Insulin Enhances Macrophage Scavenger Receptor-mediated Endocytic Uptake of Advanced Glycation End Products*

Hiroyuki Sano, Takayuki Higashi, Kenshi Matsumoto, Jukka MelkkoDagger , Yoshiteru Jinnouchi, Kazuyoshi Ikeda, Yousuke Ebina§, Hideichi Makino, Bård SmedsrødDagger , and Seikoh Horiuchipar

From the Department of Biochemistry, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan, the Dagger  Department of Experimental Pathology, University of Tromsø, N-9037 Tromsø, Norway, the § Department of Enzyme Genetics, Institute for Enzyme Research, University of Tokushima, Tokushima 770, Japan, and the  Department of Laboratory Medicine, Ehime University School of Medicine, Onsen-Gun 791, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Hyperglycemia accelerates the formation and accumulation of advanced glycation end products (AGE) in plasma and tissue, which may cause diabetic vascular complications. We recently reported that scavenger receptors expressed by liver endothelial cells (LECs) dominantly mediate the endocytic uptake of AGE proteins from plasma, suggesting its potential role as an eliminating system for AGE proteins in vivo (Smedsrød, B., Melkko, J., Araki, N., Sano, H., and Horiuchi, S. (1997) Biochem. J. 322, 567-573). In the present study we examined the effects of insulin on macrophage scavenger receptor (MSR)-mediated endocytic uptake of AGE proteins. LECs expressing MSR showed an insulin-sensitive increase of endocytic uptake of AGE-bovine serum albumin (AGE-BSA). Next, RAW 264.7 cells expressing a high amount of MSR were overexpressed with human insulin receptor (HIR). Insulin caused a 3.7-fold increase in endocytic uptake of 125I-AGE-BSA by these cells. The effect of insulin was inhibited by wortmannin, a phosphatidylinositol-3-OH kinase (PI3 kinase) inhibitor. To examine at a molecular level the relationship between insulin signal and MSR function, Chinese hamster ovary (CHO) cells expressing a negligible level of MSR were cotransfected with both MSR and HIR. Insulin caused a 1.7-fold increase in the endocytic degradation of 125I-AGE-BSA by these cells, the effect of which was also inhibited by wortmannin and LY294002, another PI3 kinase inhibitor. Transfection of CHO cells overexpressing MSR with two HIR mutants, a kinase-deficient mutant, and another lacking the binding site for insulin receptor substrates (IRS) resulted in disappearance of the stimulatory effect of insulin on endocytic uptake of AGE proteins. The present results indicate that insulin may accelerate MSR-mediated endocytic uptake of AGE proteins through an IRS/PI3 kinase pathway.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Prolonged incubation of proteins with glucose leads, through the formation of early stage products, such as Schiff base and Amadori rearrangement products, to the formation of advanced glycation end products (AGE),1 compounds that have unique properties, such as fluorescence, browning, and cross-linking. Accumulation of AGE proteins has been identified in several tissues in association with aging and age-enhanced disease states, including diabetic complications (1-3), atherosclerosis (4), hemodialysis-related amyloidosis (5), and Alzheimer's disease (6, 7). AGE-modified proteins are known to induce several cellular responses, such as mitogenic activity for macrophages (8), and a chemotactic activity for vascular smooth muscle cells (9). Therefore, the presence and/or accumulation of AGE proteins in arterial walls (4, 10) is thought to play an active role in the pathogenesis of diabetic microvascular and macrovascular complications (3, 11). However, under physiological conditions, most AGE-modified proteins in plasma should undergo rapid plasma clearance. Thus, following intravenous injection in normal rats, AGE proteins are rapidly cleared from the circulation (12). Such clearance is largely achieved by active endocytic uptake by hepatic sinusoidal cells such as endothelial and Kupffer cells (12, 13). It might be hypothesized therefore that the formation of AGE proteins beyond physiological levels or impairment of the AGE elimination system, potentially result in accumulation of AGE in tissues.

Insulin therapy reduces high plasma levels of early stage products, such as hemoglobin A1c and glycated albumin, as well as those of AGE proteins (14). Normalization of glycated proteins levels may thus delay the onset and slow the progression of diabetic complications (15). Insulin treatment may protect the tissues from AGE accumulation indirectly by reducing AGE formation due largely to normalization of plasma glucose levels. However, since AGE proteins are recognized as ligands by AGE receptors in vivo, it is also possible that insulin plays a more direct role in AGE elimination from plasma or tissues by regulating the AGE receptor system.

Three types of AGE receptors have been identified so far, including MSR (16, 17), RAGE (receptor for AGE) (18), and the receptor complex of OST-48, 80K-H, and galectin-3 (previously called as p60 and p90) (19). Our recent study using peritoneal macrophages from MSR knockout mice has indicated that the endocytic capacity for AGE-BSA by these cells was reduced to approximately 20-30% of those of wild litter mate mice (20, 21), suggesting a major role for MSR in endocytic uptake of AGE proteins by macrophages. Our recent study also showed that liver endothelial cells (LECs), which expressed a large amounts of MSR (20), were responsible for elimination of 60-65% of intravenously injected AGE proteins from plasma and that the endocytic uptake of AGE proteins by cultured LECs was significantly inhibited by the ligands for MSR, suggesting a major contribution of MSR to endocytosis of AGE proteins by LECs (13). Therefore, in the present study, we focused on MSR as an elimination system for AGE proteins and investigated the effect of insulin on endocytic uptake of AGE proteins by MSR.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Chemicals and Materials-- F-12 medium, RPMI 1640 medium, penicillin G, streptomycin, and G418 were purchased from Life Technologies, Inc., hygromycin B and wortmannin from Wako (Osaka, Japan), LY294002 and rapamycin from Biomol Research Laboratories (Plymouth Meeting, PA), and human recombinant insulin from Sigma. Na125I and 125I-human insulin was purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, United Kingdom). Other chemicals were of the best grade available from commercial sources. Culture dishes coated with type IV collagen (35 mm in diameter) were purchased from Becton Dickinson (Bedford, MA). Bovine type II MSR expression vector pXSRII was a kind gift from Dr. Tatsuhiko Kodama (Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo).

Ligand Preparation and Iodination-- AGE-BSA was prepared as described previously (12) except for incubation for 40 weeks. Human LDL (d = 1.019-1.063 g/ml) was isolated by sequential ultracentrifugation of human plasma from normal lipidemic subjects after overnight fasting and dialyzed against 0.15 M NaCl and 1 mM EDTA (22). Acetylated LDL (acetyl-LDL) was prepared by chemical modification of LDL with acetic anhydride as described previously (23). AGE-BSA was labeled with 125I by IODO-GEN (Bio-Rad) (12), and acetyl-LDL was labeled as described by McFarlane (24) to a specific radioactivity of 850 and 420 cpm/ng, respectively.

Isolation and Culture of LECs-- The preparation of pure cultures of functionally intact LECs from a single rat liver has been detailed in the previous paper (25). After collagenase perfusion of the liver, and isopycnic centrifugation of the resulting dispersed cells through Percoll (Pharmacia), pure monolayer cultures of LECs were established by selective attachment to substrates of type IV collagen.

Isolation and Culture of Transfected Cell Lines-- Human insulin receptor (HIR) cDNA nucleotides and amino acids were numbered according to the system of Ebina et al. (26). RAW 264.7 cells were transfected with the wild-type HIR expression vector (SRalpha IR) and pSV2neo by the lipofection method according to the protocol recommended by the manufacturer (Lipofectin, Life Technologies, Inc.). Clones resistant to 0.4 mg/ml G418 were assayed for the expression of HIR by using 125I-insulin binding assay as described previously (27). One of five positive clones selected was used for the experiment as HIR-transfected RAW (RAW-HIR) cells. To coexpress HIR and MSR in CHO cells, we used MSR-transfected CHO cells (CHO-MSR cells) as a starting cell, as described in our previous study (17). CHO-MSR cells were isolated according to the original method of Freeman et al. (28). Briefly, bovine type II MSR expression vector, pXSRII, was transfected to CHO cells using polybrene method. To select an MSR-positive clone, cells were cultured in F-12 medium containing 5% fetal calf lipoprotein-deficient serum, 250 µM mevalonic acid, 3 µg/ml acetyl-LDL, and 40 µM compactin. We selected one of the positive clones with a high activity for incorporation of acetyl-LDL labeled by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. CHO-MSR cells thus obtained were transfected with the wild-type HIR expression vector (SRalpha IR) and pSV2hph by the lipofection method. Clones resistant to 0.4 mg/ml hygromycin B were selected for the expression of HIR in the same way. CHO-MSR cells were also transfected with mutant-type HIR expression vector in which Lys1030 or Tyr972 was replaced by methionine (29) and phenylalanine (30) (SRalpha IR1030M and SRalpha IR972F), respectively. Clones expressing wild- or mutant-type HIR obtained by hygromycin B selection were named as CHO-MSR-HIR, CHO-MSR-HIR1030M, and CHO-MSR-HIR972F cells, respectively. HIR-transfected CHO (CHO-HIR) cells were obtained by transfecting SRalpha IR and pSV2neo to parent CHO cells and selected against 0.4 mg/ml G418. Each clone expressing wild or mutant HIR thus obtained was subjected to the binding assay using 125I-insulin. Briefly, cells in each well were incubated for 4 h at 4 °C with 0.03 nM 125I-insulin in the presence of various concentrations (0-200 nM) of unlabeled insulin, washed three times, and the cell-bound radioactivity was measured as described previously (27). The number of cell surface receptors was calculated by Scatchard analysis. Clones expressing 1.7-3.1 × 105 receptors/cell were used in the present experiments.

Cellular Assays-- Except for the binding study, all cellular experiments were performed at 37 °C in a humidified atmosphere of 5% CO2. The endocytic uptake of 125I-AGE-BSA by LECs was measured as described previously (12) with some modifications. Briefly, 5 × 106 of LECs were seeded and maintained in serum-free RPMI 1640 medium in 35-mm diameter wells, washed, and supplied with fresh medium containing 3% BSA and labeled ligands. The cells were incubated for 60 min with or without 10 nM insulin and indicated concentrations of 125I-AGE-BSA (1.25-10 µg/ml) with or without 100-fold unlabeled AGE-BSA in 1.0 ml of KRH buffer (136 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO4, 1.25 mM CaCl2, 20 mM Hepes, 1 mg/ml glucose, and 30 mg/ml BSA). By contrast, the cells were incubated for 60 min with 2 µg/ml 125I-AGE-BSA and indicated concentrations of human insulin with or without 200 µg/ml unlabeled AGE-BSA in 1.0 ml of KRH buffer. Additionally, the cells were incubated with 10 nM wortmannin (31) for 30 min before and after the addition of 2 µg/ml 125I-AGE-BSA and 10 nM insulin. After 60-min incubation with 125I-AGE-BSA, 0.75 ml of the culture medium was taken from each well and mixed with 0.3 ml of 40% trichloroacetic acid in a vortex mixer. To this solution we added 0.2 ml of 0.7 M AgNO3, followed by centrifugation. The resulting supernatant (0.5 ml) was used to determine trichloroacetic acid-soluble radioactivity, which was taken as an index of cellular degradation. The remaining cells in culture dishes were washed three times with PBS containing 1% BSA and three more times with PBS. The cells were lysed at 37 °C for 30 min with 1.0 ml of 0.1 N NaOH. One portion was used to determine the radioactivity as the cell-associated ligand, while the other portion was used to determine cellular proteins by BCA protein assay reagent (Bio-Rad).

The endocytic uptake of 125I-AGE-BSA in each transfected cell line was measured. RAW-HIR cells and mock-transfected RAW (RAW-mock) cells were cultured for 24 h on type IV collagen-coated wells (35 mm in diameter) in 1.0 ml of RPMI containing 10% fetal calf serum (FCS) at the final cell density of 8 × 105 cells/well. CHO-MSR-HIR cells, CHO-MSR cells, CHO-HIR cells, parent CHO cells, CHO-MSR-HIR1030M cells, and CHO-MSR-HIR972F cells were cultured for 24 h on type IV collagen-coated wells in 1.0 ml of F-12 medium supplemented with 10% FCS at a final cell density of 5 × 105 cells/well. After serum starvation for 5 h, the cells were incubated for 60 min with 2 µg/ml 125I-AGE-BSA and selected concentrations of human insulin with or without 200 µg/ml unlabeled AGE-BSA in 1.0 ml of KRH buffer. Cell-associated ligands and ligands degraded by those cells were measured as exactly described above.

To determine the effects of wortmannin, LY294002 (32), and rapamycin (33), CHO-MSR-HIR cells were pretreated for 30 min in 0.8 ml of KRH buffer with various concentrations of wortmannin, LY294002, rapamycin, and 0.1% of dimethyl sulfoxide as a vehicle, respectively. RAW-HIR cells were pretreated with these reagents in the same manner except for LY294002. In the next step, we added to the cells in each culture well 0.2 ml of KRH buffer containing labeled ligands to a final concentration of 2 µg/ml 125I-AGE-BSA in the absence or presence of 10 nM insulin. The endocytic degradations of 125I-AGE-BSA and cell-associated 125I-AGE-BSA were determined as described above. The endocytic uptake (degradation and cell association) of 125I-acetyl-LDL by CHO-MSR-HIR cells was determined with 2 µg/ml 125I-acetyl-LDL similar to 125I-AGE-BSA.

For the cellular binding study, the cells were cultured and subjected to serum starvation as described for the endocytic uptake assay. The cells in each well were preincubated for 60 min at 37 °C with or without 10 nM insulin, washed with ice-cold KRH buffer, and replaced with 1.0 ml of KRH buffer containing 1.25-20 µg/ml 125I-AGE-BSA, in the absence or presence of 50-fold excess amounts of unlabeled ligand (50-1000 µg/ml). After incubation of the cells for 2 h at 4 °C, each well was washed three times with 1.0 ml of ice-cold PBS containing 1% BSA and three more times with PBS. The cells were lysed, and the cell-bound radioactivity and cellular proteins were determined as described above.

Statistical Analysis-- Values are expressed as means ± S.E. Statistical significance was determined by unpaired Student's t test. Differences between insulin-stimulated and control groups were considered significant at p < 0.05.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Endocytic Uptake of AGE-BSA by LECs-- As was shown previously (13), 125I-AGE-BSA underwent effective receptor-mediated endocytosis by cultured LECs (Fig. 1, A and B). The endocytic uptake of 125I-AGE-BSA by these cells was significantly enhanced by the presence of 10 nM insulin; at ligand concentrations from 1.25 to 10 µg/ml, cell-associated 125I-AGE-BSA was increased by 122-141%, and the amount of 125I-AGE-BSA degraded was similarly increased by 113-134% above control (Fig. 1, A and B). When the dose-dependent effect of insulin was examined in the fixed amount of 125I-AGE-BSA, the amounts of cell-associated 125I-AGE-BSA, as well as those degraded by LECs, were increased with insulin concentrations up to 1 nM and became a plateau somewhere between 1 and 10 nM, followed by gradual decline at an insulin concentration of 100 nM; cell-associated 125I-AGE-BSA was increased significantly from 775 to 1108 ng/mg of cell protein/h, and amounts of 125I-AGE-BSA degraded were also increased significantly from 131 to 173 ng/mg of cell protein/h (Fig. 1, C and D). Under the identical conditions, the effect of 10 nM wortmannin, a reagent for PI3 kinase inhibitor, on the endocytosis was examined in the presence of 10 nM insulin. This drug completely reversed the insulin-induced increase of endocytosis to basal level, but slightly less than that of control (Fig. 1, C and D). These results suggest a potential link of an insulin signaling pathway to the endocytic system of LECs. Recent studies indicated that MSR expressed by these cells play a major role in the endocytic uptake of chemically modified proteins such as acetyl-LDL (21) and AGE proteins (13). Therefore, to understand the mechanism of insulin-enhanced endocytic uptake of AGE-BSA, we thought it reasonable to use RAW cells, a macrophage-like cell line that is known to express a high level of endogenous MSR (34).


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Fig. 1.   Endocytic uptake of 125I-AGE-BSA by LEC. Cultured LEC were incubated for 60 min with (square ) or without (black-square) 10 nM insulin and indicated concentrations of 125I-AGE-BSA with or without 100-fold unlabeled AGE-BSA in 1.0 ml of KRH buffer containing 1% BSA. Next, the cells were incubated with 2 µg/ml 125I-AGE-BSA for 60 min at 37 °C in the selected concentration of insulin, with or without 200 µg/ml unlabeled AGE-BSA in 1 ml of KRH buffer containing 1% BSA. In particular, the effect of 10 nM wortmannin for the endocytosis was examined in the presence of 10 nM insulin (triangle ). The culture medium was used to determine 125I-AGE-BSA degraded by these cells, and the cells were washed to determine the cell-associated 125I-AGE-BSA as described under "Experimental Procedures." Specific cell association (A, C) and specific degradation (B, D) were plotted after correcting for nonspecific cell association and degradation, respectively. Values represent mean ± S.E. of triplicate determinations. Results are representative of three independent experiments. *, p < 0.05 as compared with the control.

Endocytic Uptake of AGE-BSA by RAW-HIR Cells-- We first introduced HIR expression vector to macrophage-like RAW 294.7 cells, since recent studies have shown the MSR-mediated endocytic uptake of AGE proteins by macrophages and macrophage-derived cells (17, 20). The level of insulin receptor expressed in RAW-HIR cells was 1 × 105 receptors/cell, about 100 times higher than RAW cells; the endogenous level of insulin receptor in RAW cells was negligibly low (<103 receptors/cell) (data not shown). Endocytic uptake of 125I-AGE-BSA by RAW-HIR cells were examined in the presence of various concentrations of insulin. As shown in Fig. 2A, insulin increased the amount of 125I-AGE-BSA associated with RAW-HIR cells from 128 to 154 ng/mg of cell protein, whereas RAW-mock cells were not influenced by the presence of insulin. The amount of 125I-AGE-BSA degraded by RAW-HIR cells increased proportionately with insulin concentrations up to 1 nM, but decreased sharply at insulin concentrations of 10 and 100 nM (Fig. 2B). The maximum level reached in the presence of 1 nM insulin was 90 ng/mg of cell protein, whereas the corresponding control level was 30 ng/mg of cell protein (Fig. 2B). RAW-mock cells did not show such an insulin-dependent enhancement of endocytic degradation of 125I-AGE-BSA. These results indicate that endocytic uptake and degradation of 125I-AGE-BSA by RAW-HIR cells is regulated by insulin. Since MSR is expected to mediate the endocytic degradation of AGE proteins by RAW cells, it is possible that insulin may act on MSR of RAW-HIR cells via transfected HIR. To test such possibility, CHO cells known to express a negligible level of MSR were coexpressed with MSR and HIR.


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Fig. 2.   Endocytic uptake of 125I-AGE-BSA by RAW-HIR cells. RAW-HIR cells (square ) RAW-mock cells (black-square) were cultured for 24 h on type IV collagen-coated wells in 1.0 ml of RPMI containing 10% FCS at a final cell density of 8 × 105 cells/well. After serum starvation for 5 h, the cells were incubated for 60 min with 2 µg/ml 125I-AGE-BSA and a selected concentration of human insulin, with or without 200 µg/ml unlabeled AGE-BSA in 1.0 ml of KRH buffer. The culture medium was used to determine 125I-AGE-BSA degraded by these cells, and the cells were washed to determine the cell-associated 125I-AGE-BSA as described under "Experimental Procedures." Specific cell association (A) and specific degradation (B) were plotted after correcting for nonspecific cell association and degradation, respectively. Values represent mean ± S.E. of duplicate determinations. Results are representative of three independent experiments.

Endocytic Uptake of AGE-BSA by CHO-MSR-HIR Cells-- CHO-MSR-HIR cells expressing 2.8 × 105 insulin receptors/cell (Table I) were used for endocytic uptake study. Fig. 3B shows the endocytic degradation of 125I-AGE-BSA by these cells in the presence or absence of insulin. While the degradation activity of 125I-AGE-BSA by control cells, such as CHO cells or CHO-HIR cells, was negligible, that of CHO-MSR cells was significant; the amount of 125I-AGE-BSA degraded by CHO-MSR cells for 60 min was about 6 ng/mg of cell protein and was not influenced by insulin. CHO-MSR-HIR cells showed the same level of degradation activity for 125I-AGE-BSA at insulin concentrations less than 0.01 nM. However, endocytic degradation by these CHO-MSR-HIR cells was markedly enhanced in the presence of insulin in a dose-dependent manner; the amount of 125I-AGE-BSA degraded increased from 6.0 to 8.7 ng/mg of cell protein by 1 nM insulin, reaching a plateau of 10 ng/mg of cell protein at 10 nM insulin. Thus, the maximal enhancement of degradation activity for 125I-AGE-BSA by CHO-MSR-HIR cells was about 1.7-fold. Since insulin-induced enhancement of endocytic degradation of 125I-AGE-BSA occurred only when CHO cells were coexpressed with MSR and HIR, it is likely that both MSR and HIR play an important role in insulin-sensitive endocytic uptake of 125I-AGE-BSA.

                              
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Table I
Insulin binding to CHO cells expressing wild-type or mutant-type insulin receptors
After serum starvation for 24 h at 37 °C, the cells in each well were incubated for 4 h at 4 °C with 0.03 nM 125I-insulin in the presence of various concentrations (0-200 nM) of unlabeled insulin, washed three times, and the cell-bound radioactivity was measured as described previously (27). The number of insulin receptors expressed on the surface membranes was calculated according to the Scatchard plot. Data are the means of triplicate measurements obtained from three independent experiments.


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Fig. 3.   Effect of insulin on endocytic uptake of 125I-AGE-BSA by CHO-MSR-HIR cells. After serum starvation for 5 h, CHO-MSR-HIR cells (square ), CHO-MSR cells (black-square), CHO-HIR cells (triangle ), or parent CHO cells (black-triangle) were incubated for 60 min in 1.0 ml of KRH buffer with 2 µg/ml 125I-AGE-BSA in the absence or presence of unlabeled AGE-BSA and a selected concentration of human insulin. After incubation, the specific cell-associated 125I-AGE-BSA (A) and specific degradation of 125I-AGE-BSA (B) were determined as described in the legend to Fig. 1 and under "Experimental Procedures." Values represent mean ± S.E. of duplicate determinations. Results are representative of three independent experiments.

The cell-associated 125I-AGE-BSA was also determined in these cells under identical conditions (Fig. 3A). The amount of cell-associated 125I-AGE-BSA in CHO-MSR cells (36 ng/mg of cell protein) was significant; about 4-fold that of CHO and CHO-HIR cells (8-10 ng/mg of cell protein). The amount of cell-associated 125I-AGE-BSA in CHO-MSR cells was not influenced by insulin. Similar to endocytic degradation of 125I-AGE-BSA (Fig. 3B), insulin increased the amount of 125I-AGE-BSA associated with CHO-MSR-HIR cells from 38 to 51 ng/mg of cell protein (Fig. 3A). This phenomenon is directly related to the insulin-enhanced endocytic degradation of 125I-AGE-BSA in CHO-MSR-HIR cells, since cell association and subsequent lysosomal degradation constitute a continuous set of the endocytic pathway.

Cellular Binding of AGE-BSA to CHO-MSR-HIR Cells-- It is generally accepted that cell-associated ligands include those bound to cell surfaces as well as those internalized into the cells. Since insulin significantly increased the amount of cell-associated AGE-BSA in CHO-MSR-HIR cells (Fig. 3A), we performed the binding assay to test whether such increase was due to an increase in cell surface receptors. As shown in Fig. 4, the binding of 125I-AGE-BSA to CHO-MSR-HIR cells exhibited a saturable binding, but the amount of cell-bound 125I-AGE-BSA did not change when these CHO-MSR-HIR cells were incubated with 10 nM insulin (Fig. 4).


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Fig. 4.   Cellular binding of 125I-AGE-BSA to CHO-MSR-HIR cells. After serum starvation, the cells in each well were preincubated for 60 min at 37 °C with (black-square) or without (square ) 10 nM insulin, washed with ice-cold KRH buffer, and replaced with 1.0 ml of KRH buffer containing 1.25-20 µg/ml 125I-AGE-BSA in the absence or presence of 50-fold excess amounts of unlabeled ligand (50-1000 µg/ml). Following incubation for 2 h at 4 °C, each well was washed, and the cell-bound radioactivity and cellular proteins were determined as described under "Experimental Procedures." Specific binding was plotted after correcting for nonspecific binding. Values represent mean ± S.E. of duplicate determinations. Results are representative of three independent experiments.

Endocytic Uptake of AGE-BSA by CHO-MSR-HIR1030M and CHO-MSR-HIR972F Cells-- To examine whether HIR signaling is necessary for activating MSR function, we transfected two types of mutant HIR expression vectors to CHO-MSR cells. CHO-MSR cells were stably transfected with HIR1030M in which lysine 1030 as an ATP binding site was replaced by methionine, thus exhibiting the kinase-deficient mutant (29). The other mutant used was HIR972F, which possessed normal kinase activity but was unable to bind to IRS, because tyrosine 972 serving as a binding site for IRS was replaced with phenylalanine (30). In CHO-MSR-HIR cells, insulin enhanced the cell association (Fig. 5A) as well as endocytic degradation (Fig. 5B) of 125I-AGE-BSA. However, no such response to insulin was evident in CHO-MSR-HIR1030M or CHO-MSR-HIR972F cells. These results suggest that the kinase activity of insulin receptor, especially IRS-1, -2, -3, or -4 pathway (35-40), plays a key role in insulin-enhanced endocytic uptake of AGE proteins by MSR.


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Fig. 5.   Effect of insulin on endocytic uptake of 125I-AGE-BSA by CHO-MSR-HIR, CHO-MSR-HIR1030M and CHO-MSR-HIR972F cells. After serum starvation for 5 h, CHO-MSR-HIR cells (WT), CHO-MSR-HIR1030M cells (1030 M), or CHO-MSR-HIR972F cells (972F) were incubated in 1.0 ml of KRH buffer with 2 µg/ml 125I-AGE-BSA in the presence () or absence (black-square) of 10 nM insulin with or without 50-fold excess of unlabeled AGE-BSA. After incubation for 60 min, the specific cell-associated 125I-AGE-BSA (A) and specific degradation (B) were determined as described under "Experimental Procedures." Values are expressed as percent of control (no insulin stimulation). Data are typical results of a representative example of three independent experiments.

Effect of Wortmannin, LY294002, and Rapamycin on Insulin-enhanced Endocytosis by CHO-MSR-HIR and RAW-HIR Cells-- To examine the insulin signaling pathway to MSR, we examined the effects of several inhibitors on insulin-enhanced endocytic degradation of AGE-BSA in CHO-MSR-HIR cells and RAW-HIR cells. As shown in Fig. 6, insulin-enhanced endocytic degradation of 125I-AGE-BSA by CHO-MSR-HIR cells was inhibited dose-dependently by wortmannin, a potent PI3 kinase inhibitor, with a 50% inhibitory concentration (IC50) at 1 nM. Consistent with this, the phenomenon observed in CHO-MSR-HIR cells was similarly inhibited by another PI3 kinase inhibitor, LY294002 (Fig. 7A). Furthermore, the insulin-enhanced endocytic degradation of AGE-BSA by RAW-HIR cells disappeared completely by pretreatment with 10 nM wortmannin (Fig. 7C). These results suggest a possible involvement of insulin signaling via PI3 kinase in the insulin-enhanced MSR-mediated endocytic uptake of AGE-BSA.


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Fig. 6.   Effect of wortmannin on insulin-enhanced endocytic uptake of 125I-AGE-BSA by CHO-MSR-HIR cells. CHO-MSR-HIR cells were cultured for 24 h in a medium containing 10% FCS and for an additional 5 h without serum. The cells were then pretreated for 30 min with various concentrations of wortmannin and 0.01% dimethyl sulfoxide and incubated for 60 min with 2 µg/ml 125I-AGE-BSA in the presence of 10 nM insulin. The endocytic activity for AGE-BSA was determined as described under "Experimental Procedures." The specific cell association of 125I-AGE-BSA (black-square) and its specific degradation (square ) were obtained by correcting for nonspecific cell association and degradation. Values are expressed as percent of insulin-induced maximum increase in cell association or degradation. Data are results of a representative example of three independent experiments.


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Fig. 7.   Effect of wortmannin, LY294002, and rapamycin on insulin-enhanced endocytic uptake of 125I-AGE-BSA or 125I-acetyl-LDL by CHO-MSR-HIR cells and RAW-HIR cells. CHO-MSR-HIR cells were cultured for 24 h in 1.0 ml of F-12 containing 10% FCS. After serum starvation for 5 h, the cells were pretreated for 30 min with 10 nM wortmannin, 100 mM LY294002, or 20 ng/ml rapamycin, and 0.1% dimethyl sulfoxide as a vehicle, followed by incubation for 60 min with 2 µg/ml 125I-AGE-BSA, with or without 10 nM insulin. The specific cell association of 125I-AGE-BSA (black-square) and specific degradation () were obtained after correcting for nonspecific cell association and degradation (A). Experiments were performed in parallel in the same way except that 125I-AGE-BSA was replaced with 2 µg/ml 125I-acetyl-LDL in the absence or presence of 50-fold excess of acetyl-LDL (see above and "Experimental Procedures") (B). RAW-HIR cells were cultured in RPMI containing 10% FCS, and the assay was performed in the same way (see above and "Experimental Procedures") (C). Values of specific cell-association (black-square) and degradation () are expressed as percent of control (cells without insulin and other effectors) and the average ± S.E. of duplicate determinations. Data are results of representative examples of three independent experiments.

In addition to PI3 kinase, we tested the effects of pp70-S6 kinase (pp70S6K) on MSR function, since this kinase is located downstream of PI3 kinase (41). Similar to wortmannin and LY294002, rapamycin inhibits the activation of pp70S6K, but has no effect on PI3 kinase (42). As shown in Fig. 7, rapamycin did not influence insulin-induced MSR function for endocytic uptake of AGE-BSA in CHO-MSR-HIR cells (Fig. 7A) as well as RAW-HIR cells (Fig. 7C). Insulin-enhanced endocytic uptake was also observed with 125I-acetyl-LDL, an authentic ligand for MSR, in CHO-MSR-HIR cells (Fig. 7B) and RAW-HIR cells (data not shown).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

More than 90% of intravenously injected AGE-BSA is eliminated within 15 min by sinusoidal liver cells such as LECs and Kupffer cells (13), whereas native BSA is cleared from the circulation with a half-life of about 2 days in healthy rats (43). It is therefore evident that rapid elimination of AGE-BSA by rat sinusoidal cells is not due to the species specificity of the albumin, but to the specific recognition of the modified structure(s) of AGE-BSA by these cells. It is also shown that 60-65% of the elimination of AGE-BSA is explained by LECs and 24-28% by Kupffer cells, while contribution of hepatocytes is negligible (<0.1%) (13). Active endocytic activity for AGE-BSA was also confirmed by in vitro experiments using cultured LECs (13).

The recent study using MSR knockout mice revealed that 80% of endocytic uptake of acetyl-LDL by cultured LECs is mediated by MSR (21). Moreover, the endocytic uptake of AGE proteins by cultured LECs was inhibited by 50-70% by the ligands for MSR (13). These results likely suggest a significant role of MSR in endocytic uptake of these modified proteins by LECs. During the course of our studies of endocytic uptake of AGE proteins by rat cultured LECs, we came across the interesting observation that endocytic uptake of AGE-BSA by cultured LECs is significantly enhanced by insulin, which was completely reversed by 10 nM wortmannin (Fig. 1, C and D). For the reasons described above, we thought that a possible explanation would be some interaction of insulin signaling with MSR function. Therefore, we next tested with RAW cells, a macrophage cell line that has a high MSR activity, followed by CHO cells coexpressed with HIR and MSR.

The present study using the transfected cells has clearly shown that insulin up-regulates MSR-mediated endocytic uptake of AGE proteins through the IRS/PI3 kinase pathway in which IRS signaling following insulin receptor autophosphorylation plays an important role. Insulin-enhanced MSR-mediated endocytic uptake of 125I-AGE-BSA was inhibited by wortmannin and LY294002, but not by rapamycin, indicating that PI3 kinase may be involved in this phenomenon. Thus, it is likely that, although PI3 kinase activity is required for both the activation of pp70S6K and MSR-mediated endocytic uptake of AGE-BSA, the pathway leading to insulin-enhanced MSR-mediated endocytosis of AGE-BSA may branch at some point downstream of PI3 kinase but upstream of pp70S6K. Insulin-stimulated activation of PI3 kinase has been exemplified in several insulin-induced cellular responses, such as GLUT4 translocation (41, 47-49), activation of glycogen synthesis (50, 51), membrane ruffling (52), trafficking of transferrin receptors (53), and activation of pp70S6K kinase (41, 42). However, no previous study has reported that insulin functionally regulates MSR-mediated endocytic uptake of AGE proteins or modified LDL. Thus, to our knowledge, this is the first demonstration of the presence of functional coupling between insulin-stimulated PI3 kinase and activation of MSR-mediated endocytic uptake of ligands.

MSR gene expression is enhanced via PU.1 and AP-1/ets transcription factors along with macrophage colony-stimulating factor-induced differentiation from human monocytes into macrophages (54-56). Phorbol ester, platelets-derived growth factor-BB, and platelets-derived growth factor-AB are also able to induce MSR gene expression of vascular smooth muscle cells, but no such effect has been noted for insulin (57). The present study showed that insulin enhanced the endocytic uptake of AGE-BSA by CHO-MSR-HIR cells (Fig. 3), but it failed to increase the number of cell surface MSR expressed by these cells (Fig. 4), indicating that insulin-enhanced endocytic uptake of 125I-AGE-BSA is not due to the increased number of cell-surface MSR, but rather to certain post-binding events. The exact mechanism is not known at present. However, it could be due to an insulin-induced acceleration of the rate of endocytic degradation of AGE-BSA by these cells (Fig. 8). A similar phenomenon was observed with insulin-induced GLUT4 recruitment from the intracellular membrane vesicular pool to the surface membrane (47-49).


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Fig. 8.   Schematic representation of a functional link between insulin signaling and MSR activity. Binding of insulin to insulin receptor induces autophosphorylation that activates PI3 kinase via IRS. Activation of PI3 kinase leads to acceleration of MSR-mediated endocytic degradation pathway of AGE proteins (endocytic internalization, vesicle transport, lysosomal degradation, and subsequent exocytosis) without increasing the number of cell surface MSR.

In RAW-HIR and CHO-MSR-HIR cells, insulin stimulation may accelerate exocytosis of degraded products following the classical coated pit/endosomal pathway (58). Several receptors, such as LDL receptor and MSR, are known to be enriched in coated pits as clusters, from where ligand-receptor complexes are internalized into the cell, thus inducing the formation of endosomes. Endosomes are also formed constitutively to some extent even under nonstimulated conditions. Wortmannin and LY294002 did not affect the constitutive type of endocytic uptake both in CHO-MSR-HIR cells (Fig. 7) nor CHO-MSR cells (data not shown). This suggests that PI3 kinase is mainly coupled with an insulin-inducible type of endocytic uptake of ligands (Fig. 8), but not with a constitutive type. Further studies will help elucidate the mechanism by which insulin receptor signaling may enhance MSR-mediated endocytosis of ligands (Fig. 8). Insulin receptors are also known to form endosomes to be internalized on binding of insulin, which is inhibited by mutant insulin receptors or PI3 kinase inhibitors. So it still remains to be solved whether MSR utilize the HIR vesicles or insulin signal induce MSR vesicles trafficking independently of HIR vesicles.

A certain degree of insulin resistance may be explained by dysfunction of the insulin receptor signaling pathway. Cells transfected with mutant HIR, such as CHO-MSR-HIR1030M or CHO-MSR-HIR972F cells did not show insulin-enhanced endocytic uptake of AGE-BSA (Fig. 5). It is therefore possible that insulin resistance state in non-insulin-dependent diabetes mellitus could influence MSR function to some extent through its impaired-insulin signaling. Under such conditions, the insulin-inducible elimination of circulating AGE proteins by LECs in vivo may be reduced or impaired.

It can be hypothesized that atherosclerosis might develop as a consequence of a disability of the hepatic scavenger receptor to eliminate atherosclerotic substances from circulation. If the generation of ligands for the scavenger receptor(s) of LECs and Kupffer cells exceeds the clearance capacity of these cells, or if their endocytic activities are modulated, for example, by insulin resistance, atherogenic molecules might escape hepatic sequestration and reach cells of extra hepatic vessels, causing the development of atherosclerosis.

Recent immunological studies using anti-AGE antibodies demonstrated positive immune reactions in atherosclerotic lesions in human coronary arteries (10) and aorta (4) and aorta in streptozotocin-induced diabetic rats (59). AGE accumulations were found extracellularly as well as intracellularly in monocyte/macrophage-derived foam cells during the early stages of atherosclerosis as well as smooth muscle cell-derived foam cells in the advanced stages of atherosclerosis (4). This suggests that AGE proteins either infiltrate from the blood into the intimal layer or are formed in the subendothelial layer of the vascular wall. Speaking of chronic reaction, AGE proteins of subendothelial space may be endocytosed first by monocyte-derived macrophages and later by smooth muscle cells that migrate from the medial layer. In addition, AGE accumulation was also demonstrated in microvascular lesions, such as the nodular lesions of diabetic glomeruli (1), and intravenous administration of AGE proteins in normal rats induced focal glomerulosclerosis and albuminuria (60). Considered together, these findings suggest that AGE may play an active role in both macrovascular and microvascular diabetic complications either by inducing cellular responses (3, 8, 9) or by AGE accumulation in the tissues or cells (1, 4, 5-7, 10) following endocytosis of AGE proteins by cells of extra hepatic vessels mediated by AGE receptors (9, 12, 61).

In conclusion, although normalization of high glucose levels by insulin may help decrease AGE formation and subsequent accumulation in extrahepatic vessels, the present results obtained from in vitro cellular experiments suggest that insulin is directly involved in LECs-mediated elimination of plasma AGE proteins by enhancing MSR function of these cells.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Akira Endo at the University of Tokyo Bio-Science and Technology for donating compactin. We also thank Dr. F. G. Issa at the Department of Medicine, University of Sydney, Sydney, Australia for reading and editing the manuscript.

    FOOTNOTES

* This work was supported by Grants-in-aid 05670871 and 06671041 for Scientific Research from the Ministry of Education, Science and Culture of Japan.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.

par To whom correspondence should be addressed: Dept. of Biochemistry, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan. Tel.: 81-96-373-5068; Fax: 81-96-364-6940; E-mail: horiuchi{at}gpo.kumamoto-u.ac.jp.

1 The abbreviations used are: AGE, advanced glycation end product(s); LEC(s), liver endothelial cell(s); MSR, macrophage scavenger receptor; HIR, human insulin receptor; CHO, Chinese hamster ovary; BSA, bovine serum albumin; LDL, low density lipoproteins; acetyl, acetylated; FCS, fetal calf serum; PBS, phosphate-buffered saline; IRS, insulin receptor substrate; PI3, phosphatidylinositol-3-OH.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Makino, H., Shikata, K., Hironaka, K., Kushiro, M., Yamasaki, Y., Sugimoto, H., Ota, Z., Araki, N., and Horiuchi, S. (1995) Kidney Int. 48, 517-526[Medline] [Order article via Infotrieve]
  2. McCance, D. R., Dyer, D. G., Dunn, J. A., Bailie, K. E., Thorpe, S. R., Baynes, J. W., and Lyons, T. J. (1993) J. Clin. Invest. 91, 2470-2478[Medline] [Order article via Infotrieve]
  3. Vlassara, H., Bucala, R., and Striker, L. (1994) Lab. Invest. 70, 138-151[Medline] [Order article via Infotrieve]
  4. Kume, S., Takeya, M., Mori, T., Araki, N., Suzuki, H., Horiuchi, S., Kodama, T., Miyauchi, Y., and Takahashi, K. (1995) Am. J. Pathol. 147, 654-667[Abstract]
  5. Miyata, T., Oda, O., Inagi, R., Iida, Y., Araki, N., Yamada, N., Horiuchi, S., Taniguchi, N., Maeda, K., and Kinoshita, T. (1993) J. Clin. Invest. 92, 1243-1252[Medline] [Order article via Infotrieve]
  6. Yan, S. D., Yan, S. F., Chen, X., Fu, J., Chen, M., Kuppusamy, P., Smith, M. A., Perry, G., Godman, G. C., Nawroth, P., Zweier, J. L., and Stern, D. (1995) Nat. Med. 1, 693-699[Medline] [Order article via Infotrieve]
  7. Vitek, M. P., Bhattacharya, K., Glendening, M., Stopa, E., Vlassara, H., Bucala, R., Manogue, K., and Cerami, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4766-4770[Abstract]
  8. Yui, S., Sasaki, T., Araki, N., Horiuchi, S., and Yamazaki, M. (1994) J. Immunol. 152, 1943-1949[Abstract/Free Full Text]
  9. Higashi, T., Sano, H., Saishoji, T., Ikeda, K., Jinnouchi, Y., Kanzaki, T., Morisaki, N., Rauvala, H., Shichiri, M., and Horiuchi, S. (1997) Diabetes 46, 463-472[Abstract]
  10. Nakamura, Y., Horii, Y., Nishino, T., Shiiki, H., Sakaguchi, Y., Kagoshima, T., Dohi, K., Makita, Z., Vlassara, H., and Bucala, R. (1993) Am. J. Pathol. 143, 1649-1656[Abstract]
  11. Horiuchi, S. (1996) Trends Cardiovasc. Med. 6, 21-26
  12. Takata, K., Horiuchi, S., Araki, N., Shiga, M., Saitoh, M., and Morino, Y. (1988) J. Biol. Chem. 263, 14819-14825[Abstract/Free Full Text]
  13. Smedsrød, B., Melkko, J., Araki, N., Sano, H., and Horiuchi, S. (1997) Biochem. J. 322, 567-573[Medline] [Order article via Infotrieve]
  14. Makita, Z., Vlassara, H., Rayfield, E., Cartwright, K., Friedman, E., Rodby, R., Cerami, A., and Bucala, R. (1992) Science 258, 651-653[Medline] [Order article via Infotrieve]
  15. The Diabetes Control and Complications Trial Research Group (DCCT) (1993) N. Engl. J. Med. 329, 976-986
  16. Kodama, T., Freeman, M., Rohrer, L., Zabrecky, J., Matsudaira, P., and Krieger, M. (1990) Nature 343, 531-535[CrossRef][Medline] [Order article via Infotrieve]
  17. Araki, N., Higashi, T., Mori, T., Shibayama, R., Kawabe, Y., Kodama, T., Takahashi, K., Shichiri, M., and Horiuchi, S. (1995) Eur. J. Biochem. 230, 408-415[Abstract]
  18. Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D., Wang, F., Pan, Y. C., Elliston, K., Stern, D., and Shaw, A. (1992) J. Biol. Chem. 267, 14998-15004[Abstract/Free Full Text]
  19. Li, Y. M., Mitsuhashi, T., Wojciechowicz, D., Shimizu, N., Li, J., Stitt, A., He, C., Banerjee, D., and Vlassara, H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11047-11052[Abstract/Free Full Text]
  20. Suzuki, H., Kurihara, Y., Takeya, M., Kamada, N., Kataoka, M., Jishage, K., Ueda, O., Sakaguchi, H., Higashi, T., Suzuki, T., Takashima, Y., Kawabe, Y., Cynshi, O., Wada, Y., Honda, M., Kurihara, H., Doi, T., Matsumoto, A., Azuma, S., Noda, T., Toyoda, Y., Itakura, H., Yazaki, Y., Horiuchi, S., Takahashi, K., van Berkel, T. J. C., Steinbrecher, U. P., Ishibashi, S., Maeda, N., Gordon, S., and Kodama, T. (1997) Nature 386, 292-296[CrossRef][Medline] [Order article via Infotrieve]
  21. Ling, W., Lougheed, M., Suzuki, H., Buchan, A., Kodama, T., and Steinbrecher, U. P. (1997) J. Clin. Invest. 100, 244-252[Abstract/Free Full Text]
  22. Miyazaki, A., Sakai, M., Suginohara, Y., Hakamata, H., Sakamoto, Y., Morikawa, W., and Horiuchi, S. (1994) J. Biol. Chem. 269, 5264-5269[Abstract/Free Full Text]
  23. Sakai, M., Miyazaki, A., Hakamata, H., Sasaki, T., Yui, S., Yamazaki, M., Shichiri, M., and Horiuchi, S. (1994) J. Biol. Chem. 269, 31430-31435[Abstract/Free Full Text]
  24. McFarlane, A. S. (1958) Nature 182, 53
  25. Pertoft, H., and Smedsrød, B. (1987) in Cell Separation, Methods, and Selected Applications (Pretlow, T. G., II, and Pretlow, T. P., eds), Vol. 4, pp. 1-24, Academic Press, New York
  26. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J. H., Masiartz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., and Rutter, W. J. (1985) Cell 40, 747-758[Medline] [Order article via Infotrieve]
  27. Hayashi, H., Miyake, N., Kanai, F., Shibasaki, F., Takenawa, T., and Ebina, Y. (1991) Biochem. J. 280, 769-775[Medline] [Order article via Infotrieve]
  28. Freeman, M., Ekkel, Y., Rohrer, L., Penman, M., Freeman, N., Chisolm, G. M., and Krieger, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4931-4935[Abstract]
  29. Ebina, Y., Araki, E., Taira, M., Shimada, F., Mori, M., Craik, C. S., Siddle, K., Pierce, S. B., and Roth, R. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 704-708[Abstract]
  30. White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R. (1988) Cell 54, 641-649[Medline] [Order article via Infotrieve]
  31. Yano, H., Nakanishi, S., Kimura, K., Hanai, N., Saitoh, Y., Fukui, Y., Nonomura, Y., and Matsuda, Y. (1993) J. Biol. Chem. 268, 25846-25856[Abstract/Free Full Text]
  32. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994) J. Biol. Chem. 269, 5241-5248[Abstract/Free Full Text]
  33. Chung, J., Kuo, C. J., Crabtree, G. R., and Blenis, J. (1992) Cell 69, 1227-1236[Medline] [Order article via Infotrieve]
  34. Fraser, I., Hughes, D., and Gordon, S. (1993) Nature 364, 343-346[CrossRef][Medline] [Order article via Infotrieve]
  35. White, M. F., and Kahn, C. R. (1994) J. Biol. Chem. 269, 1-4[Free Full Text]
  36. Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S. (1994) Nature 372, 182-186[CrossRef][Medline] [Order article via Infotrieve]
  37. Araki, E., Lipes, M. A., Patti, M. E., Bruning, J. C., Haag, B., III, Johnson, R. S., and Kahn, C. R. (1994) Nature 372, 186-190[CrossRef][Medline] [Order article via Infotrieve]
  38. Sun, X. J., Wang, L. M., Zhang, Y., Yenush, Y., Myers, M. J., Jr., Glasheen, E., Lane, W. S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177[CrossRef][Medline] [Order article via Infotrieve]
  39. Lavan, B. E., Lane, W. S., and Leinhard, G. E. (1997) J. Biol. Chem. 272, 11439-11443[Abstract/Free Full Text]
  40. Lavan, B. E., Fantin, V. R., Chang, E. T., Lane, W. S., Keller, S. R., and Leinhard, G. E. (1997) J. Biol. Chem. 272, 21403-21407[Abstract/Free Full Text]
  41. Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J., and Kahn, C. R. (1994) Mol. Cell. Biol. 14, 4902-4911[Abstract]
  42. Chung, J., Grammer, T. C., Lemon, K. P., Kazlauskas, A., and Blenis, J. (1994) Nature 370, 71-75[CrossRef][Medline] [Order article via Infotrieve]
  43. Baynes, J. W., and Thorpe, S. R. (1981) Arch. Biochem. Biophys. 206, 372-379[Medline] [Order article via Infotrieve]
  44. Deleted in proof
  45. Deleted in proof
  46. Deleted in proof
  47. Kanai, F., Nishioka, Y., Hayashi, H., Kamohara, S., Todaka, M., and Ebina, Y. (1993) J. Biol. Chem. 268, 14523-14526[Abstract/Free Full Text]
  48. Clark, J. F., Young, P. W., Yonezawa, K., Kasuga, M., and Holman, G. D. (1994) Biochem. J. 300, 631-635[Medline] [Order article via Infotrieve]
  49. Verhey, K. J., Yeh, J. I., and Birnbaum, M. J. (1995) J. Cell Biol. 130, 1071-1079[Abstract]
  50. Welsh, G. I., Foulstone, E. J., Young, S. W., Tavare, J. M., and Proud, C. G. (1994) Biochem. J. 303, 15-20[Medline] [Order article via Infotrieve]
  51. Cross, D. A., Alessi, D. R., Vandenheed, J. R., McDowell, H. E., Hundal, H. S., and Cohen, P. (1994) Biochem. J. 303, 21-26[Medline] [Order article via Infotrieve]
  52. Kotani, K., Yonezawa, K., Hara, K., Ueda, H., Kitamura, Y., Sakaue, H., Ando, A., Chavanieu, A., Calas, B., Grigorescu, F., Nishiyama, M., Waterfield, M. D., and Kasuga, M. (1994) EMBO J. 13, 2313-2321[Abstract]
  53. Shepherd, P. R., Soos, M. A., and Siddle, K. (1995) Biochem. Biophys. Res. Commun. 211, 535-539[CrossRef][Medline] [Order article via Infotrieve]
  54. Wu, H., Moulton, K., Horvai, A., Parik, S., and Glass, C. K. (1994) Mol. Cell. Biol. 14, 2129-2139[Abstract]
  55. Moulton, K., Semple, K., Wu, H., and Glass, C. K. (1994) Mol. Cell. Biol. 14, 4408-4418[Abstract]
  56. Ishibashi, S., Inaba, T., Shimano, H., Harada, K., Inoue, I., Mokuno, H., Mori, N., Gotoda, T., Takaku, F., and Yamada, N. (1990) J. Biol. Chem. 265, 14109-14117[Abstract/Free Full Text]
  57. Inaba, T., Gotoda, T., Shimano, H., Shimada, M., Harada, K., Kozaki, K., Watanabe, Y., Hoh, E., Motoyoshi, K., Yazaki, Y., and Yamada, N. (1992) J. Biol. Chem. 267, 13107-13112[Abstract/Free Full Text]
  58. Willinngham, M. C., and Pastan, I. (1980) Cell 21, 67-77[Medline] [Order article via Infotrieve]
  59. Meng, J., Sakata, N., Takebayashi, S., Asano, T., Futata, T., Araki, N., and Horiuchi, S. (1996) Diabetes 45, 1037-1043[Abstract]
  60. Bucala, R., Makita, Z., Vega, G., Grundy, S., Koschinsky, T., Cerami, A., and Vlassara, H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9441-9445[Abstract/Free Full Text]
  61. Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5588-5592[Abstract]


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