Role of Insulin Action and Cell Size on Protein Expression Patterns in Adipocytes
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m404570200
ISSN1083-351X
AutoresMatthias Blüher, Leanne Wilson-Fritch, John Leszyk, Palle G. Laustsen, Silvia Corvera, C. Ronald Kahn,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoMice with a fat-specific insulin receptor knock-out (FIRKO) exhibit a polarization of white adipose tissue into two populations of cells, one small (diameter 100 μm), accompanied by changes in insulin-stimulated glucose uptake, triglyceride synthesis, and lipolysis. To characterize these subclasses of adipocytes, we have used a proteomics approach in which isolated adipocytes from FIRKO and control (IR lox/lox) mice were separated by size, fractionated into cytosolic and membrane subfractions, and analyzed by sucrose gradient, SDS-PAGE, and mass spectrometry. A total of 27 alterations in protein expression at key steps in lipid and energy metabolism could be defined, which were coordinately regulated by adipocyte cell size, impaired insulin signaling, or both. Nine proteins, including vimentin, EH-domain-containing protein 2, elongation factor 2, glucose-regulated protein 78, transketolase, and succinyl-CoA transferase were primarily affected by presence or absence of insulin signaling, whereas 21 proteins, including myosin non-muscle form A, annexin 2, annexin A6, and Hsp47 were regulated in relation to adipocyte size. Of these 27 alterations in protein expression, 14 changes correlated with altered levels of mRNA, whereas the remaining 13 were the result of changes in protein translation or turnover. These data suggest an intrinsic heterogeneity in adipocytes with differences in protein expression patterns caused by transcriptional and post-transcriptional alterations related to insulin action and cellular lipid accumulation. Mice with a fat-specific insulin receptor knock-out (FIRKO) exhibit a polarization of white adipose tissue into two populations of cells, one small (diameter 100 μm), accompanied by changes in insulin-stimulated glucose uptake, triglyceride synthesis, and lipolysis. To characterize these subclasses of adipocytes, we have used a proteomics approach in which isolated adipocytes from FIRKO and control (IR lox/lox) mice were separated by size, fractionated into cytosolic and membrane subfractions, and analyzed by sucrose gradient, SDS-PAGE, and mass spectrometry. A total of 27 alterations in protein expression at key steps in lipid and energy metabolism could be defined, which were coordinately regulated by adipocyte cell size, impaired insulin signaling, or both. Nine proteins, including vimentin, EH-domain-containing protein 2, elongation factor 2, glucose-regulated protein 78, transketolase, and succinyl-CoA transferase were primarily affected by presence or absence of insulin signaling, whereas 21 proteins, including myosin non-muscle form A, annexin 2, annexin A6, and Hsp47 were regulated in relation to adipocyte size. Of these 27 alterations in protein expression, 14 changes correlated with altered levels of mRNA, whereas the remaining 13 were the result of changes in protein translation or turnover. These data suggest an intrinsic heterogeneity in adipocytes with differences in protein expression patterns caused by transcriptional and post-transcriptional alterations related to insulin action and cellular lipid accumulation. Adipose tissue plays a central role in the pathogenesis of diabetes and obesity. White adipose tissue provides the primary site of energy storage in the body and also serves as an important endocrine cell through secretion of hormones such as leptin, adiponectin (ACRP30), tumor necrosis factor α, resistin, and other cytokines (1Saltiel A.R. Nat. Med. 2001; 7: 887-888Google Scholar). Brown adipose tissue is the major site of energy expenditure through expression of uncoupling protein 1 and its role in thermogenesis (2Lowell B.B. Spiegelman B.M. Nature. 2000; 404: 652-660Google Scholar). Insulin signaling plays an important role in lipid storage and the process of adipogenesis for both white and brown adipocytes. The loss of insulin action selectively in adipose tissue in mice with a fat-specific insulin receptor knock-out (FIRKO) 1The abbreviations used are: FIRKO, fat-specific insulin receptor knock-out; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-pro-panesulfonic acid; ESI, electrospray ion trap; GRP, glucose-regulated protein; MALDI-TOF, matrix-assisted laser desorption ionization time- of-flight; STAT, signal transducers and activators of transcription. leads to profound changes in adipocyte function, including changes in glucose metabolism, lipid metabolism, and protein expression (3Blüher M. Michael M.D. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Google Scholar). FIRKO mice have reduced fat mass and are protected against age- and diet-related obesity and its associated metabolic abnormalities, including glucose intolerance. In addition, these mice have increased longevity, despite normal or increased food intake (4Blüher M. Kahn B.B. Kahn C.R. Science. 2003; 299: 572-574Google Scholar). Adipose tissue-specific insulin receptor knock-out in FIRKO mice also causes heterogeneity of white adipose tissue with polarization into small (diameter 150 μm) subclasses of adipocytes (3Blüher M. Michael M.D. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Google Scholar). Western blot analysis of candidate molecules reveals changes in the expression of several key adipocyte proteins, such as ACRP30, fatty acid synthase, sterol regulatory element-binding protein (SREBP)-1, CCAAT/enhancer-binding protein α (C/EBP-α), and GLUT1 glucose transporter (3Blüher M. Michael M.D. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Google Scholar), suggesting that knock-out of the insulin receptor unmasks a intrinsic heterogeneity of cells in fat tissue. Analysis of gene expression using oligonucleotide microarrays has demonstrated that this is accompanied by changes in mRNA levels of 111 known cDNAs as well as many unknown expressed sequence tags, some of which are related in insulin signaling alterations and others to differences in adipocyte size (5Blüher M. Patti M.-E. Gesta S. Kahn B.B. Kahn C.R. J. Biol. Chem. 2004; 279: 31891-31901Google Scholar). The recent development of unbiased proteomic analysis using gel electrophoresis and other separation techniques coupled with mass spectrometry, however, now allows the characterization of thousands of proteins from complex samples to reveal previously unrecognized connections between biochemical processes and protein expression patterns (6Aebersold R. Mann M. Nature. 2003; 422: 198-207Google Scholar). In the present study, we have used a multidimensional proteomics approach to test the hypothesis that insulin receptor knock-out in FIRKO mice unmasks a naturally occurring heterogeneity of adipocytes that causes differential lipid storage, resulting in subsets of small and large adipocytes. By comparing these findings with mRNA expression we can define unique and complementary levels of protein and mRNA regulation (7Ideker T. Thorsson V. Ranish J.A. Christmas R. Buhler J. Eng J.K. Bumgarner R. Goodlett D.R. Aebersold R. Hood L. Science. 2001; 292: 929-934Google Scholar). Animals—IR (lox/lox) mice were derived as described previously (8Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Hörsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Google Scholar) and maintained on a mixed (C57BL/6 × 129/Sv) genetic background. Adipose tissue or FIRKO mice were derived by crossing IR lox/ox mice with mice that were heterozygous for the IR and were heterozygous of a transgene expressing the Cre recombinase under the control of the fatty acid-binding protein aP2 promoter/enhancer (aP2-Cre-IR(lox/+)) (3Blüher M. Michael M.D. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Google Scholar). Animals were housed in virus-free facilities on a 12-h light/dark cycle (0700 on to 1900 off) and were fed a standard rodent chow (Mouse Diet 9F, PMI Nutrition International) and allowed water ad libitum. All protocols for animal use and euthanasia were reviewed and approved by the Animal Care Committee of the Joslin Diabetes Center and were in accordance with National Institutes of Health guidelines. Genotyping was performed by PCR using genomic DNA isolated from the tail tip as described previously (3Blüher M. Michael M.D. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Google Scholar). Histology—Tissues were fixed in 10% buffered formalin and imbedded in paraffin. Multiple sections (separated by 70–80 μm each) were obtained from gonadal fat pads and analyzed systematically with respect to adipocyte size and number. Staining of the sections was performed with hematoxylin and eosin. For each genotype and gender at least 10 fields (representing ∼100 adipocytes)/slide were analyzed. Images were acquired using BX60 microscope (Olympus) and an HV-C20 television camera (Hitachi, Japan) and were analyzed using Image-Pro Plus 4.0 software. Isolation of Adipocytes and Separation into Small and Large Adipocytes—Animals were sacrificed, and epididymal fat pads were removed. Adipocytes were isolated by 1 mg/ml collagenase digestion. Separation of cells into small and large adipocytes was achieved by filtering the adipocyte suspension through a 75-μm pore size nylon mesh screen (see Fig. 1). Aliquots of adipocytes were fixed with osmic acid and counted in a Coulter counter (9Cushman S.W. Salans L.B. J. Lipid Res. 1978; 19: 269-273Google Scholar). Adipocyte size was determined by dividing the lipid content of the cell suspension by the cell number (9Cushman S.W. Salans L.B. J. Lipid Res. 1978; 19: 269-273Google Scholar). Cell Fractionation—Isolated small and large adipocytes were layered with 5 ml of dinonylphthalate and centrifuged for 5 min at 500 rpm in Beckman GPKR centrifuge. Adipocytes were removed and washed twice, once in Dulbecco's modified Eagle's medium and once in cytosol buffer (cytosol buffer: 25 mm Hepes, pH 7.0, 125 mm potassium acetate, 2.5 mm magnesium acetate, 0.2 m sucrose, 1 mm ATP, 5 mm creatine phosphate, 1 mm dithiothreitol, 1 mm benzamidine, 4 μg/ml leupeptin, 0.01 mg/ml tosyl-l-arginine methyl ester, 0.2 mm phenylmethylsulfonyl fluoride, 1 mm phenanthrine). Buffer was removed and fresh cytosol buffer added to the cells at a volume half of the cell volume. Adipocytes were homogenized by 10 passes at 500 rpm using a motor-driven Teflon pestle homogenizer (Schuett, Germany). After a 5-min spin at 500 × g, the postnuclear supernatant was centrifuged at 72,000 rpm in a TLA 100.3 rotor (Beckman) for 15 min at 4 °C. The supernatant was removed and saved as the cytosol fraction. The postnuclear pellet was resuspended in 100 μl of cytosol buffer and incubated with 2.5 m KCl to a final concentration of 0.5 m KCl on ice for 10 min. The suspension was centrifuged at 72,000 rpm in a TLA 100.3 rotor (Beckman) for 15 min at 4 °C. The supernatant was removed as the KCl extract. The pellet was solubilized in cytosol buffer supplemented with CHAPS to a final concentration of 6 mm, vortexed, and cytosol buffer added to bring the final concentration of CHAPS to 3 mm. The suspension was vortexed continuously for 10 min, and protein determination was performed using the Bradford assay (Bio-Rad). Equal concentrations of protein were layered on top of a 1.8-ml 10–30% sucrose gradient. The sucrose gradients were centrifuged at 35,000 rpm in a TLS55 rotor (Beckman) for 20 h at 4 °C. Each gradient was fractionated into 10 200-μl fractions from the top and then analyzed on 7.5–15% SDS-polyacrylamide gels. Gels were stained with SYPRO Ruby protein stain (Molecular Probes) and photographed with a charge-coupled device camera after illumination with a 302 nm wavelength Ultra-Lum electronic UV transilluminator. Mass Spectrometry—Proteins bands were excised from the polyacrylamide gel and digested in-gel with trypsin (0.1 mg/ml trypsin (sequence grade, Promega) in 25 mm NH4HCO3, pH 8, for 12–16 h at 37 °C). The hydrolysates were then analyzed by either electrospray ion trap mass spectrometry (ESI) or matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF). To analyze by ESI mass spectrometry, the digests were run out on a LC Packings Ultimate nano high performance liquid chromatography with a 100-μm C18PM column in a solvent A (0.1% formic acid, 3.5% acetonitrile) and solvent B (0.1% formic acid in 70/30 acetonitrile/water). Peptides were eluted with a linear gradient from 100% solvent A to 60% solvent B in 40 min at a flow of 500 nl/min. Peptides were eluted directly into the LCQ Deca ESI ion trap (liquid chromatography/mass spectrometry) mass spectrometer equipped with data-dependent acquisition and a high resolution scan performed. A higher energy tandem mass spectrometry scan was performed following the initial scan to verify peptide identifications. Peptides were searched using the Sequest software developed by John Yates and Jimmy Eng (University of Washington). To analyze by MALDI-TOF, the digested samples were concentrated further and desalted with Millipore Zip Tip C18 microtips. Peptide masses were determined using a Kratos Analytical Axima CFR MALDI-TOF spectrometer equipped with a curved field reflectron. Peptide masses were searched against the nonredundant protein data base using MS-Fit of the Protein Prospector program, a program available from the World Wide Web site of the Mass Spectrometry Facility of the University of California San Francisco. Fragmentation data from individual peptides via post source decay analysis were searched against the nonredundant protein data base using the Protein Prospector program MS-Tag. Immunoprecipitation and Western Blot Analysis—Immunoprecipitations and Western blot analyses were performed on homogenates from isolated small and large adipocytes. Samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, which were blocked with 1% bovine serum albumin in Tris-buffered saline containing 20% Tween, and incubated with the indicated primary antibodies. At least three blots of samples from four (controls) to eight animals (FIRKO) of each genotype were scanned using a Molecular Dynamics Storm PhosphorImager and quantified using ImageQuant version 4.0 software. All values are expressed as mean ± S.E. unless otherwise indicated. Statistical analyses were carried out using two-tailed Student's unpaired t test and between more than two groups by analysis of variance. Significance was rejected at p ≥ 0.05. Microarray Analysis of mRNA Levels—This work is described in detail in the accompanying paper (5Blüher M. Patti M.-E. Gesta S. Kahn B.B. Kahn C.R. J. Biol. Chem. 2004; 279: 31891-31901Google Scholar). In brief, total RNA was isolated from isolated pooled small and large adipocytes from at least eight FIRKO mice and at least four IR lox/lox mice. Double-stranded cDNA synthesis was reverse transcribed from 15 μg of isolated mRNA by using the SuperScript choice system (Invitrogen) in addition to using an oligo(dT) primer containing a T7 RNA polymerase promoter site. Double-stranded cDNA was purified with Phase Lock Gel (Eppendorf). Biotin-labeled cRNA was transcribed by using a BioArray RNA transcript labeling kit (Enzo). A hybridization mixture containing 15 μg of biotinylated cRNA, adjusted for possible carryover of residual total RNA, was prepared and hybridized to mouse Affymetrix MG-U74A-v2 chips. The chips were washed, scanned, and analyzed with GENECHIP MAS version 4.0. For each condition (FIRKO small and large, IR lox/lox small and large) five chips were analyzed. All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies. The background and the scaled noise of each of the chips were averaged. The data analysis was performed as described by Yechoor et al. (10Yechoor V.K. Patti M.E. Saccone R. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10587-10592Google Scholar). Adipocyte Heterogeneity in FIRKO Mice—Adipose tissue-specific insulin receptor knock-out causes heterogeneity of white adipose tissue with a polarization into small (diameter 100 μm) subclasses of adipocytes. Histological sections representative of white adipose tissue in FIRKO and IR lox/lox mice demonstrate a relatively uniform and normally distributed adipocyte size in white adipose tissue from IR lox/lox mice, whereas the fat pads from FIRKO mice contain a mixed population of large and small adipocytes (Fig. 1). We have previously shown that the polarization of adipocytes from FIRKO mice is accompanied by differences between small and large adipocytes in insulin-stimulated glucose uptake, glucose metabolism, triglyceride synthesis, and lipolysis as well as in the expression of candidate proteins of adipocyte differentiation and metabolism (3Blüher M. Michael M.D. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Google Scholar). Insulin receptor expression in both large and small adipocytes of FIRKO mice was reduced by 85–99% compared with the controls, indicating that the heterogeneity in adipocyte size in FIRKO mice was not caused by differences in efficiency of insulin receptor gene recombination in small and large adipocytes. The three littermate control groups: wild type, IR (lox/lox), and aP2-Cre mice were indistinguishable with regard to physiologic and metabolic parameters and possessed the same mixed genetic background as the FIRKO mice, including C57BL/6J, 129Sv, and FVB. Therefore, for all subsequent proteomics and genomics studies, IR lox/lox mice were used as controls. Proteomic Analysis—To characterize protein expression at the whole proteome level further, adipocytes were isolated from epididymal fat pads from 3-month-old male FIRKO and IR lox/lox mice and separated into small ( 75 μm) cells by filtration through nylon mesh screen. Each population of cells was fractionated further into membrane and cytosol fractions, and protein from each was loaded onto 10–30% sucrose gradients, centrifuged for 20 h, and then separate on 7–15% SDS-polyacrylamide gels. Resolved proteins were visualized by SYPRO Ruby protein stain, and differentially expressed proteins bands were excised from the gel, tryptically digested, and analyzed by either sequencing ESI mass spectrometry or peptide mass mapping fingerprinting (MALDI-TOF) as described under "Materials and Methods." A total of three experiments were performed for each cell subfraction. Each experiment consisted of the comparison between FIRKO small and large, and IR lox/lox small and large adipocyte protein extracts in the KCl fraction of ionic bound membrane proteins, the CHAPS fraction of integrated membrane proteins, and the cytosol fraction (Fig. 1). Comparing the protein expression profiles of small and large adipocytes from FIRKO and IR lox/lox mice, 27 differentially expressed proteins were identified (Table I). For 14 of the proteins identified by proteomics, additional Western blot analysis was performed, and in each case this confirmed the protein expression pattern as detected by SDS-PAGE (Figs. 2, 3, 4).Table IComparison between proteomic and genomic analysis of large and small adipocytes of FIRKO and control mice Differences in protein and mRNA levels in large versus small adipocytes from FIRKO and IR lox/lox mice are shown. Up- and down-regulated protein expression was detected by proteomics and quantified by Western blotting. mRNA expression was assessed using either Affymetrix micorarrays or real time PCR (designated by *) or both.Accession no.Protein / geneProteomicsGenomicsKO large/smallIR lox/lox large/smallLarge KO/controlSmall KO/controlKO large/smallIR lox/lox large/smallLarge KO/controlSmall KO/controlP35579MHC, non-muscle A> 20> 200.9Not detectedNAaNA, not applicableNANANAP19096Fatty acid synthase*3.343.751.140.782.992.460.810.67NP_037062CPT 2*2.271.750.840.661.020.931.010.91P04117aP2*3.051.671.110.51.031.040.890.89P11442Clathrin HC4.953.61.331.5NANANANAXP_193625Annexin A61.641.180.90.67NANANANANP_033955HSP470.678.680.950.151.180.620.980.51P19226HSP601.540.950.980.621.361.410.920.95P07356Annexin 21.022.11.042.08NANANANANP_034249EHD-containing protein1.21.050.120.09NANANANANP_031669CD 36*1.721.470.50.271.951.970.780.79P58252Elongation factor 21.071.110.60.670.891.000.951.077NP_035831Vimentin1.090.616.69.51.650.568.843.03P20029GRP780.980.162.2816.61.090.673.151.95NP_031632Carbonic anhydrase 32.92.41.060.810.951.251.011.31NP_032934Cyclophilin A1.622.310.831.190.841.041.021.26NP_059062Acyl-CoA DH*> 3Not changedNot changed< 0.30.960.951.011.00NP_060720Citrate Synthase> 2.5Not changedNot changed< 0.3NANANANANP_031409Acetyl CoA DH> 3Not changedNot changed< 0.51.631.051.000.65Q00519Xanthine DH> 2Not changedNot changed< 0.31.20.881.030.76P00009Cytochrome c> 2Not changedNot changed< 0.51.011.30.961.24NP_005304GRP58> 4Not changedNot changed< 0.3NANANANAS23506Pyruvate DH*Not changed> 3Not changed> 30.811.980.932.3Q60597Succinyl-CoA DH*Not changedNot changed< 0.3< 0.30.720.90.640.79AAG44988MMSD DH*Not changedNot changed< 0.5< 0.5NANANANANP_033786Aldehyde DH 2*Not changedNot changed< 0.3< 0.30.950.930.520.52NP_033414TransketolaseNot changedNot changed< 0.3< 0.30.891.110.510.64a NA, not applicable Open table in a new tab Fig. 3Heterogeneity of protein expression patterns depending on the adipocyte size. Differential protein expression in isolated adipocytes depending on their diameter from 3 month-old male IR (lox/lox) and FIRKO mice is shown. Adipocytes from epididymal fat pads of four IR lox/lox and eight FIRKO mice were isolated by digestion with collagenase I, pooled, and separated into two different subsets of small (IR lox S, FIRKO S) and large (IR lox L, FIRKO L) adipocytes using a nylon mesh of 75-μm pore size. Three patterns of differential protein expression in small and large adipocytes from FIRKO and IR lox/lox mice were observed: decreased levels in small adipocytes of both FIRKO and IR lox/lox mice (carnitine palmitoyl transferase 2, aP2, clathrin heavy chain (HC), fatty acid synthase (FAS)) (A); decreased levels only in small FIRKO adipocytes, with indistinguishable protein levels between small and large IR lox/lox adipocytes (annexin A6, Hsp47, Hsp60) (B); decreased protein levels only in small IR lox/lox adipocytes, with indistinguishable protein expression in small and large FIRKO adipocytes (annexin 2) (C). For each of the three patterns a representative Western blot and quantitative analysis (mean ± S.E.) from at least three independent experiments are shown.View Large Image Figure ViewerDownload (PPT)Fig. 4Differential protein expression in isolated adipocytes in 3-month-old male FIRKO and IR lox/lox mice independently from the adipocyte diameter. Adipocytes from epididymal fat pads of four IR lox/lox and eight FIRKO mice were isolated and fractionated as described in Fig. 3. Two patterns of different protein expression in small and large FIRKO and small and large IR lox/lox mice were observed: decreased protein expression in both small and large FIRKO adipocytes compared with small and large IR lox/lox adipocytes (EH domain-containing protein 2, CD36, elongation factor 2) (A); decreased protein expression in both small and large IR lox/lox adipocytes compared with FIRKO adipocytes in both cell size groups (vimentin, GRP78) (B). For each of the three patterns a representative Western blot and quantitation of the data (mean ± S.E.) from at least three independent experiments are shown.View Large Image Figure ViewerDownload (PPT) Analysis revealed a regulation of protein expression both as a function of adipocyte size, i.e. different protein expression patterns in large and small adipocytes independent from the insulin receptor knock-out in adipocytes from FIRKO mice, as well as a function of impaired insulin signaling, i.e. in FIRKO adipocytes compared with adipocytes from IR lox/lox mice independent of size. A coordinated regulation of protein levels with decreased protein expression in small adipocytes compared with large adipocytes, as well as decreased protein expression in adipocytes from FIRKO mice compared with the controls could be identified for several proteins of lipid metabolism (fatty acid synthase, carnitine palmitoyl transferase 2, acyl-CoA dehydrogenase, aP2, CD36, methylmalonate semialdehyde dehydrogenase), as well as for mitochondrial proteins (cytochrome c, succinyl-CoA transferase, citrate synthase, Hsp60) (Table I). Protein Expression in Function of the Adipocyte Size—Comparison of the gradient fractions from small and large FIRKO and IR lox/lox adipocytes by SDS-PAGE revealed major differences between small and large adipocytes both from FIRKO and IR lox/lox mice (Fig. 2). In three independent experiments analyzing "integral" membrane proteins (the CHAPS extract of membranes), myosin heavy chain, non-muscle form A, appeared only in large adipocytes from both FIRKO and IR lox/lox mice (Fig. 2). This differential expression, with no detectable myosin heavy chain, non-muscle form A protein expression in small adipocytes and easily detectable levels in extracts of large adipocytes in both FIKRO and control mice was confirmed by Western blotting with a specific antibody (Fig. 2). Within the limits of detection and this approach, this was the only protein exclusively detected in one cell type versus the other. Significantly decreased protein expression in small compared with large adipocytes from both FIRKO and IR lox/lox mice was detected for several other proteins and could be categorized into three different patterns (Fig. 3, A–C). The first pattern is characterized by decreased protein expression in small adipocytes of both FIRKO and IR lox/lox mice compared with large adipocytes. This was the case for the expression of the key enzyme in fatty acid synthesis, the fatty acid synthase, carnitine palmitoyl transferase 2 (CPT-2), the fatty acid binding protein aP2, clathrin heavy chain, carbonic anhydrase 3, and cyclophilin A (Fig. 3A). The second pattern represented proteins that were decreased only in small adipocytes of FIRKO mice, but not changed in small adipocytes from IR lox/lox mice. This pattern included annexin A6, Hsp47 and Hsp60, acyl-CoA dehydrogenase, citrate synthase, acetyl-CoA dehydrogenase, xanthine dehydrogenase, cytochrome c, and the glucose-regulated protein (GRP) 58 (Fig. 3B). The third pattern of protein expression was characterized by proteins whose levels were decreased in small adipocytes of IR lox/lox mice but normally expressed in small adipocytes of the FIRKO mouse. This pattern was observed for annexin II and pyruvate dehydrogenase (Fig. 3C). Thus, proteomic analysis demonstrated that key enzymes and other molecules involved in lipid and fatty acid metabolism are commonly decreased in small adipocytes compared with large adipocytes. This could explain, at least in part, our observation that there are differences in triglyceride storage and triglyceride and fatty acid synthesis between large and small adipocytes (3Blüher M. Michael M.D. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Google Scholar). Consequences of Impaired Insulin Signaling on Protein Expression Patterns—To futher elucidate the effect of impaired insulin signaling on different protein expression patterns, we analyzed proteins that might be regulated in response to the insulin receptor knock-out. This includes proteins with either decreased levels in both large and small FIRKO adipocytes (Fig. 4A) or increased expression in both large and small FIRKO adipocytes (Fig. 4B) compared with their respective controls. Decreased protein expression in both FIRKO large and small adipocytes was observed for the fatty acid translocase CD36, the EH domain-containing protein 2, elongation factor 2, succinyl-CoA transferase, and methylmalonate semialdehyde dehydrogenase (Fig. 4A). Conversely, an expression pattern with increased protein levels in both large and small FIRKO adipocytes was identified for vimentin, GRP78, aldehyde dehydrogenase 2, and transketolase (Fig. 4B). It is worth noting that the lack of insulin signaling resulting from knock-out of the insulin receptor in FIRKO adipocytes caused alterations in proteins involved in both adipocyte differentiation and adipocyte metabolism. Differentially regulated proteins that could be associated with the adipocyte differentiation process included proteins involved in cytoskeletal function (vimentin), protein processing (GRP78), and protein synthesis (elongation factor 2). Other proteins with differential expression were related to fatty acid metabolism (CD36, methylmalonate semialdehyde dehydrogenase), glycolysis (transketolase), and other metabolic pathways (aldehyde dehydrogenase 2, succinyl-CoA transferase). Thus, the changes in protein expression pattern suggest that the phenotype of the adipose tissue in FIRKO mice might be the result of both changes in the adipocyte differentiation program and adipocyte metabolism in response to the insulin receptor knock-out. Differentially Regulated Protein Expression Is at Least in Part Confirmed by Microarray Analysis of Gene Expression Patterns—To investigate whether the observed changes in protein expression identified by proteomics were secondary to changes at the mRNA level or the result of post-transcriptional control, protein expression patterns were compared with mRNA levels determined by Affymetrix oligonucleotide microarrays. Some proteins, including the non-muscle form A of myosin heavy chain, clathrin heavy chain, annexin A6, annexin II, EH domain-containing
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