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Departments of Clinical and Experimental Medicine (L.P., E.I., R.M., M.V., P.T.) and Biological Chemistry (G.A.), University of Padova, 35128 Padova, Italy; and Protein Technology Wallenberglab (G.A., P.J.), SE 22007 Lund, Sweden
Address all correspondence and requests for reprints to: Professor Paolo Tessari, Dipartimento di Medicina Clinica e Sperimentale, Università di Padova, via Giustiniani 2, 35128 Padova, Italy. E-mail: paolo.tessari{at}unipd.it.
Abstract
Context: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes.
Objective: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls.
Design: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry.
Results: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and 
-actinin 4 and a decrease of tubulin-ß2 and splice isoform 2 of tropomyosin ß-chain were detected.
Conclusions: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.
ALTHOUGH THE HUMAN genome has been recently sequenced (1), neither the genomic sequence nor the transcriptional profile may fully and directly correlate with protein expression (2). Therefore, protein profiling has become increasingly important in the characterization of a disease’s phenotype. Because proteins are involved in most cellular processes, their cumulative expression profile may better reflect cellular activities and provide a clue to the understanding of the pathophysiology of a variety of disorders.
Type 1 diabetes mellitus (T1DM) is a disease with a strong genetic background (3). Should genetic trait(s), associated with T1DM, be expressed in representative cell(s), their phenotype will likely be maintained in primary cultures of cells derived from T1DM patients, even after several passages under controlled conditions. The protein phenotype of such cells can be investigated using a proteomic approach. Cultured skin fibroblasts have been proven a useful tool for the investigation of pathophysiological aspects of cellular metabolism (4).
In the present study, we used a wide-search proteomic approach to study total protein expression in cultured fibroblasts obtained by skin biopsy from T1DM patients without micro- and macrovascular complications and compare them with that of healthy subjects. Proteome analysis was performed using two-dimensional electrophoresis (2-DE) for protein isolation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for their identification.
Subjects and Methods
Subjects
Five normotensive T1DM patients with a long disease duration, without micro- and macrovascular complications, and five age- and sex-matched healthy volunteers without a family history of hypertension and diabetes were recruited (Table 1
). The patients were selected on the basis of absence of other diseases as well as diabetic complications, assessed according to established clinical and laboratory criteria (24 h microalbuminuria, retinal examination with direct ophthalmoscopy, clinical examination, carotid echo-Doppler ultrasonography, Winsor index). The aims of the study were explained in detail, and each subject signed an informed consent. The protocol was approved by the Ethical Committee of the Medical Faculty at the University of Padova, Italy, and was performed according to the Helsinki Declaration (1983 revision).
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Skin biopsies were taken by excision under local anesthesia from the anterior surface of the forearm, and the fibroblasts were cultured in normal glucose (6 mmol/liter) as described previously (5). The growth medium was changed with quiescent medium (serum free) 24 h before the protein extraction. Cells were used between the seventh and eighth passage.
Sample preparation
The quiescent medium was aspirated, the fibroblasts were washed with phosphate buffer (pH 7.4), and proteins were extracted with 250 µl of 8 M urea, 4% 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate, 2% immobilized pH gradient (IPG) buffer (pH 3–10 or pH 4–7) (Amersham Pharmacia Biotech, Uppsala, Sweden) plus a cocktail of proteinase inhibitors (Sigma-Aldrich, St. Louis, MO). The samples were freeze thawed, sonicated, concentrated, and then desalted by ultrafiltration (Centricon YM-3; Millipore Corp., Bedford, MA). Protein concentration was determined by modified Lowry assay (6). There were no relevant differences in protein concentration between any cell groups.
2-DE
The isoelectric focusing (IEF) was initially carried out on Immobiline DryStrips with a linear pH range of 3–10. Subsequently the IEF runs were repeated using a linear pH range of 4–7 (see Results). A constant amount of solubilized proteins (1000 µg in 3–10 pH range strips; 1500 µg in 4–7 pH range strips) was diluted to 450 µl with a rehydration solution [8 M urea, 2% 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate, 0.5% IPG buffer, 1% dithiothreitol (DTT)]. All the IEF runs were performed using an Ettan IPGphor IEF unit (Amersham Pharmacia) for 38 kVh. Each sample was run in duplicate. Twenty-four-centimeter-long strips were used, which provided the highest sample load and the best resolution in the second dimension of the 2-DE.
After IEF completion, the strips were equilibrated for 15 min in a buffer containing 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate, 50 mM Tris-HCl (pH 8.8), and 1% DTT, and then for 15 min in a similar buffer containing 2.5% iodoacetamide instead of DTT. The second dimension was carried out in an Ettan DALT six large vertical electrophoresis system (Amersham Pharmacia) on a 12% polyacrylamide gel. The gels were stained with 0.1% Coomassie Brilliant Blue G250. The spots in the gels were digitally recorded using an Epson Expression 1680 Pro scanner with 16-bit dynamic range and 300-dpi resolution. The reproducibility of protein profiles was good (average matching efficiency of about 75%), and the quantitative comparison of the spots was analyzed using the Proteomweaver software (Bio-Rad, Hercules, CA). The spot volume, after subtraction of background, was expressed as a numeric value of OD. Spot intensities were normalized automatically by Proteomweaver to make them comparable between different gels. Normalization was run by an algorithm designed for numerical analysis not requiring any internal standard. It computes for every gel an intensity factor that makes all the normalization factors as close to 1 as possible. For every gel match, the ratio between the pair matched spots was calculated. The normalization factor is the median of these ratios.
MALDI-MS analysis
The spots were manually excised from the gel, and protein digestion was performed in the gel according to Gharahdaghi et al. (7). The samples were then desalted using C18 ZipTip (Millipore) and the digested proteins were analyzed using a MALDI-time of flight mass spectrometer (Micromass) and -cyano-4-hydroxycinnamic acid as matrix (2.5 mg/ml in acetonitrile/0.1% formic acid, 50:50) (Sigma-Aldrich). The spectra were analyzed using Mascot engine search (Matrix Science, London, UK) and PIUMS (www.hh.se/staff/bioinf) searching against the human session of the International Protein Index (http://www.ebi.ac.uk/IPI/IPIhelp.html) with a mass tolerance window of 100 ppm. Enzyme specificity was set to trypsin with up to one missed cleavage using carbamidomethylcystein as fixed modification and methionine oxidation as variable modification. The proteins were considered correctly identified when the two softwares yielded the same identification with a P < 0.05 and when the coverage of the sequence was at least 30%. The samples that were not identified directly by protein mass fingerprinting analysis were further subjected to peptide mass fingerprinting analysis using an AP-MALDI-Ion Trap (ThermoFinnigan, San Jose, CA). The MS/MS spectra were analyzed with Mascot engine search and manually inspected for further confirmation, and the identification was considered correct when at least three peptides were identified from the same protein with an individual P < 0.05.
Statistical analysis
After completion of spot matching, the normalized intensity values of individual protein spots were used to compare the protein quantitative levels between the two groups by statistical analysis. The Student’s two-tailed t test for unpaired data was used, and P < 0.05 was considered statistically significant.
Results
Using the 3–10 pH range strips, although most proteins were clearly separated, the MS analysis revealed more than one protein in some apparently single spots. A typical 2-DE protein map of these experiments is shown in Fig. 1A. As an example, in spot 19 the MALDI-time of flight-MS analysis revealed the presence of tropomyosin 4 and tropomyosin 4-ALK fusion oncoprotein type 2, whereas in spot 68 the heat shock cognate 71-kDa protein and vimentin were identified. Therefore, all cell samples were run again using the pH 4–7 strips (Fig. 1B). This pH range also enabled us to detect proteins with lower abundance, i.e. beneath the expression level of housekeeping protein, by scaling up protein load and resolution.
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, we report the list of all the identified proteins. Notably, all these proteins were found in both the normal and the diabetic samples. A total of 125 proteins were isolated and identified. Some spots represented different isoforms of the same molecule, probably due to posttranslational modifications (PTMs), and 72 unique proteins were identified, belonging to structural, enzymatic, and regulatory proteins (Fig. 2). Twenty-seven proteins had never been reported previously in 2-DE maps of human fibroblast, also including 20 proteins never reported in human skin fibroblasts (Table 2).
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). In T1DM patients, compared with normal subjects, we found a significant increase of triosephosphate I isomerase (no. 36), two splice isoforms of pyruvate kinase (no. A98, 26), -actinin 4 (no. A3), whereas tubulin-ß2 (no. A75) and the splice isoform 2 of tropomyosin ß-chain (no. A100) were significantly decreased.
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We have generated 2-DE protein reference maps of human cultured skin fibroblasts from T1DM patients and compared them with those of healthy controls. Such a database may constitute a valuable resource for the investigation of the molecular basis of pathophysiology of T1DM. There are only limited reports on 2-DE proteome profiles of cultured human fibroblasts from healthy subjects (8, 9, 10), and no such data had been published for T1DM patients so far.
The 2-DE approach allows us to detect clearly both the isoforms and PTMs of the same protein. The in-depth study of the nature of these modifications is not trivial because both PTMs and isoforms are likely to occur widely, and their detection requires a specific study. Moreover, because many isoforms of the same protein can occur, this may be considered a limit for the total quantification of the protein, but it could also represent an advantage because different isoforms of a protein may play different physiological roles within the cell.
Most of the identified proteins were cytoskeletal and cytoskeletal-related proteins, enzymes involved in the regulation of protein and energy metabolism, and proteins involved in the responses to stress (Table 2
and Fig. 2). Moreover, we identified 27 proteins not previously reported in other human skin fibroblast 2-DE maps as well as 20 proteins never reported previously in the literature for this cell type (8, 9, 10, 11, 12). These data enrich the protein database of cultured human fibroblasts, thus expanding the current limited knowledge on this cell type, widely used in many research fields. These newly identified proteins could become useful for further studies on cell homeostasis, protein processing, cytoskeleton, and energy metabolism.
Although most identified proteins did not show quantitative differences between the two groups, statistically significant differences were observed as concerns some glycolytic enzymes and cytoskeletal proteins.
With regard to glycolysis, an increase of triosophosphate I isomerase (TPI) (no. 36) (Fig. 3) and two splice isoforms of pyruvate kinase M2 (PK) (no. A98, 26) were observed in T1DM patients with respect to control subjects. The increase of these enzymes is compatible with an increased glycolytic flux, in agreement with data obtained in skeletal muscle from T1DM subjects (13). An increased glycolysis may reflect a preferential use of glucose through the nonoxidative pathway because glucose oxidation is impaired in diabetes, particularly in the presence of insulin resistance (14). Also in other insulin-resistant states, i.e. obesity and type 2 diabetes, increased glycolysis and reduced oxidative enzyme activity have been reported in skeletal muscle (15, 16).
TPI is a glycolytic enzyme, which catalyzes the interconversion of D-glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate. GAP is further oxidized to 1,3 diphosphoglycerate by GAP-dehydrogenase (GAPDH). GAPDH activity was reduced in both human and experimental T1DM as well as cells exposed to high glucose (17). A decreased GAPDH activity would increase the glycolytic intermediates that are upstream of this enzymatic step (18), thus activating two other pathways found to be altered in diabetes, i.e. the advanced glycosylation end products pathway and the protein kinase C pathway. Although GAP concentration is normally much lower than that of its isomer dihydroxyacetone phosphate (<10 vs. > 90%, respectively) (19), a decreased GAP oxidation could increase its concentration and allosterically activate TPI to enhance GAP disposal through an alternative route. This may explain why the expression of TPI is increased in the fibroblasts from the T1DM subjects.
PK is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP. Literature data on PK activity in diabetes are contrasting. PK activity and mRNA expression were decreased in both human and experimentally induced T1DM (20, 21, 22, 23). However, also mildly increased (24) or unchanged PK levels (25) were reported. Because insulin activates PK (26), we suggest that the increased PK expression in the fibroblasts of T1DM subjects may represent a compensatory mechanism to counteract a possibly decreased PK activity. Whether PK structure is altered in T1DM, leading to a decreased activity, cannot be established from our data. In addition, a possibly different effect of hyperglycemia on PK activity between T1DM and control subjects should be tested.
No differences in the expression of enzymes involved in the redox regulation of the cell, such as Cu,Zn superoxide dismutase (no. 7), peroxiredoxine-2 (no. 16) peroxiredoxine-6 (no. 81), and heat shock proteins (no. A31, 58, A30, 6, 14, A71, 55, A4, 56, 53), were observed between the two groups. Our results agree with recent data (27) showing that antioxidant enzyme activities and mRNA expression were not different between fibroblasts from T1DM patients and normal subjects cultured under euglycemic conditions.
As concerns cytoskeleton or cytoskeletal-related proteins, in the fibroblasts from T1DM patients, compared with those of normal subjects, we found an increase of -actinin 4 (no. A3) (Fig. 3) and a decrease of tubulin-ß2 (no. A75) and splice isoform 2 of tropomyosin ß-chain (no. A100). The -actinin 4 (no. A2) isoform did not change. This may reflect changes in function of different isoforms, although we cannot provide a proof for that. On the other hand, neither posttranslational modifications nor size or isoelectric point affect staining intensity.
Insulin itself plays a tonic regulatory role in controlling the actin microfilament network (28) and the microtubules (29), and it may differentially regulate these proteins. Our data suggest an association between T1DM and alterations of the cytoskeleton in cultured fibroblasts.
The long-term T1DM patients studied here were free from chronic diabetic micro- and macrovascular complications. Because the development of complications (mostly microvascular) in T1DM may be at least in part genetically determined (30) and such a predisposition may be expressed also on the phenotype of protein expression, our data provide skin fibroblast maps in patients without such a predisposition. Similar studies also should be performed in patients with long-lasting diabetes and chronic microvascular complication in the search for a possible phenotype associated with microangiopathy.
In conclusion, the present study provides a qualitative analysis of the skin fibroblast proteome from both normal and T1DM subjects. Further investigations will require the development of improved steps to increase the enrichment of low-abundance proteins, possibly not evidenced in the present study. Proteome analysis technologies can be applied for monitoring protein expression in cultured fibroblasts to investigate the molecular mechanisms involved in the pathogenesis of diabetes and, possibly, of its complications.
Acknowledgments
We thank Dr. Roberto Trevisan for his invaluable contribution in the collection of the skin biopsies.
Footnotes
This work was supported by a research grant from the University of Padova (year 2004), the Italian Ministry of Education, University and Research ("Ministero dell’Istruzione, dell’Università e della Ricerca") (Fondo per gli Investimenti della Ricerca di Base grant, year 2003), the Knut and Alice Wallenberg Foundation (to P.J.), and a Ph.D. program of the "Fondazione Cassa di Risparmio di Padova e Rovigo."
First Published Online July 5, 2006
Abbreviations: 2-DE, Two-dimensional electrophoresis; DTT, dithiothreitol; GAP, D-glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IEF, isoelectric focusing; IPG, immobilized pH gradient; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PK, pyruvate kinase; PTM, posttranslational modification; T1DM, type 1 diabetes mellitus; TPI, triosephosphate I isomerase.
Received February 7, 2006.
Accepted June 22, 2006.
References
function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism. Diabetes 50:2472–2480
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