The Physiologic Effects of Caloric Restriction Are Reflected in the in Vivo Adipocyte-Enriched Proteome of Overweight/Obese Subjects

The Physiologic Effects of Caloric Restriction Are Reflected in the in Vivo Adipocyte-Enriched Proteome of Overweight/Obese Subjects Freek G. Bouwman,† Mandy Claessens,† Marleen A. van Baak,† Jean-Paul Noben,‡ Ping Wang,† Wim H. M. Saris,† and Edwin C. M. Mariman*,† NUTRIM School for Nutrition, Toxicology and Metabolism, Department of Human Biology, Maastricht University Medical Centre+, P.O. Box 616, NL-6200MD Maastricht, The Netherlands, and Hasselt University, Biomedical Research Institute and Transnational University Limburg, School of Life Sciences, Diepenbeek, Belgium Received July 10, 2009 We have applied our recently designed proteomics apparoach to search for protein changes in the in vivo adipocyte-enriched proteome from 8 overweight/obese subjects who underwent an intervention of 5 weeks of a very low calorie diet followed by 3 weeks of a normal diet. On average, persons lost 9.5 kg body weight largely contributed by the loss of fat mass (7.1 kg). Various parameters of adiposity and lipid metabolism changed significantly. Proteomics analysis revealed 6 significantly changed proteins. Analysis indicates that fructose-bisphosphate aldolase C and tubulin beta 5 are potential biomarkers for the present intervention. Further, identified proteins indicate a reduced intracellular scaffolding of GLUT4 (ALDOC, TUBB5, ANXA2), an increased uptake of fatty acids (FABP4), an improved inflammatory profile of the adipose tissue (ApoA1, AOP1) and a change in fat droplet organization (vimentin). Correlation analysis between changes in protein spot intensities and parameters of adiposity or lipid metabolism points to a link between aldo-ketoreductase 1C2 and parameters of adiposity, between FABP4 and parameters of lipid metabolism, and between proteins for beta-oxidation (HADH, ACADS, ACAT1) and FFA levels. Altogether, our findings underscore the potential value of in vivo proteomics for human intervention studies. Keywords: Caloric restriction • Obesity • Proteomics • adipose tissue • lipid metabolism • physiologic effects 1. Introduction The worldwide increasing prevalence of obesity and its consequences for human health request novel ways of prevention and treatment. A better insight in the underlying physiologic and molecular processes is therefore required. Obesity is characterized by the accumulation of excessive fat mass in the body, which is associated with morphological, histological and functional changes of the adipose tissue including fibrosis, infiltration of macrophages and changes in the adipokine profile.1-5 Some of those changes are believed to increase the risk for obesity-associated diseases like type II diabetes and cardiovascular disorders. Caloric restriction is one way to (partly) ameliorate those adverse conditions.6-8 The application of proteomics techniques is a welcome new approach to obtain an integrative view of the molecular dynamics of adipose tissue during weight regulation. However, tissue samples like adipose tissue biopsies are collections of various cell types, each with specific functions. As such, the * Corresponding author: Prof. Dr. Edwin C.M. Mariman, Dept. Human Biology, NUTRIM, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel: +31 43 3882893; Fax: +31 43 3670976; E-mail: e.mariman@hb.unimaas.nl. † Maastricht University Medical Centre+. ‡ Hasselt University, Biomedical Research Institute and Transnational University Limburg. 5532 Journal of Proteome Research 2009, 8, 5532–5540 Published on Web 10/15/2009 use of tissue samples interferes with the possibility to study cell-type specific processes. Obtaining cell-type specific information from a tissue sample is one of the major challenges of experimental omics-approaches. Recently, we have designed a proteomics approach that looks more specifically at adipocyte protein regulation in human adipose tissue biopsies by defining the adipocyte-enriched spots in the 2D-tissue proteome using a subtraction protocol.9 In this protocol, protein spots on a 2D-gel of the fat biopsy are regarded adipocyte-derived if the spots are present on a matched 2D-gel of purified adipocyte, but not present on a matched 2D-gel of blood cells. Further, in the present study, adipocyte-enriched spots were checked for absence of platelet-derived proteins.10 In that way, differential proteins were identified in adipose tissue that complied with the physiological differences categorizing subjects as high- or low fat-oxidizers.9 In the present study, we have used this protocol to search for diet-induced changes in the in vivo adipocyte-enriched proteome. To this end, abdominal subcutaneous adipose tissue biopsies were taken from overweight/obese persons who were subjected for 5 weeks to a very low calorie diet (VLCD) followed by 3 weeks on a normal diet. Here, we report the analysis of the subcutaneous adipocyte-enriched proteome before and after this 8 weeks intervention in relation to the physiological changes. 10.1021/pr900606m CCC: $40.75  2009 American Chemical Society Physiologic Effects of Caloric Restriction in Adipocytes 2. Experimental Procedures 2.1. Subjects Selection and Experimental Design. Four male and four female overweight and obese subjects (BMI g 27 kg/m2), aged 30-60 years, willing to undergo adipose tissue biopsies, were recruited from a study that investigated the role of dietary protein content for long-term weight maintenance after weight loss. An extensive description of the design of this study has been published previously.11 In short, subjects underwent a brief medical screening examination, including a medical history, routine physical examination and a fasting blood sample was collected. Subjects had to be weight stable over the 2 months before enrollment. Subjects were excluded if fasting glucose (>6 mmol/L), triglycerides (>2.3 mmol/L) or total cholesterol levels (>6.5 mmol/L) were increased, or when diastolic blood pressure exceeded 100 mmHg. Furthermore, subjects were excluded during the study when they were unable to lose at least 5% of their initial body weight (BW) during the weight loss period. Body composition was determined by measuring body weight in air and underwater on a digital balance. Lung volume was measured simultaneously with the helium dilution technique using a spirometer. The body density was used to calculate body fat according to the two-compartment model as described by Siri12 The Medical Ethics Committee of the Maastricht Academic Hospital and University approved the study and all subjects gave their written informed consent before entering the study. After baseline measurements of anthropometric and physiologic parameters, collection of fasting blood samples and a biopsy from the abdominal subcutaneous adipose tissue, subjects started a 5-week VLCD period. During this period, they consumed a diet providing only 500 kcal per day (Modifast, Nutrition et Sante’, France). Subjects were allowed to eat an unrestricted amount of vegetables (all vegetables except pulse crops). During week 6, the VLCD was gradually replaced by normal ad libitum meals and protein or carbohydrate supplements were gradually introduced. All subjects received dietary counseling by a dietician and were advised to limit their fat intake to approximately 30% of energy intake. Measurements of anthropometric and physiological variables were performed and fasting blood samples were collected in week 6. To make the comparison in an energy balanced situation before and after the weight loss period, the second adipose tissue biopsy was taken 3 weeks after returning to a normal diet at week 8 in the morning after an overnight fast. 2.2. Fat Biopsy. Abdominal subcutaneous adipose tissue biopsies (approximately 1.5 g) were obtained from the paraumbilical region by needle liposuction under local anesthesia (2% lidocaine with adrenaline 1:80 000, AstraZeneca BV, The Netherlands). The tissue was immediately washed in cold saline, frozen in liquid nitrogen, and stored at -80 °C until protein isolation. 2.3. Sample Preparation. 2.3.1. Fat Tissue Biopsy. About 350 mg of tissue from the biopsy was washed in PBS to get rid of the major part of blood, frozen again in liquid nitrogen and grinded in a mortar. The powder was dissolved in 200 µL of 8 M urea, 2% (w/v) CHAPS, 65 mM DTT per 100 mg of biopsy. The homogenate was vortexed for 5 min and centrifuged at 20 000g for 30 min at 10 °C. The supernatant containing the adipose tissue proteome was carefully collected and aliquots were stored at -80 °C. 2.3.2. Purified Adipocytes. From a biopsy of a subject not taking part in the intervention study, adipocytes to be used in research articles the subtraction procedure were isolated exactly as described before.9 These isolated purified adipocytes were resuspended in 8 M urea, 2% (w/v) CHAPS, and 65 mM DTT. Adipocytes were lysed by subjecting them to three cycles of freeze-thawing in liquid nitrogen. The homogenate was vortexed for 1 min and centrifuged at 20 000g for 30 min at 10 °C. The supernatant was carefully collected and aliquots were stored at -80 °C. 2.3.3. Blood Cells. From an EDTA containing blood sample of a subject not taking part in the intervention study, blood cells were isolated to be used in the subtraction procedure. First, the blood sample was centrifuged at 1000g at 4 °C for 10 min. Afterward, plasma was discarded and erythrocytes were mixed with the buffy coat. This mixture was washed 3 times with 0.9% NaCl buffered with PBS. Blood cells were resuspended in 8 M urea, 2% (w/v) CHAPS, and 65 mM DTT and they were lysed by subjecting them to three cycles of quick freezing in liquid nitrogen and subsequent thawing. The homogenate was vortexed for 1 min and centrifuged at 20 000g for 30 min at 10 °C. The supernatant was carefully collected and aliquots were stored at -80 °C. Protein concentration in all samples was determined by a Bradford based protein assay.13 2.4. 2D-Electrophoresis. From all 16 biopsy samples, 150 µg of total protein was loaded for the first-dimension separation. One gel was run with the protein from purified adipocytes and from blood cell proteins. Isoelectric focusing was performed on an IPG PHOR electrophoresis unit (Amersham Biosciences) at 20 °C. Immobiline Dry Strips (pH 3-10 Linear, 24 cm long) were rehydrated overnight in 500 µL of 8 M urea, 2% (w/v) CHAPS, 65 mM DTT, and 0.5% (v/v) IPG buffer pH 3-10 Linear at 30 V. Isoelectric focusing was performed using the following program: 500 V for 1 h, 1000 V for 1 h, 1000-8000 V for 2 h and a final step of 8000 V for 6.5 h. After focusing, IPG strips were equilibrated for 15 min in 50 mM Tris-HCl, pH 6.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 1% (w/v) DTT and for 15 min in 50 mM Tris-HCl, pH 6.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 2.5% (w/v) iodoacetamide, and were placed onto a slab gel and sealed with a 0.5% (w/v) agarose solution in Laemmli buffer with a trace of bromophenol blue. The second-dimension run was carried out on 12.5% SDSPAGE gels. Electrophoresis was conducted at a constant voltage of 200 V for 5 h in a 24 cm Dodeca Cell (Bio-Rad).14-16 These gels were stained with Flamingo fluorescent gel stain according to the manufacturer’s protocol. Gel images were obtained with a FX Molecular Imager (Bio-Rad). Spot detection and matching was performed with the PDQuest v7.3 software package (Bio-Rad). Gel images were normalized to the adipocyte-enriched spots. Fold changes were obtained by dividing the average spot intensity (n ) 8) of the after diet group by that of the before diet group. Molecular weight values were estimated using standard MW-markers. 2.5. In-Gel Digestion. Protein spots were excised from gels using an automated spot cutter (Bio-Rad) and processed on a MassPREP digestion robot (Waters, Manchester, U.K.). A solution of 50 mM ammonium bicarbonate in 50% (v/v) acetonitrile (ACN) was used for destaining. Cysteines were reduced with 10 mM DTT in 100 mM ammonium bicarbonate for 30 min followed by alkylation with 55 mM iodoacetamide in 100 mM ammonium bicarbonate for 20 min. Spots were washed with 100 mM ammonium bicarbonate to remove excess reagents and were subsequently dehydrated with 100% ACN. Trypsin (6 ng/µL) in 50 mM ammonium bicarbonate was added to the gel plug and incubation was performed at 37 °C for 5 h. Journal of Proteome Research • Vol. 8, No. 12, 2009 5533 research articles Peptides were extracted in 30 µL of 1% (v/v) formic acid/2% (v/v) ACN in water for 30 min at room temperature. A second extraction was performed using 24 µL of 50% (v/v) ACN in water for 20 min at room temperature.16,17 2.6. Mass Spectrometry. For MALDI-TOF mass spectrometry, 1.5 µL of peptide mixture and 0.5 µL of matrix solution (2.5 mg/mL R-cyano-4-hydroxycinnamic acid in 50% ACN/ 0.1% TFA) were spotted automatically onto a 96 well-format target plate. Spots were allowed to air-dry for homogeneous crystallization. Spectra were obtained using an M@LDI-LR mass spectrometer (Waters). The instrument was operated in positive reflector mode. Acquisition mass range was 800-3500 Da. The instrument was calibrated on 10-12 reference masses from a tryptic digest of alcohol dehydrogenase. In addition, a near point lockmass correction for each sample spot was performed using adrenocorticotropic hormone fragment 18-39 (MH+ 2465.199) to achieve maximum mass accuracy. Typically 120 shots were combined and background subtracted. A peptide mass list was generated by Masslynx v4.0 for the subsequent database search.16,17 Samples that could not be identified via MALDI-TOF MS were further analyzed by nano liquid chromatography tandem mass spectrometry (LC-MSMS) on a LCQ Classic (ThermoFinnigan).18 De novo sequencing of ApoA1 was preformed on a MALDI-TOF/TOF mass spectrometer (4800 MALDI TOF/TOF analyzer, Applied Biosystems). 2.7. Database Search. The peptide mass list was searched with the Mascot search engine (version 2.2.04; Matrix Science, London, U.K.) against the Swiss-Prot database (Swiss-Prot release 56.5; 402 482 sequences) for protein identification. One miss-cleavage was tolerated, carbamidomethylation was set as a fixed modification and oxidation of methionine as an optional modification. The peptide mass tolerance was set to 100 ppm. No restrictions were made on the protein molecular weight and the isoelectric point. A protein was regarded identified when it had a significant Mascot probability score (p < 0.05).17 2.8. Western Blotting. Samples with equal amount of protein were run on a 12% SDS polyacrylamide gel (180 V, Criterion Cell; Bio-Rad, Hercules, CA), then were transferred (90 min, 100 V, Criterion blotter; Bio-Rad) to 0.45-mm nitrocellulose membranes. After Ponceau S staining and destaining, membranes were blocked in 5% nonfat dry milk power (NFDM; Bio-Rad) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. Thereafter, the three blots were incubated with the primary antibodies against ApoA1 (1:1000 dilution, Santa Cruz), Fructose-bisphosphate aldolase C (1:250 dilution, Santa Cruz) and Tubulin beta (1:500 dilution, Cell signaling) in 5% NFDM-TBST overnight at 4 °C on a shaker. Thereafter, the blots were washed three times for 10 min in TBST, then incubated for 1 h with a 1:10 000 dilution of the horseradish peroxidaseconjugated secondary antibody (DAKO) in 5% NFDM-TBST. The blots were washed three times for 10 min in TBST. A CCD camera (XRS-system, Biorad) was used to detect immunoreactive bands using chemiluminescent substrate (SuperSignal CL; Pierce). The quantification was performed with the program Quantity One version 4.6.5 (Bio-Rad). β-Actin was used to standardize for the amount of protein loaded.19 2.9. Statistical Analysis. Physiological data are presented as mean ( SEM. The changes of physiological data and spot intensities between before and after diet intervention groups were analyzed by paired-samples t tests. Changes in leptin concentrations were log-transformed because of non-normal distribution. All other changes in physiologic measurements 5534 Journal of Proteome Research • Vol. 8, No. 12, 2009 Bouwman et al. Table 1. Physiologic Measurements (Mean ( SEM) before and after the Diet Intervention (n ) 8) variable Body weight (kg) Fat mass (kg) Fat-free mass (kg) BMI (kg/m2) Waist circumference (cm) Hip circumference (cm) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Glucose (mmol/L) Insulin (µU/mL) Glucagon (pg/mL) Total cholesterol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Triglycerides (mmol/L) FFA (mmol/L) Leptin (ng/mL) Adiponectin (µg/mL) a week 0 week 6 P-valuea 99.7 ( 6.5 37.5 ( 2.8 62.3 ( 4.9 32.6 ( 1.1 111.6 ( 4.3 115.9 ( 3.8 138.1 ( 8.9 90.2 ( 6.0 30.4 ( 3.0 59.8 ( 4.1 29.5 ( 1.2 101.9 ( 4.2 107.5 ( 4.2 132.3 ( 9.5 < 0.001 < 0.001 0.025 < 0.001 < 0.001 0.011 0.372 91.3 ( 5.0 86.3 ( 1.9 0.251 5.10 ( 0.33 17.6 ( 2.5 73.5 ( 11.2 4.58 ( 0.40 4.73 ( 0.30 13.1 ( 2.0 53.2 ( 6.7 3.84 ( 0.36 0.011 0.069 0.027 0.007 1.01 ( 0.08 1.02 ( 0.06 3.03 ( 0.39 2.28 ( 0.31 1.68 ( 0.27 1.16 ( 0.15 0.781 ( 0.122 0.415 ( 0.043 47.2 ( 20.2 22.5 ( 11.3 12.8 ( 3.6 16.1 ( 4.4 0.887 0.003 0.116 0.007 0.032 0.304 Paired-sample t test week 0 vs week 6. were normally distributed. For the associations between the change of protein level and change of physiological parameters, Pearson correlation coefficients were calculated. A P-value <0.05 was considered statistically significant. These statistical analyses were done using the SPSS v15.0 statistical software. The FDR of Multiple testing was calculated using the statistical software R v2.8.1 package fdr tool.20 3. Results 3.1. Subject Characteristics. Adipose tissue biopsies were derived from eight overweight/obese subjects, four males and four females, before and after a dietary intervention that consisted of a VLCD for 5 weeks followed by 3 weeks of adaptation to a weight maintenance diet. Physiological parameters were determined at the start of the intervention and at week 6, that is, after 1 week adaptation to a weight maintenance diet to avoid the influence of a negative energy balance. The physiological results are summarized in Table 1. A significant reduction of BMI was noticed due to an average weight loss of 9.5 kg. Of this, roughly 7 kg had to be ascribed to loss of fat mass and 2.5 kg to loss of fat-free mass (Table 1). This was accompanied by a reduction of the leptin level. There was a significant decrease in plasma glucose together with a trend for a lower insulin level. Total plasma cholesterol, LDLcholesterol and FFA levels were significantly lower after the intervention. No significant difference was noticed for the plasma HDL-cholesterol and triglyceride levels. No gender effects were observed. Altogether, this indicates an increase in insulin sensitivity and an improved lipid profile due to the VLCD intervention. 3.2. 2D-Gel Electrophoresis. Proteins were isolated from adipose tissue biopsies and separated by 2D-gel electrophoresis as described above. On average, 516 valid protein spots were detected on the gel. In parallel, proteins isolated from purified human adipocytes and from total blood cells were separated (Figure 1). The latter patterns were then used to select the spots with an increased likelihood to be derived from adipocyteexpressed proteins.9 In this way, 101 adipocyte-derived spots Physiologic Effects of Caloric Restriction in Adipocytes research articles Figure 1. Identified proteins marked on a 2D gel from a subcutaneous fat tissue biopsy (A), from primary adipocytes purified from a subcutaneous fat tissue biopsy (B) and from isolated blood cells (C). Numbers on the gel images correspond to the spot numbers in Table 2. were selected, which were subsequently used for normalizing the individual gel-patterns. All 101 spots were cut from the gels and subjected to tryptic digestion followed by mass spectrometry to identify the protein. From the 101 spots, for 40 the protein could be identified (Table 2) belonging to 34 different proteins. In Table 2, the identified proteins are divided in three groups according to their change in relative abundance. Seven spots showed a significant change (p < 0.05) with the following as identified proteins: tubulin beta 5 (TUBB5), apolipoprotein A1 (ApoA1), fatty acid binding protein 4 (FABP4), thioredoxindependent peroxide reductase (AOP1), annexin A2 (ANXA2, N-term.) and fructose-bisphosphate aldolase C (ALDOC). Despite the fact that ApoA1 is not produced by adipocytes, two spots of ApoA1 that changed in relative abundance after the intervention were detected in adipose tissue (spots 4 and 12 in Figure 2A,B). Therefore, we looked in detail for the presence of those spots in total blood cells and isolated adipocytes. As can be seen in Figure 2C,D, the spots are not observed in blood cells, but do appear in the sample of isolated adipocytes. 3.3. Western Blotting. To confirm our findings with 2D-GE, Western blotting was performed for three significantly changed proteins. Individual samples were blotted. The result of the ApoA1 blot (Figure 3A) indeed confirms the results of the 2D analysis. The concentration of ApoA1 in the adipose tissue is significantly higher after the diet intervention than before (p ) 0.017). For fructose-bisphosphate aldolase C (Figure 3B), a trend for reduction was observed (p ) 0.061) in keeping with the 2D analysis. An antibody specific for tubulin beta-5 is not available. Therefore, we used an antibody against tubulin beta in general, but with this antibody, the 2D-GE results could not be confirmed (Figure 3C). 3.4. Detailed Analysis of Individual Data. For the enzyme fructose-bisphosphate aldolase C, two spots were detected by 2D-GE (spots 9 and 27 in Table 2), which may reflect different isoforms. With the pooled 2D-data, both spots showed a significant decrease in abundance. More detailed analysis revealed a consistent decrease of all individual 2D-values for both spots (Figure 4A,B), further corroborating a link with the intervention. A similar analysis of tubulin beta-5 and the N-terminal fragment of annexin A2 showed that also here all the individual 2D-values consistently decreased (data not shown), but in the latter case, data from only 5 subjects were available. Although the pooled 2D-data of the other three proteins resulted in a significant change after the intervention, the spot intensity for some individuals increased while for others it decreased. An example can be seen in Figure 4C. To find out whether changes in physiological parameters, in particular those related to adipose tissue function, were quantitatively linked with changes of the differential proteins, correlation analysis was performed between the changes of those parameters and changes of the 40 spot intensities. Table 3 lists the significant correlations with p-values of less than 0.05. Since the number of subjects is only 8, after correction for multiple testing (FDR), no significance was reached for any of the calculations. However, we reasoned that, when a parameter correlates with more proteins from the same molecular pathway, this may still represent a genuine link. As such, the positive correlations between changes in FFA level and changes in three mitochondrial enzymes of β-oxidation (short chain specific acyl-CoA dehydrogenase (ACADS), short chain 3-hydroxyacylCoA dehydrogenase (HADH) and acetyl-CoA acetyltransferase (ACAT1)) may be relevant. Similarly suggestive is a situation in which the change of a protein correlates with the change of several physiological parameters. The change in aldo-keto reductase family 1 member C2 (AKR1C2) was found to correlate positively with changes in parameters of adiposity, that is, weight, BMI and waist. The change in fatty acid binding protein (FABP4) was found to inversely correlate with changes in several parameters of lipid metabolism, that is, plasma total Journal of Proteome Research • Vol. 8, No. 12, 2009 5535 research articles Bouwman et al. Table 2. Protein Identification of Adipocyte-Specific Spots and Expression Difference before and after Diet Intervention difference spot g1.5 g1.2-<1.5 <1.2 a p e 0.05. b b expression exp/theorMw after/before accession (kDa) intervention P-value Q-value number 0.005a 0.157 0.087 0.014a 0.053 0.032b 0.095 0.082 0.042b 0.072 P07437 P00558 P07355 P02647 P52895 -1.76 0.070 0.078 P14550 52/58 -1.79 0.088 0.082 Q02252 8 9 10 11 12 13 14 15 49/53 43/40 35/38 46/42 26/30 47/50 12/14 32/35 -2.22 -2.62 1.41 1.38 1.33 1.33 1.30 1.29 0.059 0.002a 0.035a 0.144 0.061 0.325 0.017a 0.150 0.074 0.025b 0.062 0.093 0.075 0.152 0.043b 0.094 P08670 P09972 P07355 P42765 P02647 P68104 P15090 P63244 16 17 18 19 20 21 22 23 24 25 26 27 28 33/38 12/15 43/45 42/40 27/24 39/38 48/54 51/56 18/17 47/53 21/20 43/40 24/28 1.26 1.24 1.24 1.23 1.23 1.23 1.22 -1.25 -1.30 -1.31 -1.32 -1.48 -1.48 0.508 0.095 0.533 0.441 0.135 0.437 0.353 0.366 0.154 0.293 0.150 0.014a 0.044a 0.194 0.083 0.198 0.180 0.091 0.180 0.160 0.163 0.094 0.143 0.094 0.042b 0.067 P07355 P09382 P24752 P04075 P52565 P04083 P08670 P06576 O14558 P08670 P02511 P09972 P30048 29 33/34 1.19 0.100 0.084 Q16836 30 31 32 33 34 35 36 29/30 29/32 22/25 35/33 57/61 45/42 45/46 1.17 1.17 1.13 1.08 1.08 1.08 1.03 0.510 0.175 0.561 0.513 0.654 0.438 0.840 0.194 0.101 0.206 0.195 0.232 0.180 0.280 P35232 P22676 P04179 Q99685 P10809 P60709 O75874 37 42/45 -1.00 0.922 0.299 P16219 38 25/70 -1.10 0.786 0.267 O14975 39 37/36 -1.15 0.785 0.266 P04406 40 37/36 -1.18 0.429 0.178 P40926 1 2 3 4 5 38/50 45/45 37/38 26/30 38/37 1.86 1.69 1.63 1.62 1.54 6 40/36 7 Tubulin beta 5 Phosphoglycerate kinase 1 Annexin A2 Apolipoprotein A-I Aldo-keto reductase family 1 member C2 Alcohol dehydrogenase [NADP+] Methylmalonate-semialdehyde dehydrogenase Vimentin Fructose-bisphosphate aldolase C Annexin A2, N-term AcetylCoA acyltransferase, Mt Apolipoprotein A-I Elongation factor 1-alpha 1 FABP4 Guanine nucleotide-binding protein beta-2-like 1 Annexin A2 Galectin-1 Acetyl-CoA acetyltransferase, Mt Fructose-bisphosphate aldolase A Rho GDP-dissociation inhibitor 1 Annexin A1 Vimentin ATP synthase subunit beta Heat shock protein beta-6 Vimentin Alpha-Crystallin B Fructose-bisphosphate aldolase C Thioredoxin-dependent peroxide reductase, Mt Short chain 3-hydroxyacyl-CoA dehydrogenase, Mt Prohibitin Calretinin Superoxide dismutase [Mn], Mt Monoglyceride lipase 60 kDa heat shock protein beta Actin Isocitrate dehydrogenase [NADP] Cyt Acyl-CoA dehydrogenase, short-chain specific, Mt Very-long-chain acyl-CoA synthetase, partial Glyceraldehyde-3-phosphate dehydrogenase, liver Malate dehydrogenase, Mt matched/ Mascot sequence unmatched score coverage % peptides 97 98 113 70 90 23 52 27 28 15 9/27 21/149 8/11 7/31 5/5 69 18 5/9 100 20 8/12 234 80c 96 110 80 70c 107 76 56 39 24 18 40 16 59 25 34/113 15/235 8/15 8/15 10/100 6/10 7/18 7/30 72 73 72 75 94 94 226 88 72 187 90 72 71 29 41 39 14 29 28 56 28 30 51 45 29 25 9/42 5/15 16/124 5/7 7/39 8/19 31/119 11/76 5/63 31/141 8/33 7/46 6/18 76 16 6/7 76 94 71 91 104 100 74 21 26 20 26 28 35 19 5/8 8/17 4/5 7/10 13/44 9/26 8/22 75 26 10/35 72 8 5/5 78 38 9/57 88 28 7/15 q e 0.05. c Also confirmed with LC-MSMS. cholesterol, LDL-cholesterol and triglyceride levels. However, it should be kept in mind that those parameters of lipid metabolism or adiposity cannot be regarded as independent. 4. Discussion In this study, we analyzed subcutaneous fat biopsies taken from subjects before and after an intervention of 5 weeks on a very low calorie diet followed by 3 weeks of adaptation to a weight-maintaining normal diet in order to prevent influences of a negative energy balance. Using 2D gel separation of biopsy proteins, we searched for differential 5536 protein description Journal of Proteome Research • Vol. 8, No. 12, 2009 proteome differences complying with general physiological observations. Indeed, even 3 weeks after returning to a normal diet, differential proteins were observed, thus, reflecting established changes at the level of gene expression due to weight reduction. Six of the identified proteins showed a significant change in abundance (p < 0.05). Combining pooled and individual data revealed that fructose-bisphosphate aldolase C (ALDOC) and tubulin beta 5 (TUBB5) are potential markers for the present intervention which includes both the weight loss (5 weeks) and weight maintenance (3 weeks) period. Physiologic Effects of Caloric Restriction in Adipocytes research articles Figure 2. Magnification of ApoA1 region: (A) Fat tissue biopsy before diet intervention; (B) fat tissue biopsy after diet intervention; (C) isolated blood cells and (D) purified adipocytes from fat tissue biopsy. The spot numbers refer to identifications in Table 2. Two spots for ALDOC were detected and both were reduced in abundance after the intervention. ALDOC is an enzyme of the glycolysis. Two other glycolytic enzymes, phosphoglycerate kinase 1 and fructose-bisphosphate aldolase A, were found upregulated and one other glycolytic enzyme, glyceraldehyde-3phosphate dehydrogenase, was down-regulated, but all not significantly. Therefore, no conclusion can be drawn for a major change in glycolysis. Interestingly, ALDOC has been shown to function as a structural component of the actin cytoskeleton. Moreover, it is able to mediate the association of F-actin with the glucose transporter GLUT4.21 It was proposed that ALDOC is partly responsible for the intracellular sequestration of GLUT4. Both insulin stimulation and the substrates fructose1,6-bisphosphate and glyceraldehyde-3-phosphate lead to the release of GLUT4 boosting glucose uptake by its translation to the membrane. In this regard, lower ALDOC after the intervention may promote increased levels of GLUT4 in the cell membrane accompanied by increased uptake of glucose necessary for triglyceride synthesis and storage. As such, a decrease of ALDOC might contribute to a decrease in plasma levels of glucose after the intervention (Table 1). Not much is known about the exact function of the beta-5 isoform of tubulin. In general, beta-tubulin forms dimers with alpha-tubulin. Interestingly, ALDOC activity can be inhibited by its binding to the C-terminal region of alpha-tubulin.22 Rearrangement of the tubulin filaments by the significant upregulation of tubulin beta 5 might thus lead to a functional reduction of the already reduced amount of ALDOC. Similarly, not much is known about the function of annexin A2 in adipocytes. A significant increase of the N-terminal part indicates increased production of the mature protein. In 3T3L1 cells, annexin A2 has been shown to support GLUT4 translocation.23 Altogether, our findings with the three consistently up- or down-regulated proteins suggest changes in glucose uptake in adipocytes by the intervention. Similarly, the uptake of fatty acids seems improved because on average there is a 40% increase in the abundance of FABP4 after the intervention. Taken together, this provides evidence that weight reduction, in particular loss of fat mass, stimulates the basal function of triglyceride storage by adipocytes. However, since Figure 3. Expresion differences of the protein blots before and after diet intervention, (A) ApoA1 blot; (B) fructose-bisphosphate aldolase C blot; and (C) Tubulin beta blot. we were not able to directly measure glucose and fatty acid uptake, this remains speculative. Since tubulin beta 5 and annexin A2 are on the list of 44 generally detected differential proteins,24 part of this stimulation may come from a change in cellular stress in the adipocytes due to metabolic effects of decreased energy supply. ApoA1 is believed to be produced by liver and intestine, but not by adipocytes. Yet, ApoA1 was detected in the proteome of purified adipocytes. One explanation could be that during adipocyte purification some macrophages remain attached to the adipocytes. Macrophages have a high affinity for ApoA1 leading to copurification of this protein with the adipocytes. On the other hand, the increase in ApoA1 may reflect a genuine biological function as it has been reported that adipocytes can process HDL particles.25,26 ApoA1 has been recognized as an anti-inflammatory factor.27-29 Thus, the increased concentration of ApoA1 after the intervention indicates an improved Journal of Proteome Research • Vol. 8, No. 12, 2009 5537 research articles Bouwman et al. Figure 4. Differences of ODs from the 2D before and after diet intervention of each subject. Table 3. Pearson Correlation Coefficients of Spot Intensity Changes with Changes in Physiologic Parametersa spot no. 4 5 10 12 14 18 19 23 24 25 29 36 37 38 39 a protein description Apolipoprotein A-I Aldo-keto reductase family 1 member C2 Annexin A2, N-term Apolipoprotein A-I FABP4 Acetyl-CoA acetyltransferase, Mt Fructose-bisphosphate aldolase A ATP synthase subunit beta Heat shock protein beta-6 Vimentin Short chain 3-hydroxyacyl-CoA dehydrogenase, Mt Isocitrate dehydrogenase [NADP] Cyt Acyl-CoA dehydrogenase, short-chainspecific, Mt Very-long-chain acyl-CoA synthetase, partial Glyceraldehyde-3-phosphate dehydrogenase, liver Body weight 0.717b No significant correlation was obtained with Fat mass. BMI waist 0.770b 0.752b 0.843c Journal of Proteome Research • Vol. 8, No. 12, 2009 HDL LDL TG FFA 0.709b 0.723b 0.803b 0.834c (log) leptin -0.741b 0.755b -0.809b -0.920c -0.858c -0.764b 0.745b b 0.727 0.746b -0.846c 0.708b -0.788b 0.752b 0.710b 0.733b -0.731b 0.710b 0.894c -0.712b -0.764b b 0.719b P < 0.05. c P < 0.01. inflammatory profile of the adipose tissue. Interestingly, ApoA1 in adipocytes was seen as two differentially expressed spots (4 and 12 in Figure 2). It has been reported that ApoA1 can become palmitoylated.30 This post-translational modification of serine and cysteine residues allows proteins to attach to the cell membrane.31 It is tempting to assume that one of the spots represents this membrane binding form. Alternatively, ApoA1 is known to be processed from a precursor by the removal of a 6-amino acid N-terminal propeptide.32,33 Therefore, the two spots could represent the processed and nonprocessed form. MALDI-TOF/TOF de novo sequencing did not allow us to 5538 chol decide on this matter. The presence of ApoA1 in the adipocytesenriched proteome needs further investigation. Another spot with significant differential expression is that of mitochondrial thioredoxin-dependent peroxide reductase (fold change -1.48), also known as peroxiredoxin-3 or antioxidant protein 1 (AOP1). Analysis of the individual samples shows a reduction of high individual values (Figure 4C). The window of spot values before the intervention (1300-4400 units) is reduced by more than 4× after the intervention (1300-2000 units) as if weight loss induces normalization of this protein to a basal level. It has been reported that this Physiologic Effects of Caloric Restriction in Adipocytes research articles protein can bind to leucine zipper-bearing kinase (LZK) and this interaction was shown to enhance the LZK-induced activation of NF-κB, a well-known mediator of inflammatory pathways.34 Therefore, the reduction of this protein may indicate a reduced level of oxidative stress inside the adipocytes after weight loss, but may also reflect the inflammatory status of the adipose tissue. Although not reaching significance, three spots belonging to vimentin displayed a fold changes of -2.22 (p ) 0.06), +1.22 (p ) 0.35) and -1.31 (p ) 0.29), respectively, suggesting that the vimentin filaments undergo rearrangement during the intervention. This seems plausible, because vimentin filaments have been shown to be linked to the fat droplets.35 A change in vimentin thus might be in line with a change in fat droplet organization in the adipocytes.36 In a previous study, we have compared the adipocyteenriched proteome of human adipose tissue between physiologically distinguished low and high fat-oxidizing obese subjects.9 There we found a 2.4-fold higher abundance of ALDH6A1 in low fat-oxidizers, which was suggested to promote the input of carbon atoms into the TCA-cycle via succinyl-CoA as a compensation for decreased oxaloacetate formation. Present analysis of the individual samples shows that the overall effect is due to the extreme reduction of the enzyme level in half of the individuals (Figure 4D). It is tempting to speculate that those subjects would be physiologically classified as low-fat oxidizers. In this respect, the 2-fold down-regulation of ALDH6A1 after the present intervention might indicate sufficient formation of oxaloacetate from pyruvate in line with improved uptake of glucose. Loss of fat mass is generally associated with decreased fatty acid oxidation.37,38 There may even be a mechanistic link, because fatty acid oxidation is correlated with lipolysis resulting in decreased extrusion of FA into the plasma.39 Correlation analysis between changes in spot intensities and physiological parameters showed that three enzymes of the fatty acid oxidation pathway (HADH, ACADS, ACAT1) are positively correlated with the decrease in the plasma FFA level. Although suggestive of a functional link, the outcome of this analysis does not allow conclusions about the regulation of fatty acid oxidation. No correlation was found between the FFA level and another enzyme of fatty acid oxidation: acetyl-CoA acyltransferase. Remarkably, this enzyme is involved in the breakdown of FA from the n16-stage on, whereas the other three only catalyze the catabolic steps down from the n6-stage. Other interesting correlations were those of AKR1C2 and FABP4 with parameters of adiposity or lipid metabolism, respectively. It has already been reported that the level of the mRNA AKR1C2 expression in adipose tissue of females positively correlates with adiposity.40 FABP4 inversely correlates with total plasma and LDL-cholesterol levels, and at lower significance with the plasma triglyceride level. It has been indicated that the cholesterol level in adipocytes is linked to their metabolic activity, such as fatty acid uptake. Therefore, a significantly decreased cholesterol supply from the bloodstream, especially of LDL-cholesterol, may require adaptive upregulation of FABP4 to maintain a normal fatty acid uptake function of adipocytes.41,42 In short, after 5 weeks of a very low calorie diet followed by 3 weeks of a normal diet, changes in the adipocyte-enriched proteome can be detected. Those changes suggest a reduced intracellular scaffolding of GLUT4 (ALDOC, TUBB5, ANXA2), an improved inflammatory status (ApoA1, AOP1), a higher uptake of fatty acids (FABP4), a change in fat droplet organization (vimentin), and a correlation between plasma FFA-level and fatty acid oxidation (HADH, ACADS, ACAT1). Additional studies can now be initiated to confirm and deepen the role of specific proteins and their molecular pathways indicated by the present results. Overall, our results underscore the potential of in vivo proteomics to provide insight in physiologic effects of human intervention studies. In the present study, we successfully analyzed subcutaneous adipose tissue. The same analyses can now be applied to visceral adipose tissue, which is less accessible but not less relevant in the context of weight regulation. Abbreviations: BMI, body mass index; BW, body weight; VLCD, very low calorie diet; KRBH, Krebs Ringer bicarbonate buffer supplemented with HEPES; NFDM, nonfat dry milk power; 2D-GE, 2D-gel electrophoresis; ALDH6A1, methylmalonate semialdehyde dehydrogenase; TUBB5, tubulin beta 5; ApoA1, apolipoprotein A1; ALDOC, fructose-bisphosphate aldolase C; ANXA2, annexin A2; ACADS, short chain specific acylCoA dehydrogenase; HADH, short chain 3-hydroxyacyl-CoA dehydrogenase; ACAT1, acetyl-CoA acetyltransferase; AOP1, antioxidant protein 1; GLUT4, glucose transporter 4; AKR1C2, aldo-keto reductase family 1 member C2; FABP4, fatty acid binding protein 4. Acknowledgment. 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