Six lean and six obese healthy volunteers were studied. Their clinical characteristics are shown in . None of the participants had a family history of diabetes or other endocrine disorders or were taking medications. Their body weights were stable for at least 2 months before the biopsies. Compared with the nonobese volunteers, the obese volunteers were heavier (93.4 vs. 77.4 kg; P < 0.03) and had more body fat (40.7 vs. 19.9 kg; P = 0.004) but had the same fat-free mass (57.6 vs. 57.6 kg) and were insulin resistant (1/homeostasis model assessment 0.44 vs. 0.29; P < 0.05). Informed written consent was obtained from all subjects after explanation of the nature, purpose, and potential risks of these studies. The study protocol was approved by the institutional review board of Temple University Hospital.
The subjects were admitted to the Temple University Hospital Clinical Research Center on the day before the studies. At ~8:00 a.m.
on the day after admission, a venous blood sample was obtained and an open fat biopsy was performed by a surgeon. Fat biopsies were obtained from the lateral aspect of the upper thigh (~15 cm above the patella) under local anesthesia, as described (4
). The excised fat was dropped immediately into isopentane and kept at its freezing point (−160°C) by liquid nitrogen. The frozen fat was stored at −80°C until analyzed.
Sample preparation for two-dimensional gel analysis.
Frozen adipose tissue from six nonobese and six obese nondiabetic volunteers was individually processed by grinding with a mortar and pestle cooled with liquid nitrogen. Frozen powders from each individual tissue block were thawed by adding 0.8 ml of cold lysis buffer (7 mol/l urea, 2 mol/l thiourea, 4% CHAPS, 40 mmol/l Tris, and 60 mmol/l DTT) and sonicated in an ice bath for ~1 min (Sonic Dismembrator; Fisher Scientific). Sonicated solutions were centrifuged at 10,000g for 12 min at 4°C. Supernatants were collected and acetone added to precipitate proteins. Precipitated proteins were resolubilized using the above lysis buffer. Protein concentrations in the extracts were measured in triplicate using a Bio-Rad Bradford-based protein assay with BSA as the standard.
Two-dimensional gel electrophoresis.
Adipose tissue lysates were processed individually (six from each group of subjects). For the first-dimension separation, 50 μg of sample protein was diluted in 125 μl of rehydration buffer and loaded onto an immobilized pH gradient (IPG) strip by overnight passive in-gel rehydration. A global view of the proteome was obtained initially using IPG strips of pI 3–10 (data not shown). To enhance resolution and sensitivity, narrow-range IPG strips (pI 4–7 and 6–10) were subsequently used. The rehydration buffer contained 8 mol/l urea, 2% CHAPS, 0.2% carrier ampholytes, and 10 mmol/l DTT for pI 4–7 linear and pI 3–10 nonlinear IPG strips. The pI 6–10 IPG strips were rehydrated with the rehydration buffer containing 15 mg/ml Destreak reagent substitute for 10 mmol/l DTT. Isoelectric focusing within the strips was performed at 20°C with a Ettan IPGphor system Amersham (Piscataway, NJ) using a total of 12,000 V/h with a maximum of 8,000 V.
For two-dimensional separation, the IPG strips were soaked for 15 min in 10 ml of equilibration buffer (6 mol/l urea, 30% glycerol, 2% SDS, 1% DTT, and 0.05 mol/l Tris, pH 8.8) followed by 15 min in 10 ml of a second equilibration buffer (with 2.5% iodoacetamide substituted for 1% DTT) and positioned against 10–14% SDS polyacrylamide gels in a Bio-Rad Mini-PROTEAN 3 System at 200 V for 45 min. Polyacrylamide gels were then fixed twice using 50% methanol, 7% acetic acid, and balance water. The resolved protein spots in the gels were visualized with Sypro-Ruby fluorescence total protein stain.
Image analysis of two-dimensional gels.
Fluorescence images of individual gels from the 12 adipose tissue lysates were captured with a FLA-5000 Fluor Imager (Fuji Photo Film, Tokyo, Japan) and analyzed using PDQuest software (version 8.0). After automatic detection of spots by PDQuest software, the files were also inspected manually to assess accuracy of computer-generated images. The software calculated individual spot “volumes” in each gel by density/area integration. To control for slight differences in protein loading across gels, the spot volume obtained from each individual fat lysate was automatically calculated by image analysis software and normalized to total spot volume on that gel.
In-gel trypsin digestion.
Differentially expressed spots were excised using an Xcise automated robotic system (Shimadzu Biotech, Columbia, MD). Destaining of excised gel pieces was performed by two 30-min washes with 50% acetonitrile containing 50 mmol/l ammonium bicarbonate. Following dehydration with 100% acetonitrile, 10 μl of 12.5 ng/μl sequencing grade trypsin (Promega, Madison, WI) was added to the gel pieces and incubated overnight at 37°C. Resulting tryptic peptides were extracted twice with 15 μl (5% formic acid, 50% acetonitrile, and balance water) for 20 min, and the pooled extracts were desalted with ZipTips C18 (Millipore, Billerica, MA).
The desalted peptides from each spot were mixed 1:1 with matrix solution (1% α-cyano-4-hydroxy cinnamic acid in 50% acetonitrile and 50% 0.1% trifluoroacetic acid), and 1.0-μl volumes were applied to wells of an AnchorChip sample target plate used for the Bruker Auto-flex MALDI-TOF/TOF instrument. Peptide mass fingerprints were obtained using the reflective and positive ion mode. Mass spectra were collected from the sum of 100–400 laser shots, and monoisotopic peaks were generated by FlexAnalysis software with signal-to-noise ratio of two to one. Mass peak value calculations used two trypsin autodigestion peptides with M + H values 842.509 and 2211.104 as internal standards. Proteins were identified by matching the calibrated peptide mass values within Swiss-Prot protein database for homosapiens using an in-house version of Mascot Server 2.1 imbedded in Bruker's Biotool software. Match variances allowed were a mass tolerance of 50 ppm, one missed trypsin cleavage, fixed modification of carbamidomethyl cysteine, and variable modification of methionine oxidation. For the samples that did not produce a “hit” with a confident score, peptide peaks with good signal were further fragmented using laser-induced decomposition to obtain LIFT-TOF/TOF spectra, and these tandem mass spectra data alone or combined with the previously produced mass spectra data were used to search against the protein database through the Mascot.
Western blot analysis.
Proteins (30–80 μg) from the same adipose tissue lysates as used for the two-dimensional gels were separated by 10–14% gradient SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane in a semidry blotting chamber according to the manufacturer's protocol (Bio-Rad, Hercules, CA).
Blots were blocked with 5% milk in Tris-buffered saline solution (pH 7.6) containing 0.05% Tween-20 and probed with the following rabbit anti-human antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) at a concentration of 0.4 μg/ml: protein disulfide isomerase A3 (PDI) (SC-20132), calreticulin (CRT) (SC-11398), and calnexin (CNX) (SC-11397). In addition, a rabbit antiserum that detects phospho c-jun NH2-terminal kinase (JNK)-1, -2, and -3 (Cell Signaling Technology, Danvers, MA) and a rabbit antiserum that detects total JNK were used. Blots were incubated with primary antibody overnight at 4°C with gentle shaking and then incubated with a mouse anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:10,000) (Biomeda, Foster City, CA) for 1 h at room temperature. Blots were exposed using a chemiluminescent detection method (Enhanced ECL Detection System; GE Healthcare BioSciences, Piscataway, NJ).
Total RNAs were isolated from frozen adipose tissues, and real-time RT-PCR was performed with a SYBR Green One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA) and a Light-Cycler (Roche, Indianapolis, IN). Primers for X-box binding protein (XBP)-1s (NM-005080) were sense TTGAGAACCAGGAGTTAAG and antisense CTGCACCTGCTGCGGACT.
Two-dimensional gel analysis was performed by PDQuest software (Bio-Rad), version 8.0. Each gel from obese and lean fat samples was enumerated and analyzed for spot detection, background subtraction, and protein spot volume quantification. Of six samples per group, the gel image showing the highest quality of spots and the best protein pattern was chosen as a reference template, and spots in a reference gel were then matched across all gels. Manual corrections were performed to validate the matches automatically generated by the software. Spot volume values were normalized in each gel by dividing the raw quantity of each spot by the total volume of all the spots included in the same gel. For each protein spot, the average spot volume values and its SD in each group were determined. The match spots were subjected to Student's t test in order to determine the spots that were differentially expressed. Only those spots that show a statistically significant difference with a confidence level of 0.05 were chosen for identification. To test for intergel reproducibility, two-dimensional gel analysis was performed in triplicate using one representative sample. Equal amounts of high-quality protein spots (345 for pI 4–7) were detected. The average variance (coefficient of variation) of the normalized spot volume was determined to be 19.25%.