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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 3 1301-1305
Copyright © 2001 by The Endocrine Society

Original Studies

Hyperglycemia Acutely Increases Monocyte Extracellular Signal-Regulated Kinase Activity in Vivo in Humans

Giulio Ceolotto, Alessandra Gallo, Michelangelo Sartori, Roberto Valente, Elisabetta Baritono, Andrea Semplicini and Angelo Avogaro

Department of Clinical and Experimental Medicine, University of Padova, 35100 Padova, Italy

Address all correspondence and requests for reprints to: Angelo Avogaro, M.D., or Andrea Semplicini, M.D., Department of Clinical and Experimental Medicine, Via Giustiniani 2, 35100 Padova, Italy. E-mail: avogaro{at}ux1.unipd.it or asempl@ux1.unipd.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glycemic spikes may negatively affect the long-term prognosis of patients with diabetes. Extracellular signal-regulated kinases (ERKs) are intracellular mediators of cell proliferation, and they can be activated in response to high glucose levels. However, the modifications of their activity in response to hyperglycemia have been poorly investigated, in vivo, in humans. Thus, we sought to determine in circulating monocytes: 1) the role of hyperglycemia in ERKs activity and phosphorylation, and 2) whether hyperglycemia affects mitogen-activated protein kinase kinase (MEK) activity and mitogen-activated protein phosphatase-1 (MKP-1) expression. These goals were performed in five normal subjects. Baseline monocyte ERKs activity was 60 ± 5 pmol/min·mg protein; when exogenous hyperglycemia was induced, both monocyte ERKs activity (81 ± 11 pmol/min·mg protein; P < 0.05) and phosphorylation significantly increased (P < 0.01). MEK activity was significantly increased by hyperglycemia (1251 ± 136 vs. 2000 ± 42 cpm; P = 0.0017), whereas no changes were observed in MKP-1 expression. We conclude that hyperglycemia acutely stimulates ERKs activity and phosphorylation in human monocytes by the MEK pathway in vivo. These findings may be relevant in understanding the negative role of acute hyperglycemia on monocyte pathophysiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPERGLYCEMIA IS A risk factor for both micro- and macrovascular complications of diabetes and can cause long-term complications by inducing hyperosmolarity, oxidant formation and diacylglycerol-protein kinase C (PKC) activation (1, 2). It is likely that glucose mediates its adverse effects by altering the various signal transduction pathways. We and others have recently demonstrated that the activation of PKC, especially the ß isoform, could be responsible for some of the vascular dysfunctions observed in the diabetic state (3, 4). However, it has been suggested that the risk of diabetic complications may be more highly dependent on the extent of postprandial glycemic excursions (5); these may increase oxidative stress (6), the expression of adhesion molecules on endothelial surfaces (7), and coagulation activation (8).

Extracellular signal-regulated protein kinases (ERKs) are a conserved family of serine/threonine protein kinases that play a central role in intracellular signaling (9). These proteins are activated by phosphorylation on tyrosine and threonine residues by the dual specificity mitogen-activated protein kinase kinase (MEK) and are inactivated by dephosphorylation by a mitogen-activated protein phosphatase-1 (MKP-1) (10). Therefore, ERKs phosphorylation results from the balance between kinases and phosphatases.

Recent studies have shown that hyperglycemia can influence the regulation of ERKs activity (11, 12). However, information on ERKs activity in vivo in humans and, specifically, on the role of hyperglycemia in their regulation by MEK and MKP-1 is scanty. A report on the effect of hyperglycemia on monocyte ERKs from Goldfine et al. found that in type 2 diabetic patients, therapy with sodium metavanadate, which mimics the effect of insulin, increases by 4-fold the basal ERKs activity in circulating mononuclear cells (13). On the contrary, very recently Cusi et al. have shown in skeletal muscle that in euglycemic insulin-resistant type 2 diabetic patients insulin resistance does not affect the ERKs signaling pathway (14). However, hyper- rather than euglycemia is the metabolic signal that appears to negatively influence mitogenic activity in the cells.

For these reasons, we chose circulating monocytes as a cell model to assess the intracellular modifications induced by acute hyperglycemia. Recent studies indicated that several abnormalities in leukocyte-endothelium interaction of diabetes can be related to hyperglycemia (15, 16). Furthermore, investigation of the role of ERKs in monocytes appears to be of importance because these cells play a crucial role in the earliest events of atherosclerotic lesion, especially in the presence of high glucose concentrations. ERKs in monocytes also appear to be important mediators of immunological response, as the activation of MEK/ERKs is critical for cytokine and PGE2 production in response to lipopolysaccharide (17). Therefore, in this study we sought to determine the effects of hyperglycemia on the regulation of ERKs activity in monocytes from normal controls both in baseline conditions and after 5 h of exogenously induced hyperglycemia.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
We recruited for this study five control volunteers (two females and three males) who participated in the study after giving written informed consent; their mean age and body mass index were 29 ± 3 yr and 22.5 ± 1.6 kg/m2, respectively. Their serum total cholesterol was 4.45 ± 0.17 mmol/L, high density lipoprotein cholesterol was 1.22 ± 0.07 mmol/L, and triglyceride level was 1.52 ± 0.13 mmol/L. Plasma insulin and C peptide were 36 ± 6 pmol/L and 0.73 ± 0.14 nmol/L, respectively. They were in good health. For at least 3 days before the study, each subject consumed a diet containing more than 250 g carbohydrate. The Local Ethical Committee approved the study protocol.

Experimental procedures

Subjects were studied after an overnight fast. At 0730 h, after baseline sampling for monocyte preparation was accomplished, a combined exogenous iv continuous infusion of glucose (10%, wt/vol) and somatostatin (250 µg/h) was begun to raise plasma glucose from a normal mean of 4.7 mmol/L to a mean of 13.05 mmol/L. Plasma glucose was determined every 10 min. Five hours after the beginning of glucose and somatostatin infusions, blood sampling for the determination of ERKs activity and immunoblotting and MEK expression in circulating monocytes was repeated.

Laboratory analysis

Plasma glucose was measured using a glucose oxidase method on a glucose analyzer (Beckman Coulter, Inc. Palo Alto, CA). Plasma insulin and C peptide were measured by polyclonal RIA (18). Cholesterol and triglycerides in plasma were measured by enzymatic methods. High density lipoprotein cholesterol was determined according to the method of Kostner et al. (19).

Determination of ERKs activity in circulating monocytes

Monocyte preparation. Mononuclear cells were prepared from heparinized blood by centrifugation (400 x g for 30 min at room temperature) over Histopaque-1077 (Sigma, St. Louis, MO), as previously described (20). Mononuclear cells were washed once with saline phosphate buffer (PBS) and twice with RPMI 1640 medium, then resuspended in glucose-deficient RPMI 1640 medium supplemented with 5% FCS, 2 mmol/L L-glutamine, 2 mmol/L glucose, 100 U/mL penicillin, and 10 µg/mL streptomycin. Cells were plated at 1 x 107 cells/mL in 60-mm culture dishes incubated for 1 h at 37 C in humidified atmosphere with 5% CO2. Nonadherent cells were removed by washing with ice-cold PBS, and adherent cells were kept in serum-free RPMI 1640 for 1–2 h. More than 90% of adherent cells were monocytes by morphological examination and by staining with Diff-Quik. More than 95% of monocytes was viable as determined by trypan blue exclusion.

Cell fractionation. Monocytes were rapidly lysed by the addition of ice-cold extraction buffer containing 12.5 mmol/L Tris-HCl (pH 7.4), 250 mmol/L sucrose, 2 mmol/L ethylenediamine tetraacetate, 2 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 25 mmol/L ß-glycerol phosphate, 2 mmol/L sodium vanadate, 10 µmol/L phenylmethylsulfonylfluoride, 1 µg/mL leupeptin, and 5 µg/mL aprotinin. Cells were disrupted with a Dounce homogenizer (20 strokes; Kontes Co., Vineland, NJ) on ice for 1 min. The homogenate was centrifuged at 800 x g for 10 min at 4 C to remove cell debris and nuclei. The homogenate was then centrifuged at 14,000 x g for 15 min at 4 C in an Eppendorf centrifuge. The resultant supernatant was removed and stored at -80 C.

Protein assay. The protein concentration of the supernatant was determined with Bradford’s method using BSA as standard.

Immunoblot analysis of ERKs, MEK, and MKP-1 expression

Lysate fraction (40 µg) from human monocytes was immunoblotted with rabbit polyclonal antibodies for ERKs, MEK, and MKP-1 detection (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). It was solubilized in Laemmli buffer and then separated by electrophoresis through a 10% polyacrylamide gel. Proteins separated on the gels were electroblotted onto nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) in blotting buffer containing 48 mmol/L Tris, 39 mmol/L glycine, 0.037% SDS, and 20% (vol/vol) methanol for 2 h at 100 V in the cold using a Transblot cell (Elettrofor, Padova, Italy). The membranes were blocked overnight at 4 C in PBS containing 0.05% (vol/vol) Tween and 5% BSA. Membranes were exposed to primary antibody (1:3000 dilution) for anti-ERKs, (1:3000 dilution) anti-MEK (1:2000 dilution) and MKP-1 overnight at 4 C. Membranes were washed (four times, 20 min each time) with the same buffer and then incubated with 1:4000 goat antirabbit antibody conjugate to horseradish peroxidase. Detection was made using the enhanced chemiluminescence system from Amersham Pharmacia Biotech. Blots were scanned and quantified with Chemiluminescence Molecular Imaging Systems (Bio-Rad Laboratories, Inc., Richmond, CA), and results were expressed relative to the control(s) on the same blot, which was set at 100%.

Assessment of p42/p44 ERKs phosphorylation

Samples (40 µg) for phospho-ERKs immunoblot analysis were electrophoresed on 10% polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane as described above. The membrane was blocked at 4 C overnight in PBS containing 0.05% (vol/vol) Tween and BSA and then incubated with anti-phospho-ERK-specific antibody (Santa Cruz Biotechnology, Inc.; 1:1500 dilution) overnight at 4 C. The membrane was then washed four times and incubated with antimouse antibody conjugate to horseradish peroxidase (1:4000 dilution) at room temperature for 1 h. The bands were detected using the enhanced chemiluminescence system (Amersham Pharmacia Biotech).

ERKs activity assay

ERKs activity was determined using a MAPK assay kit (Upstate Biotechnology, Inc., Lake Placid, NY). The assay is based on phosphorylation of the specific substrate myelin basic protein (MBP), using the transfer of the {gamma}-phosphate of [32P]ATP ([{gamma}-32P]ATP) by ERKs. The methodology was based on the manufacturer’s instructions. Briefly, 10 µL of each substrate mixture, inhibitor mixture, and cell lysate (10 µg/mL protein) were aliquoted into a microcentrifuge tube. The substrate mixture contained 2 mg/mL MBP in assay dilution buffer containing 20 mmol/L MOPS (pH 7.2), 5 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 25 mmol/L ß-glycerol phosphate, 1 mmol/L sodium orthovanadate, 2 µmol/L protein kinase A inhibitor peptide, and 200 µmol/L PKC inhibitor peptide.

The reaction was initiated by the addition of 10 µL Mg2+/ATP mixture (75 mmol/L MgCl2 and 500 µmol/L ATP in assay dilution buffer) containing [{gamma}-32P]ATP (3000 Ci/mmol) for 10 min at 30 C. Reactions were terminated by spotting a 25-µL aliquot onto phosphocellulose paper (P81, Whatman, Clifton, NJ). 32P-Labeled proteins were counted by a liquid scintillation counter after four washes in 75 mmol/L phosphoric acid. ERKs activity was expressed as picomoles of ATP incorporated into MBP per mg cell extract/min.

MEK activity assay

MEK activity was measured using a recombinant kinase-inactive glutathione-S-transferase-MEK1 as substrate (Upstate Biotechnology, Inc.) after immunoprecipitation of the kinase. Cell lysate (300 µg protein) was incubated with 5 µL anti-MEK antibody for 2 h at 4 C. The immunoprecipitate was recovered by incubation with Protein A/G Plus agarose (50 µL; Santa Cruz Biotechnology, Inc.) overnight at 4 C, then centrifugation and washing three times with cell lysis buffer and once with the kinase buffer containing 25 mmol/L HEPES (pH 7.4), 25 mmol/L ß-glycerolphosphate,15 mmol/L MgCl2, 50 µmol/L ATP, 10 µCi [{gamma}-32P]ATP, and 2 µg MEK1 substrate. The immunoprecipitate was incubated with the peptide for 15 min at 30 C. The reaction was terminated by spotting a 25-µL aliquot onto phosphocellulose paper (Whatman P81). 32P-Labeled proteins were counted by a liquid scintillation counter after four washes in 75 mmol/L phosphoric acid. The reaction blank was subtracted from all values.

Statistics

The nonparametric test Wilcoxon sign rank test was used to analyze data with GraphPad Software, Inc. (version 2.00) statistical software.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Metabolic parameters

Plasma glucose was raised from a mean of 4.72 ± 0.36 to 13.21 ± 0.92 mmol/L (P < 0.001). During somatostatin infusion plasma insulin and C peptide concentrations slightly, but not significantly, decreased [from 37 ± 5 to 25 ± 8 pmol/L (P = 0.2391) and from 0.59 ± 0.09 to 0.38 ± 0.06 nmol/L (P = 0.0882), respectively].

Basal condition

We first examined ERKs and MEK protein expression by immunoblotting cell extracts and detecting ERKs and MEK expression using specific polyclonal antibodies. Figure 1AGo shows a typical blot for ERKs expression in human monocytes. The antibody for ERKs kinases detected both bands, with molecular masses corresponding to 44 kDa (ERK1) and 42 kDa (ERK2). Figure 1BGo shows a typical blot for MEK expression in human monocytes. The antibody for MEK detected the band at 45 kDa.



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  		Figure 1. Immunoblot of ERK1 and ERK2 isoforms in monocytes from two control subjects. The upper panel shows representative immunoblot of the 44- and 42 kDa isoforms of ERKs. Protein samples (40 µg) were resolved on 10% polyacrylamide gels and transferred to a nitrocellulose membrane. The blots were probed with anti-ERK-specific antibody. The bottom panel shows a representative immunoblot of MEK kinase in monocytes from the same subjects.

 
We then investigated the effects of acute induced hyperglycemia in vivo in five normal subjects on ERKs activity and ERKs phosphorylation. As shown in Fig. 2Go, hyperglycemia induced a marked and significant increase in ERKs activity (from 60.2 ± 6.9 to 80.6 ± 10; P < 0.05).



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  		Figure 2. Effect of acutely induced hyperglycemia on ERKs kinase activity in vivo. ERKs activity was determined in monocytes of normal controls during euglycemia ({circ}) and during exogenously induced hyperglycemia (•). ERKs activity was measured as described in Materials and Methods, with duplicate determinations in each experiment.

 
This effect was associated with the phosphorylation of the two isoforms of ERKs, ERK1 and ERK2, identified by the specific phospho-p44/p42 ERK antibody (Fig. 3AGo). No difference were found in ERK1 and ERK2 protein levels in normal subjects after hyperglycemia (Fig. 3BGo). Densitometric analysis indicated that acute hyperglycemia lead to about 2-fold increase in p44/p42 phosphorylation (P < 0.01; Fig. 3CGo).



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  		Figure 3. The upper panel shows representative immunoblots of phosphorylated ERK1 and ERK2 in monocyte lysates from two controls during euglycemia (Eu) and during hyperglycemia (Hyp) using an antiphospho-ERKs antibody as described in Materials and Methods. Equal amounts of protein (40 µg) were separated by 10% polyacrylamide gel and immunoblotted with p-ERKs antibody. The same sample used for the ERKs phosphorylation study blotted with anti-ERKs antibody is shown in the middle panel. Densitometric analysis of ERKs phosphorylation is shown in the lower panel. Data (mean ± SE; n = 5) are expressed relative to the euglycemic state, assigning a value of 100% to the control value. *, P < 0.01, euglycemia vs. hyperglycemia.

 
The signaling pathway leading to ERKs activation was dependent upon the upstream effectors, in particular, MEK activity (21). To assess the contribution of MEK to the stimulation of ERKs by acute hyperglycemia, we determined its activity in an immunocomplex assay. As shown in Fig. 4Go, acute hyperglycemia markedly increased the activity of MEK.



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  		Figure 4. Effect of acutely induced hyperglycemia on MEK kinase activity in human monocytes in vivo. MEK activity was assayed using anti-MEK immunoprecipitates as described in Materials and Methods. {square}, Basal values during euglycemia; {blacksquare}, basal values during hyperglycemia. Data are presented as the mean ± SE. **, P < 0.01.

 
The effect of acute hyperglycemia was also investigated on MKP-1 expression in human monocytes. As shown in Fig. 5Go, MKP-1 was detectable at 50 kDa in monocytes, and its expression was strongly induced after 60 min of phorbol 12-myristate 13-acetate stimulation. However, acute hyperglycemia did not significantly modify the expression of MKP-1 in human monocytes.



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  		Figure 5. Effect of acutely induced hyperglycemia on MKP-1 phosphatase expression in vivo. The upper panel shows a representative immunoblot of MKP-1 phosphatase in monocyte lysates from normal subjects during euglycemia (Eu) and during hyperglycemia (Hyp). A sample of human monocytes was also pretreated for 1 h with 1 µmol/L phorbol 12-myristate 13-acetate. The lower panel reports the densitometric analysis of MKP-1 expression. Data (mean ± SE; n = 5) are expressed relative to the euglycemia state, assigning a value of 100% to the control mean. {square}, Euglycemia; {blacksquare}, hyperglycemia.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ERKs are potentially involved in the tissue responses to hyperglycemia-associated cellular changes and organ damage. These effects have been recently reviewed (7), yet the bulk of the data gathered is derived mainly from in vitro studies. The present study shows that in vivo exogenously induced acute hyperglycemia significantly increases ERKs and MEK activities.

ERKs play a key role in cell growth and in diabetic complications as glucose transducers (11). Their stimulation, in fact, increases the activity of transcription factors by phosphorylation. These can alter the balance of gene expression, leading to altered insulin secretion and insulin resistance in patients with diabetes. However, it is not known yet which transcription signals are activated by the persistently activated ERKs in chronic hyperglycemia or diabetes.

In vitro, several studies have confirmed the relationship between ERKs and hyperglycemia (22, 23). Furthermore, Ishiki et al. found that in mesangial cells troglitazone, an insulin-sensitizing agent, normalized the increased PKC-MAPK activity induced by hyperglycemia and in diabetic rats prevented glomerular dysfunction without changing blood glucose levels, but inhibiting the activation of the PKC-ERKs pathway (24).

As far as we know this is the first study to demonstrates that acute hyperglycemia induces an increase in ERKs activity in vivo in humans. Furthermore, the finding of increased activity and phosphorylation of ERKs during hyperglycemia is nicely explained by the parallel increase in MEK activity as assessed by the immunocomplex assay. MEK is a serine/threonine-tyrosine kinase that is the direct upstream activator of ERKs (21). Therefore, hyperglycemia has the ability to enhance ERKs activity via MEK stimulation.

Recently, we found that hyperglycemia induced translocation of the isoform ß2 of PKC from the cytosol to the membrane in human monocytes (3). These results suggest that the glucose-induced activation of the PKC/MEK/ERKs pathway could be a putative mediator by which glucose causes toxic effects in pathological condition. An up-regulation, specifically in human monocytes, of this pathway can lead to an increased production of cell adhesion molecules and to increased monocyte capture/tethering to endothelial cells. It has been reported, in fact, that sera from diabetic patients with hyperglycemia had significant adhesive responses compared with sera from normal subjects (25).

Two aspects of this study deserve attention. First, to induce hyperglycemia we infused somatostatin, which may modulate, through a family of seven transmembrane receptor domain G protein-coupled receptors, ERKs kinase, at least in the central nervous system (26). However, it has been recently shown that somatostatin does not affect to a significant extent both p44/p42 ERKs and p38 activities in two human pancreatic cancer cell lines (27). Thus, the contribution of the infused somatostatin cannot be excluded, but appears unlikely. Second, the effect of insulin on ERKs activity can be ruled out, at least in insulin-sensitive subjects. Moreover, the infusion of somatostatin caused a slight decrease in insulin concentration; therefore, we tried to keep the experimental protocol as simple as possible to assess the effect of hyperglycemia per se on these intracellular events. Therefore, studies are needed to determine the individual roles of both hyperglycemia and hyperinsulinemia in insulin-resistant patients.

Another aspect of our study was to investigate the role of MKP-1 in ERKs regulation during acute hyperglycemia infusion in human monocytes. MKP-1 is a phosphotyrosine and phosphothreonine phosphatase that is able to dephosphorylate ERKs and turn off ERKs signaling (28). Our results demonstrated that the expression of MKP-1 is unchanged in human monocytes after 5 h of hyperglycemia.

MKP-1 is principally regulated at the transcriptional level, and a rapid increase in its expression is caused by growth factors or agents that cause oxidative stress and heat shock (29). Furthermore, a physiological concentration of insulin rapidly induced an increase in MKP-1 expression (30). On the contrary, hyperglycemia does not seem to influence its expression, at least acutely.

In conclusion, our findings show that monocyte ERKs activity and phosphorylation are significantly increased by acutely induced hyperglycemia in vivo in normal controls. These high glucose-induced modifications of monocyte ERKs activities may be relevant to the vascular damage induced by hyperglycemia and in the development of the atherogenic process. It will be important to assess this intracellular signal transduction in monocytes from subjects with insulin resistance.

Received August 16, 2000.

Revised October 31, 2000.

Accepted November 2, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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