Glucose regulates insulin mitogenic effect by modulating SHP-2 activation and localization in JAr cells.

Glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6 mM glucose (LG), proliferation and thymidine incorporation were induced by serum, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I), but not by insulin. At variance, at 25 mM glucose (HG), proliferation and thymidine incorporation were stimulated by insulin, serum, EGF and IGF-I, to a comparable extent, while basal levels were 25% lower than those in LG. HG culturing also enhanced insulin-stimulated insulin receptor (IR) and insulin receptor substrate 1 (IRS1) tyrosine phosphorylations while decreasing basal phosphorylations. These actions of glucose were accompanied by an increase in cellular tyrosine phosphatase activity. The activity of SHP-2, in HG-treated JAr cells, was 400% of that measured in LG-treated cells. SHP-2 co-precipitation with IRS1 was also increased in HG-treated cells. SHP-2 was mainly cytosolic in LG-treated cells. However, HG culturing largely redistributed SHP-2 to the internal membrane compartment, where tyrosine phosphorylated IRS1 predominantly localizes. Further exposure to insulin rescued SHP-2 cytosolic localization, thereby preventing its interaction with IRS1. Antisense inhibition of SHP-2 reverted the effect of HG on basal and insulin-stimulated IR and IRS1 phosphorylation as well as that on thymidine incorporation. Thus, in JAr cells, glucose modulates insulin mitogenic action by modulating SHP-2 activity and intracellular localization.


INTRODUCTION
Glucose controls a variety of cellular functions. Changes in the extracellular concentrations of glucose may determine either increased cell proliferation or cell death (1)(2)(3). For instance, beta-cell mitogenesis is stimulated by glucose through a protein kinase C-dependent mechanism (4,5). At variance, in human endothelial cells, high glucose concentrations activate JNK signalling and trigger apoptosis (6). Previous evidence indicates that glucose modulates the transduction pathways of many growth factors (7)(8)(9)(10)(11)(12)(13). However, the mechanisms responsible for glucose control of growth signalling have not been extensively investigated.
Chronic abnormalities in glucose concentration in the extracellular fluids may lead to impaired tissue homeostasis (14). High blood glucose levels in pregnant women with diabetes often causes placenta hypercellularity, resulting in larger and heavier placentas (15). There is evidence that placenta growth is regulated by glucose as well as by growth factors (16)(17)(18)(19)(20). In vitro, epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1) and transforming growth factor α (TGFα) stimulate proliferation of cultured human cytotrophoblast cells and their ability to produce human chorionic gonadotropin and progesterone (21). Insulin also induces proliferative responses in placenta, likely due to the activation of Insulin Receptor Substrate (IRS)/Mitogen Activated Protein Kinase (MAPK) pathway (20). In several cell types, insulin signalling is regulated by glucose at different levels (7,22). But, whether and how glucose affects insulin mitogenic signalling in placenta is unclear.
Hepes, 250mM sucrose, 1 mM EDTA, 5mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1µM leupeptin, pH 7.4. Subcellular fractions were prepared by a modification of the methods of Simpson (24). Briefly, the cells were washed with ice-cold buffer A (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 255 mM sucrose, 1 mM PMSF, 10 mM NaF, 100 µM Na 3 VO 4 , 1 mM NaP 2 O 7 , 5 µg/ml aprotinin, 5 µg/ml leupeptin) and broken in ice-cold buffer A by passing through a 27-gauge needle and centrifuged at 800 x g for 5 min at 4°C to pellet nuclei. Supernatants were centrifuged at 16,000 x g for 20 min at 4°C. Pellets were applied on a 1.12 M sucrose cushion and centrifuged at 212,000 x g for 70 min for preparation of the plasma membrane fraction. Supernatants were further centrifuged at 212,000 x g for 20 min for preparation of the internal membrane (pellet) and cytosol (supernatant) fractions (24). Purity of the subcellullar fractions was assessed by western blot analysis of cytocrhome c oxidase (internal membrane), tubulin (cytosol) and by determination of 5'-nucleotidase activity (plasma membrane) according to (25), (26), and (27), respectively.

[H]thymidine incorporation
-For this assay, the cells were plated in 24-well plates (10 5 cells/well) in DMEM supplemented with 10% FBS. Upon 12h, the medium was substituted with glucose free DMEM supplemented with 0.25% BSA for 16h. The cells were then incubated in the medium supplemented with 6 mM (LG) or 25 mM (HG) glucose with or without insulin (100 nM), IGF-1 (100 nM), EGF (100 ng/ml), FBS (10%) for the indicated times. In control experiments the cells were incubated in glucose free DMEM containing 6 or 25mM sucrose or fructose. 5 µCi/ml 3 [H]-thymidine were added and the incubation was prolonged for 4 more hours. The cells were then lysed in 1N NaOH and radioactivity was counted by liquid scintillation counting.
Cell proliferation and DNA fragmentation assay -For proliferation assay, JAr cells were plated in 24-well multiplates (2 x 10 4 cells/plate) in DMEM supplemented with 10% FBS. 12 h later, this medium was replaced with serum-free DMEM containing 0,25% BSA. Upon 12 additional hours, the medium was substituted with DMEM containing either 6 or 25 mM glucose and growth factors for the indicated times. For DNA fragmentation analysis, JAr cells were deprived from serum and glucose for 12 h and then incubated for 48h in DMEM containing 6 or 25 mM glucose in the absence or the presence of 100nM insulin and 25 ng/ml TNFα. Cells (2x10 6 ) were lysed in 0.5 ml lysis buffer (10 mM Tris, pH 7.5, 0.6 % SDS, 10 mM EDTA). The RNAse solution was added to a concentration of 15 µg/ml, and cell lysates were incubated for 20 min at 37C. NaCl was then added to 1 M (final concentration) followed by incubation at 4C for 2 h. Samples were centrifuged at 14,000 x g (4C for 30 min), and supernatant DNA was extracted by phenol-chloroform and ethanol precipitation at -20C overnight. Upon centrifugation at 14,000 x g, the DNA pellet was air dried and dissolved in 20 µl TE buffer (10 mM Tris, 10 mM EDTA). Identical amounts of DNA were electrophoresed on 1.5% agarose gels containing 0.5 µg/ml ethidium bromide and visualized by UV light as described in (28).
Insulin receptor binding and insulin degradation -Insulin receptor binding was performed by radioreceptor assay as described in (29). For this assay, JAr cells were deprived from serum and glucose for 16 h and then incubated in DMEM supplemented with 6 or 25 mM glucose for 48, 96 or 128 h. The cells were then incubated for 12h at 4C in 1 ml of binding buffer (pH 7.8, 25 mM Tris-HCl, 120 mM NaCl, 1.2 mM MgSO 4 , 2.5 mM KCl, 2% BSA, 1 mg Bacitracin) containing 20,000 cpm 125 [I]-Insulin. Non specific binding was determined in the presence of 8.5 x 10 -5 insulin and was subtracted from total bound radioactivity to yield specific binding. Binding data were analysed using the LIGAND program for curve fitting and parameter estimation (30). Insulin degradation was determined by TCA precipitation as described in (31).
IR and IRS1 phosphorylation -For IR phosphorylation studies, JAr cells were deprived from serum and glucose for 12 h and then incubated in DMEM supplemented with 6 or 25 mM glucose for 48 h.
MAPK phosphorylation and PTPase assay -JAr cells were incubated in 6 or 25 mM glucose and stimulated with insulin as described above. The cells were then lysed in TA buffer supplemented with 0.1% Triton-X-100 (TAT). Lysates were separated on 12% SDS-PAGE, transferred on nitrocellulose filter and then blotted with phospho-MAPK or MAPK antibodies according to (33).
Alternatively, to determine protein tyrosine phosphatase activity (PTPase) the cells were incubated with or without 2 mM sodium vanadate in phosphate-free buffer for 20 min at 37C, according to (34). Incubation with 100 µM pervanadate instead of 2mM vanadate yielded identical results. The cells were lysed in 0.5% Triton-X-100 and then immunoprecipitated with IR, IRS1 and SHP-2 specific antibodies. Pellets were resuspended in 50 mM Hepes, pH 7.0 and reactions initiated by the addition of 20 mM p-nitrophenyl phosphate (pNPP) at 37°C for 15 min. Reactions were stopped with 1N NaOH and release of p-nitrophenol was spectrophotometrically quantitated at 410 nm.
PTPase (vanadate-sensitive activity) was expressed as the difference between the total phosphatase activity (measured in the absence of vanadate) and the vanadate-resistant activity.
PTPs IRS1 association -JAr cells were incubated in 6 or 25 mM glucose and stimulated with insulin as described above. Cells were then lysed with TAT buffer and extracts incubated with sepharose-bound IR or IRS1 antibodies at 4C for 3 h. The beads were washed with HNT buffer (TA containing 0.1% Triton X-100) and bound proteins released by heating at 65C for 5 min with SDS sampling buffer (4% SDS, 10% glycerol, 100 mM Tris pH 6.8, 1 mM EDTA, 10 mM dithiothreitol, 8M urea). Released proteins blotted with SHP-2, PTP1B or LAR antibodies and revealed by ECL and autoradiography. In different cell types, glucose affects apoptosis (35,36). However, no DNA laddering was observed in JAr cells cultured with LG or HG, either in the absence or in the presence of insulin (data not shown).

Glucose action on insulin mitogenic signalling.
To explore the mechanism of glucose action on insulin mitogenesis, we investigated insulin binding and early signalling events in JAr cells cultured with different glucose concentrations. Based on Western blotting with IR Ab, IR protein content was almost identical in LG-and HG-cultured JAr cells (Fig. 3A, top panel). Binding levels and Kds also did not show significant differences in cells maintained in LG and in HG (Table I) To further address the molecular mechanism of glucose effect on insulin action, we next investigated phosphorylation of IRS1 in the JAr cells. As shown in Fig. 4 (panel A), insulinindependent IRS1 phosphorylation was 50% lower in HG-as compared to LG-treated cells (p< 0.05). However, insulin increased IRS1 phosphorylation by 400% of control in HG-cultured cells (p< 0.01) but elicited no significant effect in LG cells. The differences in IRS1 phosphorylation in cells cultured in LG and HG were accompained by similarly-sized differences in MAPK phosphorylation (Fig. 4, panel B). There were no changes in IRS1 and MAPK protein levels in LG and HG cells (Fig 4, panel C).
Effect of glucose on protein-tyrosine phosphatase activity. We next investigated whether glucose effect on IR and IRS1 phosphorylation is mediated by changes in cellular tyrosine phosphatase (PTPase) activity. As shown in Table II, exposure to HG increased PTPase activity in JAr cell extracts by almost 300% (p< 0.01). Insulin, inhibited PTPase activity in LG cells by about 30% (not statistically significant) and almost completely abolished the effect of HG (p<0.05). PTPase activity was also measured in IR and IRS1 immunoprecipitates from the cells. Neither glucose nor insulin affected the IR-co-precipitated PTPase, however. At variance, IRS1-associated PTPase activity was 350% higher in HG than in LG cells (p < 0.01) and was blocked by insulin stimulation.
To verify whether specific PTPase(s) are involved in glucose regulation of IR signalling we evaluated the expression of SHP-2, PTP1B and LAR in JAr cells. All of these PTPases are involved in insulin signalling (37)(38)(39) and, as shown in Interestingly, in HG cultured cell extracts but not in those from LG cells, SHP-2 co-precipitated with IRS1 (Fig. 5, panel B). Further exposure of HG-treated cells to insulin completely abolished IRS1-SHP-2 co-precipitation. No IR-SHP-2 co-precipitation was detected either in HG or in LG cells. LAR and PTP1B association with IRS1 was barely detectable in JAr cells and was not modified by glucose or insulin treatment (Fig. 5, panel C). As shown in Fig. 6 (panel B), phosphatase activity in SHP-2 immunoprecipitates was increased by 400% upon exposure of the cells to HG. Same as in the IRS1 precipitates, insulin stimulation of the cells completely inhibited PTPase activity in the SHP-2 precipitates.
To prove SHP-2 involvement in glucose action on insulin mitogenesis, we have transiently transfected SHP-2 antisense oligonucleotides (SHP-2-AS) in the JAr cells. As shown in Fig. 6 (panel A), the antisense reduced SHP-2 expression by >70% (p<0.001). Consistently, SHP-2 activity was barely detectable in SHP-2 precipitates from cells transfected with SHP-2-AS but unmodified by control oligonucleotides (Fig. 6, panel A). Also, the SHP-2-AS completely inhibited SHP-2-IRS1 co-precipitation in extracts from HG-treated cells (Fig. 7A, top panel). In HG-cultured cells, block of SHP-2 expression increased insulin-independent IR and IRS1 phosphorylation, respectively, by 30% and 50% (p< 0.01). The effect of the antisense was accompanied by a 300% decrease in insulin-stimulated phosphorylations of IR and IRS1. SHP-2-AS treatment of HG cells, also returned basal and insulin stimulated thymidine incorporation to levels comparable to those observed in LG cells (Fig. 7B). SHP-2-AS had no effect on basal and insulin-dependent IR and IRS1 phosphorylation (Fig. 7A, middle and bottom panel, lanes E-F vs lanes A-B) as well as thymidine incorporation (Fig. 7B) in LG-exposed cells.
To address the tissue-specificity of glucose action on SHP-2 activity and insulin signalling, we have further compared SHP-2 activity in the choriocarcinoma BeWo cells, and in 3T3 fibroblasts.
As shown in Fig. 8, HG exposure of BeWo cells increased SHP-2 activity, similarly as in JAr cells.
Also, in the BeWo, as well as in the JAr cells, insulin blocked glucose activation of SHP-2. By contrast SHP-2 activity was identical in 3T3 cells whether cultured in HG or in LG and increased upon insulin exposure by 500 % of control both upon LG and HG treatment. In addition, in LGcultured BeWo cells, insulin failed to increase DNA synthesis. HG culturing determined a 20% reduction of thymidine incorporation compared to LG, but allowed a > 300% increase in DNA To this end, we prepared internal membrane (IM) and cytosolic fractions (Cy) from JAr cells. The purity of the different fractions is shown in Table III. As shown in Fig. 9 (panel A)

DISCUSSION
We have investigated glucose regulation of cell growth in the JAr human choriocarcinoma cell line.
These cells maintain in culture many features characteristic of human first trimester trophoblast, including chorionic gonadotropin and progesterone secretion (21). Because of these features, JAr cells have been widely used to investigate glucose and insulin action in placenta (19,20). We found that insulin does not induce proliferation of JAr cells when these are maintained in low glucose medium. This finding is consistent with previous reports showing that, in vivo, insulin is a weak mitogenic factor in placenta when glycemic control is achieved (17,40). As recently reported by Glucose co-operation with growth factor signalling has already been described in different cell types (5,41). For istance, in insulinoma cells, glucose induces a dose-dependent increase of nerve growth factor effect on insulin secretion, but the mechanisms remain elusive (5,41). However, the data in the present report show, for the first time, that extracellular glucose also controls insulin mitogenic action. Our study shows that glucose does not affect insulin receptor (IR) number or affinity in the JAr cells. Still, chronic exposure to high glucose concentrations slightly reduced the basal activation state of the IRS1/MAPK cascade but significantly increased insulin-stimulated signalling along this pathway. Thus, at least in part, glucose potentiates insulin mitogenic signalling in JAr cells by inducing the IRS1/MAPK pathway. We envisioned two possible mechanisms for this glucose regulatory effect. Firstly, in JAr cells, glucose may affect PKC activity. These Ser/Thr kinase phosphorylate several insulin signalling molecules and inhibit their subsequent tyrosine phosphorylation and function (42,43). Hence, our previous work in muscle cells showed that glucose rapidly causes reverse translocation of PKC alpha from the plasma membrane to the cytoplasm. Reverse translocation of PKC alpha reduces PKC alpha-IR association and IR ser/thr phosphorylation and acutely activates the insulin signalling system in these cells (42). A similar mechanism is unlikely to account for glucose effect on insulin mitogenic signalling in the JAr cells, however. In fact, treatment of JAr cells with the PKC inhibitor bisindolylmaleimide does not affect IR and IRS1 phosphorylation in the presence of either low or high glucose concentrations (data not shown). Alternatively, in the JAr cells, glucose may regulate the action of tyrosine phosphatase(s) (PTPases) on key elements of the insulin signalling pathway (37,44). Consistent with this hypothesis, we show here that the activity of the SHP-2 PTPase is increased in extracts of JAr as well as in those from BeWo placenta cells cultured in high glucose medium. In addition, we have shown that SHP-2 but not PTP1B or LAR, coprecipitated with IRS1 in JAr cell extracts. SHP-2-IRS1 association only occurred in HG cultured cells. Importantly, antisense block of SHP-2 expression in HG cells returned IRS1 phosphorylation and insulin mitogenesis to levels comparable to those of cells maintained in low glucose medium indicating that SHP-2 mediates the permissive effect of glucose on insulin signalling through the IRS1/MAPK pathway.

Acute stimulation of HG-cultured cells with insulin prevented SHP-2-IRS1 co-precipitation.
Thus, in JAr cells, insulin causes the release of the SHP-2-IRS1 association induced by the chronic exposure to HG levels. We propose that the SHP-2 association is responsible for the reduced IRS1 phosphorylation levels we have observed in HG-cultured JAr cells. Subsequent exposure of these cells to insulin releases the association and enables IRS1 to undergo tyrosine phosphorylation more effectively than in LG cells, which feature higher basal levels of IRS1 phosphorylation. Enhanced phosphorylation of IR/IRS1 in HG-cultured cells, in turn, more actively conveys insulin signal through the MAPK/mitogenic pathway. Thus, by fostering SHP-2 association, glucose maintains IRS1 in a low phosphorylation state. Insulin then release the association enabling IRS1 to achieve higher levels of phosphorylation by IR kinase. The changes in IRS1 phosphorylation determined by HG exposure were paralleled by similar changes in the phosphorylation of IR kinase. At variance with IRS1 however, no glucose-and insulin-dependent IR-SHP-2 co-precipitation was detectable in the JAr cells. SHP-2 association with the IR may be weaker than with IRS1, preventing coimmunoprecipitation. In addition, previous work by Solow et al. evidenced that IRS1 may determine the activation state of the IR through a PTPase-dependent mechanism (45). Thus, HG effects on IR tyrosine phosphorylation may also reflect the regulation of IRS1 activity by SHP-2.
SHP-2 is a cytosolic PTPase (46). However, in the present report, we show that exposure of JAr Previous studies evidenced that SHP-2 plays an important role in transducing insulin mitogenic signals (37,47). Consistent with these reports, we describe that antisense inhibition of SHP-2 expression in JAr cells led to a significant inhibition of insulin-induced DNA synthesis.
Interestingly, however, we have also found that HG exposure slightly inhibits DNA synthesis, but increases SHP-2 activity. Thus, in JAr cells, SHP-2 activation may not be sufficient to elicit growth stimulatory responses. Hence, when the cells are stimulated with insulin in the presence of high glucose levels, coincidence of SHP-2-IRS1 dissociation and induction of MAPK and DNA synthesis suggests that SHP-2 might play a regulatory rather than a direct causal role in JAr cell proliferation, by controlling the phosphorylation state of signalling molecules. In 3T3 cells, the expression of a catalytically inactive SHP-2 causes a reduction in MAPK phosphorylation and insulin mitogenesis with no effect on IR and IRS1 phosphorylation (47). At variance, in JAr cells SHP-2 modulates the insulin mitogenic signalling cascade by acting at the level of IR and IRS1 phosphorylation. It appears therefore that the function of SHP-2, as well as that of glucose, feature tissue-specificity. Regulation of SHP-2 also appears to involve different mechanisms in different cell types. In fact, previous evidence indicates that insulin stimulates SHP-2 activity in fibroblasts (47). Here, we show that both the JAr and BeWo placenta cells feature glucose but not insulin activation of SHP-2. Glucose itself impinges on molecular mechanisms in a tissue-specific fashion.
In conclusion, in the present report, we have shown that glucose exerts a permissive action on insulin mitogenesis in placenta cells. Glucose-induced activation and intracellular de-localization of SHP-2 is a key mechanism controlling insulin signal transduction through the IRS1/MAPK pathway.        cells. BeWo and 3T3 cells were cultured in either LG or HG media. The cells were then stimulated with insulin for 10 min and SHP-2 activity was assayed as described under Materials and Methods (A). Alternatively, the cells were incubated with 100 nM insulin and then assayed for thymidine incorporation as outlined in the legend to Fig. 8 (B). Bars represent the mean ± S.D. of duplicate determinations in three (panel A) and four (panel B) independent experiments.   PTPase activity was measured from total cell lysates (50 µg protein) and from IR and IRS1 precipitates (500 µg proteins, as determined before the immunoprecipitation) using p-nitrophenylphosphate as substrate. The activity is expressed as nmol mg 1 hr 1 as described under Materials and Methods. Data are the means ± S.D. of three independent experiments.