(Received for publication, May 23, 1994; and in revised form, December 12, 1994 )
From the
The mechanisms for the insulin resistance induced by
hyperglycemia were investigated by studying the effect of high glucose
concentration (HG) and its modulation by thiazolidine derivatives, on
insulin signaling using Rat 1 fibroblasts expressing human insulin
receptors (HIRc). Incubating HIRc cells in 27 mMD-glucose for 4 days impaired the insulin-stimulated
phosphorylation of pp185 and receptor -subunits. Both protein
kinase C activities and phorbol dibutyrate binding to intact cells were
unchanged; however, cytosolic protein-tyrosine phosphatase (PTPase)
activity increased within 1 h prior to the impairment of insulin
receptor kinase in HG cells (Maegawa, H., Tachikawa-Ide, R., Ugi, S.,
Iwanishi, M., Egawa, K., Kikkawa, R., Shigeta, Y., and Kashiwagi,
A.(1993) Biochem. Biophys. Res. Commun. 197, 1078-1082).
Increased PTPase activity was consistent with a 2-fold increase in the
amount of PTP1B, and anti-PTP1B antibody inhibited this increment of
cytosolic PTPase activity in HG cells. Co-incubating cells with
pioglitazone prevented these abnormalities in cytosolic PTPase, the
PTP1B content and the impaired phosphorylation of pp185 and receptor
subunits in HG cells. Finally, HG cells had impaired
insulin-stimulated
-aminoisobutyric acid uptake, which was
ameliorated by exposure to thiazolidine derivatives. In conclusion,
exposing cells to high glucose levels desensitizes insulin receptor
function, and thiazolidine derivatives can reverse the process via the
normalization of cytosolic PTPase, but not of protein kinase C.
Non-insulin-dependent diabetes mellitus (NIDDM) ()is
characterized by insulin resistance in insulin-sensitive peripheral
tissues, particularly the skeletal muscles(1) . Several studies
on insulin receptor function of skeletal muscles in NIDDM have revealed
a decrease in the kinase activity of insulin receptors, which may be
partially responsible for the decreased action of insulin in patients
with NIDDM(2, 3, 4) . Therefore, the
enhancement of insulin sensitivity in patients with NIDDM is one
treatment modality. The thiazolidine derivatives, pioglitazone and
troglitazone, have been tested for use as oral anti-diabetic
drugs(5, 6, 7, 8) . We reported that
pioglitazone may increase insulin sensitivity by activating the
tyrosine kinase of skeletal muscle insulin receptors isolated from not
only insulin-resistant Wistar fatty rats, but also from rats given a
high-fat diet(9, 10) .
Hyperglycemia per se induces insulin resistance in experimental animal models, based upon the finding that the correction of hyperglycemia with phlorizin normalizes in vivo insulin sensitivity in diabetic rats(11) . The in vitro studies of Müller et al.(12) have shown that insulin receptor kinase activity in rat adipocytes is modulated by incubating the cells in high glucose medium. Similarly, we also reported (13) that in NIDDM patients without hyperinsulinemia, there is a reverse relationship between fasting plasma glucose levels and the insulin receptor kinase activities of the skeletal muscles. Thus, hyperglycemia per se may induce in vivo insulin resistance by desensitizing insulin receptors in insulin-sensitive tissues. To study how high glucose affects insulin receptor function in cell cultures, we used Rat 1 fibroblasts that overexpress human insulin receptors (HIRc). We found that high glucose impaired the insulin receptor kinase activity and that pioglitazone, a thiazolidine derivative, normalized the insulin receptor kinase level in culture(14) .
Kellerer et al.(15) have also reported that the thiazolidine derivative, troglitazone, can normalize the insulin receptor dysfunction induced by incubating cells in high glucose medium. They have shown that protein kinase C (PKC) activity is increased in HIRc cells exposed to high glucose and that troglitazone can normalize the PKC activation. Their results suggest that the effect of troglitazone on insulin receptor kinase activity is mediated by the normalization of PKC activity. However, in a preliminary study, we did not find significant PKC activation in HIRc cells exposed to high glucose, although pioglitazone significantly improved the receptor function(14) . Therefore, the mode of action of thiazolidine derivatives needs further clarification. Protein-tyrosine phosphatase (PTPase) is considered to be an important modulator in the desensitization of insulin receptor function(16) . Thus, in this study, we investigated whether the activities of PKC and PTPase were altered in HIRc cells cultured in high glucose, whether they led to a dysfunction in insulin receptors, and if so, whether thiazolidine derivatives prevented these processes in high glucose-induced insulin resistance.
Figure 1:
Insulin-stimulated phosphorylation in
NG and HG cells with or without pioglitazone. We performed Western
blotting of phosphotyrosine from the insulin-stimulated cells. Cellular
proteins were immunoprecipitated with PY20. Bound proteins were
resolved by SDS-PAGE and transferred to an Immobilon membrane
(Millipore) using standard procedures. Immunoblotting was carried out
using
PY20, and blots were visualized using anti-rabbit antiserum
and enhanced chemiluminescence (ECL, Amersham
Corp.).
Figure 2:
Protein-tyrosine phosphatase (PTPase)
activities in NG and HG cells. A, PTPase activities were
detected using P phosphorylated insulin receptors in NG
and HG cells. Autoradiogram of dephosphorylated insulin receptors
incubated with the cytosolic fraction obtained from NG and HG cells. No
treatment (lanes1 and 9), NG (lanes2 and 8), HG (lanes3-7), 0.1 µM pioglitazone (AD; lanes4 and 6), 1 µM H7 (lane5). B, PTPase activities
determined by an immunoenzymic assay in NG and HG cells PTPase
activities were determined using an immunoenzymic assay system that
included a phosphorylated insulin receptor (150 fmol) and a monoclonal
phosphotyrosine antibody (
PY20). PTPase activities time- and
dose-dependently increased within the range between 15 and 45 min and
between 10 and 50 µg of protein, respectively (data not shown).
Dephosphorylation was initiated by adding 20 µg of protein of the
cytosolic and particulate fractions from NG and HG cells. After a
30-min incubation at 22 °C, PTPase activities were measured using
PY20 and peroxidase-conjugated anti-rabbit antibody. The receptor
dephosphorylation was calculated as the ratio of the A
of dephosphorylated receptors (measured by incubating insulin
receptors in the presence of phosphatases) over that of control
receptors (incubated in the absence of phosphatases). Each value is
presented as the means of five separate experiments in quadruplicate
(± S.E.). *, p < 0.01 versus PTPase
activity in the other groups, and *, p < 0.01 versus PTPase activity compared with the normal glucose group by
Scheffe's multiple comparison test.
Figure 3: Time course of changes in PTPase activities in HG cells. Cytosolic and particulate PTPase activities were measured after the indicated time periods. *, p < 0.05;** p < 0.01 versus cytosolic PTPase activity in NG cells (time 0); #, p < 0.05 versus particulate PTPase activity in NG cells (time 0). Each point is presented as the mean ± S.E. of three to five experiments. Inset shows time course within 6 h.
PTP1B is considered to be involved in insulin action. As shown in Fig. 4, the amount of PTP1B in the cytosolic fraction of HG cells was significantly elevated, which was consistent with the increased PTPase activity in the cytosolic fraction in HG cells. On the other hand, the change in the PTP1B content in the particulate fraction was not significant between NG and HG cells, even though PTP1B was dominantly localized in the particulate fraction(28) . Furthermore, the cells exposed to pioglitazone completely inhibited the increase in the PTP1B content in the cytosolic fractions of HG cells. To investigate how much PTP1B contributes to PTPase activities in HG cells, we studied the effects of anti-PTP1B antibody on PTPase activities. As shown in Fig. 5, adding anti-PTP1B (125 µg/tube) to the assay system efficiently inhibited the increment of cytosolic PTPase activity in HG cells. We observed a similar tendency in the particulate fraction, but it was not statistically significant.
Figure 4:
Western
blotting of PTP1B in the cytosolic and particulate fractions from NG
and HG cells. A, samples from NG and HG cells were separated
into cytosolic and particulate fractions, and PTP1B from both fractions
(40 and 20 µg of cytosolic and particulate fractions, respectively)
was immunoprecipitated using anti-PTP1B antibody. The
immunoprecipitated proteins were resolved by SDS-PAGE and transferred
to an Immobilon membrane (Millipore) by standard procedures.
Immunoblotting was carried out using anti-PTP1B antibody, and proteins
were visualized by means of enhanced chemiluminescence (ECL kit,
Amersham Corp.) using a anti-rabbit antiserum. B, PTP1B
content from each fraction was quantified by densitometric scanning.
, NG cells;
, HG cells; &cjs2108;, HG cells with 0.1
µM pioglitazone; &cjs2108;, NG cells with 0.1 µM pioglitazone. Each column is presented as mean ± S.E. of
three to five experiments. Statistically significance was determined by
unpaired Student's t test.
Figure 5:
Effects of PTP1B antibody in PTPase
activities in HG cells. PTPase activities were measured in the presence
of either 125 µg of PTP1B antibody or preimmune IgG using the
immunoenzymic assay. Each column is presented as the mean ± S.E.
of three to four separate experiments. *, p < 0.01 versus other high glucose groups, and
, p <
0.01 versus normal glucose groups by Scheffe's multiple
comparison test.
Figure 6:
Insulin-stimulation of AIB uptake in NG
(), HG (
), HG cells co-incubated with 0.1 µM pioglitazone (
), and HG cells co-incubated with 4.5
µM troglitazone (
). After an incubation for 3 h, the
uptake of [
H]AIB (8 µM, 0.5
µCi/tube) was determined in the cells over 12 min. A,
insulin dose-response curve. B, absolute uptake rate (pmol/mg
of protein/12 min) at basal (
) and maximal stimulation in 167
nM insulin (
). #, p < 0.01 basal uptake in
HG cells compared with that under other conditions;
, p < 0.01 maximal uptake in HG cells compared with that under
other conditions by Scheffe's multiple comparison
test.
We studied hyperglycemia-induced insulin resistance by
measuring the in vitro autophosphorylation and the tyrosine
kinase activities of WGA-purified insulin receptors obtained from cells
cultured in HG for 4 days. We found that the relatively chronic
exposure of HIRc cells to high glucose led to impaired
autophosphorylation and tyrosine kinase activity. Furthermore, the
thiazolidine derivative, pioglitazone, completely ameliorated the
receptor dysfunction in HG cells. These effects were caused by 16
mM glucose (32% of the maximal effect at 27 mM) and
were within 24 h (40% of the maximal effect at 4-day high glucose
culture), indicating that this glucose effect depended on both the
incubation period and D-glucose concentration in the media.
Furthermore, insulin receptor kinase activity was not affected by
co-incubating the cells with 5.5 mMD-glucose and
21.5 mM raffinose as a high osmotic control group, suggesting
that the effect was specific for D-glucose
metabolism(14) . In this study, we further confirmed that the in vivo insulin-stimulated phosphorylation of pp185 and
subunit of the insulin receptor was also impaired in HG cells and that
pioglitazone completely ameliorated these abnormalities in HG cells.
Concerning the molecular mechanism for dysfunction of insulin
receptor kinase in HG cells, a short-term, high glucose concentration
(within 24 h) may activate PKC (29) and then impair the
tyrosine kinase activity of insulin receptors as found in rat
adipocytes(12) . Furthermore, Berti et al.(30) have reported that high glucose medium induces
insulin receptor dysfunction via the activation of PKC in Rat 1
fibroblasts that overexpress human insulin receptors. They observed the
effect within 30 min via the translocation of several PKC isoforms
(,
,
, and
) to the plasma membrane within 1 min.
This is in accordance with the direct activation of PKC by phorbol
ester leading to the serine and threonine phosphorylation of insulin
receptors, resulting in the impairment of receptor kinase
activity(20, 31) . Furthermore, their results indicate
that troglitazone, another thiazolidine derivative, prevents the rapid
deactivation of insulin receptor kinase in HIRc cells by preventing PKC
activation in HG cells (15) . However, we could not find
increased PKC activities in HG cells nor a pioglitazone effect upon PKC
activities using two methods. Furthermore, we did not see any change in
PKC activities in the presence of 4.5 µM troglitazone
(data not shown). Consistent with these results, an orthophosphate
labeling study showed that the serine and threonine residues in the
insulin receptor did not increase by exposing cells to high glucose
(data not shown). Furthermore, co-incubating the cells with 1
µM H7, a potent PKC inhibitor, failed to prevent the
impairment of insulin receptor kinase. Although the possibility that
the early transient activation of PKC (at most 15% increase in PDBu
binding after 30 min-high glucose culture) has some effect on insulin
receptor kinase afterward cannot be discarded, there was no persistent
activation of PKC in HG cells.
PTPase is considered to be an important regulator of insulin action. Furthermore, its activation may produce insulin resistance(16) , and abnormal regulation of PTPase has been reported in animals and patients resistant to insulin(32, 33, 34) . In this study, we found that PTPase activity in the cytosolic fraction in HG cells was increased 2-fold compared with that of NG cells using two methods. This activation of cytosolic PTPase occurred within 1 h, and reached a plateau at 72 h. On the other hand, we reported that no changes in receptor kinase occurred within 1 h, and receptor kinase defects were observed only after 24 h (40% of the maximal effect)(14) . These results suggested that the activation of PTPase occurs prior to the impairment of insulin receptor kinase. The amount of PTP1B, a PTPase involved in insulin action(16), was significantly increased in the cytosolic fraction in HG cells. Furthermore, the presence of anti-PTP1B antibody in the PTPase assay system significantly inhibited the increased cytosolic PTPase activities in HG cells, but preimmune IgG did not. On the other hand, anti-PTP1B antibody had little effect on PTPase activities in NG cells. These results suggested that PTP1B does not significantly contribute to PTPase activities in NG cells. Although it is unclear whether other PTPases are also activated in HG cells, it is evident that at least the activity and content of PTP1B was increased in the cells exposed to high glucose. Furthermore, one of the thiazolidine derivatives, pioglitazone normalized the increased PTPase activities with a coincident decrease in level of PTP1B in the cytosolic fractions of HG cells. Therefore, cytosolic PTPase activity may be stimulated in the presence of high glucose, resulting in the decreased autophosphorylation of the insulin receptor and its kinase activity, and these agents may reverse these abnormal insulin receptor functions. Concerning the regulation of PTPase activity in high glucose, the activation of PKC induced by TPA can stimulate PTPase activity in the soluble fraction of human erythroleukemia cells(35) . However, in rat adipocytes, TPA has no effect on PTPase activity. In our study, TPA had no effect on cytosolic PTPase activity in HIRc cells. Furthermore, incubating the cells with 1 µM H7, a potent PKC inhibitor, failed to normalize the increased PTPase activity in HG cells. Although the regulatory mechanisms for gene expression of PTPases are not completely understood, the activation of PKC by TPA, insulin, and insulin-like growth factor I can promote the gene expression of PTP1B(36, 37) . Currently, there is no clear explanation for the mechanism that may be responsible for the stimulation of cytosolic PTP1B activity in HG cells. Further investigations are required to clarify the regulation of PTP1B activity including not only de novo synthesis of PTP1B protein but also its activation, which may be another mechanism of glucose-induced insulin resistance.
In our study, the relatively chronic exposure of
HIRc cells to high glucose condition led to impaired insulin-stimulated
AIB uptake accompanied by a decrease in the level of insulin receptor
kinase and the activation of cytosolic PTPase activity. Pioglitazone
normalized both the increased PTPase activity and the increase in PTP1B
content in the cytosolic fractions of HG cells and ameliorated the
insulin sensitivity on AIB uptake with normalization of the insulin
tyrosine kinase activities. Furthermore, troglitazone, another
thiazolidine derivative also ameliorated insulin resistance in AIB
uptake (Fig. 6). These results indicated that high glucose can
impair insulin signaling via the activation of PTPase, as well as the
insulin stimulation of both autophosphorylation of -subunits of
insulin receptor and phosphorylation of pp185. It is possible that the
regulation of PTPase activity is a crucial step in high glucose-induced
insulin resistance, and thiazolidine derivatives specifically improve
the high glucose-induced desensitization of insulin receptor signaling
via the normalization of PTPase activity.
Although further investigation is required to clarify the exact mechanisms of hyperglycemia-induced insulin resistance, these agents may be useful tools with which to clarify the mechanism of the impaired insulin receptor signaling in diabetes mellitus.