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J. Biol. Chem., Vol. 275, Issue 27, 20880-20886, July 7, 2000

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Selective Attenuation of Metabolic Branch of Insulin Receptor Down-signaling by High Glucose in a Hepatoma Cell Line, HepG2 Cells^*

Koji Nakajima, Keishi Yamauchi, Satoshi Shigematsu, Sachiko Ikeo, Mitsuhisa Komatsu, Toru Aizawa, and Kiyoshi Hashizume

From the Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan

Received for publication, July 13, 2000, and in revised form, April 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The effects of a high concentration of glucose on the insulin receptor-down signaling were investigated in human hepatoma (HepG2) cells in vitro to delineate the molecular mechanism of insulin resistance under glucose toxicity. Treatment of the cells with high concentrations of glucose (15-33 mM) caused phosphorylation of serine residues of the insulin receptor substrate 1 (IRS-1), leading to reduced electrophoretic mobility of it. The phosphorylation of IRS-1 with high glucose treatment was blocked only by protein kinase C (PKC) inhibitors. The high glucose treatment attenuated insulin-induced association of IRS-1 and phosphatidylinositol 3-kinase and insulin-stimulated phosphorylation of Akt. A metabolic effect of insulin, stimulation of glycogen synthesis, was also inhibited by the treatment. In contrast, insulin-induced association of Shc and Grb2 was not inhibited. Treatment of the cells with high glucose promoted the translocation of PKC and PKC from the cytosol to the plasma membrane but not that of other PKC isoforms. Finally, PKC and PKC directly phosphorylated IRS-1 under cell-free conditions. We conclude that a high concentration of glucose causes phosphorylation of IRS-1, leading to selective attenuation of metabolic signaling of insulin. PKC and PKC are involved in the down-regulation of insulin signaling, and they may lie in a pathway regulating the phosphorylation of IRS-1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In diabetes mellitus, chronic hyperglycemia develops as a consequence of decreased insulin action due to impaired insulin secretion and insulin resistance (1). Once chronic hyperglycemia is established, however, hyperglycemia in turn damages pancreatic beta cell and aggravates insulin resistance, forming a vicious circle that is collectively called glucose toxicity (2). The pathophysiological importance of such self-perpetuating effects of hyperglycemia is well recognized; however, the underlying mechanism of glucose toxicity is still largely unknown. In the important insulin target tissues, the adipose tissue and skeletal muscle, previous studies have suggested that an increased flux through the hexosamine biosynthetic pathway eventually suppresses membrane expression of the GLUT4 glucose transporter, which is responsible for increased insulin resistance (3). Indeed, by continuous exposure of the cell to glucosamine per se, insulin resistance could be created (4). More recently, however, Hrekso et al. (5) found that glucosamine-induced insulin resistance may result from intracellular ATP-depletion. In another insulin target tissue, the liver, glucose transport across the plasma membrane cannot be a rate-limiting step of glucose metabolism due to abundant membrane expression of the GLUT2 glucose transporter with a high K_m value (6), irrespective of the presence or absence of insulin. In the hepatocyte, therefore, insulin resistance results from impaired insulin receptor down-signaling. Nevertheless, the molecular basis of hyperglycemia-induced impairment of insulin receptor-down signaling in the hepatocyte has mostly been unknown. On the other hand, the pathological importance of hepatic insulin resistance in glucose homeostasis has recently been reappraised (7). With these facts as background, we systematically examined in the present study how hyperglycemia disturbs insulin receptor down-signaling in the hepatocyte.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- Human recombinant insulin was obtained from Eli Lilly Co. (Indianapolis, IN). D-Glucose, sucrose, PMA,¹ phosphatidylserine, diacylglycerol, and wortmannin were purchased form Nakalai Tesque (Kyoto, Japan), H7 and H89 from Seikagaku Kogyo (Tokyo, Japan), and PD98059 and GF109203X from Calbiochem (La Jolla, CA). FK-366, an aldose reductase inhibitor, was a gift from Fujisawa Pharmaceutical Co. (Osaka, Japan). All culture media, the serum, and the reagents for the cell culture were purchased from Life Technologies, Inc. Anti-phosphotyrosine monoclonal antibody (4G10), a carboxyl-terminal anti-IRS-1 polyclonal antibody (IRS-1), anti-p85 polyclonal antibody (p85), and rat recombinant IRS-1 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY); anti-Shc monoclonal antibody (Shc), anti-Shc polyclonal antibody (Shc-poly), anti-Grb2 monoclonal antibody (Grb2), and anti-PKC monoclonal antibodies against PKC, PKC, PKC PKC, and PKC were from Transduction Laboratories (Lexington, KY); anti-Akt polyclonal antibody (Akt) and anti-phospho specific Akt polyclonal antibody (pAkt), which recognizes threonine-308 phosphorylation, were from New England Biolabs (Beverly, MA). Recombinant human PKC and PKC were obtained from BIOMOL (Plymouth Meeting, PA).

Cell Culture-- HepG2 cells, a human hepatoma cell line, were obtained from Riken Cell Bank (Tokyo, Japan) and maintained in Dulbecco's modified Eagle's medium plus 10% heat-inactivated fetal bovine serum and 10% heat-inactivated calf serum. Two days after plating, the medium was changed to F-12K containing 7 mM D-glucose and 10% fetal bovine serum, and the culture was continued for 2 more days. The cells were then cultured in serum-free F-12K medium containing 7 mM D-glucose for 12 h. Subsequently, the experimental treatment was carried out for the indicated periods with various concentrations of D-glucose. PKC inhibitors (H7 and GF109203X), a PI 3-kinase inhibitor (wortmannin), a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor (PD98059), a protein kinase A inhibitor (H89), or an aldose reductase inhibitor (FK-366) was included in some experiments as needed. In some experiments, the cells were exposed to 100 nM insulin for 10 min after 6 h of incubation with 33 mM D-glucose.

Western Blot Analysis-- Whole cell extracts were prepared by exposing the cells to 200 µl of lysis buffer (20 mM Hepes, pH 7.4, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5 µM pepstatin) for 1 h at 4 °C as described (8). The cell extracts were then subjected to Western blotting using antibodies such as 4G10, IRS-1, p85, Shc, Grb2, Akt, pAkt, and PKCs, visualized with the ECL detection system (Amersham Pharmacia Biotech), and quantified by densitometry.

Immunoprecipitations-- For immunoprecipitation, the whole cell extracts were 5× diluted with the lysis buffer without Triton X-100 and incubated with 4 µg of IRS-1 or Shc-poly for 2 h at 4 °C. Fifty microliters of protein A-agarose was then added, and incubation was carried out for another 1 h at 4 °C. The resulting immunoprecipitates were then subjected to SDS-polyacrylamide gel electrophoresis and Western blotted as described above.

Phosphoamino Acid Analysis-- ³²P-Labeled phosphoamino acid analysis was performed as described previously (9). In brief, proteins were eluted from the gel using electrophoresis and were subjected to acid hydrolysis in 6 M HCl for 2 h at 110 °C. Phosphoamino acids were separated on the thin layer cellulose plates by electrophoresis at pH 3.5. As standard phosphoamino acids, phosphoserine, phosphothreonine, and phosphotyrosine were added to all radioactive samples analyzed. The ³²P-labeled phosphoamino acids were visualized by autoradiography and quantified by densitometry.

Assay of Glycogen Synthesis-- The accumulation of glycogen was determined as described previously (10). After serum deprivation and treatment with 7 or 33 mM glucose for 6 h, the cells were washed three times with serum-free F-12K medium and incubated with 7 mM glucose and D-[U-¹⁴C]glucose (2 µCi/well) in the presence or absence of insulin for 2 h. Then, the cells were washed three times with ice-cold phosphate-buffered saline and solubilized in 30% KOH, and the radioactivity was counted as an index of incorporation of labeled glucose into glycogen.

Subcellular Fractionation-- The cytosolic and membrane fractions were obtained by ultracentrifugation as described previously (11) with minor modifications. Briefly, HepG2 cells were removed from the plates using a rubber policeman and homogenized in ice-cold buffer containing 20 mM Tris-HCl, pH 7.6, 10 mM EDTA, 10 mM EGTA, 1 mM NaHCO₃, 5 mM MgCl₂, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5 µM pepstatin. Then, the homogenate was centrifuged at 100,000 × g for 60 min at 4 °C, and the supernatant was obtained as cytosolic fraction. The pellet was washed with the same buffer, resuspended in the buffer containing 1% Triton X-100, and recentrifuged at 100,000 × g for 60 min at 4 °C. The resultant supernatant was collected as the membrane fraction. To the 100-µl cytosolic or membrane fractions, 50 µl of 3× Laemmli buffer was added; this mixture was boiled for 5 min and Western blotted after electrophoresis. Protein was measured with the Pierce BCA protein assay kit (Rockford, IL) using bovine serum albumin as a standard.

Phosphorylation of IRS-1 by Recombinant PKC under Cell-free Conditions-- Immunoprecipitated IRS-1 was prepared from ten 100-mm dishes of HepG2 cells according to the immunoprecipitation protocol as described above, except that protein phosphatase inhibitors, such as sodium fluoride, sodium pyrophosphate, and sodium orthovanadate, were not included here. Phosphorylation of IRS-1 by PKCs was quantified by the incorporation of ³²P from [r-³²P]ATP into IRS-1 (12). One microgram of immunoprecipitated IRS-1 or 1 µg of recombinant rat IRS-1 was incubated with 1 µg of PKC or PKC in the absence or presence of 200 µg/ml phosphatidylserine and 20 µg/ml diacylglycerol in Hepes buffer containing 20 mM Hepes, pH 7.4, 0.1 mM EGTA, and 0.03% Triton X-100. Phosphorylation was initiated by incubation with 10 mM MgCl₂ and 100 µM [-P³²]ATP (3 µCi/nmol) at 30 °C. The reaction was terminated 3 min later by addition of 10 mM ATP and 30 mM EDTA for 3 min. The resulting samples were then subjected to SDS-polyacrylamide gel electrophoresis, and the autoradiography was taken.

For mobility shift assay, recombinant IRS-1 incubated with PKC or PKC as described above except using nonradioactive ATP. The resulting samples were then subjected to Western blotting using IRS-1 antibody.

Statistical Analysis-- Statistical significance of difference was evaluated by analysis of variance with Fisher's protected least significance difference (StatView, SAS Institute Inc., Cary, NC). p < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of a High Concentration of D-Glucose on the Electrophoretic Mobility of IRS-1 and Shc-- As shown in Fig. 1A, electrophoretic mobility of IRS-1 was decreased by the treatment of the cells with high (33 mM) D-glucose for 6, 12, and 24 h. The shift was not observed in the lysate prepared from the cells treated with 33 mM D-glucose for 48 h (Fig. 1A). The glucose effect on the mobility shift of IRS-1 was clearly concentration dependent (Fig. 1B). One hundred nanomolar insulin reduced electrophoretic mobility of IRS-1 in the presence of 7 mM D-glucose (Fig. 1B, second lane from left).

View larger version (41K): [in this window] [in a new window]   Fig. 1.   High D-glucose decreases the electrophoretic mobility of IRS-1 in a time- and concentration-dependent manner. HepG2 cells were incubated with 33 mM D-glucose for various durations (A) or with various concentrations of D-glucose for 6 h (B). Whole cell lysates were prepared as described under "Experimental Procedures" and immunoblotted with an antibody directed against IRS-1. A decrease in IRS-1 mobility was detected in the cells exposed to 33 mM D-glucose for 6-24 h (A). The glucose effect was clearly concentration-dependent (B).

To rule out the possibility that the glucose effect on the electrophoretic mobility shift of IRS-1 is due to increased medium osmolarity, the effects of sucrose and glucose were compared in parallel. Twenty-six millimolar sucrose in the presence of 7 mM D-glucose did not at all affect the electrophoretic mobility of IRS-1, and 33 mM D-glucose significantly reduced the mobility of IRS-1 (Fig. 2A). The result implies that the electrophoretic mobility shift of IRS-1 caused by high D-glucose is due to specific modification of IRS-1 by the sugar, and the hyperosmolar effect was ruled out.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Effects of high concentration of D-glucose, sucrose, and insulin on the electrophoretic mobility of IRS-1. HepG2 cells were incubated with 7 mM D-glucose, 7 mM D-glucose + 23 mM sucrose, or 23 mM D-glucose for 6 h and were chased with 100 nM insulin for 5 min. Whole cell lysates were prepared as described under "Experimental Procedures" and immunoblotted with an antibody directed against IRS-1 (A) or Shc (B).

In marked contrast to IRS-1, the electrophoretic mobility of Shc was not at all altered by the high glucose treatment of the cell (Fig. 2B).

Effect of Protein Kinase Inhibitors on the Electrophoretic Mobility Shift of IRS-1 and Phosphoamino Acid Analysis of IRS-1-- We considered that high glucose-induced reduction of electrophoretic mobility of IRS-1 may be due to phosphorylation of IRS-1 molecule, as in the case of insulin stimulation. It is well established that insulin stimulation reduces electrophoretic mobility of IRS-1 due to phosphorylation of IRS-1 (13). Accordingly, the possibility of high glucose-induced phosphorylation of IRS-1 was explored in the following experiments.

First, involvement of protein kinases in the process of glucose-induced reduction of the electrophoretic mobility of IRS-1 was examined by the use of inhibitors of various protein kinases and an inhibitor of aldose reductase at effective concentrations (10, 14-17). As shown in Fig. 3, 33 mM D-glucose-induced reduction of electrophoretic mobility of IRS-1 was inhibited by appropriate concentration of H7 (10 µM) and GF109203X (200 nM) (PKC inhibitors). However, a low concentration (10 nM) of GF109203X, which inhibits conventional but not novel PKC, was without effect. High glucose-induced electrophoretic mobility shift of IRS-1 was not at all affected by wortmannin (PI 3-kinase inhibitor), PD98059 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor), or H89 (protein kinase A inhibitor). An aldose reductase inhibitor (FK-366) was also without effect on the high glucose-induced mobility shift of IRS-1, implying that intracellular accumulation of polyol is not responsible for it.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Effects of inhibitors of protein kinases and aldose reductase on high glucose-induced reduction of the electrophoretic mobility of IRS-1. HepG2 cells were incubated with 7 or 33 mM D-glucose in the presence or absence of GF109203X, H7 (PKC inhibitors), H89 (protein kinase A inhibitor), wortmannin (PI 3-kinase inhibitor), PD98059 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor), and FK-366 (aldose reductase inhibitor) for 6 h. Whole cell lysates were prepared as described under "Experimental Procedures" and immunoblotted with an antibody directed against IRS-1. Neither H89 (100 nM), wortmannin (10 nM), PD98059 (10 µM), nor FK-366 (100 µM) affect high glucose-induced IRS-1 mobility shift. On the other hand, the PKC inhibitors H7 (10 µM) and GF109203X (200 nM) eliminated high glucose-induced electrophoretic retardation of IRS-1.

As a next step, phosphorylation of IRS-1 by high glucose was directly analyzed. As shown in Fig. 4, serine and threonine residues of IRS-1 were phosphorylated by the treatment of the cells with 33 mM D-glucose for 6 h. In order to confirm the evidence, we examined the effect of PMA that is expected to phosphorylate serine and threonine residues of IRS-1 through activation of PKC. A significant increase in phosphorylation of these amino acids was observed after treatment with 100 nM PMA (Fig. 4). Only a trace amount of phosphoamino acids was detected in IRS-1 derived from the cells incubated with a basal concentration (7 mM) of D-glucose (Fig. 4).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Phosphoamino acid analysis of IRS-1 from 32P-labeled HepG2 cells. PMA and high glucose phosphorylated serine and threonine residues of IRS-1. The cells were labeled with [32P]orthophosphate and exposed to 100 nM PMA or 33 mM D-glucose for 6 h. Phosphoamino acids were separated by thin layer electrophoresis, and 32P-labeled amino acids were visualized by autoradiography.

Effects of High Glucose on Insulin Stimulation of Tyrosine Phosphorylation of IRS-1 and Shc and Their Interaction with the Secondary Signaling Molecules, p85 and Grb2, Respectively-- In the cells treated with 33 mM D-glucose for 6 h, insulin-induced tyrosine phosphorylation of IRS-1 took place normally, as in the control cells (Fig. 5A). However, insulin-induced association of IRS-1 and p85 was severely inhibited in the glucose-treated cells (Fig. 5B). On the other hand, insulin-induced tyrosine phosphorylation of Shc and association of it with Grb2 were both unaffected by the treatment of the cell with high glucose (Fig. 5C).

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Effects of high glucose on insulin-induced phosphorylation of IRS-1 and Shc, and insulin-stimulated association of IRS-1 and Shc with p85 and Grb2, respectively. Insulin-induced phosphorylation of IRS-1 and Shc were not affected by high glucose (A), whereas high glucose decreased insulin-induced association of IRS-1 and p85 (B) and that of IRS-1 and Grb2 (C). On the other hand, high glucose did not attenuate insulin-induced association of Shc and Grb2 (C). HepG2 cells were incubated with 33 mM D-glucose for 6 h and subsequently stimulated with 100 nM insulin for 5 min. Whole cell lysates were prepared, and IRS-1 and Shc were immunoprecipitated with IRS-1 antibody and Shc antibody, respectively (A). The immunoprecipitates were then immunoblotted with antibodies directed against phosphotyrosine (4G10) (A), p85 (B), and Grb2 (C). Each set of data is a representative of three independent experiments. The densitometry data are presented as relative density compared with the density at the basal (untreated) condition. Values are means ± S.E. NS, not significant. *, p < 0.05; **, p < 0.01.

Effects of High Glucose on Insulin-induced Phosphorylation of Akt and Glycogen Synthesis-- Akt is a key molecule mediating the metabolic effects of insulin in the liver. It lies downstream of PI 3-kinase and facilitates glycogen synthesis by inactivating glycogen synthase kinase-3 (18, 19). As shown in Fig. 6A, the treatment of the cells with a high concentration of glucose clearly blocked the insulin-induced phosphorylation of Akt. As expected from reduced phosphorylation of Akt, the insulin-induced glycogen synthesis was markedly suppressed by the treatment with high glucose (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effects of high glucose on insulin-induced activation of Akt and glycogen synthesis. HepG2 cells were incubated with 33 mM D-glucose for 6 h and subsequently stimulated with 100 nM insulin for 10 min. A, whole cell lysates were immunoblotted with pAkt (IB: pAkt) and Akt (IB: Akt). Insulin stimulation of phosphorylation of Akt was inhibited by high glucose (A, IB: pAkt). This is a representative set of data from three independent experiments. For assay of glycogen synthesis, HepG2 cells were incubated with 7 or 33 mM D-glucose for 6 h followed by 100 nM insulin treatment. D-[U-14C]glucose incorporation into glycogen in the cells (B) was determined as described under "Experimental Procedures." Insulin stimulation of glycogen synthesis was also inhibited by high glucose. This is a representative set of data from three independent experiments. Values are means ± S.E. NS, not significant. *, p < 0.05; **, p < 0.01.

High Glucose-induced Translocation of PKC-- Activation of PKC is expected to induce translocation of it from the cytosol to the plasma membrane (11); therefore, membrane translocation of PKC upon exposure to high glucose was examined. As shown in Fig. 7, treatment of the cells with 33 mM D-glucose promoted membrane translocation of PKC and PKC but not of PKC, PKC, PKC, or PKC.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effects of high glucose on subcellular localization of PKC isoforms. HepG2 cells were incubated with 7 or 33 mM D-glucose for 6 h. Whole cell lysates were prepared, fractionated, and immunoblotted as described under "Experimental Procedures." High glucose caused translocation of PKC and PKC but not of PKC, PKC, PKC, and PKC. This is a representative set of data from three independent experiments.

Direct Phosphorylation of IRS-1 by PKC and PKC under Cell-free Conditions-- Immunoprecipitated IRS-1 of HepG2 cell was incubated with recombinant PKC or PKC. As shown in Fig. 8A, upon addition of diacylgycerol and phosphatidylserine, PKC and PKC phosphorylated themselves as described previously. At the same time, PKC and PKC phosphorylated the immunoprecipitated IRS-1 (Fig. 8A). Recombinant rat IRS-1 was also phosphorylated by the PKCs, as shown in Fig. 8B. PKC phosphorylation of IRS-1 was associated with decreased electrophoretic mobility, as shown in Fig. 8C.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Phosphorylation of IRS-1 by PKC and PKC under cell-free conditions. Immunoprecipitated IRS-1 of HepG2 cells (A) was incubated with recombinant PKC or PKC as described under "Experimental Procedures." PKC and PKC clearly phosphorylated immunoprecipitated IRS-1. Recombinant rat IRS-1 (B) was also phosphorylated by PKC and PKC. Phosphorylation of IRS-1 by PKC or PKC caused electrophoretic mobility shift of IRS-1 (C). Autophosphorylation of PKC and PKC was also demonstrated (A and B). This is a representative set of data from two independent experiments. PS, phosphatidylserine; DAG, diacylglycerol.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Insulin resistance in diabetes, especially in type 2 diabetes, was discovered more than 30 years ago (20), and it is now well established that insulin resistance occurs in all insulin target tissues, such as skeletal muscle, adipose tissue, and the liver (7, 21). Through extensive research on the molecular basis of insulin receptor down-signaling (23, 24), the complexity of intracellular insulin signaling network has been gradually delineated. The two important biological actions of insulin, metabolic and growth-promoting actions, are mediated by activation of the PI 3-kinase (23) and Shc-Grb2 pathways (26), respectively, although there appears to be a certain overlap between the two. IRS-1 is a proximal signaling molecule to all of these, and tyrosine phosphorylation of it by insulin facilitates association of IRS-1 and PI 3-kinase and association of IRS-1 and Shc. This leads to activation of the signaling cascades. Despite such advancement of the research in insulin receptor down-signaling, the locus of glucose toxicity in producing insulin resistance was not clear, especially in the liver. In this study, using HepG2 cells, a human hepatoma cell line, we investigated how hyperglycemia preferentially impairs the metabolic branch of insulin receptor-down signaling.

Although it is ideal to carry out the study using the primary hepatocytes, it is practically impossible to analyze phosphorylation of the signaling molecules using the primary hepatocytes, most likely due to potent phosphatase activity of the hepatocytes under experimental conditions such as those used with the primary cell culture.

We found that long term exposure of the cells to high concentration of glucose results in serine/threonine phosphorylation of IRS-1, most likely through activation of PKC and PKC. The phosphorylation of IRS-1 accompanied selective inhibition of insulin-stimulated association of IRS-1 and p85 (a subunit of PI 3-kinase), and inhibition of phosphorylation of Akt, a key regulator of glycogen synthesis. Also, we demonstrated that high glucose inhibited insulin-induced glycogen synthesis in this study. In sharp contrast, high glucose did not at all attenuate insulin-induced tyrosine phosphorylation of Shc or association of it with Grb2. PI 3-kinase was implicated to stimulate glycogen synthesis and prevent apoptosis through phosphorylation of Akt (27, 28), whereas signaling through Shc-Grb2 pathway activates the Ras-mitogen-activated protein kinase system to enhance cellular proliferation (29). Thus, our data are rather straightforward. High glucose-induced serine/threonine phosphorylation of IRS-1 selectively impairs metabolic down-signaling of insulin primarily by inhibition of IRS-1/p85 association. As far as we are aware, this is the first report showing the molecular mechanism of glucose toxicity in the liver-derived cell. We speculate that similar mechanism is operating in other insulin target tissues, the skeletal muscle and the adipose tissue, leading to insulin resistance. Phosphorylation of IRS-1 by high glucose was previously reported in pancreatic beta cells, but the functional significance of it was unknown (30).

We demonstrated that high glucose induces electrophoretic retardation of IRS-1 due to phosphorylation of the molecule, indicating that a certain protein kinase is activated by high glucose in the cell. Although we did not directly show glucose-responsive protein kinase activity, PKC inhibitors prevented the high glucose-induced electrophoretic mobility shift of IRS-1, strongly suggesting the involvement of PKC. A known activator of PKC, PMA, mimicked the effect of high glucose. Furthermore, translocation of PKC and PKC, but not other isoforms of PKC, from cytosol to the plasma membrane was found upon exposure to high glucose. A low concentration of the PKC inhibitor (10 nM GF109203X), which selectively inhibits conventional PKC (31), did not attenuate high glucose-induced IRS-1 mobility shift. But a high concentration of GF109203X (200 nM), which inhibits both conventional and novel PKCs (32), reduced the mobility shift. These results suggested that novel PKCs play an important role for IRS-1 phosphorylation induced by high concentration of glucose. The mechanism of activation of novel PKCs as PKC and PKC by high glucose may be due to de novo synthesis of diacylglycerol (33). As PKCs activate many intracellular protein kinases, such as Raf-1 kinase (34), we examined whether PKC directly phosphorylates IRS-1 or phosphorylates it via activation of other kinase(s). We showed that PKC and PKC directly phosphorylate IRS-1 under the cell-free conditions and decreased its electrophoretic mobility. Thus, we provided a strong evidence in support of the idea that IRS-1 is phosphorylated by PKC in the cells treated with high glucose, although we did not directly demonstrate it.

We observed that IRS-1-PI 3-kinase system is selectively attenuated by high glucose. In other words, signaling for the growth-promoting effect of insulin remained intact with ambient high glucose. Because insulin secretion by the pancreatic beta cells is mainly regulated by plasma glucose concentration, a combination of mild hyperinsulinemia and hyperglycemia is common in type 2 diabetes. Provided that growth-promoting action of insulin is not impaired under that circumstance, hyperinsulinemia will excessively stimulate cell proliferation. Accordingly, we speculate that selective attenuation of metabolic branch of insulin signaling by glucose toxicity contributes to the development and progression of diabetic vascular complications.

Tumor necrosis factor  (TNF) is known to be one of the mediators of insulin resistance (22). Treatment of cultured adipocytes with TNF induced serine phosphorylation of IRS-1, and the phosphorylated IRS-1 in this setting acted as an inhibitor of the insulin-receptor tyrosine kinase activity in vitro (25). Although we did not measure the concentration of TNF in the medium, the addition of TNF antibody to the culture media at a concentration sufficient to neutralize 10 nM TNF did not at all block the observed high glucose effects in HepG2 cells (data not shown). Also, high glucose did not decrease insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 5A). In addition, overproduction of TNF by the hepatocyte upon exposure to high glucose has not been reported. Therefore, we consider that involvement of TNF in the in the process of hepatic glucose toxicity is most unlikely.

Increased intracellular hexosamine, through impairment of membrane translocation of GLUT4 glucose transporter, has been claimed to be responsible for insulin resistance in the primary adipocytes, 3T3L adipocytes, and the skeletal muscle. However, we think this is not the case in our experiments, in which insulin resistance was studied in the liver-derived cells. First, in contrast to the adipocytes and skeletal muscles, glucose transport at the membrane is not the rate-limiting step of glucose metabolism. Second, we did not see retardation in electrophoretic mobility of IRS-1 in 3T3L1 adipocytes (data not shown).

In summary, we demonstrated that a high concentration of glucose selectively attenuates the metabolic branch of insulin receptor down-signaling in a hepatoma cell line, HepG2 cells. Highly selective alteration in phosphorylation status and functional coupling of the signaling molecules involved in the metabolic action of insulin were shown. We consider that a similar, if not identical, mechanism may be operating in the liver of the patients with diabetes in whom hepatic insulin resistance is one of the important contributing factors for sustained hyperglycemia.

	ACKNOWLEDGEMENT

FK-366, an aldose reductase inhibitor, was a generous gift from Fujisawa Pharmaceutical (Osaka, Japan).

	FOOTNOTES

^* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to K. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-263-37-2686; Fax: 81-263-37-2710; E-mail: keishi@hsp.md.shinshu-u.ac.jp.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M905410199

	ABBREVIATIONS

The abbreviations used are: PMA, phorbol 12-myristate-13-acetate; IRS-1, insulin receptor substrate 1; PKC, protein kinase C; PI 3-kinase, phosphatidylinositol 3-kinase; TNF, tumor necrosis factor .

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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