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J. Biol. Chem., Vol. 279, Issue 30, 31902-31909, July 23, 2004
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¶
From the
Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 and the Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received for publication, April 26, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, resistin, and other cytokines (1). Brown adipose tissue is the major site of energy expenditure through expression of uncoupling protein 1 and its role in thermogenesis (2).
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)1 leads to profound changes in adipocyte function, including changes in glucose metabolism, lipid metabolism, and protein expression (3). 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 (4). Adipose tissue-specific insulin receptor knock-out in FIRKO mice also causes heterogeneity of white adipose tissue with polarization into small (diameter <50 µm) and large (diameter >150 µm) subclasses of adipocytes (3). 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 (C/EBP-), and GLUT1 glucose transporter (3), 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 (5). 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 (6).
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 (7).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 (9). Adipocyte size was determined by dividing the lipid content of the cell suspension by the cell number (9).
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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 (5). 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. (10).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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) and large (>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).
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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 (3).
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 protein 2, citrate synthase, methylmalonate semialdehyde dehydrogenase, and GRP58, were not present on the array, and therefore real time PCR analysis was performed to obtain corresponding mRNA expression profiles for these proteins (Table I). Fatty acid synthase, pyruvate dehydrogenase, xanthine dehydrogenase, acetyl-CoA dehydrogenase, succinyl-CoA dehydrogenase, aldehyde dehydrogenase 2, transketolase, GRP78, vimentin, and CD36 were all detectable on the microarray, and in every case, the change in proteins was accompanied by a parallel change in mRNA expression (Table I). However, the magnitude of the mRNA expression changes was in general lower than that in protein expression, suggesting that post-transcriptional modulation might contribute additionally to the expression changes observed by proteomics. The remaining nine proteins (carnitine palmitoyl transferase 2, aP2, Hsp47, Hsp60, elongation factor 2, carbonic anhydrase 3, cyclophilin A, acyl-CoA dehydrogenase, cytochrome c) were also represented on the array; however, no differences in the mRNA expression could be identified for these, indicating that the change in protein level was due primarily or exclusively to post-transcriptional events.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (3). These alterations in the protein expression pattern could contribute to the heterogeneity of adipocytes and the beneficial effects of the insulin receptor knock-out in adipose tissue on whole body glucose homeostasis. Heterogeneity in adipocyte cell size is also observed in mice with a deficiency of the enzyme hormone-sensitive lipase (12, 13) and mice with a genetic knock-out of perilipin (14). Based on these and other observations, we have postulated that adipose-specific disruption of the insulin receptor unmasks a naturally occurring heterogeneity of adipocytes with differential protein and gene expression patterns and different roles in lipid storage and other functions.
In this study we have used a multidimensional proteomics approach to detect differentially expressed proteins in isolated adipocyte samples that might play a role in the regulation of adipocyte biology and contribute to the heterogeneity of adipose tissue in FIRKO and normal mice. We find that protein expression patterns are regulated in adipocytes, both as a function of cell size and as a function of impaired insulin signaling. These two categories of changes may define adipocyte subclasses and suggest that insulin resistance in adipocytes may cause profound changes in protein expression profiles. Furthermore, although only 25% of adipocytes from control mice versus 50% of FIRKO adipocytes are smaller than 75 µm (3), the data of the present study show changes in protein expression in small adipocytes, which in some cases are present in both genotypes, whereas in others affect adipocytes of only the knock-out or control genotype. Thus, although small adipocytes could simply represent the natural precursors of large adipocytes (and some almost certainly do), the patterns of differential protein expression suggest that this is not strictly the case.
The only protein that was detected exclusively in large adipocytes of both genotypes in this study was the non-muscle myosin heavy chain, form A. Mutations of MYH9, the gene for the non-muscle myosin heavy chain causes the Fechtner syndrome, a rare inherited condition characterized by progressive nephritis, macrothrombocytopenia, Dohle-like leukocyte inclusions, deafness, and cataract (15). Although the exact role of non-muscle myosin heavy chain in adipocyte biology is not known, this protein might be involved in the regulation of the integrity of cytoskeleton and cell shape (16), such that the lack of non-muscle myosin heavy chain could contribute to the smaller cell size. Recently, Bose et al. (17) have also suggested a role for non-muscle myosin, Myo1c, in glucose transport regulation. Myo1c binds to actin filaments, which are associated with actin-based tubulovesicular membranes containing GLUT4. These investigators also showed that decreased expression of endogenous Myo1c inhibits insulin-stimulated glucose uptake via a phosphatidylinositol 3-kinase-independent insulin signaling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane (17). In the current study, reduced levels of the non-muscle type A form of myosin are found in the small adipocytes, suggesting that the absence of this form of myosin correlates with insulin sensitivity rather than insulin resistance. Clearly defining the role of each of these forms of non-muscle myosin in adipocytes will require further study.
We further demonstrate a coordinated down-regulation of enzymes of 
-oxidation (acyl-CoA dehydrogenase, acetyl-CoA dehydrogenase, carnitine palmitoyl transferase 2) and fatty acid synthesis (fatty acid synthase) in small adipocytes compared with large adipocytes. The common down-regulation of these enzymes of fatty acid metabolism was confirmed by parallel mRNA expression changes as determined by microarray analysis. These protein expression changes are in agreement with functional analysis of these same populations, which shows decreased triglyceride storage in small adipocytes (3). Whether these protein expression changes are intrinsic to small adipocytes or secondary effects of an impaired substrate uptake needs to be further elucidated.
In contrast to our previous studies using whole cell lysates, which demonstrated no difference in the expression of aP2 protein in small adipocytes (3), the current study finds that this fatty acid-binding protein is decreased in small adipocytes in both genotypes, but only in the membrane fraction. Thus, defining the specific compartmentalization of the protein adds another dimension to the proteomic analysis. Uysal et al. (18) have shown that genetically obese mice, which lack aP2, have improved lipid and glucose metabolism. Thus, decreased membrane-associated aP2 protein in small adipocytes might contribute to the improved lipid and glucose metabolism in these cells compared with the population of larger adipocytes (3).
A general down-regulation of energy metabolism in small adipocytes is suggested further by the finding that cytochrome c, citrate synthase, pyruvate dehydrogenase, xanthine dehydrogenase protein levels are all decreased at the protein level in small adipocytes. Clathrin protein expression was also decreased in small adipocytes from FIRKO and control mice. This is may also be important in the metabolic phenotype because clathrin has been shown to be involved in intracellular vesicle transport including GLUT4 (19) and insulin receptor (20) endocytosis. Furthermore, subcellular distribution of clathrin has been shown to be regulated by insulin (21).
Besides these proteins that are involved in adipocyte metabolism, we found differential expression of several proteins not previously related specifically to adipocyte function. Annexin II and cyclophilin A modulate the transport of cholesterol ester from caveolae to internal membranes as part of a caveolinannexin II lipid-protein complex (22). Both annexin II and cyclophilin A proteins levels were decreased in small adipocytes, and this could contribute to a decreased substrate availability in small adipocytes. Carbonic anhydrase 3, which is rich in skeletal muscle and adipocytes, was decreased at the protein level in small adipocytes. Carbonic anhydrase 3 has been shown to be the most abundant protein in rat adipocytes (23). The observation that the concentration of this enzyme decreases in the Zucker fatty rat with obesity and insulin resistance (23) is contrary to our finding that large adipocytes have higher levels of carbonic anhydrase 3 but are more insulin-resistant than small adipocytes (3). Clearly insulin sensitivity is determined by many factors, and the specific role of carbonic anhydrase 3 needs to be determined. Protein levels of GRP58, which is involved in the STAT signal transduction in response to growth factors and cytokines (24), are also lower in small FIRKO adipocytes. It is possible that this produces alterations in growth factor and cytokine signaling, which contribute to these differences heterogeneity in adipocyte size. Whether the lower levels of carnitine anhydrase 3, annexin II, and cyclophilin A protein expression represent intrinsic differences contributing to adipocyte heterogeneity or are secondary to adipocyte heterogeneity needs to be further investigated.
In addition to the differences in protein expression dependent on adipocyte size, our data also indicate differences in protein expression because of a loss of insulin signaling in adipocytes from FIRKO mice. The major changes detected in these cells which lack insulin signaling were decreases in levels of several proteins involved in lipid and energy metabolism, including the fatty acid translocase CD36, succinyl-CoA transferase, and methylmalonate semialdehyde dehydrogenase. This suggests that the expression of these proteins is directly regulated by insulin, and this is confirmed by the finding of parallel regulation at the mRNA level. The finding that elongation factor 2 protein is also decreased in both small and large FIRKO adipocytes is in accordance with the observation that insulin rapidly induces the biosynthesis of elongation factor 2 (25). EH domain-containing protein 2 is also decreased in FIRKO fat cells. This protein is known to be involved in membrane dynamics and growth factor signaling and could therefore play a role in adipocyte differentiation processes (25, 26).
An opposite pattern with increased protein expression in FIRKO adipocytes was observed for vimentin, GRP78 (Hsp70), aldehyde dehydrogenase 2, and transketolase, suggesting that insulin negatively regulates the expression of these proteins. Vimentin has been considered an early marker of adipogenesis because vimentin regulates lipid droplet content during differentiation (26, 27). Moreover, vimentin was identified by mass spectrometry in intracellular GLUT4-enriched membranes, suggesting a role for this protein in glucose transport (28). Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide causes dispersion of perinuclear GLUT4 to peripheral regions of the cells (28). Thus, the increased vimentin in FIRKO cells could contribute to altered trafficking of glucose transporters (3), leading to heterogeneity of adipocyte size in FIRKO mice. GRP78 belongs to the Hsp70 family and is expressed during early organogenesis (29). Moreover, although no direct insulin effect could be detected, GRP78 expression has been shown to be regulated similarly to GLUT1 in response to stress (30), and in our previous studies Western blotting for candidate proteins, we showed that GLUT1 protein levels were decreased markedly in both large and small FIRKO adipocytes compared with controls (3).
In conclusion, the knock-out of the insulin receptor in fat appears to unmask an intrinsic heterogeneity in adipocytes accompanied by changes in cell size and mRNA and protein expression patterns. Using a proteomics approach, we have identified multiple differentially expressed proteins that participate in these alterations, including proteins that are involved in the regulation of adipocyte differentiation, triglyceride storage, and adipocyte metabolism. Approximately half of the changes observed at the protein level would have been undetected by mRNA analysis, and others were detected only by fraction of cells into specific compartments. These protein alterations provide insight into the nature of adipocyte heterogeneity and could represent potential novel therapeutic targets for modulation of adipose mass and the treatment or prevention of obesity.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2635; Fax: 617-732-2487; E-mail: c.ronald.kahn{at}joslin.harvard.edu.
1 The 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.
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ACKNOWLEDGMENTS |
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