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Am J Physiol Gastrointest Liver Physiol 291: G744-G750, 2006. First published June 15, 2006; doi:10.1152/ajpgi.00551.2005
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HORMONES AND SIGNALING

EGF-induced inhibition of glucose transport is mediated by PKC and MAPK signal pathways in primary cultured chicken hepatocytes

Min Young Lee,1 Soo Hyun Park,1 Yun Jung Lee,1 Jung Sun Heo,1 Jang Hern Lee,2 and Ho Jae Han1

1Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju; and 2College of Veterinary Medicine, Seoul National University, Seoul, Korea

Submitted 6 December 2005 ; accepted in final form 27 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
EGF is a regulator of a wide variety of processes in various cell systems. Hepatocytes are important sites in the body's metabolism and function. Glucose transporter 2 (GLUT2) is a major transporter that is expressed strongly in hepatocytes. Therefore, this study examined the effect of EGF on GLUT2 and its related signal cascades in primary cultured chicken hepatocytes. EGF decreased [3H]deoxyglucose uptake in a dose- and time-dependent manner (>10 ng/ml, 2 h). AG-1478 (an EGF receptor antagonist) and genistein and herbimycin A (tyrosine kinase inhibitors) blocked the EGF-induced decrease in [3H]deoxyglucose uptake, which correlated with the GLUT2 expression level. In addition, the EGF-induced decrease in GLUT2 protein expression was inhibited by staurosporine, H-7, or bisindolylmaleimide I (PKC inhibitors), PD-98059 (a MEK inhibitor), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor), suggesting a role of both PKC and MAPKs (p44/42 MAPK, p38 MAPK, and JNK). In particular, EGF increased the translocation of PKC isoforms (PKC-{alpha}, -beta1, -{gamma}, -{delta}, and -{zeta}) from the cytosol to the membrane fraction and increased the activation of p44/42 MAPK, p38 MAPK, and JNK. Moreover, PKC inhibitors blocked the EGF-induced phosphorylation of three MAPKs. In conclusion, EGF decreases the GLUT2 expression level via the PKC-MAPK signal cascade in chicken hepatocytes.

epidermal growth factor; glucose transporter 2; protein kinase C; mitogen-activated protein kinases

GLUCOSE is a major fuel for energy metabolism in hepatocytes and is transported into hepatocytes via glucose transporters (GLUTs). Facilitative GLUT2 is the major subtype that is expressed in hepatocytes (6, 20, 26, 51). Altered transport of glucose into hepatocytes may be another factor that might help understanding the physiological action of EGF. In chickens and mammals, D-glucose uses the GLUT2 mechanism to cross the basolateral membrane (9). EGF is a potent mitogen that has been shown to promote DNA synthesis and transcription of RNA leading to protein synthesis (9, 18). EGF has also been shown to regulate several transporters such as GLUTs and glycylsarcosine transporters (19, 29). EGF is a key regulatory polypeptide in the function of hepatocytes that are rich in EGF receptors (EGFRs) (21, 23, 24, 43).

EGF exerts its actions by binding to its receptor with a tyrosine kinase residue (14). The activation of the EGFR may trigger the phosphatidylinositol pathway such as through the activation of PKC and inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] as well as by increasing the intracellular cytoplasmic calcium concentration. It is well known that MAPKs are activated in response to external stimuli in many cell types and play a key role in many signal transduction pathways. ERKs are activated by the phosphorylation of threonine and tyrosine residues (34). In addition, several reports have suggested that other MAPK cascades also drive specific cell responses to extracellular stimuli, including p38 and JNK, which can affect the function of hepatocytes (31, 50). These reports also suggested that EGF stimulates MAPK cascades in chicken hepatocytes. Recent reports have demonstrated that EGF activates p44/42 MAPK, p38 MAPK, or JNK/stress-activated protein kinase (SAPK) kinase in hepatocytes. However, the involvement of MAPKs in the EGF-induced alteration of GLUTs in chicken hepatocytes has not been determined.

Chickens have many differences in their lipid, glucose, and glycogen metabolism as well as differences in the regulation of hormones compared with mammals (35). Studies on the metabolism in chicken hepatocytes compared with those of mammals are quite interesting. A primary culture of hepatocytes has been used for many biophysiological studies on liver function because a primary culture of hepatocytes retains many of the liver-specific functions and responds to various hormones by the expression of liver-specific functions. The primary chicken hepatocytes culture system utilized in this study has also been recognized to retain in vitro the differentiated phenotype typical of the liver, including albumin expression (17), cytochrome P-450 1A induction (17), tyrosine aminotransferase expression (39), and ascorbate recycling (40). Moreover, chickens are characterized by a blood glucose level that is twice as high as that in most mammals (1). The cellular glucose level is modulated by facilitative GLUTs. Among the many isoforms of the GLUT family, GLUT2 is expressed predominantly in the liver of chickens and mammals, which can be regulated by phloretin (25, 48) and can play a crucial role in chicken whole body glucose homeostasis. Therefore, this study examined the effect of EGF on GLUT2 activity and its related signaling pathways in primary cultured chicken hepatocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Two-week-old white Leghorn male chickens were obtained from Dae Han Experimental Animal (Chungju, Korea). All the procedures for animal management followed the standard operation protocols of Seoul National University. The appropriate management of experimental samples and quality control of the laboratory facility and equipment were maintained. The class IV collagenase and soybean trypsin inhibitors were purchased from Life Technologies (Grand Island, NY). Fetal bovine serum was purchased from Biowhittaker (Walkersville, MD). EGF, AG-1478, herbimycin A, genistein, PD-98059, SB-203580, and SP-600125 were obtained from Sigma Chemical (St. Louis, MO). Phospho-p44/42 MAPKs, p44/42 MAPKs, phospho-p38 MAPK, and p38 MAPK antibodies were purchased from New England Biolabs (Herts, UK). The antibodies to total EGFR, phospho-EGFR, SAPK/JNK, and GLUT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The liquiscint was obtained from National Diagnostics (Parsippany, NY, USA). Goat anti-rabbit IgG was purchased from Jackson Immunoresearch (West Grove, PA). All other reagents were of the highest purity commercially available.

Primary culture of chicken hepatocytes. Chicken liver cells were prepared and maintained in a monolayer culture as described previously (12). Briefly, chicken hepatocytes were isolated by perfusion with 0.05% collagenase from a liver that had been starved for 3 h. Hepatocytes with >90% viability, as verified by a trypan blue exclusion test, were used for subsequent plating. Hepatocytes were plated (5.0 x 105 cells/60-mm collagen-coated dish) with incubation medium (basal medium Eagle supplemented with essential amino acids) containing 75 U/ml penicillin, 75 U/ml streptomycin, 1 µg/ml insulin, 10–12 M dexamethasone, 5 µg/ml transferrin, 10–8 M thyroxine (T3), and 5% calf serum and incubated for 4 h at 37°C in 5% CO2. The medium was subsequently replaced with fresh incubation medium, and hepatocytes were incubated for a further 20 h to achieve the monolayer culture. The experiments were then carried out.

Uptake experiments. The 2-[3H]deoxyglucose (2-DG) uptake experiments were carried out using a modification of the method described by Henriksen et al. (20). To study the 2-DG uptake, the culture medium was removed by aspiration, and cells were gently washed twice with uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM KH2PO4, 10 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 5 mM L-alanine, 5 µM indomethacin, and 10 mM HEPES-Tris; pH 7.4). After being washed, cells were incubated in uptake buffer containing 1 µCi/ml 2-DG at 37°C for 30 min. At the end of the incubation period, cells were washed three times with ice-cold uptake buffer and dissolved in 1 ml of 0.1% SDS. The level of 2-DG uptake incorporated intracellulary was determined by removing 900 µl of each sample and measuring the radioactivity in a liquid scintillation counter (LS 6500, Beckman Instruments, Fullerton, CA). The remainder of each sample was used to determine the protein level (2). The radioactivity counts in each sample were then normalized with respect to the protein and corrected for time 0 uptake per milligram of protein. All uptake measurements were carried out in triplicate.

Membrane preparation for Western blot analysis. The medium was then removed, and cells were washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF, and 10 µg/ml leupeptin; pH 7.5). The resuspended cells were lysed mechanically on ice by trituration with a 21.1-gauge needle. Lysates were first centrifuged at 1,000 g for 10 min at 4°C. Supernatants were centrifuged at 100,000 g for 1 h at 4°C to prepare the cytosolic and total particulate fractions. The particulate fractions containing the membrane fraction were washed twice and resuspended in buffer A containing 1% Triton X-100. The protein level in each fraction was quantified using the Bradford procedure (2).

RNA isolation and RT-PCR. Total RNA was extracted from the cells using STAT-60, which is a monophasic solution of phenol and guanidine isothiocyanate purchased from Tel-Test (Friendwood, TX). RT was carried out with 3 µg RNA using a RT system kit (AccuPower RT PreMix) with oligo-dT18 primers. Five microliters of RT products were then amplified with a PCR kit (AccuPower PCR PreMix) using the following procedure: denaturation at 94°C for 5 min and 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by a 5-min extension at 72°C. The primers used were as follows: 5'-TGTTCAGCTCCTCCAAGTACC-3' (sense) and 5'-ACAACGAACACATACGGTCC-3' (antisense) for GLUT2 (523 bp). PCR of beta-actin was also performed as a control to confirm the quantity of RNA. RT-PCR products were separated and visualized on 1.2% agarose gels. PCR products were analyzed by calculating the quantitative values of the EGF treatment group relative to the control group and standardizing the data relative to beta-actin.

Western blot analysis. Cell homogenates (20 µg protein) were separated by 10% SDS-PAGE and transferred to nitrocellulose paper. Blots were then washed with H2O, blocked with 5% skim milk powder in Tris-buffered saline-Tween 20 [10 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween-20] for 2 h, and incubated with the primary polyclonal antibody at the dilutions recommended by the supplier. The membrane was then washed, and the primary antibodies were detected with goat anti-rabbit IgG conjugated to horseradish peroxidase. Bands were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech).

Statistical analysis. Results are expressed as means ± SE. All experiments were analyzed by ANOVA. In some experiments, a comparison of the treatment means was made with the control using the Bonnferroni-Dunn test. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Time- and dose-dependent effects of EGF on 2-DG uptake. The time-dependent effect of EGF on 2-DG uptake was determined by exposing chicken hepatocytes to 100 ng/ml EGF for various times (0–24 h). EGF significantly inhibited 2-DG uptake over a 2-h period and exhibited the maximum inhibitory effect between 8 and 12 h (Fig. 1A). There was no significant difference between 8 and 24 h. The dose-dependent effect of EGF on 2-DG uptake was also determined (Fig. 1B). Therefore, chicken hepatocytes were exposed to various concentrations of EGF for 8 h. EGF (≥10 ng/ml) significantly inhibited 2-DG uptake. The maximum effect was observed between 100 ng/ml and 1 µg/ml EGF. Therefore, the following experiments used 100 ng/ml EGF for 8 h. GLUT2 gene and protein expression levels were downregulated in response to EGF (Fig. 1C).


Figure 1
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  		Fig. 1. Time (A) and dose (B) response of EGF on 2-[3H]deoxyglucose (2-DG) uptake. Chicken hepatocytes were treated with EGF (100 ng/ml) for different times (0–24 h) or doses of EGF (1–1,000 ng/ml) before the uptake experiments. Values are represented as means ± SE of 3 independent experiments with triplicate dishes. C, top: glucose transporter 2 (GLUT2) gene (left) and protein (right) expression levels were determined by RT-PCR and Western blot analysis, respectively. Hepatocytes were treated with EGF (100 ng/ml) for 8 h. Genes were 523 bp for GLUT2 and 350 bp for beta-actin. Membrane protein blots were probed with GLUT2- or beta-actin-specific antibodies. Bands represent 50~60 kDa of GLUT2 and 41 kDa of beta-actin, respectively. C, bottom: bars indicating means ± SE of 3 experiments for each condition determined from densitometry relative to beta-actin. *P < 0.05 vs. control (Con).

 
Receptor and tyrosine kinase dependency of EGF on 2-DG uptake. To determine whether the effect of EGF was dependent on EGFR, chicken hepatocytes were treated with AG-1478 (10–5 M) before the EGF treatment. As shown in Fig. 2B, there was no inhibitory effect of EGF. The EGF-induced inhibition of 2-DG uptake was also blocked by either herbimycin A or genistein (10–6 M) (tyrosine kinase inhibitors; Fig. 2A). Indeed, EGF increased phosphorylation of EGFR over 5 min, and its effect was sustained for 8 h (Fig. 2B). These results suggest that the inhibitory effect of EGF on 2-DG uptake requires the activation of the EGFR with the tyrosine kinase residue.


Figure 2
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  		Fig. 2. Effect of the EGF receptor (EGFR) antagonist AG-1478 and tyrosine kinase inhibitors genistein and herbymycin A in EGF-induced 2-DG uptake in chicken hepatocytes. A: chicken hepatocytes were treated with AG-1478 (10–5 M) or with genistein or herbimycin A (10–6 M) for 30 min before 100 ng/ml EGF treatment for 8 h. Values are represented as means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone. B: phosphorylated (p-)EGFR was detected in response to EGF at various time points.

 
Involvement of PKC in the EGF-induced decrease of 2-DG uptake. The relationship between PKC in the EGF-induced decrease in 2-DG uptake was examined. Staurosporine, H-7, or bisindolylmaleimide I (PKC blockers, 10–7 M) blocked the EGF-induced decrease in 2-DG uptake (Fig. 3A). To certify this result, PKC translocation was examined using the pan-PKC antibody. Indeed, the translocation of PKC from the cytosolic to membrane compartment was observed after an 8-h treatment with 100 ng/ml EGF (Fig. 3B). The Western blot analysis of PKC isoforms revealed that PKC-{alpha}, -beta1, -{gamma}, -{delta}, and -{zeta} were translocated from the cytosolic to membrane compartment, suggesting a role of these isoforms in the effect of EGF. The correlation between the EGF-induced decrease in 2-DG uptake and the GLUT2 expression level was examined. Figure 4 shows the EGF-induced decrease of the GLUT2 expression level, which was blocked by AG-1478, herbimycin, staurosporine, and bisindolylmaleimide I, thus supporting the role of PKC.


Figure 3
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  		Fig. 3. Effect of PKC inhibitors on EGF-induced 2-DG uptake and effect of EGF on PKC activation. A: chicken hepatocytes were treated with staurosporine, H-7, or bisindolylmaleimide I (10–7 M) for 30 min before EGF treatment (100 ng/ml) for 8 h. Values are represented as means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone. B: PKC protein levels, which were present in either the cytosolic and membrane fractions, were then detected using Western blot analysis as described in MATERIALS AND METHODS. The arrow indicates the 82-kDa band corresponding to PKC. beta-Actin was used as a control. C: effect of EGF on the expression of PKC isoforms. PKC-{alpha}, -beta1, -{gamma}, -{delta}, and -{zeta} were detected in response to EGF.

 

Figure 4
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  		Fig. 4. Effect of EGFR antagonist, tyrosine kinase inhibitor, and PKC inhibitors on EGF-induced GLUT2 expression. Chicken hepatyctes were treated with AG-1478, herbimycin A, or staurosporine for 30 min before EGF treatment for 8 h. Membrane protein was then subjected to SDS-PAGE and blotted with GLUT2 antibody (top). Bottom: bars indicating the means ± SE of 5 experiments for each condition determined from densitometry relative to beta-actin. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

 
Involvement of MAPKs in the EGF-induced decrease of 2-DG uptake. To determine whether MAPKs are involved in the EGF-induced decrease in 2-DG uptake, chicken hepatocytes were treated with PD-98059 (MEK inhibitor), SB-203580 (p38 MAPK inhibitor), or SP-600125 (SAPK/JNK inhibitor) for 30 min before the EGF treatment. Figure 5A shows that SB-203580, PD-98059, and SP-600125 significantly prevented the EGF-induced decrease in 2-DG uptake (P < 0.05). In addition, EGF enhanced phosphorylation of p44/42 MAPKs, p38 MAPK, and JNK. Maximal peaks appeared at 10 min for p44/42 MAPKs, 30 min for p38 MAPK, and 30 min for JNK after EGF treatment, respectively (Fig. 5B). Total protein levels of these MAPKs by Western blot analysis were similar. As shown in Fig. 5C, AG-1478, herbimycin A, or bisindolylmaleimide I blocked the EGF-induced phosphorylation of p44/42 MAPKs, p38 MAPK, and JNK. This suggests that all the MAPK pathways including p44/42 MAPKs, p38 MAPK, and SAPK/JNK are involved in the EGF-induced decrease in 2-DG uptake. Figure 6 shows that EGF decreased the expression of GLUT2. This effect was blocked by SB-203580, PD-98059, and SP-600125, which supports the role of all MAPKs.


Figure 5
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  		Fig. 5. Effect of MAPK inhibitors on EGF-induced 2-DG uptake and effect of EGF on the activation of MAPKs. A: chicken hepatocytes were treated with PD-98059, SB-203580, or SP-600125 (10–6 M) for 30 min before EGF treatment (100 ng/ml). B: chicken hepatocytes were treated with EGF (100 ng/ml) for different time points (0–90 min). Phosphorylation of p44/42 MAPKs, p38 MAPK, and JNK were detected as described in MATERIALS AND METHODS. C: chicken hepatocytes were treated with AG-1478, herbimycin A, or bisindolymaleimide I for 30 min before EGF treatment (100 ng/ml), and the phosphorylation of p44/42 MAPKs, p38 MAPK, and JNK was then detected. Each example shown is a representative of 3 experiments. Values are represented as means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

 

Figure 6
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  		Fig. 6. Effect of the MEK inhibitor PD-98059, p38 MAPK inhibitor SB-203580, and JNK inhibitor SP-600125 on EGF-induced GLUT2 expression. Chicken hepatoyctes were treated with PD-98059, SB-203580, or SP-600125 for 30 min before EGF treatment for 8 h. Membrane protein was subjected to SDS-PAGE and blotted with GLUT2 antibody (top). Bottom: bars indicating means ± SE of 4 experiments for each condition determined from densitometry relative to beta-actin. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
GLUTs are regulated by various factors including mitogens, oncogenes, cytokines, and growth factors. This study examined the EGF signal transduction pathway that mediates the alteration in GLUT activity in primary cultured chicken hepatocytes. Several lines of evidence have suggested that EGF affects GLUTs in a short time period in various cells (16, 46). However, in the present study, EGF did not affect glucose transport over a short time (<1 h). Unexpectedly, it was found that EGF caused a time-dependent (>2 h) and dose-dependent decrease in GLUT activity. These inhibitory effects of GLUT activity were mediated by the long-term action of EGF via the decrease of mRNA/protein levels in GLUT2, which is the major isoform in chicken hepatocytes (25). We also observed that EGF downregulated GLUT1 expression, which contributed to the inhibitory effect of GLUTs (data not shown). In conflict with these results, EGF stimulates GLUT activity in enterocytes (8). This discrepancy may be due to the difference in species (chickens vs. rabbits) or cell specificity (hepatocytes vs. enterocytes). Avian GLUTs are regulated via different mechanisms from those in mammals. The control of GLUTs by the v-src oncogene in chicken embryo fibroblasts appears to be different from that in rat embryo fibroblasts (49). Moreover, the expression levels of high-affinity GLUT isoforms are ubiquitous in all chicken tissues compared with mammals. This difference can explain the different functional role of GLUT1 and GLUT3 in chickens compared with mammals (47). Although the physiological levels of EGF were defined at 3–10 ng/ml, high EGF concentrations (100, 500, and 1,000 ng/ml) have been used under various experimental conditions and different cell types (5, 36). In this study, the decrease in effective 2-DG uptake appeared at a wide range of EGF concentrations (10, 100, and 1,000 ng/ml). These variations may be due to difference in cell types, marker indexes, or experimental conditions.

EGF signaling is initiated by binding to its receptor. This receptor is activated by autophosphorylation, after which it interacts with a variety of proteins in the signal transduction pathway leading to the inhibition of GLUTs. Hardy et al. (13) reported similar results in that the signaling domains in the carboxy terminus of the EGFR are essential for this receptor to induce glucose transport. This study found that the EGFR-induced decrease in glucose transport requires a phosphotyrosine motif in the EGFR because an EGFR antagonist and tyrosine kinase inhibitors blocked the EGF-induced decrease in glucose transport. This result is also supported by a report showing that hepatocytes have an EGFR with a tyrosine phosphorylation residue (28, 42).

EGF-stimulated tyrosine kinase activity has been shown to activate phospholipase C (PLC)-{gamma} (22, 32). Once activated, this enzyme catalyzes the hydrolysis of phosphatidylinositol in the plasma membrane into diacylglycerol (DAG) and Ins(1,4,5)P3. Signal transduction within the cell then continues down two pathways. DAG activates PKC, and Ins(1,4,5)P3 releases Ca2+ from intracellular stores to increase the Ca2+ concentration. In accordance with these reports, EGF was found to activate the PKC family downstream of the signaling by PLC, and PKC activation is involved in the EGF-induced decrease in 2-DG uptake. These effects of EGF are restricted to chicken hepatocytes but not to rat hepatocytes, because EGF did not stimulate DAG synthesis and the EGF-induced mitogenic effect is PKC independent in rat hepatocytes (4). In the present study, we identified that PKC-{alpha}, -beta1, -{gamma}, -{delta}, and -{zeta} are translocated from the cytosolic to membrane fraction in response to EGF in chicken hepatocytes. However, Marino et al. (27) reported that EGF translocated only PKC-{alpha} and -{epsilon} among the PKC isoforms from the cytosolic to membrane fraction in chicken embryo hepatocytes. The discrepancy may be due to the difference of developmental stage (postnatal vs. prenatal).

Three principal MAPKs were expressed in hepatocytes: p44/42 MAPKs, JNK, and p38 MAPK (30, 45). In this study, EGF increased three MAPKs in a time-dependent manner in chicken hepatocytes. It was interesting that in this study, unlike in other reports, p44/42 MAPKs and JNK have one band instead of two. The reason for this is unclear but may be due to the specificity of species. Surprisingly, all the inhibitors of the three MAPKs blocked the EGF-induced decrease in glucose uptake and GLUT2 expression. These results are in agreement with those of a study in rat hepatocytes in which the effect of EGF was inhibited completely by PD-98059 and partially by SB-203580 (30). A recent report has also revealed the involvement of p38 MAPK in the downregulation of GLUT4 expression (3). Furthermore, this study provided other evidence that SAPK/JNK also is involved in the effect of the EGF-induced decrease in glucose transport. Although the signal pathways between the three MAPKs are different, there may be cross-talk between the three MAPKs. A recent report also demonstrated cross-talk between the MAPKs (41). Of interest is that EGF-induced activation of these MAPKs is involved in the transcriptional regulation of GLUT2. A recent report has suggested that the chronic activation of p38 MAPK, p44/42 MAPKs, and p38 MAPK can induce the alteration of GLUT mRNA levels (7). Other investigators have also demonstrated that the activation of p44/42 MAPKs, p38 MAPK, or JNK is involved in the effect of GLUT1 mRNA levels in various cells (38, 52). Although these reports did not reveal direct evidence, they support our hypothesis that EGF can decrease the level of GLUT2 mRNA via activation of three MAPKs.

The relative importance of PKC in the activation of the p44/42 MAPK pathway has been shown to depend on the agonist and cell types (37, 44). In hepatocytes, PKC-dependent activation of p44/42 MAPKs is involved in ischemic preconditioning (8). Moreover, there is some controversy regarding the role of PKC in the EGF stimulation of the p44/42 MAPK pathway (11, 33). This study demonstrated that PKC plays a role in the EGF-induced activation of p44/42 MAPKs. In addition, evidence was provided to show that PKC activation is also responsible for the EGF-induced stimulation of p38 MAPK and JNK. To our knowledge, this is the first report of a new mechanism showing that EGF induced the phosphorylation of EGFR and caused the activation of PKC, thereby leading the activation of three MAPKs (p38 MAPK, p44/42 MAPKs, and JNK) that are involved in the inhibition of GLUTs in chicken hepatocytes. These results can provide a better understanding of the regulation of EGF in GLUTs and the involvement of various signaling molecules in chicken hepatocytes. In conclusion, EGF inhibited GLUT activity via the PKC-MAPK signal cascade in primary cultured chicken hepatocytes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the Korean Rural Development Administration (BioGreen 21 Program), and the authors acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development through the Brain Korea 21 project, Republic of Korea.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Han, Dept. of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National Univ., Gwangju 500-757, Korea (e-mail: hjhan{at}chonnam.ac.kr)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Belo PS, Romsos DR, and Leville GA. Blood metabolites and glucose metabolism in the fed and fasted chicken. J Nutr 106: 1135–1143, 1976.[Abstract/Free Full Text]
  2. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  3. Carlson CJ, Koterski S, Sciotti RJ, Poccard GB, and Rondinone CM. Enhanced basal activation of mitogen-activated protein kinases in adipocytes from type 2 diabetes: potential role of p38 in the downregulation of GLUT4 expression. Diabetes 52: 634–641, 2003.[Abstract/Free Full Text]
  4. Dajani OF, Sandnes D, Melien O, Rezvani F, Nilssen LS, Thoresen GH, and Christoffersen T. Role of diacylglycerol (DAG) in hormonal induction of S phase in hepatocytes: the DAG-dependent protein kinase C pathway is not activated by epidermal growth factor (EGF), but is involved in mediating the enhancement of responsiveness to EGF by vasopressin, angiotensin II, and norepinephrine. J Cell Physiol 180: 203–214, 1999.[CrossRef][ISI][Medline]
  5. D'Onofrio M, Arcella A, Bruno V, Ngomba RT, Battaglia G, Lombari V, Ragona G, Calogero A, and Nicoletti F. Pharmacological blockade of mGlu2/3 metabotropic glutamate receptors reduces cell proliferation in cultured human glioma cells. J Neurochem 84: 1288–1295, 2003.[CrossRef][ISI][Medline]
  6. Eisenberg ML, Maker AV, Slezak LA, Nathan JD, Sritharan KC, Jena BP, Geibel JP, and Andersen DK. Insulin receptor (IR) and glucose transporter 2 (GLUT2) proteins form a complex on the rat hepatocyte membrane. Cell Physiol Biochem 15: 51–58, 2005.[CrossRef][ISI][Medline]
  7. Fujishiro M, Gotoh Y, Katagiri H, Sakoda H, Ogihara T, Anai M, Onishi Y, Ono H, Abe M, Shojima N, Fukushima Y, Kikuchi M, Oka Y, and Asano T. Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes. Mol Endocrinol 17: 487–497, 2003.[Abstract/Free Full Text]
  8. Gao Y, Shan YQ, Pan MX, Wang Y, Tang LJ, Li H, and Zhang Z. Protein kinase C-dependent activation of p44/42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning. World J Gastroenterol 10: 1019–1027, 2004.[Medline]
  9. Garriga C, Moreto M, and Planas JM. Hexose transport across the basolateral membrane of the chicken jejunum. Am J Physiol Regul Integr Comp Physiol 272: R1330–R1335, 1997.[Abstract/Free Full Text]
  10. Goodlad RA and Wright NA. Epidermal growth factor (EGF). Baillieres Clin Gastroenterol 10: 33–47, 1996.[CrossRef][ISI][Medline]
  11. Han HJ, Park JY, Lee YJ, and Park SH. Effect of epidermal growth factor on phosphate uptake in renal proximal tubule cells: involvement of PKC, MAPK, and cPLA2. Kidney Blood Press Res 26: 315–324, 2003.[CrossRef][ISI][Medline]
  12. Hardin JA, Wong JK, Cheeseman CI, and Gall DG. Effect of luminal epidermal growth factor on enterocyte glucose and proline transport. Am J Physiol Gastrointest Liver Physiol 271: G509–G515, 1996.[Abstract/Free Full Text]
  13. Hardy RW, Gupta KB, McDonald JM, Williford J, and Wells A. Epidermal growth factor (EGF) receptor carboxy-terminal domains are required for EGF-induced glucose transport in transgenic 3T3-L1 adipocytes. Endocrinology 136: 431–439, 1995.[Abstract]
  14. Harris RC. EGF receptor activation by G-protein coupled receptors. Kidney Int 58: 898–899, 2000.[CrossRef][ISI][Medline]
  15. Henriksen EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, and Holloszy JO. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol Endocrinol Metab 259: E593–E598, 1990.[Abstract/Free Full Text]
  16. Horvath K, Hill ID, Devarajan P, Mehta D, Thomas SC, Lu RB, and Lebenthal E. Short-term effect of epidermal growth factor (EGF) on sodium and glucose cotransport of isolated jejunal epithelial cells. Biochim Biophys Acta 1222: 215–222, 1994.[Medline]
  17. Hou DX, Kunitake T, Kusuda J, and Fujii M. Primary culture of chicken hepatocytes as an in vitro model for determining the influence of dioxin. Biosci Biotechnol Biochem 65: 218–221, 2001.[Medline]
  18. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, and Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 284: 31–53, 2003.[CrossRef][ISI][Medline]
  19. Kato Y, Hamada Y, Ito S, Okumura T, and Hioki K. Epidermal growth factor stimulates the recovery of glucose absorption after small bowel transplantation. J Surg Res 80: 315–319, 1998.[CrossRef][ISI][Medline]
  20. Kim JW, Kim YK, and Ahn YH. A mechanism of differential expression of GLUT2 in hepatocyte and pancreatic beta-cell line. Exp Mol Med 30: 15–20, 1998.[ISI][Medline]
  21. Kimura M, Inoue H, Hirabayashi K, Natsume H, and Ogihara M. Glycyrrhizin and some analogues induce growth of primary cultured adult rat hepatocytes via epidermal growth factor receptors. Eur J Pharmacol 431: 151–161, 2001.[CrossRef][ISI][Medline]
  22. Klein C, Gensburger C, Freyermuth S, Nair BC, Labourdette G, and Malviya AN. A 120 kDa nuclear phospholipase C{gamma}1 protein fragment is stimulated in vivo by EGF signal phosphorylating nuclear membrane EGFR. Biochemistry 43: 15873–15883, 2004.[CrossRef][Medline]
  23. Komuves LG, Feren A, Jones AL, and Fodor E. Expression of epidermal growth factor and its receptor in cirrhotic liver disease. J Histochem Cytochem 48: 821–830, 2000.[Abstract/Free Full Text]
  24. Kong M, Mounier C, Wu J, and Posner BI. Epidermal growth factor-induced phosphatidylinositol 3-kinase activation and DNA synthesis. Identification of Grb2-associated binder 2 as the major mediator in rat hepatocytes. J Biol Chem 275: 36035–36042, 2000.[Abstract/Free Full Text]
  25. Kono T, Nishida M, Nishiki Y, Seki Y, Sato K, and Akiba Y. Characterisation of glucose transporter (GLUT) gene expression in broiler chickens. Br Poult Sci 46: 510–515, 2005.[CrossRef][ISI][Medline]
  26. Lachaal M, Rampal AL, Ryu J, Lee W, Hah J, and Jung CY. Characterization and partial purification of liver glucose transporter GLUT2. Biochim Biophys Acta 1466: 379–389, 2000.[Medline]
  27. Marino M, Mele R, Spagnuolo S, Pulcinelli FM, Mangiantini MT, and Leoni S. Epidermal growth factor regulates amino acid transport in chick embryo hepatocytes via protein kinase C. Exp Physiol 85: 363–369, 2000.[Abstract]
  28. Musallam L, Ethier C, Haddad PS, and Bilodeau M. Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes. Am J Physiol Gastrointest Liver Physiol 280: G1360–G1369, 2001.[Abstract/Free Full Text]
  29. Nielsen CU, Amstrup J, Steffansen B, Frokjaer S, and Brodin B. Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line. Am J Physiol Gastrointest Liver Physiol 281: G191–G199, 2001.[Abstract/Free Full Text]
  30. Nilssen LS, Hege Thoresen G, Christoffersen T, and Sandnes D. Differential role of MAP kinases in stimulation of hepatocyte growth by EGF and G-protein-coupled receptor agonists. Biochem Biophys Res Commun 291: 588–592, 2002.[CrossRef][ISI][Medline]
  31. Nishina H, Wada T, and Katada T. Physiological roles of SAPK/JNK signaling pathway. J Biochem (Tokyo) 136: 123–126, 2004.[Abstract/Free Full Text]
  32. Nogami M, Yamazaki M, Watanabe H, Okabayashi Y, Kido Y, Kasuga M, Sasaki T, Maehama T, and Kanaho Y. Requirement of autophosphorylated tyrosine 992 of EGF receptor and its docking protein phospholipase C{gamma}1 for membrane ruffle formation. FEBS Lett 536: 71–76, 2003.[CrossRef][ISI][Medline]
  33. Pan M, Meng QH, Wolfgang CL, Lin CM, Karinch AM, Vary TC, and Souba WW. Activation of intestinal arginine transport by protein kinase C is mediated by mitogen-activated protein kinases. J Gastrointest Surg 6: 876–882, 2002.[CrossRef][ISI][Medline]
  34. Peak M, Rochford JJ, Borthwick AC, Yeaman SJ, and Agius L. Signalling pathways involved in the stimulation of glycogen synthesis by insulin in rat hepatocytes. Diabetologia 41: 16–25, 1998.[CrossRef][ISI][Medline]
  35. Pearce J. Some differences between avian and mammalian biochemistry. Int J Biochem 8: 269–275, 1977.[CrossRef]
  36. Perry JE, Grossmann ME, and Tindall DJ. Epidermal growth factor induces cyclin D1 in a human prostate cancer cell line. Prostate 35: 117–124, 1998.[CrossRef][ISI][Medline]
  37. Robin P, Boulven I, Bole-Feysot C, Tanfin Z, and Leiber D. Contribution of PKC-dependent and -independent processes in temporal ERK regulation by ET-1, PDGF, and EGF in rat myometrial cells. Am J Physiol Cell Physiol 286: C798–C806, 2004.[Abstract/Free Full Text]
  38. Santalucia T, Christmann M, Yacoub MH, and Brand NJ. Hypertrophic agonists induce the binding of c-Fos to an AP-1 site in cardiac myocytes: implications for the expression of GLUT1. Cardiovasc Res 59: 639–48, 2003.[CrossRef][ISI][Medline]
  39. Sasaki K, Kitaguchi Y, Fukuda T, and Aoyagi Y. Ascorbic acid supplementation to primary culture of chicken hepatocytes with non-serum medium. Int J Biochem Cell Biol 32: 967–973, 2000.[CrossRef][ISI][Medline]
  40. Sasaki K, Kitaguchi Y, Koga K, Narita R, Fukuda T, and Aoyagi Y. Dehydroascorbic acid reduction in several tissues and cultured hepatocytes of the chicken. Biosci Biotechnol Biochem 65: 2288–2290, 2001.[Medline]
  41. Sharma GD, He J, and Bazan HE. p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades. J Biol Chem 278: 21989–21997, 2003.[Abstract/Free Full Text]
  42. Shinohara M, Kodama A, Matozaki T, Fukuhara A, Tachibana K, Nakanishi H, and Takai Y. Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1. J Biol Chem 276: 18941–18946, 2001.[Abstract/Free Full Text]
  43. Skarpen E, Oksvold MP, Grosvik H, Widnes C, and Huitfeldt HS. Altered regulation of EGF receptor signaling following a partial hepatectomy. J Cell Physiol 202: 707–716, 2005.[CrossRef][ISI][Medline]
  44. Stariha RL and Kim SU. Protein kinase C and mitogen-activated protein kinase signalling in oligodendrocytes. Microsc Res Tech 52: 680–688, 2001.[CrossRef][ISI][Medline]
  45. Thevananther S, Sun H, Li D, Arjunan V, Awad SS, Wyllie S, Zimmerman TL, Goss JA, and Karpen SJ. Extracellular ATP activates c-jun N-terminal kinase signaling and cell cycle progression in hepatocytes. Hepatology 39: 393–402, 2004.[CrossRef][ISI][Medline]
  46. Tonb D, Mehta R, Wang H, Tung J, and Mehta DI. Short-term effect of epidermal growth factor on glucose uptake in endoscopic biopsies. Dig Dis Sci 48: 1614–1618, 2003.[CrossRef][ISI][Medline]
  47. Wagstaff P, Kang HY, Mylott D, Robbins PJ, and White MK. Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts. Mol Biol Cell 6: 1575–1589, 1995.[Abstract]
  48. Wang MY, Tsai MY, and Wang C. Identification of chicken liver glucose transporter. Arch Biochem Biophys 310: 172–179, 1994.[CrossRef][ISI][Medline]
  49. White MK and Weber MJ. Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms. Mol Cell Biol 8: 138–144, 1988.[Abstract/Free Full Text]
  50. Yamamoto T, Kojima T, Murata M, Takano K, Go M, Hatakeyama N, Chiba H, and Sawada N. p38 MAP-kinase regulates function of gap and tight junctions during regeneration of rat hepatocytes. J Hepatol 42: 707–718, 2005.[CrossRef][ISI][Medline]
  51. Zheng Q, Levitsky LL, Mink K, and Rhoads DB. Glucose regulation of glucose transporters in cultured adult and fetal hepatocytes. Metabolism 44: 1553–1558, 1995.[CrossRef][ISI][Medline]
  52. Zhou J, Deo BK, Hosoya K, Terasaki T, Obrosova IG, Brosius FC 3rd, and Kumagai AK. Increased JNK phosphorylation and oxidative stress in response to increased glucose flux through increased GLUT1 expression in rat retinal endothelial cells. Invest Ophthalmol Vis Sci 46: 3403–3410, 2005.[Abstract/Free Full Text]



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