Proteomics-Based Identification of Differentially-Expressed Proteins ...

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Jan 6, 2009 - Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, Lanzhou Institute of Biological Products. (LIBP), The China National ...

Proteomics-Based Identification of Differentially-Expressed Proteins Including Galectin-1 in the Blood Plasma of Type 2 Diabetic Patients XiaoJun Liu,†,‡ QiPing Feng,†,§ Yong Chen,| Jin Zuo,‡ Nishith Gupta,⊥ YongSheng Chang,*,‡ and FuDe Fang*,‡ National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005, China, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, Lanzhou Institute of Biological Products (LIBP), The China National Biotech Group (CNBG), Lanzhou University, Lanzhou, 730046, China, and Department of Molecular Parasitology, Humboldt University, Philippstrasse 13, House 14, Berlin, 10115, Germany Received July 15, 2008

Type 2 diabetes (T2D) is a very heterogeneous and multifactorial disease. The pathophysiology of T2D is presumed to occur with an alteration in the levels of plasma proteins. To identify these differentially expressed proteins, plasma samples from normal and T2D humans were subjected to two-dimensional gel electrophoresis, quantitative densitometry, and mass spectrometry. Up to 200 protein spots were visible on each gel, of which 57 appeared modulated in diabetic individuals. Subsequently, 31 spots with g2-fold change in their expression were analyzed by MALDI-TOF mass spectrometry leading to the identification of 11 proteins with average sequence coverage of ∼38%. The expression of apolipoprotein A-I was reduced by 4.2-fold, and galectin-1 was increased 4.8 times in diabetic samples. Induction of galectin-1 in T2D samples was confirmed by ELISA. In addition, the dose-dependent treatment of rat L6 skeletal muscle cells with glucose resulted in an upregulation of galectin-1. These data implicate the association of galectin-1 with the pathophysiology of diabetes and identify galectin-1 as a novel diagnostic marker protein in T2D patients. Keywords: type 2 diabetes • two-dimensional gel electrophoresis • matrix-assisted laser desorption ionization time-of-flight mass spectrometry • galectin-1

Introduction Type 2 diabetes (T2D) is one of the most prevalent diseases worldwide. Together with an uncertain mode of inheritance, the complex onset of T2D involves the genetic and environmental determinants. Typically, it is characterized by hyperglycemia that is due to defects in insulin secretion and its molecular action.1 Elevated glucose levels during the fasting period and persistent postprandial hyperglycemia can inflict chronic health issues that are usually systemic in nature. Initially, the effect of T2D is limited to the detrimental loss of insulin-producing pancreatic β-cells. Subsequent reduction in insulin synthesis, however, may lead to multiple malfunctions such as heart disease, high blood pressure, kidney failure and nerve damage. In addition, the pathogenesis of T2D is quite * To whom correspondence should be addressed. Prof. FuDe Fang and Associate Prof. YongSheng Chang, National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, No.5, Dongdan Santiao, Beijing 100005, China. Tel.:+86-10-65296424. Fax: +86-10-65253005. E-mail: [email protected] and [email protected] † These authors contributed equally to this work. ‡ Chinese Academy of Medical Sciences and Peking Union Medical College. § Mayo Clinic. | Lanzhou University. ⊥ Humboldt University. 10.1021/pr800850a CCC: $40.75

 2009 American Chemical Society

Table 1. Clinical and Metabolic Parameters of the Diabetic and Normal Individuals Participating in This Investigation (Values Are Mean ( SD) parameters

Number of Subjects Gender Male Female Age (years) Age (range) Weight (kg) BMI (kg/m2)

diabetic individuals

17 7 10 61.0 ( 13.6 35-79 68.53 ( 9.35 25.94 ( 4.13

normal individuals

15 9 6 44.6 ( 5.73 35-55 65.47 ( 10.3 22.54 ( 2.72

complex due to its very heterogeneous and multifactorial nature, where the expression of many genes and their products is also altered.1,2 Proteomics has been used to identify novel disease markers that are differentially expressed during a pathological state compared to healthy individuals.3 The presence of distinguished protein markers in the blood plasma of a patient is a requisite for an explicit diagnosis. However, the high abundance of a few proteins, for example, albumin, immunoglobulin and transferrin in the plasma that account up to 80% of the total plasma proteins, often negatively impact the identification of the differentially expressed but low-abundance proteins.4 Journal of Proteome Research 2009, 8, 1255–1262 1255 Published on Web 01/06/2009

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Figure 1. Two-dimensional gel electrophoresis images (silver-stained) of the resolved plasma proteins from diabetic (A) and normal (B) subjects. The pI range of separation (pH 3-10) and the molecular masses (kDa) are depicted on the top and left sides, respectively. The images shown here are representative of three independent experiments. (C) Enlarged views of spot #8 and #3 from (A) and (B).

Consequently, the progress in plasma proteomics to identify the markers of many diseases including T2D has been hampered. The systemic nature of T2D implicates the modulation of proteins in the blood plasma that may be the cause or consequence of the pathophysiology of this disease. Previously, the proteins associated with the differentiation of NIH-3T3 adipocytes have been identified by 2-D PAGE.5,6 The expression of other proteins was altered in obese Zucker rats, when compared to lean rats.7 Differential protein expression was also observed in the plasma of normal glucose-tolerant and T2DMindividuals.8 However, the plasma proteomics of T2D has been very limited and requires further investigation to find reliable diagnostic protein markers. In this study, we have employed two-dimensional gel electrophoresis coupled with MALDI-TOF MS to demonstrate the changes in plasma proteins of T2D individuals as well as to identify the novel diagnostic marker proteins.

Materials and Methods Chemicals. All chemicals, IPG strips and ampholytes were purchased from GE Healthcare (Piscataway, NJ). The cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). Goat polyclonal antigalectin-1 (sc-19276) and all secondary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-Actin antibody was obtained from Sigma-Aldrich (St. Louis, MO). Sample Collection. This investigation was conducted following the informed consent of all participating individuals, and approved by the Ethical Committee of the Peking Union Medical College Hospital. In total, 17 type 2 diabetic and 15 healthy subjects were evaluated. The criteria to assess the presence of type 2 diabetes mellitus (T2DM) were based on 1256

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the guidelines proposed by the World Health Organization. All T2D patients had typical diabetic symptoms along with a single fasting plasma glucose level of g7.0 mmol/L or 2 h postglucose or casual postprandial plasma glucose level of g11.1 mmol/L. Healthy individuals with their fasting blood glucose below 5.5 mmol/L were selected as controls. Other clinical and metabolic parameters of T2DM and normal controls are depicted in Table 1. The plasma samples of all subjects were kept frozen in liquid nitrogen until their proteome analysis. Protein Extraction and Gel Electrophoresis. The plasma samples were prepared by mixing equal proportion of 17 diabetic or 15 normal subjects to average the biological variation. Briefly, the samples were homogenized on ice and diluted in 10 mM Tris-Cl (pH 7.4). They were then adsorbed to Cibacron Blue 3GA pre-equilibrated with 10 mM Tris-Cl (pH 7.4) and shaken for 40 min to remove albumin. The supernatants were collected (2000g × 2 min) and treated with protein A (pre-equilibrated with 10 mM Tris-Cl (pH 7.4)) for 40 min to remove the excess IgG. The resulting supernatants (500g × 5 min) were subjected to protein determination by Bradford’s method using BSA standard. The equal amounts of individual samples were pooled to yield two samples representative of T2DM and control groups. Commercial IPG strips (GE Healthcare), 24 cm in length with a nonlinear range of pH 3-10, were rehydrated overnight in 450 µL of a solution containing 8 M urea, 2% (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) IPG buffer, 0.002% (w/v) bromophenol blue and 100 µg of the homogenized samples. Electro-focusing was performed for 64 kVh using IPGphor (GE Healthcare) at 20 °C following the manufacturer’s instruction. Prior to the second dimension electrophoresis, IPG strips were conditioned in: (i) 50 mM Tris-Cl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% (w/v) bromphenol blue and 1% (w/v) DTT

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Galectin-1 in the Blood Plasma of Type 2 Diabetic Patients

Table 2. Plasma Proteins of T2D and Healthy Individuals As Identified by MALDI-TOF Mass Spectrometry

spot number

protein identified

accession number

peptide sequence molecular isoelectric identified/ MASCOT coverage weight point peptide score (%) (Da) (pI) simulated










Hypothetical protein gi|7269700















Unknown Protein (IMAGE:3934797)










Chain M, Crystal gi|48425 Structure Of A Broadly Neutralizing Anti-Hiv-1 Antibody In Complex With A Peptide Mimotope

Hypothetical protein gi|41410097 MAP3999c


gi|77702 17
















matched peptides


fold position change

9-20 10-20 10-23 45-61 62-82 75-82 86-100 87-100 157-172 173-193 218-229





17-29 19-29 -4.152 30-46 34-46 52-65 66-83 103-112 103-113 114-122 123-129 125-137 130-137 147-155 147-157 160-166 167-177 213-221 222-232 233-244


6-26 6-26 19-26 30-44 31-44 101-116 117-137




109-127 110-127 128-143 204-212 209-215




19-33 70-84


27-39 -5.7 93-113 106-113 118-131 179-187





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Table 2. Continued

spot number


accession number

Chain A, X-Ray gi|42542977 Crystal Structure Of Human Galectin-1





protein identified

Ig kappa chain NIG26 precursor

Anti-Entamoeba histolytica immunoglobulin kappa light chain

EF hand domain protein




peptide sequence molecular isoelectric identified/ MASCOT coverage weight point peptide score (%) (Da) (pI) simulated




















fold position change




29-48 37-48 49-63 64-73




109-127 110-127 128-143




109-127 110-127 128-143




126-137 197-204 205-218





The protein spot T1 exists only in the sample of T2D subjects and the protein spot N1 and N2 exist only in the sample of normal subjects.

on a rocking shaker for 15 min, followed by (ii) 50 mM Tris-Cl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% (w/v) bromophenol blue and 2.5% (w/v) iodoacetamide for 15 min. The electrophoresed strips were loaded on 12% acrylamide Laemmli gels (26 × 20 cm) and run for 0.5 h at 0.5 W/gel, and then at 15 W/gel until the dye front reached the gel bottom (Ettan DALT Twelve system, GE Healthcare). Proteins were visualized by silver staining.9 Gels were fixed in methanol-acetic acid (40%/10%) for 30 min followed by washing in water for 30 min. They were then sensitized with 0.02% (w/v) sodium thiosulfate for 1 min, rinsed 3× with water for 10 min each, and incubated in chilled 0.1% (w/v) silver nitrate for 20 min. Afterward, gels were rinsed 2× with distilled water for 1 min each, and developed in 0.04% (v/v) formalin (37% formaldehyde in water) in 2% (w/v) sodium carbonate until the desired intensity of staining. Gels were incubated with 5% acetic acid for 10 min to stop the development and then rinsed with water for 5 min. Gel Quantification. All silver-stained gels were digitized using the image scanner (300 dpi) and analyzed by ImageMaster 2D Platinum (GE Healthcare). The spot detection parameters were: smooth factor, 2; saliency, 100; and minimal area, 60. The spots were detected automatically and images were visually screened for any undetected or incorrect bands. Small and round spots with tight intensity were selected as the landmarks for automatic matching, and incorrect spots were corrected manually. Usually, the average spot intensity was normalized to the total spot volume and multiplied with a factor of 100. Differences in intensity were calculated for each spot on every pair of gels. The cutoff level for a modulated protein was defined as at least a 2-fold change in its intensity. Statistically significant (p < 0.05) differences for each protein spot between both groups were computed by t-test. 1258


matched peptides

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Protein Digestion and Mass Spectrometry Analysis. Protein spots were excised from 2DE gels followed by destaining and dehydration in 50% acetonitrile/50 mM NH4HCO3 and 100% acetonitrile. Proteins were reduced in 10 mM DTT/25 mM NH4HCO3 at 56 °C for 1 h and then alkylated in 55 mM iodoacetamide for 45 min in the dark at room temperature. The samples were extensively washed with 25 mM ammonium bicarbonate in water/acetonitrile (50/50) and then dried in a Speedvac. Proteins were digested in a modified trypsin solution (10 ng/µL in 25 mM ammonium bicarbonate) overnight at 37 °C. Tryptic digests were extracted from the gel matrix and concentrated to 5-10 µL using SpeedVac. The resultant peptides were analyzed by MALDI-TOF mass spectrometer (Bruker Daltonics) using an AutoFlex (Bruker Daltonics, Billerica, MA). HCAA (R-cyano-4-hydroxycinnamic acid) suspended in acetonitrile/water/TFA (50:49.9:0.1) was used as the matrix. Tryptic peptides obtained from in-gel digestion were mixed with HCAA suspension in the ratio of 1:1 and applied onto the target well followed by drying at room temperature.10 The instrument was calibrated with human angiotensin II tryptic peptides ([M + H]+ 1046.5417) and adreno-corticotropin fragment ([M + H]+ 2465.1989). The ion masses were accurate within 100 ppm following the calibration. All spectra were collected in the reflection mode by summing 200 laser shots with an ion source voltages of 19 kV and 16.27 kV, 100 ns delay and monoisotopic peptides peak gate at m/z 600. The data were submitted to the Mascot search engine ( and compared with the human subset database of NCBInr (www.ncbi.nlm.; 120 740 sequences). The analysis parameters included: (1) protein expectation with P < 0.05; (2) complete cysteine modification by iodoacetamide (57-Da), partial methionine oxidation (16-Da) at the N-terminus and 1 missed cleavage site; (3) identification was assumed as positive when monoisotopic

Galectin-1 in the Blood Plasma of Type 2 Diabetic Patients

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Figure 2. MALDI-TOF mass spectra of the trypsin-digested plasma proteins from the diabetic individuals corresponding to the spot #8 (galectin-1) and #3 (apolipoprotein A-I) on 2-D gels. Both proteins were differentially expressed between the normal and diabetic samples.

masses of a minimum of 6 peptide matched a particular. In addition, the correlation of theoretical molecular weight and of the pI value with the 2D gel segment was also considered to validate the positively identified hits. Enzyme-Linked Immunosorbent Assay. All sera obtained from diabetic and normal subjects were diluted in plasma diluent (1:40) and applied to triplicate wells. In brief, 100 µL aliquot of antigalectin-1 antibody (sc-19276, 1 µg/mL) in 0.1 M PBS buffer was used for the overnight coating of 96-well microtiter plates at 4 °C. The wells were washed 3× in PBS supplemented with 0.1% Tween 20 (PBST), and then saturated with PBS containing 5% bovine plasma and 0.1% Tween 20 (PMT) for 1 h at 37 °C. 100 µL of the 0.1% bovine plasma PMT-

diluted plasma samples were allowed to react with the primary antibody for 1 h at 37 °C followed by 4 washes with PMT. Subsequently, the samples were treated with 100 µL of the secondary antibody (0.25 µg/ml) in PMT for 1 h at 37 °C. Following three additional washes, 100 µL of streptavidinperoxidase solution diluted in PMT (1:500) was added to the well and incubated for 1 h at 37 °C. The samples were subjected to 3 more washes in PMT followed by treatment with 2.5 mM H2O2 and 2 mM of 2,2′-azino-di(ethyl-benzthiazoline) sulfonic acid (30 min; Sigma Chemical Co.). Optical densities were monitored at 405 nm using the ELISA reader (Molecular Devices). Journal of Proteome Research • Vol. 8, No. 3, 2009 1259

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Figure 3. Differential expression of galectin-1 in normal and type 2 diabetic plasma samples as confirmed by ELISA. * p < 0.05.

Cell Culture. The rat L6 skeletal muscle cells were maintained (5% CO2 at 37 °C) in DMEM (high glucose) containing the fetal bovine serum (10%), glutamine (2 mM), penicillin (100 U) and streptomycin (0.1 mg/mL). Confluent L6 cells were preincubated in serum-free medium for 6 h followed by treatment with glucose for 24 h. The experiments involving HepG2, Hela, A375, 293ET and H4 cells were performed as described for L6 cells. For BJAB, GH3 and MCF-7 cells, DMEM wassubstitutedbyRPMI1640,DMEM/F12andMEM,respectively. SDS-PAGE and Immunoblotting. The protein samples were resolved by SDS-PAGE (10%) and transferred to polyvinylidine difluoride membrane that was blocked in Tris-buffered saline (TBST) supplemented with 5% milk powder and 0.05% Tween 20 for 2 h at room temperature. The blot was treated with antigalectin-1 antibody overnight at 4 °C or with anti-β-Actin antibody for 1 h at room temperature, washed three times in TBST (10 min each), and incubated with the horseradish peroxidase-conjugated IgG for 1 h at room temperature. The membrane was washed 3x with TBST, and proteins were visualized by enhanced chemiluminescence.11

Results Differential Expression of Plasma Proteins in Diabetic and Normal Individuals. Control and T2D samples were prepared by an equivalent mixing of the plasma proteins that ensured an equal representation of 15 healthy or 17 T2D subjects in their respective samples. The enormous potential of plasma proteomics is usually undermined by the fact that few abundant proteins, for example, immunoglobulin and albumin can obscure the detection and quantification of other proteins with low abundance. To overcome this problem, plasma albumin and IgG were removed by treatment with Cibacron Blue 3GA and protein A, respectively, as also described elsewhere.12 The use of IPG strips with a pH range of 3-10 was indispensable to investigate the differences of plasma proteins in T2D and control samples. The majority of protein spots were localized in the pH range of 4-7 (Figure 1A, B). The molecular weight of most proteins ranged from 10 to 100 kDa. The 2-DE technique is prone to “gel to gel” variation;13,14 hence, three independent gel images were analyzed to define the protein spots that were common to all gels. In total, 203 and 216 protein spots were visualized in the plasma of T2D and control samples, respectively. Of these, 57 proteins were differentially expressed in both samples. Intensity of 30 protein spots was decreased 1260

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Liu et al. in T2D samples compared to control, of which, 12 proteins exhibited more than 3-fold reduction in their expression, and 18 were suppressed between 2 to 3-fold. The expression of 27 proteins was increased in T2D samples with 11 proteins demonstrating >3-fold induction and 16 spots between 2 to 3-fold. Finally, we selected 23 spots with 3 fold change and 8 proteins with 2-3 fold change in T2D samples for ensuing analysis by the MALDI-TOF mass spectrometry. Figure 1C depicts the enlarged views of two selected spots (#8 and #3) that were differentially expressed in T2D and control samples. Identification of 2-DE Spots by Mass Spectrometry. Identification of the gel-excised proteins was performed by MALDITOF MS. All qualified proteins ranked in the top two hits, matched at least six peptide sequences with a total coverage of more than 15%. Finally, we were able to identify 11 proteins from 31 spots by their comparison with the NCBInr entries. These 11 proteins were reproducible representative of three independent analyses (Table 2). Their sequence coverage was more than 15% and averaged at 38%. The MS results of the spot #8 and #3 are depicted in Figure 2. There were 5 peptides for the spot #8 matching with 12 peptides that were yielded by the simulation of trypsin digestion. On the other hand, 19 sequences were similar to 38 predicted peptides for the spot #3. The sequence coverage for the spot #8 and #3 was 41% and 63%, respectively. The protein spot #8 revealed 4.8-fold higher intensity in T2D subjects and it was identified as galectin-1. In contrast, the spot #3 was diminished in its intensity by 4.2fold in diabetic individuals and corresponded to apolipoprotein A-I. Their theoretical molecular weights as well as the pI values are also similar to the corresponding spots. The identification results and statistical significance of other spots given in Table 2 were also in accordance to the spot #8 and #3. The ELISA using antigalectin-1 antibody further validated the MS-based identification and induction of galectin-1 in T2D samples where it was clearly amplified in T2D sera samples by 1.8-fold (Figure 3). Glucose Induces the Galectin-1 Expression in L6 Skeletal Muscle Cells. Because galectin-1 is expressed in various tissues and its expression is variable, we tested its protein content in multiple cell lines. No apparent expression of galectin-1 was observed in BJAB, GH3 and 293ET cells (Figure 4). However, its presence was obvious in gland and secretion cells. The modulation of glucose level is the most important physiological change in T2D patients. Hence, we stimulated L6 skeletal muscle cells with high glucose and monitored its effect on the expression of galectin-1. In fact, glucose treatment augmented the amount of galectin-1 protein (Figure 5). Intriguingly, the induction of galectin-1 by glucose appears to be nonlinear.

Discussion Proteomics of the human diseases typically adopt a comparative analysis method that is defined by the differential expression of proteins under different biological states such as control versus treated or healthy versus unhealthy individuals. Since T2D is polygenic in its nature and the regulation of many plasma proteins can be anticipated, we investigated the proteomics of this disease. Our 2D gel analyses revealed 203 and 216 proteins in the plasma samples of T2D and normal individuals, respectively, of which, 57 proteins demonstrated an altered expression. The intensity of 30 protein dots was decreased and of 27 proteins was increased in T2D samples. Our MS-based analysis identified 11 proteins including galec-

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Galectin-1 in the Blood Plasma of Type 2 Diabetic Patients

Figure 4. Galectin-1 expression in different cell lines. Cells were treated with RIPA lysis buffer and processed for the Western blot analysis as described in materials and methods. (1) HepG2, (2) L6, (3) A375, (4) BJAB, (5) GH3, (6) Hela, (7) MCF7, (8) 293ET, and (9) H4 cell lines.

Figure 5. Glucose-dependent induction of galectin-1 in L6 skeletal muscle cells. (A) Cells were serum-starved for 6 h followed by treatment with glucose (0, 5, 15, and 150 mM) for 24 h. They were subjected to RIPA lysis, and the Western blot analysis was performed as described in materials and methods. (B) The amounts of galectin-1 and β-Actin proteins were quantified by densitometry. The pixel intensity was normalized and a value of 1 was assigned to the basal condition. ** p < 0.01.

tin-1 and apolipoprotein A-I. Galectin-1 was increased by 4.8fold and apolipoprotein A-I was decreased 4.2-fold in T2D patients. Apolipoprotein A-I is a constituent of the high density lipoprotein that is active in promoting the cholesterol transport into the liver, where it is metabolized and then excreted from the body.15 Apolipoprotein A-I is also associated with diabetes and cardio-cerebral vascular diseases. Its altered expression is an important indicator of the cardiovascular illness.16-18 Annunziata Lapolla et al. (2008) have suggested that unglycated as well as glycated apolipoprotein A-I are induced in the plasma sample of diabetic and nephropathic patients.3 This study, however, demonstrates a reduction in apolipoprotein A-I in diabetic individuals, and is consistent with other reports.19-22 Galectins are a conserved family of lectin proteins recognized by their carbohydrate recognition domain that is responsible for β-galactoside binding. Galectin-1, the first identified protein of the galectin family is differentially expressed in various normal and pathological tissues. This protein is functionally polyvalent, and participates in a wide range of biological processes including the (1) regulation of cell growth, migration and adhesion; (2) development and differentiation of embryonic and adult tissues; (3) tumor progression and its immuneevasion; (4) nervous system pathology; (5) initiation, amplification and resolution of inflammatory responses.23 No correlation between galectin-1 and diabetes has been reported, so far. Notably, previous studies implicate galectin-1 in differentiation of the muscle cells that are also the target of insulin and glucose

action. Intracellular galectin-1 is externalized as myoblasts differentiate into myotubes, but its exact function in myogenesis remains unknown. Galectin-1 has also been shown to induce noncommitted myogenic cells in the dermis to express myogenic markers, and to stimulate the terminal differentiation of the committed myogenic cells. Moreover, it can also influence the regenerative ability of the muscles.23 The literature evidence suggest the deregulation of apoptosis in the pancreatic β-cells as an important determinant of diabetes.24,25 Galectin-1 is a secretory protein that is known to interact with its receptor on the cell surface26 and participates in the cellular proliferation,27,28 differentiation and apoptosis.28,29 Similar to these functions, galectin-1 might also facilitate the apoptosis of β-cells. Consequently, any up-regulation of galectin-1 should lead to an alteration in the cell metabolism and signal transduction. Another member of this family, galectin3, initially identified as an advanced glycation end product (AGE) binding protein, interacts with β-galactoside residues at the cell surface and with the matrix glycoproteins through its carbohydrate recognition domain. It is also capable of peptide-peptide associations that are mediated by its N-terminus domain. These structural properties enable galectin-3 to exert multiple effects such as the modulation of cell adhesion, the control of cell cycle and the mRNA splicing.30 Increasing evidence also indicate that galectin-3 might also contribute to the AGE-related pathophysiology during diabetes.31-33 This study reveals that galectin-1 is induced in the plasma of T2D individuals, in vivo. In addition, glucose can stimulate its expression in L6 skeletal muscle cells, in vitro. Taken together, we postulate galectin-1 to be an important regulator of the pathophysiology of T2D. Our research also identifies galectin-1 as a novel plasma marker protein of this disease. Further investigations would be necessary to elucidate its mechanistic role in the onset of diabetes mellitus.

Acknowledgment. This work was supported by generous grants provided by the Major State Basic Research Development Program of China (973 Program 2006CB503909), National High Technology Research and Development Program of China (863 Program 2006AA02Z192), and National Natural Science Foundation of China (30721063). References (1) Gerich, J. E. The genetic basis of type 2 diabetes mellitus: impaired insulin secretion versus impaired insulin sensitivity. Endocr. Rev. 1998, 19 (4), 491–503. (2) Gloyn, A. L. The search for type 2 diabetes genes. Ageing Res. Rev. 2003, 2 (2), 111–27. (3) Lapolla, A.; Brioschi, M.; Banfi, C.; Tremoli, E.; Bonfante, L.; Cristoni, S.; Seraglia, R.; Traldi, P. On the search for glycated lipoprotein ApoA-I in the plasma of diabetic and nephropathic patients. J. Mass Spectrom. 2008, 43 (1), 74–81. (4) Brichory, F.; Beer, D.; Le Naour, F.; Giordano, T.; Hanash, S. Proteomics-based identification of protein gene product 9.5 as a

Journal of Proteome Research • Vol. 8, No. 3, 2009 1261

research articles (5)


(7) (8)



(11) (12) (13)


(15) (16)




tumor antigen that induces a humoral immune response in lung cancer. Cancer Res. 2001, 61 (21), 7908–12. Sadowski, H. B.; Wheeler, T. T.; Young, D. A. Gene expression during 3T3-L1 adipocyte differentiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation. J. Biol. Chem. 1992, 267 (7), 4722–31. Levenson, R. M.; Blackshear, P. J. Insulin-stimulated protein tyrosine phosphorylation in intact cells evaluated by giant twodimensional gel electrophoresis. J. Biol. Chem. 1989, 264 (33), 19984–93. Lynch, C. J.; Brennan, W. A., Jr.; Vary, T. C.; Carter, N.; Dodgson, S. J. Carbonic anhydrase III in obese Zucker rats. Am. J. Physiol. 1993, 264 (4 Pt 1), E621–30. Sundsten, T.; Eberhardson, M.; Goransson, M.; Bergsten, P. The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes. Proteome Sci. 2006, 4, 22. Wang, J.; Xue, Y.; Feng, X.; Li, X.; Wang, H.; Li, W.; Zhao, C.; Cheng, X.; Ma, Y.; Zhou, P.; Yin, J.; Bhatnagar, A.; Wang, R.; Liu, S. An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions. Proteomics 2004, 4 (1), 136–50. Li, X.; Wang, X.; Zhao, K.; Zhou, Z.; Zhao, C.; Yan, R.; Lin, L.; Lei, T.; Yin, J.; Wang, R.; Sun, Z.; Xu, Z.; Bao, J.; Zhang, X.; Feng, X.; Liu, S. A novel approach for identifying the heme-binding proteins from mouse tissues. Genomics, Proteomics Bioinf. 2003, 1 (1), 78– 86. Liu, X. J.; Yang, C.; Gupta, N.; Zuo, J.; Chang, Y. S.; Fang, F. D. Protein kinase C-zeta regulation of GLUT4 translocation through Actin remodeling in CHO cells. J. Mol. Med. 2007, 85 (8), 851–61. Hu, S.; Loo, J. A.; Wong, D. T. Human body fluid proteome analysis. Proteomics 2006, 6 (23), 6326–53. Hunt, S. M.; Thomas, M. R.; Sebastian, L. T.; Pedersen, S. K.; Harcourt, R. L.; Sloane, A. J.; Wilkins, M. R. Optimal replication and the importance of experimental design for gel-based quantitative proteomics. J. Proteome Res. 2005, 4 (3), 809–19. Escoubas, P.; Chamot-Rooke, J.; Stocklin, R.; Whiteley, B. J.; Corzo, G.; Genet, R.; Nakajima, T. A comparison of matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography electrospray ionization mass spectrometry methods for the analysis of crude tarantula venoms in the Pterinochilus group. Rapid Commun. Mass Spectrom. 1999, 13 (18), 1861–8. Rader, D. J. Regulation of reverse cholesterol transport and clinical implications. Am. J. Cardiol. 2003, 92 (4A), 42J–49J. Rohrer, L.; Hersberger, M.; von Eckardstein, A. High density lipoproteins in the intersection of diabetes mellitus, inflammation and cardiovascular disease. Curr. Opin. Lipidol. 2004, 15 (3), 269– 78. Walldius, G.; Jungner, I. The apoB/apoA-I ratio: a strong, new risk factor for cardiovascular disease and a target for lipid-lowering therapy--a review of the evidence. J. Intern. Med. 2006, 259 (5), 493–519. Ginsberg, H. N.; Zhang, Y. L.; Hernandez-Ono, A. Regulation of plasma triglycerides in insulin resistance and diabetes. Arch. Med. Res. 2005, 36 (3), 232–40.

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Liu et al. (19) Yamato, K.; Tamasawa, N.; Murakami, H.; Matsui, J.; Tanabe, J.; Suda, T.; Yasujima, M. Evaluation of apolipoprotein E secretion by macrophages in type 2 diabetic patients: role of HDL and apolipoprotein A-I. Diabetes Res. Clin. Pract. 2005, 69 (2), 124–8. (20) Tamsma, J. T.; Beverdam, F. H.; Leuven, J. A.; Lemkes, H. H. Decreased interstitial apolipoprotein A-I levels in IDDM patients with diabetic nephropathy. Diabetes 1995, 44 (5), 501–5. (21) Folin, M.; Chinello, G. E.; Contiero, E.; Vaselli, G. M. Preliminary research on apolipoprotein A-I and A-II in patients with noninsulin-dependent diabetes mellitus (NIDDM). Haematologica 1993, 78 (5), 277–81. (22) Horng, Y. C.; Wei, J. S.; Chang, R. S.; Huang, H. S.; Huang, M. J.; Huang, B. Y. Apolipoprotein levels in normolipidemic non-insulindependent diabetes mellitus. J. Formosan Med. Assoc. 1992, 91 (6), 590–4. (23) Camby, I.; Le Mercier, M.; Lefranc, F.; Kiss, R. Galectin-1: a small protein with major functions. Glycobiology 2006, 16 (11), 137R– 157R. (24) Hui, H.; Dotta, F.; Di Mario, U.; Perfetti, R. Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death. J. Cell Physiol. 2004, 200 (2), 177–200. (25) Donath, M. Y.; Halban, P. A. Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications. Diabetologia 2004, 47 (3), 581–9. (26) Perillo, N. L.; Marcus, M. E.; Baum, L. G. Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death. J. Mol. Med. 1998, 76 (6), 402–12. (27) Scott, K.; Weinberg, C. Galectin-1: a bifunctional regulator of cellular proliferation. Glycoconj. J. 2004, 19 (7-9), 467–77. (28) Yang, R. Y.; Liu, F. T. Galectins in cell growth and apoptosis. Cell. Mol. Life Sci. 2003, 60 (2), 267–76. (29) Sotomayor, C. E.; Rabinovich, G. A. Galectin-1 induces central and peripheral cell death: implications in T-cell physiopathology. Dev. Immunol. 2000, 7 (2-4), 117–29. (30) Pricci, F.; Leto, G.; Amadio, L.; Iacobini, C.; Romeo, G.; Cordone, S.; Gradini, R.; Barsotti, P.; Liu, F. T.; Di Mario, U.; Pugliese, G. Role of galectin-3 as a receptor for advanced glycosylation end products. Kidney Int. Suppl. 2000, 77, S31–9. (31) Pugliese, G.; Pricci, F.; Leto, G.; Amadio, L.; Iacobini, C.; Romeo, G.; Lenti, L.; Sale, P.; Gradini, R.; Liu, F. T.; Di Mario, U. The diabetic milieu modulates the advanced glycation end productreceptor complex in the mesangium by inducing or upregulating galectin-3 expression. Diabetes 2000, 49 (7), 1249–57. (32) Pugliese, G.; Pricci, F.; Iacobini, C.; Leto, G.; Amadio, L.; Barsotti, P.; Frigeri, L.; Hsu, D. K.; Vlassara, H.; Liu, F. T.; Di Mario, U. Accelerated diabetic glomerulopathy in galectin-3/AGE receptor 3 knockout mice. Faseb J. 2001, 15 (13), 2471–9. (33) Stitt, A. W.; McGoldrick, C.; Rice-McCaldin, A.; McCance, D. R.; Glenn, J. V.; Hsu, D. K.; Liu, F. T.; Thorpe, S. R.; Gardiner, T. A. Impaired retinal angiogenesis in diabetes: role of advanced glycation end products and galectin-3. Diabetes 2005, 54 (3), 785–94.


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