The Effects of Rosiglitazone and High Glucose on Protein Expression

Nov 16, 2009 - Rosiglitazone Attenuates Endothelial Progenitor Cell Apoptosis Induced by TNF-α via ERK/MAPK and NF-κB Signal Pathways. Shengjie Xu ...
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The Effects of Rosiglitazone and High Glucose on Protein Expression in Endothelial Cells Renato Millioni,§,# Lucia Puricelli,§,# Elisabetta Iori,§ Giorgio Arrigoni,†,‡ and Paolo Tessari*,§ Department of Clinical and Experimental Medicine, Chair of Metabolism, University of Padova, Padova, Italy, Department of Biological Chemistry, University of Padova, Italy, and VIMM, Venetian Institute of Molecular Medicine, Padova, Italy Received May 15, 2009

Abstract: Rosiglitazone is a thiazolidinedione used to treat insulin resistance in diabetes. Although thiazolidinediones may also exert cardiovascular effects, contrasting results were reported. Favorable effects were shown for pioglitazone, whereas adverse reactions were suspected for rosiglitazone. Therefore, a reassessment of the molecular effects of rosiglitazone on vascular cells is required. We tested the effects of rosiglitazone on the proteome of human endothelial cells grown under either normal or high glucose levels. Protein profiles were analyzed in both membrane and cytosolic fractions. About 150 cytosolic proteins, and ≈100 membrane proteins, were detected. Two-thirds of the proteins significantly altered by high glucose were also modulated by rosiglitazone in an antagonistic way. Half of these proteins are involved in apoptosis. Using an independent assay of apoptosis based on nucleosome quantification, an ≈20% stimulation by high versus normal glucose was shown (p < 0.05). Conversely, rosiglitazone reduced apoptosis by ≈30-50% in cells exposed to either glucose conditions (p < 0.001). In addition, rosiglitazone differently modulated cytoskeleton and energy metabolism-related proteins. Our data show novel, potential sites of action of rosiglitazone through protein expression of endothelial cells. These mechanisms may foster new investigations on the overall vascular effects of this compound, and help to discriminate between desired and adverse effects. Keywords: differential proteomics endothelial cells • apoptosis

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Introduction Tiazolidinediones (TZD) are insulin sensitizer agents which are employed in the oral therapy of Type 2 Diabetes Mellitus (T2DM). Their mechanism of action is through activation of * Corresponding author: Prof. Paolo Tessari, Dipartimento di Medicina Clinica e Sperimentale, Universita` di Padova, via Giustiniani 2, 35128 Padova, Italy. E-mail: [email protected]. Fax: 0039-0498754179. Phone: 00390498211749. § Department of Clinical and Experimental Medicine, University of Padova. # These authors contributed equally to this work. † Department of Biological Chemistry, University of Padova. ‡ VIMM, Venetian Institute of Molecular Medicine.

578 Journal of Proteome Research 2010, 9, 578–584 Published on Web 11/16/2009

peroxisome proliferator activated receptors (PPAR-γ), which favors peripheral glucose utilization mostly in adipose tissue. As a consequence, by reducing lipotoxicity, they improve glucose metabolism and insulin sensitivity also in skeletal muscle. Therefore, TZD reduce hyperglycemia and hyperinsulinemia in T2DM and improve the metabolic control.1-3 TZD retain however many other effects at cellular and molecular level, which are only partially known. TZD have been shown to reduce the proliferation and the migration of smooth muscle cells, by inhibiting the activation of protein kinase C (PKC),4 to reduce platelet aggregation5 and to inhibit DNAsynthesis induced both by insulin and by angiotensin II through activation of extracellular signal-regulated kinase.6,7 TZD exert interesting and potentially favorable effects also with respect to the mechanism(s) leading to oxidative stress, which retain a widely recognized and fundamental role in both the normal functioning of cardiac and vascular cells and in the pathogenesis of cardiovascular diseases.8 Hyperglycemia increases the reactive oxygen species (ROS) synthesis, through the formation of advanced glycation products,9 the activation of the polyol pathways,10 mitochondrial dysfunction11 and the activation of PKC.12 In contrast, TZD have been shown to reduce oxidative stress after coronary reperfusion in noninsulin dependent diabetic mice.9 Recently, other in vitro effects of TZD have been detected, which could prove to be particularly useful in the prevention of diabetic chronic complications. The insulin sensitizer drug Rosiglitazone (RSG) improved nitric oxide bioavailability and reduced the progression of nephropathy in patients with microalbuminuria.13 RSG also improved the pro-thrombotic state14 of T2DM associated to insulin-resistance related to an increased platelet activity.15 Other authors16 demonstrated that RSG significantly decreases glucose-induced oxidative stress and that this effect is independent of its ability to activate PPARγ. Thus, TZD have pleiotropic effects beyond those already known, both at the molecular lever and in the entire organism. Many other effects of this agent could be still unknown. In contrast, however, with the above-reported favorable effects, RSG, at variance with pioglitazone,17 has recently been the object of a critical reassessment, based on epidemiological surveys and clinical studies, reporting adverse effects mostly at the cardiovascular level.18-20 These untoward effects require a further and deeper insight on all possible and pleiotropic molecular effects of RSG mostly at the cardiovascular level. 10.1021/pr900435z

 2010 American Chemical Society

Rosiglitazone and High Glucose on the Endothelial Cells Proteome One of such wide-search and unbiased techniques of investigation is the proteomic approach applied to the cell and/or tissue object of the investigation. The candidate cell type more closely related to cardiovascular functions is the endothelial cell (EC). Furthermore, since a single eukaryotic cell contains several thousands of proteins, a deeper analysis is based on the separation of the whole cell proteome into discrete fractions which can be assessed separately.21 Therefore, in this study, we compared the protein expression profiles of both membrane- and cytosolic-enriched fractions of human ECs, cultured under either normal or high glucose concentrations, with or without exposure to RSG. In particular, since there is evidence that RSG reduces glucose-induced ROS16 which in turn can induce apoptosis, we also evaluated independently the effect of RGS on glucose-induced apoptosis.

Experimental Procedures Cell Culture and Treatments. ECs were obtained from Clonetics, Inc. (San Diego, CA) and cultured in a Growth Medium (PromoCell, Heidelberg, Germany) with 10% fetal bovine serum (Sigma-Aldrich,), 0.02% Supplement Mix/Endothelial Cell Growth Medium (PromoCell) and maintained in a humidified 5% CO2 incubator at 37 °C. Thereafter, ECs were incubated for 48 h in the presence of either normal (5.5 mmol/ L) or elevated (20 mmol/L) glucose concentration, with or without RSG (20 µmol/L) dissolved in a 10% solution of dymethyl sulfoxide (DMSO). As controls, we used cells maintained at normal and elevated glucose concentrations and treated with DMSO 0.02%. RSG was kindly gifted by GlaxoSmithKline (U.K.). Sample Preparation. The culture medium was substituted with quiescent medium 24 h before protein extraction, and cells were washed three times with PBS. For every flask, 0.250 mL of lysis buffer, composed of TRIS-HCl 12 mM, DTT 1 mM and a cocktail of protease inhibitors, was used. The lysate was collected and submitted to three cycles of freeze-thawing in liquid N2 and sonication in ice. A sample prefractionation was accomplished by ultracentrifugation of protein lysates (at 100 000 RCF for 1 h at 10 °C). The pellet was constituted of a membrane-enriched protein fraction and was solubilized in a specific buffer for hydrophobic proteins (urea 6 M, thiourea 2 M, Triton X-100 1% (v/v), CHAPS 2% (w/v), IPG buffer 0.5%). The supernatant (cytosol-enriched protein fraction) was concentrated through ultrafiltration (Microcon-Amicon YM-3, Millipore Corporation, Bedford, MA). Proteins were quantified by Bradford method.22 Protein Separation by Two-Dimensional Gel Electrophoresis (2-DE). Treatments were repeated three times (biological replicates), and protein extracts from each culture were run in triplicate (technical replicates). A constant amount of solubilized proteins (200 µg) was diluted to 450 µL using a solution of urea 8 M, CHAPS 2%, IPG buffer 0.5%, DTT 1%, for the cytosolic fraction and a solution of urea 6 M, thiourea 2 M, CHAPS 2%, IPG buffer 0.5%, DTT 1% for the membrane fraction. Proteins were separated by 2-DE as previously reported.23 Briefly, isoelectric focusing was carried out on 24 cm long Immobiline pH gradient strips providing a linear 4-7 pH range for a total of about 35 000 Vh. The second dimension was run on a 12% polyacrylamide gel, after strip equilibration in a buffer containing urea 6 M, glycerol 30%, SDS 2%, TrisHCl 50 mM pH 8.8 and DTT 1%, and then in the same buffer containing iodoacetamide 2.5%, instead of DTT. 2-DE gels were stained with Coomassie Brilliant Blue G-250 and images were

technical notes

recorded using an Epson Expression 1680 Pro scanner (SeikoEPSON Corp., Japan) with 16 bit dynamic range and 300 dpi resolution. Gel image analyses were performed using the Proteomweaver software (BioRad, CA). After spot matching, the following parameters were taken into account to estimate gelto-gel reproducibility: the efficiency of matching, defined as the percentage of matched spot on the total of detected spots between two gels, a figure that indicates qualitative differences among gels; and the coefficient of variation of matched spot intensities, calculated on a total of 9 samples for each experimental group (see above), that reflects the quantitative differences. These correlation analyses revealed reproducible protein patterns with a an average matching efficiency of 75%, and coefficient of variation of spot intensity of 28%, good figures also in agreement with a published report24 (see Supplementary Data: Tables 1 and 2 and Figures 1 and 2). Protein Identification by Mass Spectrometry (MS). The spots were manually excised from the gel and the protein digestion was performed in gel using sequencing grade modified trypsin (Promega, Madison, WI). Briefly, gel pieces were repeatedly washed with 50% acetonitrile/50 mM NH4HCO3 and then dried under vacuum. A total of 8-10 µL of trypsin (12.5 ng/µL in 50 mM NH4HCO3) was added to each gel spot and samples were incubated for 30 min at 4 °C. Digestion was carried out at 37 °C overnight. After trypsin digestion, peptides were extracted from the gel with 3 changes (20-30 µL each time) of 75% acetonitrile/0.1% trifluoroacetic acid (TFA). The final peptide mixtures were then dried under vacuum and finally resuspended with 4 µL of 20% acetonitrile/0.1% TFA. One microliter of sample was mixed with 1 µL of matrix solution (R-cyano-4-hydroxycinnamic acid, 5 mg/mL in 70% acetonitrile/0.1% TFA) and 0.8 µL of the final mixture was spotted onto a stainless steel MALDI target plate. The samples were then analyzed using a MALDI-TOF-TOF 4800 mass spectrometer (Applied Biosystems, Toronto, Canada) operating in a data dependent mode: a full MS scan was followed by MS/MS scans on the 10 most intense peaks for each sample. MS/MS data were searched using Mascot (Matrix Science, London, U.K.) against the human section of the Swiss-Prot database (version 55.4, 19 630 entries). Enzyme specificity was set to trypsin with 1 missed cleavage using carbamidomethylcysteine as fixed modification and methionine oxidation as variable modification. The tolerance of the precursor ion was set to 50 ppm, while the tolerance of fragment ions was set to 0.3 Da. Proteins were considered as positively identified if at least 2 peptides with individual significant score (p < 0.05) were sequenced. The search was done also against the corresponding randomized database which did not return any positive identification under the same strict conditions (i.e., at least 2 peptides sequenced with individual significant score). For the 2 samples that could not be identified using the MS/MS data, peptide mass fingerprint (PMF) identification was obtained. For these samples, Mascot search was done against the same database and using the same search parameters, with a peptide tolerance of 50 ppm. All the proteins and peptides identified are reported in Tables 1 and 2 of the Supporting Information. The peak lists from MS/MS data can be downloaded from ProteomeCommons.org Tranche (https:// proteomecommons.org/tranche/), using the following Tranche hash: TSrsCjgVyXinAQfCbnIv0CKsMayAFpF/ vRdqmghdK6WWWPTKdFxLceutfTPetPpOzq/ 1wQePCvNDR0cVMN82v9pI9WwAAAAAAAABBw)). Journal of Proteome Research • Vol. 9, No. 1, 2010 579

technical notes Protein Expression by Western Blot Analysis. ECs were washed three times with ice-cold PBS and lysed in buffer containing 62.5 mM Tris-HCl pH 7.2, 10% glycerol, 10% SDS, 2% 2-mercaptoethanol and a cocktail of protease inhibitors. The protein content was measured by Bradford method.22 Protein extracts (50 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose filter (Hybond ECL Amersham Life Science). The membranes were blocked with 5% nonfat dry milk in PBS containing 0.01% Tween 20 (PBST) for 6 h at room temperature and incubated overnight at 4 °C with a primary antibody against 78 kDa glucose-regulated protein (GRP78) (Sigma) diluted 1:1000. Subsequently, membranes were washed three times for 10 min with PBST solution, and incubated for 1 h at room temperature with a horseradish peroxidase conjugated IgG (Santa Cruz Biotechnology, CA). After washing with PBST, the protein expression was visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL), according to manufacturer’s instructions. The intensity of the signals was quantified by VersaDoc Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA). The values were normalized by detection of GAPDH housekeeping protein. The same membrane was incubated with mouse anti-GAPDH antibody (Chemicon-International Temecula, CA) diluted 1: 600 with PBST and 0.5% powdered milk overnight at 4 °C. After washing with PBST, the nitrocellulose membrane was incubated with horseradish peroxidase conjugated anti-mouse antibody (Santa Cruz Biotechnology). The Western blot analyses were performed in three biological replicates. Quantification of Apoptosis. To evaluate the effect of RSG on apoptosis, ECs were incubated for 48 h with normal (5.5 mM) and elevated (20 mM) glucose concentrations in the absence or presence of 20 µM RSG. Apoptosis was quantified by detection of histone-complexed DNA fragments (mono and oligonucleosomes) from the cell cytoplasm. A commercial kit “Cell Death Detection ELISA plus” purchased from Roche (Roche, Molecular Biochemicals, Mannheim, Germany) was used according to the manufacturer’s instructions for DNA fragment detection.25 Statistical Analysis. 2-DE quantitative comparison was performed using the Proteomweaver software. The spot volume, after subtraction of background, was expressed as a numeric value of optical density. Normalization was run automatically by Proteomweaver, which computes relative spot volumes by setting total spot volume on a gel to 100% and thus making normalized spot intensities comparable among different gels without the need of an internal standard. Individual values obtained from technical replicates were then used for the quantitative analyses. For the intra group analysis, pairwise comparisons using the 2-tailed Student’s paired t test were performed (n ) 9 data for each group). A repeated measure ANOVA was used to compare the effect of RSG treatment in the presence of either normal or high glucose concentrations (Statistica 6, StatSoft Italia srl). To evaluate the RSG effect on apoptosis, the quantity of histone-complexed DNA fragments was compared at normal and high glucose concentrations using the 2-tailed Student’s paired t test. The significance for all statistical tests was set at p < 0.05. Bioinformatic Analysis. Data were elaborated with Babelomics (http://babelomics.bioinfo.cipf.es/), a complete suite of Web tools for the functional analysis of groups of proteins in high-throughput experiments, which includes the use of in580

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Figure 1. Representative 2-DE maps of the cytosolic (A) and membrane (B) fractions of ECs. Spot numbers correspond to proteins significantly altered.

formation on Gene Ontology terms, interpro motifs, KEGG pathways, Swiss-Prot keywords, analysis of predicted transcription factor binding sites, and chromosomal positions.

Results Proteome Analysis of ECs by 2-DE Analysis. In ECs, cultured under normal glucose conditions, about 150 proteins of the cytosolic fraction and about 100 of the membrane fraction were detected by means of 2-DE analysis. Representative 2-DE maps of these two cellular fractions are reported in Figure 1. The intragroup and intergroup statistical analyses allowed us to detect 28 spots (corresponding to 19 unique proteins) of the cytosolic fraction, and 13 spots (corresponding to 11 unique proteins) of the membrane fraction, which were significantly altered by our stimulations (high glucose and/or RSG). These proteins were gathered into “Molecular chaperones”, “Cytoskeleton related proteins”, “Energy metabolism” according to Swiss-Prot (www.ebi.ac.uk/swissprot/) classification. Six proteins, which did not have any common function, were classified as “Other proteins”. Detailed results of quantitative analysis, a short description of each protein and their database codes are reported in the Table 3 of Supporting Information. Western Blot Analysis. In addition to 2-DE data, Western blot analyses were carried out to further investigate GRP78 protein expression. Anti-GAPDH antibody was used to normalize the density values. Compared to cells cultured at normal glucose and in absence of RSG, the expression of GRP78 was

Rosiglitazone and High Glucose on the Endothelial Cells Proteome

technical notes

presence of RSG determined a significant reduction of apoptosis both under normal and high glucose levels (p < 0.001).

Discussion

Figure 2. Representative Western blot analysis of GRP78 in ECs cultured at 5.5 or 20 mM glucose, in the presence (+) or absence (-) of 20 µM RSG. GAPDH protein expression was used as internal control. Gray bars indicate GRP78 expression after high glucose treatment, in the presence or absence of RSG. Data expressed as mean ( SEM of three different experiments. *p < 0.05.

Figure 3. Effect of rosiglitazone on apoptosis in ECs. Endothelial cells were incubated for 48 h with 5.5 and 20 mM glucose and 0.02% DMSO, in the absence or presence of 20 µM RSG. Apoptosis was quantified by detection of histone-complexed DNA fragments (mono and oligonucleosomes) from cells. The graph shows nucleosome fold change versus 5.5 mM glucoseDMSO. Data expressed as mean ( SEM of three different experiments. *p < 0.05; **p < 0.001.

significantly increased by high versus normal glucose treatment, while in presence of RSG, GRP78 expression was the same at both glucose levels. The delta changes of high versus normal glucose levels, either without or with RSG treatment, is shown in Figure 2. Rosiglitazone-Related Changes in Apoptosis. To determine the possible contribution of RSG on apoptosis, cells were exposed to 5.5 and 20 mM glucose with and without 20 µM RSG. As shown in Figure 3, we observed a significant increase in histone-complexed DNA fragments (mono and oligonucleosomes) in cells exposed to high glucose compared to those maintained at normal glucose concentrations (p < 0.05). The

The aim of this study was to investigate whether RSG is capable of offsetting the toxic effects of high glucose on cultured ECs. The proteomic approach we employed in such a study yielded a complex set of data which need a careful evaluation. A first very important observation is that ≈70% of the spots altered by high glucose are also modulated by RSG in an antagonistic way. Among these modulated proteins, about half are involved in the regulation of apoptosis (Table 1). The antagonistic effect of RSG on the changes induced by high glucose on the proteins involved in apoptosis can be appreciated by looking at the delta values reported in Table 1. We have further assessed this process using an independent assay based on nucleosome quantification. Taken together, our data strongly support the view that RSG is capable of antagonizing the glucose-induced apoptosis on EC. This conclusion is straightforward as regards the direct assessment of apoptosis (Figure 3), whereas its interpretation is more difficult as concerns the changes of individual proteins involved in the programmed cell death and detected by the proteomic approach. In this regard, however, it is reassuring that 10 chaperone proteins involved in apoptosis, which were altered by high glucose, showed an opposite regulation after the RSG treatment (Table 1). Other two proteins (Lactoylglutathione lyase and Prohibitin) with uncommon functions but also involved in apoptosis, showed a similar behavior (Table 1). It is well-established that chronic hyperglycemia induces protein glycation, systemic low grade inflammation, and endothelial dysfunction.26 Hyperglycemia-induced endothelial dysfunction is characterized by an enhanced production of ROS,27,28 which are important factors in the development of vascular damage. Oxidative stress, and other pro-atherogenic factors, including oxidized low-density lipoproteins and angiotensin II, can induce EC apoptosis.28 Such damage to the endothelium may be an initiating event in atherogenesis since EC apoptosis may compromise vasoregulation. Recently, it has been suggested that RSG could inhibit apoptosis by regulating the expression of Bcl-2 in EC29 and in neuroblastoma cells30 and that RSG decreases glucolipotoxicity induced β-cell apoptosis and preserves β-cell morphology, and mass in OLETF rats, which are a type 2 diabetic animal model.31 NAD(P)H oxidase appears is one of the major sources of ROS production after exposure to high glucose.32 Furthermore, vascular NAD(P)H oxidase activity is increased in diabetic patients in vivo, and endothelial NAD(P)H oxidase activity is markedly increased by high glucose levels in vitro.33,34 Interestingly, our group16 has recently demonstrated that RSG prevents the hyperactivity of NAD(P)H oxidase induced by high glucose, through activation of AMPK and a possible inhibition of PKC. Therefore, RSG protects ECs against glucose-induced oxidative stress leading to apoptosis, and this effects may be mediated also through a modulation of some HSP, such as those reported in this study. Our data may indicate novel sites of action of RSG for apoptosis inhibition; it could be interesting to study these important issues more in depth since the regulation of intracellular ROS and modification of the apoptotic cascade may control apoptosis. Our findings may provide new strategies for prevention and treatment for hyperglycemia-induced endothelial dysfunction. Journal of Proteome Research • Vol. 9, No. 1, 2010 581

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Table 1. Spot Numbers, Protein Names, Protein and Gene Database Codes, Quantitative Analysis Graphs of Proteins Involved in Apoptosis and Separated by 2-DEa

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Table 1. Continued

a Short descriptions of protein functions are reported. White and black bars indicate, respectively, protein levels at normal and high glucose, gray bars indicate delta values. Data expressed as mean ( SEM. *p < 0.05; **p < 0.001.

RSG has been shown to inhibit and/or prevent apoptosis in a number of cellular models in vitro and in vivo.35,36 Therefore, our data based on a wide search approach such as proteome profiling, as well as on quantification of cytosolic histonecomplexed DNA fragments both under normal and high glucose levels (Figure 3), are in agreement with this concept. Cytoskeleton is involved in cellular plasticity. It has been suggested that actin remodelling induced by TZD drugs could have beneficial effects in the reduction of cell rigidity in the microvasculature of diabetic patients.37 We observed that RSG has an effect on additional cytoskeleton-related protein isoforms (corresponding to 7 unique proteins) (see Supporting Information Table 3). Among them, three lamin isoforms, a nuclear envelope protein interacting with chromatin, are normalized by RSG. Therefore, our data suggest that not only actin, but also other structural proteins are involved in the RSGinduced cellular plasticity under conditions of either normal or high glucose. Finally, our results show also that RSG has an effect on energy metabolism (see Supporting Information Table 3), through the modulation of three glycolytic enzymes (alpha enolase, three isoforms of pyruvate kinase M1/M2 and triosephosphate isomerase). However, the potentially more interesting result may be that of Cytochrome b-c1 complex subunit 1, involved in the respiratory chain, which is inhibited by high glucose and returned to a normal expression by RSG, suggesting an amelioration of glucose metabolism. The antagonistic effect of RSG on the glucose induced increment of GRP78 expression was confirmed by Western blot analysis (Figure 2). In our study, we have isolated about 150 spots in the cytosolic fraction, and 100 in the membrane one. We are aware that other techniques may yield a larger number of identified

proteins. This limitation can be partially overcome by using alternative approaches to 2DE, such as “gel-free” proteomics approaches that, however, require more sophisticated technologies and are more expensive overall for studies requiring large number of samples. In conclusion, in this study, we provide evidence that RSG modulates the expression of a number of proteins involved in the regulation of apoptosis as well as in cell plasticity and energy metabolism. These newly discovered effects may amplify our knowledge about the mechanism(s) and the possible pleiotropic effects of this drug. Abbreviations: 2-DE, two-dimensional electrophoresis; DMSO, dymethyl sulfoxide; EC, endothelial cell; HSP, heat shock protein; MS, mass spectrometry; PPARγ, Peroxisome proliferator-activated receptor-gamma; PKC, protein kinase C; ROS, reactive oxygen species; RSG, rosiglitazone; T2DM, Type 2 diabetes mellitus; TFA, trifloroacetic acid; TZD, thiazolidinediones.

Acknowledgment. The authors greatly thank GlaxoSmithKline for supplying Rosiglitazone and supporting the study. The Foundation for Advanced Biomedical Research and VIMM are grateful to the Veneto Banca Holding for funding the acquisition of the MALDI-TOF-TOF. Supporting Information Available: Supplemental Tables 1-5; Figure 1-2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Dumasia, R.; Eagle, K. A.; Kline-Rogers, E.; May, N.; Cho, L.; Mukherjee, D. Role of PPAR- gamma agonist thiazolidinediones in treatment of pre-diabetic and diabetic individuals: a cardio-

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