Project leaders:
PD Dr.Leonhard Mohr, Dr. María Matilde Bartolomé Rodríguez
Innere Medizin II, Labor B3, Tel 270-3509
Hugstetter Strasse 55, 79106 Freiburg
Leonhard.mohr@uniklinik-freiburg.de, Maria.bartolome@uniklinik-freiburg.de
Topic: Insulin and IGF-1 mediated signal transduction in hepatocytes
Introduction: Insulin is the key regulator of glucose and lipid metabolism, whereas IGF-1
expression is induced in hepatocytes following growth hormone stimulation. In hepatocytes,
insulin and IGF-1 signaling are involved in liver regeneration. Insulin and IGF induce
metabolic, proliferative and anti-apoptotic responses. The diverse biological effects are
mediated by a complex signal transduction pathway. Binding of insulin to its receptor (IR)
results in autophosphorylation and activation of the receptor tyrosine kinase domain. Two
major docking molecules (insulin receptor substrates 1 and 2 (IRS-1/IRS-2)) bind to the
phosphorylated IR and are subsequently phosphorylated at multiple tyrosine residues.
Phosphorylated IRS-1 and IRS-2 bind multiple signaling molecules containing SH-2 domains
including PI3 kinase. Binding of PI3 kinase to IRS-1/2 results in PI3 kinase activation, which
in turn activates PKB/Akt. PKB/Akt mediates most of the metabolic responses of insulin as
well the inhibition of apoptosis. Further binding of Shc to the IR and of Grb2/SOS to IRS1/IRS-2 activates the MAP-kinase cascade via Ras, resulting in proliferative stimuli required
for liver regeneration. Mathematical modeling of several linked processes will be performed
together with the modeling platform (Timmer, Freiburg and Gilles, Magdeburg).
Work plan:
First year:
A) Analysis of the specific functions of IRS-1 and IRS-2 in primary hepatocytes: In the
first period of the project the analysis of dose responses and kinetics of tyrosylphosphorylation for both IR and IRS-1 were performed and the obtained data were
statistically analyzed in collaboration with the FDM. In this new period, the focal
point will be IRS-2. As already performed for IRS-1, kinetics of IRS-2
phosphorylation after insulin stimulation will be characterized. In addition, kinetics of
IRS-1/IRS-2 dephosphorylation after 5 min insulin pulse and the kinetics of PI3K
binding to IRS-2 will be quantitatively analyzed. The biological question is to
characterize the precise function of each IRS-1/-2 in PMH, which are relatively similar
molecules. Since both molecules compete for the same binding site at the IR, precise
quantification and kinetic analyses are highly important for the design of appropriate
mathematical models.
B) Design of specific calibrators for IRS-1 and IRS-2 (in close collaboration with U.
Klingmüller, Project B1) for accurate quantification of IRS-1/2 binding to PI3 kinase.
C) Quantitative analysis of MAPK and PI3 kinase phosphorylation following continuous
or pulse stimulation with insulin will be performed and a mathematical model will be
etablished (Timmer, Klingmüller).
D) These experiments will be identically performed using IGF-1 to determine biological
differences between the effect of insulin and IGF-1 in PMH. Main focus in the first
year would be the analysis of dose responses and kinetics of tyrosyl-phosphorylation
for IR, IRS-1 and IRS-2.
Second year:
A) To further analyze the function of IRS-1 and IRS-2 independently for each other,
PMH from IRS-1- and IRS-2-knockout mice (B6 background) will be used. For
competition studies, reintroduction of IRS-1 in PMH from IRS-2 knockout mice by a
B)
C)
D)
E)
replication deficient, recombinant adenovirus engineered to express IRS-1 will be
performed. The IRS-1 expressing Adenovirus will be constructed using the Adeasy
system, which is routinely used in our laboratory for gene transfer studies.
Visualizing of insulin uptake using live cell imaging. For the analysis of insulin
binding to the IR on the cell surface a FITC-labeled insulin is available. Binding of
FITC-labeled insulin to hepatocytes will be determined by FACS analysis and also by
confocal microscopy in collaboration with the Nitschke and Klingmüller Groups (B1
and B8). Experiments regarding early endosome trafficking of insulin-FITC will be
also performed in collaboration with the Doodlely Group.
Careful quantification of PI3 kinase-recruitment to IRS-1 and IRS-2 will be measured
to determine differential affinities of the protein to each adaptor.
Design of a PI3 kinase-GFP fusion protein as well a PI3 kinase-GFP expressing
adenovirus (in close collaboration with Klingmüller, Doodley and Hengstler) to
analyze kinetics of PI3 kinase transport to the cell membrane.
IGF-1 signaling: main focus in the second year would be the analysis of dose
responses and kinetics of PI3 kinase binding to both IRS-1 and IRS-2 after IGF-1
stimulation.
Third year:
A) Live cell imaging of PI3 kinase transport to the cell membrane using a PI3K-GFP
fusion protein (collaboration with Nitschke and Klingmüller) and analysis of
phosphorylation by separate immunoprecipitation of cell membranes and cytoplasma
in comparation with total cell lysates. Quantitative analysis of PI3K molecules
translocating to the cell membrane in wild type PMH and in PMH from IRS-1 and
IRS-2 knockout mice. Quantitative analysis of phosphorylated versus total PI3K in
PMH of the three mouse models will be done and a mathematical modeling about the
differential stimulation of the PI3K in all three systems is planed (Timmer).
B) Analysis of PIP2 and PIP3 production in the cell with WB analysis as indicator of PI3
kinase activity. Kinetics in wildtyp and both IRS1 and IRS-2 knockout mouse to
further analyze the contribution of IRS-1 and IRS-2 for the PI3 kinase activity.
C) Kinetics of ERK phosphorilation and recruitment to the cell nucleus using live cell
imaging and immunoprecipitation of cell cytoplasma and cell nucleus.
D) To analyze kinetics of insulin and IGF-1 induced gene expression, expression of
selected target genes (e.g. c-fos) after insulin / IGF-1 stimulation will be analyzed by
real time PCR. Based on these data, gene array analysis of differential gene expression
following insulin/IGF-1 stimulation in primary hepatocytes (wild type, IRS-1 and
IRS-2 knockout) will be performed in collaboration with Donauer/Walz (B7) and
Merfort (B2) Groups.
Milestones:
1st year: Generation of calibrators for IRS-1 and IRS-2. Mathematical modeling of IR
phosphorylation and IRS-1/2 phosphorylation after insulin / IGF-1 stimulation (Timmer).
2nd year: SOPs for IRS-1 and IRS-2 knockout mice (von Weizsäcker). Establishment of viral
vectors (adenoviruses) for the expression of target genes in PMH (Klingmüller). Comparison
of the dynamics of IRS-1 and IRS-2 phosphorylation in response to insulin versus IGF-1.
Mathematical analysis of the differential functions of IRS-1 and IRS-2 in primary hepatocytes
(Timmer).
3rd year: Mathematical model of the binding of PI3 kinase to both IRS-1 and IRS-2 and
comparison to the obtained results with HGF (Klingmüller and Timmer). Mathematical model
of ERK phosphorylation kinetics as well as recruitment to the cell nucleus. IGF-1 versus
Insulin signaling: Comparison of both signaling cascades (Timmer).
Budget:
BAT IIa for Dr. María Matilde Bartolomé Rodríguez, who is an experienced postdoctoral
fellow and is essential for the continuation of the project.
BAT IV: Technician (Astrid Wäldin), already involved in the project and experienced in the
preparation and cultivation of PMH as well in the analytical techniques of insulin signal
transduction.
BAT IIa/2 NN (biological Ph.D. student): main focus in IGF-1 signal transduction.
Materials:
Antibodies, PAGE, detection kits
IRS-1 and IRS-2 knockout mice
Cell culture, glass ware:
Gene arrays (500 €/array)
Travel budget:
20000 €/year
2000 € purchase
1500 € cage fee/year
3500 €/year
4000 €
4000 €/year
References:
1. A mathematical model of metabolic insulin signaling pathways. Sedaghat et al.: Am J
Physiol Endocrinol Metab, 2002. 283: E1084.
2.The insulin signaling system and the IRS proteins. M. White: Diabetologia, 1997. 40: 2.
3.Liver regeneration: from myth to mechanism. R. Taub: Nature reviews / molecular cell
biology, 2004. 5: 836.
4.Modulation of insulin action. Pirola et al: Diabetologia, 2004. 47: 170.
5. Mohr, L., Tanaka, S., and Wands, J. R. Ethanol inhibits hepatocyte proliferation in insulin
receptor substrate 1 transgenic mice, Gastroenterology. 115: 1558-65, 1998.
6. Banerjee, K., Mohr, L., Wands, J. R., and de la Monte, S. M. Ethanol inhibition of insulin
signaling in hepatocellular carcinoma cells, Alcohol Clin Exp Res. 22: 2093-101, 1998.
7.Tanaka, S., Mohr, L., Schmidt, E. V., Sugimachi, K., and Wands, J. R. Biological effects of
human insulin receptor substrate-1 overexpression in hepatocytes, Hepatology. 26: 598-604,
1997.