 (H), antibody A1314.
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INSULIN SIGNALING IN RAT HORMONAL HEPATOCARCINOGENESIS
Table 3 Immunohistochemical pattern of insulin signal transduction proteins in foci of altered hepatocytes and hepatocellular neoplasms
Type of lesion
Protein
CCF
(n ⫽ 78)
MCF
(n ⫽ 20)
BCF
(n ⫽ 3)
HCAg
(n ⫽ 11)
HCAb
(n ⫽ 3)
HCCg
(n ⫽ 4)
HCCb
(n ⫽ 3)
Membranous
Intracytoplasmic
IRS-1
Raf-1
Mek-1
2(⫽)
1
11
11
11
⫽(2)
1(⫽)
1(⫽)
1(⫽)
1(⫽)
⫽
⫽(2)
⫽(2)
⫽(2)
⫽(2)
⫽
11
11
11
11
⫽
⫽(2)
⫽(2)
⫽(2)
⫽(2)
⫽
11
11
11
11
⫽
⫽(2)
⫽(2)
⫽(2)
⫽(2)
IR
Note. The intensity of the immunohistochemical parameters in focal lesions was estimated semiquantitatively compared with unaltered liver tissue of the same sections using five
grades: 22, strong decrease (not noticed); 2, decrease; ⫽, equal; 1, increase; 11, strong increase. If staining was not uniform in one lesion, the secondary pattern is given in
parentheses.
Abbreviations: CCF, clear cell foci; MCF, mixed-cell foci; BCF, basophilic-cell foci; HCAg, glycogen-storing hepatocellular adenoma; HCAb, basophilic hepatocellular adenoma;
HCCg, glycogen-storing hepatocellular carcinoma; HCCb, basophilic hepatocellular carcinoma.
types of foci, i.e., MCF (n ⫽ 20) or BCF (n ⫽ 3), developed after
several weeks.
Because of the hyperinsulinemic environment, FAHs, indeed,
showed an altered signaling pattern in insulin receptor immunohistochemistry when compared with the adjacent hypoinsulinemic liver
tissue. CCF showed a considerable increase of granular cytoplasmic
signals with simultaneously decreased or sometimes unaltered membrane staining, resembling the same translocation of the receptor from
membrane into cytoplasm, as it was induced in the re-fed group of the
fasting experiment (Fig. 1E and F; Table 3).
Ultrastructurally, the immunogold-labeled insulin receptor-antibody detected most of the receptor molecules on the microvilli in the
space of Disse and at the border between the sinusoidal and the lateral
cell surface in normal tissue (Fig. 2A). Adjacent collagen fibers were
free of gold particles. They were also rare in endothelial cells. The
hepatocyte nuclei, mitochondria, lipid droplets, and peroxisomes were
virtually free of gold staining; few granules were sometimes noted
within the membrane system of rough endoplasmic reticulum. Most
intracellular, i.e., non-membranous gold particles in the hepatocytes,
appeared in groups within vesicles, particularly near the sinusoidal
membrane. By contrast, in the preneoplastic hepatocytes within the
FAHs, most of the gold particles were located within these cytosolic
vesicles, and the membranous portion was decreased (Fig. 2B), supporting the results obtained by light microscopy.
Pure BCFs with a homogeneous basophilic-cell population showed
no differences in membrane-bound staining, and cytoplasmic staining
was equal or even slightly decreased when compared with the adjacent parenchyma (Table 3). The individual glycogen-storing and
basophilic hepatocytes within the MCF had the same signaling pattern
as in CCF and BCF, respectively, i.e., the clear cells showed insulin
receptor translocation, whereas the basophilic cells did not. Interestingly, all of the very few spontaneous FAHs, observed in 2-year-old
untreated animals of the control group belonged to the clear-cell type
and showed the same alterations.
The 14 HCAs and 7 HCCs of this experiment can be subdivided
into tumors composed predominantly of glycogen-storing (11 adenomas and 4 carcinomas) or basophilic cells (3 adenomas and 3 carcinomas). The glycogen-storing phenotype still predominated in the
adenomas, but was less frequent in the carcinomas. Cytoplasmic
insulin receptor expression in the clear-cell tumors was higher when
compared not only with the unaltered adjacent parenchyma but also
with the FAHs, and it was still strongly associated with increased
glycogen storage (Fig. 1G and H), while basophilic neoplasms, analogous to the preneoplasias, showed no translocation. Membranestaining was difficult to assess, because the tumors do not possess the
sinusoidal architecture that can be preserved by perfusion fixation. In
cases in which evaluation was possible, there was no difference from
the unaltered liver tissue. In addition, no difference was found to exist
between adenomas and carcinomas. Thus, the majority of tumors
showed insulin receptor overexpression. As in the fasting experiment,
one might conclude that a high insulin concentration in the sinusoidal
blood within early FAHs leads to permanent internalization of receptorbound insulin into the hepatocytes. It is of particular interest that even
in later stages, i.e., after several months, this pattern was maintained,
and no adaptive receptor down-regulation, which might have been
expected, was observed.
Insulin Receptor Substrate-1, Raf-1, Mek-1
In control animals and in the unaltered liver tissue of diabetic
rats, IRS-1, Raf-1 and Mek-1 proteins were detected immunohistochemically in hepatocytes and in nonparenchymal cells, presenting as weak granular staining of the cytoplasm. In the hepatocytes
of all of the CCF examined, there was a striking increase in protein
Fig. 2. Electron micrograph of a normal (A) and
preneoplastic (B) hepatocyte after immunogold labeling of the insulin receptor protein. Positive signals in this unaltered hepatocyte (A) is predominantly visible on the sinusoidal membrane,
particularly the microvilli. A few signals are also
seen in the adjacent portion of the cytoplasm, often
grouped within vesicles (lower left part). The covering endothelial lining (arrow) does not show positive reactions. In the preneoplastic hepatocyte (B),
virtually all of the signals were located within vesicles in the cytoplasm near the sinusoidal membrane but not on the membrane or the microvilli
(upper part) itself. Fat droplets (F) and mitochondria (M) were not stained. A, ⫻44,000; B, ⫻25,300.
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INSULIN SIGNALING IN RAT HORMONAL HEPATOCARCINOGENESIS
immunoreactivity (Fig. 3A–H; Table 3). The majority of the MCF
also showed an increase in signaling when compared with the
unaltered adjacent tissue, although the amount of protein was
smaller than in CCF and was closely correlated with the portion of
glycogen-storing cells within the MCFs (Fig. 3I–N). In the few
pure BCF, the overall extent of IRS-1 was as low as in the adjacent
unaltered parenchyma or was slightly decreased (Fig. 4A and B).
The amount of the different proteins in HCAs and HCCs was
similarly correlated with the ratio of glycogen-storing-cells to
basophilic-cells as in FAHs, and the overall increase in signal
reactivity for these proteins in the neoplasms was about the same
as for insulin receptor (Fig. 4C–F). Analogous to insulin receptor,
the few spontaneous clear-cell preneoplastic hepatocellular foci in
the 2-year-old animals of the untreated control group showed the
Fig. 3. Immunohistochemical expression of IRS-1, Raf-1, and Mek-1 in clear-cell preneoplastic foci (A–H) and a MCF (I–N). Liver tissue 6 months after islet transplantation (A–D)
containing three FAHs. In the PAS reaction (A), the FAHs were highlighted because of their increased glycogen and lipid content. Serial sections were stained with antibodies directed
for IRS-1 (B), Raf-1 (C), and Mek-1 (D) and show an increase in expression of these proteins within the FAHs, even at low magnification. In E, a clear-cell FAH in more detail is
depicted in PAS reaction. The lesion is restricted within the liver acinus (hepatic venule in the lower right part), draining the blood from the islet graft, which is located within a portal
tract (arrow). F–H, serial sections to E, with antibodies against IRS-1 (F), Raf-1 (G), and Mek-1 (H). Normal protein expression is visible in the unaltered liver tissue of the adjacent
parenchyma. Serial sections of a MCF are shown in I–N. In the PAS reaction (I), the majority of the hepatocytes within the MCF is pale, only a few single cells dispersed and a group
of cells (arrow) have maintained the glycogen-storing phenotype. Vice versa, these latter cells were not stained with the basophilic reagent cresyl-violet (J), whereas the basophilic
cell population stains deeply blue-violet. Interestingly, the glycogen-storing cells in this focus do not show the translocation of the insulin receptor from the membrane to the cytoplasm
(K), which is characteristic for the early foci of pure clear-cell phenotype and as it is depicted in Fig. 1. The IRS-1 (L), Raf-1 (M), and Mek-1 (N) overexpression is strictly connected
with the clear-cell (glycogen-storing) phenotype of the preneoplastic hepatocytes. The content of the respective proteins within the basophilic cell population is slightly decreased in
this focus, when compared with the unaltered hepatocytes of the adjacent parenchyma (lower left parts of the panels). A–D, ⫻7; E, ⫻71; F–H, ⫻47; I–N, ⫻62.
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Fig. 4. Expression of insulin signaling proteins
in a pure BCF (A–B), a well differentiated clear-cell
HCC (E–H), and a spontaneous preneoplastic focus
(G–I). This large basophilic focus (A) is composed
exclusively of basophilic cells, which are pale in
the PAS reaction. No overexpression of insulin
signaling proteins was visible in this focus, (B)
depicting exemplary the immunohistochemical reaction for Mek-1. This well-differentiated HCC
(C–F, left part) has maintained the clear-cell (glycogen-storing) phenotype (C, PAS reaction) and
also demonstrates the overexpression of IRS-1 (D),
Raf-1 (E), and Mek-1 (F). Basophilic adenomas
and carcinomas (not depicted), however, do not
show overexpression of any of these proteins, thus
corresponding to the basophilic preneoplastic foci
(A–B). Very rarely spontaneous hepatocellular foci
(G–I), always of the glycogen-storing phenotype
(G, PAS reaction), developed in single animals of
all groups, when the animals were ⬃2 years old.
Throughout, they also show the overexpression of
the intracellular insulin-signaling proteins, IRS-1
(H) and Mek-1 (I). A–B, ⫻55; C–F, ⫻71; G–I,
⫻94.
same staining pattern as did their insulin-induced counterparts
(Fig. 4G–I).
In situ hybridization for IRS-1 mRNA revealed specific positive
signals in the cytoplasm of hepatocytes in normal, preneoplastic, and
neoplastic liver tissue. Sinusoids and nuclei, however, were virtually
devoid of any signals. When compared with unaltered adjacent liver
parenchyma, the FAHs showed a slight increase in signal density (Fig.
5); in a minority of the cases, no difference was noted. As with in situ
hybridization, quantitative reverse transcription-PCR of laser-microdissected liver tissue also showed an overexpression of pooled IRS-1
mRNA in the FAHs when compared with the unaltered liver tissue in
both animals, 6.0 ⫾ 1.9-fold and 6.3 ⫾ 1.9-fold, respectively.
DISCUSSION
It is generally accepted that insulin signaling is primarily accomplished by binding to the membranous insulin-receptor, although there
is also evidence that insulin can enter the cytoplasm of hepatocytes via
fluid-phase-endocytosis (8). A recent investigation of the normal rat
liver has shown that the insulin receptor protein is predominantly
localized in hepatocytes of the perivenous zone, mainly at the plasma
Fig. 5. IRS-1 mRNA in situ hybridization. Dark field illumination after hybridization
with a 35S-labeled RNA probe, 11 days after islet transplantation. I, islet in the upper left
corner; arrows, the border between the preneoplastic focus (middle part) and the adjacent
unaltered parenchyma (right part). Within the islet, although only a few autoradiographic
grains were visible, the preneoplastic hepatocytes of this focus show a more intense
staining when compared with the unaltered tissue, corresponding to an overexpression of
IRS-1-mRNA. ⫻135.
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INSULIN SIGNALING IN RAT HORMONAL HEPATOCARCINOGENESIS
membrane (40), which is corroborated by our observation in control
tissue of normoglycemic and diabetic, i.e., systemically hyperglycemic and hypoinsulinemic, animals. In addition, we confirmed the
results obtained in earlier studies according to which, hepatocytes of
streptozotocin-diabetic rats contain more insulin receptor than do
normoglycemic animals (41– 42).
The main alteration regarding the insulin receptor in early preneoplastic CCF, which drain the blood from the islet grafts, was a
translocation from the plasma membrane into the cytoplasm, demonstrated by light and electron microscopy. At the beginning, this
appears to be the consequence of constant extracellular insulin receptor binding caused by the local hyperinsulinemia and subsequent
receptor internalization. This is supported by the finding that we were
able to induce a similar translocation of the insulin receptor by
refeeding fasted rats, which showed a high postprandial insulin concentration in their portal-venous blood. However, despite the translocation, overall protein expression of the insulin receptor was not
altered in early CCF, which stands in contrast to the observation of
insulin receptor down-regulation in (hyperinsulinemic and hyperglycemic) type 2 diabetes in humans, and to a former study, in which
insulin-treated streptozotozin-diabetic rats and rat hepatoma cells
have shown a decrease in insulin receptor mRNA or protein expression, measured indirectly by the binding of 125I-labeled insulin (43–
44). This absence of receptor down-regulation is most likely the
consequence of the systemic hyperglucagonemia caused by insulin
deficiency (45), which has been demonstrated in streptozotocindiabetic rats, and can be restored by exogenous insulin treatment or
high-dose islet transplantation (46 – 47). The absence of insulin receptor down-regulation might be important for the development and
maintenance of carcinogenesis in this model.
It also intriguing that glycogen-storing HCAs and HCCs maintain
the cytosolic pattern of insulin receptor expression and even show
increased expression. This pattern can be easily explained for the early
FAHs, which are confined to the anatomic borders of the liver acinus
and drain the hyperinsulinemic blood from the islet grafts. With the
increasing size of the lesions, particularly after neoplastic transformation, however, the often scattered islet graft remnants cannot be made
responsible for this overexpression, although they can be regularly
demonstrated within the tumors (27). Although the intralesional insulin concentration cannot be measured, it can be assumed that the
former hyperinsulinemia, induced by the islet grafts, has diminished
or is completely absent within these tumors. Thus, the insulin receptor
overexpression seen in the tumors cannot exclusively be explained as
a consequence of increased insulin internalization. Additional factors,
such as impaired or altered degradation, lack of retranslocation to the
plasmamembrane or genetic and epigenetic mechanisms have to be
taken into account.
Overexpression of insulin receptor and IRS-1 has been shown in
human and rodent HCC and in other neoplasms (12–14, 19). Moreover, the transforming potential of IRS-1 in hepatic and nonhepatic
cell lines has been well documented (16, 48). IRS-1 overexpression
also protects HCC cells against transforming growth factor-␤-induced
apoptosis (21). Vice versa, the expression of an inhibitory COOHterminal-truncated IRS-1 molecule resulted in reversal of the malignant phenotype of human HCC cells (20). Conversely, transgenic
mice overexpressing IRS-1, do not develop hepatocellular tumors,
although they show increased liver growth in the first 3 months of life
(49). Particularly interesting is the overexpression of IRS-1 as an early
event in a rat model of N-nitrosomorpholine-induced hepatocarcinogenesis, because this model is similar to ours in some aspects, particularly regarding the insulinomimetic phenotype of the preneoplastic
hepatocytes (17). In that study, the authors also observed a strong
correlation between the IRS-1 overexpression and the extent of gly-
cogen-storage in the preneoplastic and neoplastic cells, suggesting
that the phenotype of preneoplastic hepatic glycogenosis is elicited by
an overexpression of IRS-1. This was also demonstrated by the same
authors in another investigation, in which amphophilic (non-glycogen-storing) FAHs did not show overexpression of IRS-1 (18). However, it is unclear which mechanisms lead to the overexpression of
IRS-1 in this chemically induced model of hepatocarcinogenesis. In
our model, the alterations in the insulin signaling transduction cascade
have also been observed from the beginning of the formation of
preneoplastic foci, even 2 days after islet transplantation; therefore,
they should be regarded as a direct consequence of the local hyperinsulinemia. Although Ras itself has not yet been investigated, the
overexpression of several upstream and downstream proteins of the
Ras/Raf-1/Mek-1 pathway as shown in this study indicates that this
pathway is essentially involved in mediating the insulin signal in this
model, thus contributing to the alterations in cell metabolism, proliferation kinetics, and the development and maintenance of the preneoplastic phenotype. Although the phosphorylation status of the signaling proteins is difficult to establish by immunohistochemistry alone,
and the FAHs in this model are too small for Western blotting after
laser microdissection, it is very likely that the overexpression of the
proteins is accompanied by phosphorylation as has been already
shown. Toyoda et al. (24), with another model of hepatocarcinogenesis in rats, have recently demonstrated that the expression and phosphorylation statuses of IRS-1, ERK-1 and ERK-2 are altered in the
same manner so that overexpression is indeed accompanied by increased activity.
At later stages of the carcinogenic process, the FAHs become more
diverse, showing a decrease in glycogen storage in part of (MCF) or
in all preneoplastic hepatocytes (BCF), although the majority maintain
their glycogenotic appearance even after neoplastic transformation. It
is unclear, however, whether the maintenance of the glycogen-storing
preneoplastic character of the altered hepatocytes at this time point
still depends on continuous insulin signaling in principle, or on other
mechanisms that have meanwhile been activated, fixing the carcinogenic process and making it “irreversible.” In this context it is noteworthy that transforming growth factor-␣ is overexpressed in latestage foci and particularly in the glycogen-poor HCCs, and that single
HCCs in this model display strong IGF-II overexpression (27, 29).
In conclusion, we showed that in a model of low-number islet
transplantation into streptozotocin-diabetic rats, insulin induces a
morphologic and metabolic transformation of hepatocytes by activation of its intracellular signaling cascade, involving the Ras-RafMAPKK pathway. This transformation results in the clear-cell “insulinomimetic” phenotype of preneoplastic FAH, which is known to be
induced by chemical carcinogens, such as N-nitrosomorpholine. Future studies will show whether other intracellular transduction cascades of the insulin signaling network, in particular the phosphoinositol-3-kinase/AKT-pathway and related transcription factors, such as
nuclear factor ␬B or the forkhead transcription factor FKHR, are
activated, and whether this activation also contributes to the carcinogenic process in this model.
ACKNOWLEDGMENTS
The authors wish to thank Jörg Bedorf, Mathilde-Hau-Liersch, Inge Heim,
Mariana Dombrowski, Uta Schönborn, and Sybille Wolf-Kümmeth for excellent their technical assistance; Kurt Rüdel for animal care; Dr. Jens-Gerd
Scharf and Susanne Hupe for their help in establishing the IRS-1 in situ
hybridization; and Bernd Wüsthoff for editing the manuscript.
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Insulin Receptor, Insulin Receptor Substrate-1, Raf-1, and
Mek-1 during Hormonal Hepatocarcinogenesis by
Intrahepatic Pancreatic Islet Transplantation in Diabetic Rats
Matthias Evert, Jianping Sun, Sabine Pichler, et al.
Cancer Res 2004;64:8093-8100.
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