Metformin modulates insulin post-receptor signaling trans-duction in chronically insulin-treated Hep G2 cells
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Yuan L et a l / Acta Pharmac ol Sin 2 003 Jan; 24 (1): 55-60 ·55 ·
200 3, Act a Pharm acolog ica Si nica
Chin ese Phar macolog ical Soc iety
Shang hai Insti tute of Materia M edica
Chine se Academ y of Sci ences
ht tp:/ /www.Chi naPh ar.c om
Metformin modulates insulin post-receptor signaling trans-
duction in chronically insulin-treated Hep G2 cells
YUAN Li1, Reinhard ZIEGLER, Andreas HAMANN2
2
Depar tment of Endocrinology and Metabolism in Hospital of University Heidelberg, H eidelberg 69115, Germany
KEY WORDS metformin; insulin receptors; signal transduction
ABSTRACT
AIM: To study the effect of chronic insulin treatment on insulin post-receptor signaling transduction and whether
the effects of metformin are transmitted throughout the cascade of insulin signaling intermediates in a human
hepatoma cell line (Hep G2). METHODS: Hep G2 cells were incubated in serum free media containing either
insulin 100 nmol/L or insulin 100 nmol/L plus different concentrations (0.01-10 mmol/L) of metformin for 16 h and
then were stimulated with insulin 100 nmol/L for 1 min. RESULTS: Chronic treatment of insulin 100 nmol/L
induced a significant reduction in the phosphorylation and protein expression of IRß, IRS1 and IRS2, which there-
fore resulted in a downregulation of association of PI3K with IRS. Therapeutic concentrations (0.01-0.1 mmol/L)
of metformin prevented the changes induced by chronic insulin treatment in these post-receptor components of
insulin signaling pathway. Tyrosine phosphorylation of IRβ, IRS1, and IRS2 was increased by 2.7 fold (P·56 · Yuan L et al / Acta Pharmac ol Sin 2 003 Jan; 24 (1): 55-60
However, the molecular basis of metformin effect on 100 nmol/L plus metformin 0.01-10 mmol/L for 16 h
hepatocytes is largely unknown, involving the possible and then were stimulated with insulin 100 nmol/L for 1
effects of metformin on insulin signaling tranduction, min, as specified in the results section. Cells under
for instance, metformin could directly or indirectly in- basal conditions were used as controls.
fluence protein-protein interactions within the signaling Immunoprecipitation and Western blotting
cascade. Phosphorylated proteins of IRβ, IRS1, IRS2 and inter-
Chronic hyperinsulinsm can induce insulin resis- action of PI3K with IRS were determined by immuno-
[4]
tance . Therefore, in the present study, we set up an precipitation assay. protein lysates 1000 µg were sub-
insulin resistant cell model, with the Hep G2 cells jected to immunoprecipitstion using corresponding spe-
chronic exposure to high concentration of insulin. The cific antibodies (anti-IRβ, anti-IRS1, anti-IRS2) and
aim is: (1) to examine the changes in insulin signaling incubation at 4 ºC for 2 h. Protein-antibody complexes
transduction after chronic insulin treatment. (2) to de- were conjugated with protein A and Protein G for an-
termine whether the antihyperglycemic action of other 1 h. Precipitates were take up in 30 µL sample
metformin in hepatocytes is associated with effects on buffer containing T ris 62.5 mmol/L, dithiothreitol
signaling protein expression and phosphorylation. (DTT) 100 mmol/L, 10 % glycerol, 2 % SDS, 0.01 %
bromphenolblue and denatured at 95 ºC for 10 min.
MATERIALS AND METHODS Protein samples were subjected to 7.5 % SDS-PAGE.
After SDS-PAGE, electrotransfer of protein from the
Chemicals RPMI-1640 medium and fetal bovine
gel to nitrocellulose membranes was performed by
serum (FBS) were obtained from Gibco BRL (Karlsruhe,
Western blotting. Subsequently, nitrocellulose filters
Germany). Reagents for sodium dodecyl sulfate-poly-
were incubates at 4 ºC overnight with one of the fol-
acrylamide gel electrophoresis (SDS-PAGE) and
lowing antibodies (1st antibody) at a concentration of
immunoblotting were obtained from Bio-Rad (Hercules,
1 mg/L: anti-PY, anti-IRβ, anti-IRS1, anti-IRS2 or anti-
CA). Rabbit polyclonal anti-insulin receptor β-subunit
p85, each of those suspended in 2.5 % non-fat dried
(IRβ), anti-insulin receptor substrate 1 (IRS1), anti-in-
milk. Blots were then incubated with 1:3000 HRPO
sulin receptor substrate 2 (IRS2), and anti-p85 subunit
conjugate (2nd antibody) at room temperature (28 ºC)
of phosphatidylinositol 3-kinase (PI3K) antibodies used
for 1 h. Immunoreactive bands were determined with
for Western blotting were purchased from UBI (Lake
the ECL detected reagents.
Placid, NY). Anti-biotin and mouse monoclonal anti-
Immunoblotting Relative protein levels of IRβ,
phosphotyrosine (PY) antibody were from New E n-
IRS-1, IRS-2, and p85 were determined by Western-
gland Biolabs (Frankfurt am Main, Germany). Protein
immunoblotting in total cell lysates. Similar size aliquots
A and protein G sepharose and BCA-assay reagents were
of sample (150 µg) were processed for 7. 5 % SDS-
purchased from Pierce (Hamburg, Germany). HRPO-
PAGE and proteins were separated by electrophoresis.
conjugated anti-mouse and anti-rabbit antibodies, re-
Immunoblotting was performed as described above.
agents for E CL were obtained from Amersham
Statistical analysis Relative amounts of immu-
Pharmacia Biotech (Uppsala, Sweden). Human insulin
noreactive proteins were quantitated by Image Quant
(Ac trapi d) wa s obt ained from Novo Nord isk
scanning densitometry. Experimental results were ex-
(Deisenhofen, Germany). Metformin, aprotinin,
pressed as mean±SD. Statistical analysis was per-
pepstatin, leupeptin, and other reagents were from Sigma
formed by Student’s t-test. Statistical significance was
(Steinheim, Germany).
assessed at PYuan L et a l / Acta Pharmac ol Sin 2 003 Jan; 24 (1): 55-60 ·57 · residues of signaling proteins. After cells were treated with insulin 100 nmol/L for 16 h, insulin-induced autophosphorylations of IRβ, IRS1, and IRS2 were sig- nificantly decreased. By comparison with control cells, tyrosine phosphorylation of IRβ was decreased to 22 % (P
·58 · Yuan L et al / Acta Pharmac ol Sin 2 003 Jan; 24 (1): 55-60 (P
Yuan L et a l / Acta Pharmac ol Sin 2 003 Jan; 24 (1): 55-60 ·59 ·
insulin resistance in vitro[4, 5]. Thereby, in the present transport, activation of glycogen synthase, and inhibi-
study, we first set up an insulin resistant model by us- tion of PEPCK as the key enzyme of gluconeogenesis.
ing chronic treatment of Hep G2 cells with high doses Since the effect of metformin on insulin signaling pro-
of insulin. Such in vitro allows direct assessment of cesses was observed at concentrations reached in the
the effect of an additional treatment on the biological serum of metformin-treated patients, these data are
response to insulin stimulation. Since metformin ex- somewhat suggestive for the actual situation in humans.
erts its beneficiary antihyperglycemic effect primarily Thus, the primary mechanism of metformin’s action to
in liver, this should also involve an alteration of the in- restore insulin sensitivity in hepatocytes might be re-
sulin resistant state in this organ. Therefore, it appeared lated to the increased insulin post-receptor signal trans-
interesting to focus on determining relevant molecules duction linked to tyrosine phosphorylation of IRβ, IRS1
which transmit the effects of metformin within the in- and IRS2 and the activation of PI3K.
sulin signaling cascade in a liver cell model system, in In contrast to the effect of metformin at thera-
which insulin resistance had been induced. T he data peutical concentrations, pharmacological concentrations
described here have suggested that impaired insulin signal of metformin inhibit further phosphorylation of signal-
transduction linked to IRβ, IRS1, IRS2, and PI3K is ing proteins and association of IRS with PI3K. These
associated with insulin resistance, which was caused inhibitory effects may reflect the fact that higher
by chronic insulin treatment. metformin concentrations inhibit insulin action[9]. It
Metformin could interact with insulin at many should be noted that the changes in tyrosine phospho-
potential steps, including the increased binding of insu- rylation associated with metformin treatment were not
lin to its receptor[6], the intensified of insulin receptor parallel to the alterations of protein expression level,
tyrosine kinase activity[7-9], the elevated inositol-1,4,5- which were increased in the presence of pharmaco-
trisphosphate production, the augmented glycogen logical concentrations of metformin. This suggests that
synthesis, and the inhibition of PEPCK as a key enzyme effect of metformin on tyrosine phosphorylation of sig-
of gluconeogenesis[10]. In present experiment, the ef- naling protein could not be explained by the changes in
fect of chronic insulin treatment can be reversed by the level of protein expression. Whether inhibitory ef-
metformin. It suggests that metformin’s action site is fects of pharmacological concentrations of metformin
most likely located at an early post-receptor level and are correlated with either the alteration in insulin receptor,
may directly or indirectly interact with the intracellular IRS1 and IRS2 serine phosphorylation, or the direct
insulin signaling cascade. action of metformin on insulin signal transduction or
Up to now, there were only a few reports regard- other regulatory event, remains to be investigated in
ing the effect of metformin on intracellular insulin sig- future studies.
naling system and there has been a controversy in dif- In conclusion, the present data suggest that chronic
ferent experiments. Metformin was reported to increase insulin exposure of Hep G2 cells results in down-regu-
insulin signaling transduction in cholesterol-treated Hep lation of insulin signal transduction via PI3K pathway.
G2 cells[11] and to reverse chronic insulin effects on Therapeutic concentrations of metformin can reverse
insulin signaling in rat adipocytes [5 ]. However, the effect of chronic hyperinsulinism on expression and
metformin treatment had no effect on insulin signaling activation of insulin signaling molecules, and pharma-
cascade in human adipoctey[12] and skeletal muscle[13 ]. cological concentrations of metformin inhibit insulin
These different observations may be due to difference signal transduction. The effect of metformin on insulin
of tissues and cultured conditions. In the present study, signal transduction represents a primary mechanism of
therapeutic doses of metformin have reversed the re- metformin action in insulin resistant state.
duction in phosphorylation of IRβ , IRS1, and IRS2
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