Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots

Yazmín Macotela; Brice Emanuelli; Marcelo A. Mori; Stephane Gesta; Tim J. Schulz; Yu-Hua Tseng; C. Ronald Kahn

doi:10.2337/db11-1753

Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots

Macotela, Yazmín; Emanuelli, Brice; Mori, Marcelo A.; Gesta, Stephane; Schulz, Tim J.; Tseng, Yu-Hua; Kahn, C. Ronald 2012-06-15 00:00:00 ORIGINAL ARTICLE Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots Yazmín Macotela, Brice Emanuelli, Marcelo A. Mori, Stephane Gesta, Tim J. Schulz, Yu-Hua Tseng, and C. Ronald Kahn expression have also been observed in human pre- Obesity and body fat distribution are important risk factors for adipocytes from different depots (8), but exactly how this the development of type 2 diabetes and metabolic syndrome. relates to intrinsic differences in adipose tissues and the Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. In propensity for obesity and insulin resistance is unclear. the current study, we demonstrate that adipocyte precursor cells Spalding et al. (9) recently demonstrated that in humans, (APCs) isolated from visceral and subcutaneous white adipose ;10% of the adipocyte pool turns over annually and that depots of mice have distinct patterns of gene expression, the absolute number of adipocytes turning over in indi- differentiation potential, and response to environmental and viduals with obesity is approximately double from that in genetic inﬂuences. APCs derived from subcutaneous fat differ- lean individuals. Exactly how adipocytes in different de- entiate well in the presence of classical induction cocktail, pots turn over and to what extent this is inﬂuenced by whereas those from visceral fat differentiate poorly but can be genetic or environmental factors is unknown. However, induced to differentiate by addition of bone morphogenetic using lineage tracing and ﬂuorescence-activated cell protein (BMP)-2 or BMP-4. This difference correlates with major differences in gene expression signature between subcutaneous sorting (FACS) strategies, two groups (10,11) have devel- and visceral APCs. The number of APCs is higher in obesity- oped techniques to identify and isolate the adipocyte pre- prone C57BL/6 mice than obesity-resistant 129 mice, and the cursor cells (APCs) from the stromovascular fraction number in both depots is increased by up to 270% by exposure of (SVF) of white fat. In the current study, we show that APCs mice to high-fat diet. Thus, APCs from visceral and subcutaneous are dynamic and respond to different environmental and depots are dynamic populations, which have intrinsic differences genetic factors. We show that APCs from Vis versus SC fat in gene expression, differentiation properties, and responses to differ in their speciﬁc gene expression signatures and that environmental/genetic factors. Regulation of these populations may SC APCs are more adipogenic and require fewer growth provide a new target for the treatment and prevention of obesity factors, whereas Vis APCs have anti-adipogenic charac- and its metabolic complications. Diabetes 61:1691–1699, 2012 teristics and require additional growth factor stimulation to differentiate. These results show that there are intrinsic differences between preadipocytes from different depots here are two main types of white adipose tissue and that these differences contribute to the differential (WAT) in humans and rodents—subcutaneous properties and turnover of adipocytes in different depots. (SC) fat and visceral (intra-abdominal [Vis]) fat. TThese contribute differentially to disease risk. RESEARCH DESIGN AND METHODS Accumulation of Vis adipose tissue is associated with ad- Mice. C57BL/6 mice and 129 mice were obtained from The Jackson Laboratory verse metabolic outcomes, whereas increased amounts of and kept under a normal diurnal cycle in a temperature-controlled room. Mice SC fat has been viewed as neutral or even beneﬁcial in its were fed with standard chow containing 22% of calories from fat (Mouse Diet 9F metabolic effects (1–3). While part of this difference may 5020; PharmaServ) or a 60% high-fat diet (HFD) (OpenSource Diet D12492; Research Diets). For the HFD versus chow diet study, two cohorts of mice were be related to anatomical location with different patterns of used starting at aged 3 and 8 weeks. Animal care and study protocols were venous drainage, over the past few years, it has become approved by the animal care committee of Joslin Diabetes Center and were in clear that these two types of WAT differ in their intrinsic accordance with National Institutes of Health guidelines. characteristics, including levels of adipokine secretion, Adipocyte precursor isolation and ﬂow cytometry. Epididymal and ingui- insulin sensitivity, lipolysis rate, and tendency to develop nal fat pads, representing Vis and SC fat, respectively, were cut in small pieces and inﬂammation (4). We and others (5–7) recently have shown incubated with 1 mg/mL collagenase I for 30 min. The cell suspension was ﬁltered through a 150-mmol/L nylon mesh, and the SVF was isolated by low-speed that this correlates with differences in gene expression be- centrifugation. For FACS analysis, erythrocyte-free SVF cells were incubated tween adipocytes in different depots, including the expres- with a mix of antibodies against different surface markers as described previ- sion of fundamental development and patterning genes. ously (10) and sorted using an Aria ﬂow cytometer (BD Biosciences). Dead cells Differences in differentiation and developmental gene were removed using propidium iodide staining. Cells negative for Ter119, CD45, and CD31 and positive for both SCA1 and CD34 were considered as APCs. Cell culture and differentiation of APCs. Cells were grown as described previously (12) with some modiﬁcations. Medium containing 60% Dulbecco’s From the Joslin Diabetes Center, Harvard Medical School, Boston, Massachu- modiﬁed Eagle’s medium–low glucose (Invitrogen) and 40% MCDB201 (Sigma- setts. Corresponding author: C. Ronald Kahn, [email protected]. Aldrich) and supplemented with Normocin 0.1 mg/mL (InvivoGen), 10% FBS, Received 19 December 2011 and accepted 5 March 2012. 13 insulin-transferrin-selenium mix, 13 linoleic acid conjugated to BSA, DOI: 10.2337/db11-1753 1 nmol/L dexamethasone, and 0.1 mmol/L L-ascorbic acid 2-phosphate (Sigma- This article contains Supplementary Data online at http://diabetes Aldrich) was used as growth medium. This medium was further supplemented .diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1753/-/DC1. with 10 ng/mL epidermal growth factor (PeproTech), 10 ng/mL leukemia inhibitory Y.M. is currently afﬁliated with the Instituto de Neurobiología, Universidad factor (Millipore), 10 ng/mL platelet-derived growth factor BB (PeproTech), Nacional Autónoma de México, Querétaro, Mexico. and 5 ng/mL basic ﬁbroblast growth factor (Sigma-Aldrich). Once sorted, cells 2012 by the American Diabetes Association. Readers may use this article as were rinsed with medium and plated at 50,000 cells per well in 24-well plates. long as the work is properly cited, the use is educational and not for proﬁt, After 6–7 days, cells reached ;80% conﬂuence. Medium was changed every and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. other day. For differentiation, cells were seeded at 20,000 cells per well in diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1691 ADIPOCYTE PRECURSOR CELL SUBTYPES 48-well plates. After cells reached 80% conﬂuence, they were treated or not for including rosiglitazone, and differentiation was assessed 2 days with 3.3 nmol/L BMP-2 or BMP-4 (R&D Systems) in growth medium by lipid accumulation and expression of markers of ter- with 2% FBS and without growth factors. Medium was then replaced by dif- minal differentiation, including Slc2a4 (Glut-4), Adipoq ferentiation medium (growth medium with no growth factors but with 2% FBS, 1 (adiponectin), Lep (leptin), and Fabp4 (fatty acid binding mmol/L dexamethasone, 0.5 mmol/L isobutylmethylxanthine, 100 nmol/L in- protein or aP2). Using this approach, APCs from the SC fat sulin, and 1 mmol/L rosiglitazone) for 3 days, after which the medium was replaced with growth medium containing 2% FBS and 100 nmol/L insulin for 2 differentiated well within 7 days after induction, with .90% more days; for the last 2–3 days of differentiation, cells were incubated with of the cells containing large lipid droplets (Fig. 2A). In fact, growth medium with 2% FBS alone. ;10% of SC APCs differentiated in the absence of any RNA extraction and PCR. RNA was extracted from cells using RNeasy hormonal induction (Fig. 2A). By contrast, with the same (QIAGEN). Reverse transcription was performed with 0.3 mg RNA by using High protocol, ,20% of APCs from the Vis depot could be in- Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). duced to differentiate (Fig. 2A). This raised two possibili- Real-time PCR was performed using Maxima SYBR Green (Fermentas, Glen Burnie, MD) in duplicate using the ABI Prism 7900 System (Applied Biosystems) ties: one was that the Vis APCs were not really adipocyte under the following conditions: 50°C 22min, 95°C 210 min, and 40 cycles of 95°C precursors; the other was that the APCs were missing some 215 s, 60°C 220 s, and 72°C 230 s. Dissociation protocols were conducted after signals required for differentiation. every run to check for primer speciﬁcity. To obtain relative expression values, BMPs are known to induce commitment of mesenchymal 2DCt we calculated the 2 parameter for each individual sample using cycle stem cells into adipocytes, muscle, and bone (13–15). We threshold values of TATA-box-binding protein as an endogenous control. hypothesized that perhaps BMP signaling was lacking in the Microarray. Approximately 1 3 10 sorted cells per sample were used to isolate RNA for the microarray analysis. Five independent RNA samples each APCs from the Vis depot. We therefore pretreated subcon- from a pool of isolated APCs from 10 different mice from each depot were ﬂuent Vis and SC APCs with 3.3 nmol/L BMP-4 for 2 days analyzed on Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). All prior to the addition of the induction cocktail (IC) and then data were subjected to global normalization to an intensity of 1,500 using the assessed differentiation. With the SC APCs, BMP-4 pre- Gene Chip Software MAS V. 5.0. All differences reported had a P value ,0.05. treatment did not further enhance the almost complete dif- ferentiation achieved by the IC alone (Fig. 2A). However, RESULTS with the Vis APCs, BMP-4 pretreatment markedly enhanced Isolation of APCs from different WAT depots. APCs differentiation, resulting in almost 90% of cells showing typ- were isolated by FACS from SVFs of Vis and SC fat from ical lipid droplets (Fig. 2A). Pretreatment with BMP-2 in- mice as described above and in Supplementary Fig. 1. duced Vis APCs to differentiate at a similar extent as BMP-4 Cells negative for CD45, CD31, and Ter119 and positive for (Fig. 2B) but had no further effect on SC APCs (not shown). CD34 and SCA1 were considered APCs. Cells positive for These differences in differentiation potential were con- CD45, CD31, and/or Ter119 (endothelial cells, platelets, ﬁrmed by quantitative PCR analysis for expression of ter- macrophages, white blood cells, osteoclasts, and eryth- minal adipocyte differentiation genes. SC APCs 7–8 days rocytes) were designated as “other SVF.” Only live cells were after treatment with standard IC with or without BMP-4 counted. In a typical sorting, using male C57BL/6 mice aged treatment showed marked increases in expression of Fabp4 8 weeks, Vis fat contained 0.9 6 0.1 3 10 APCs and 3.5 6 (730-fold), Slc2a4 (300-fold), Adipoq (730-fold), and Lep 0.8 3 10 other SVF cells per fat pad, whereas SC fat con- (20-fold) (Fig. 2C, right three bars, each panel). On the other 5 5 tained 0.65 6 0.08 3 10 APCs and 7.1 6 0.7 3 10 other SVF hand, Vis APCs treated with standard IC showed little ac- cells per pad (Fig. 1A). Thus, the percentage of stromovas- cumulation of lipid and very small increases in expression cular cells, which are APCs, was almost three times higher in of these differentiation markers (Fig. 2C, left two bars, each Vis fat than in SC fat (21 6 3.7 vs. 7.8 6 1.7%) (Fig. 1B). panel). However, when the Vis APCs were pretreated with APCs from different fat depots differ in their BMP-4 followed by IC, the increases in gene expression differentiation requirements. To conﬁrm that the iso- were similar to those seen in SC APCs after differentiation, lated APCs were preadipocytes, we analyzed the differ- with large increases in Fabp4 (480-fold), Slc2a4 (160-fold), entiation ability of both Vis and SC APCs. Conﬂuent APC Adipoq (660-fold), and Lep (21-fold) (Fig. 2C, third bar, each cultures were treated with the typical induction protocol, panel). FIG. 1. Frequency of APCs and other SVF cells in SC vs. Vis depots. Typical FACS sorting analysis of APCs and other SVF cells from SC and Vis 2 2 depots isolated from male mice aged 7–9 weeks. Data were calculated using only live cells. Double positive CD34, SCA1 (and CD45 , CD31 , and 2 + + + Ter119 ) cells were considered APCs, whereas CD45 , CD31 , and Ter119 cells were considered as other SVF cells. A: Number of cells per depot. B: Percent of APCs in both depots relative to other SVF cells. Data are mean 6 SEM of n = 4 independent experiments. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. 1692 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES FIG. 2. Higher differentiation capacity in APCs from SC than from Vis depots. After FACS sorting, APCs from SC and Vis depots were placed in culture; upon 80% conﬂuence, they were treated with or without BMP2 or BMP4 and 2 days later, were induced to differentiate (insulin, iso- butylmethylxanthine, dexamethasone, and rosiglitazone). Seven days after induction of differentiation, they were photographed to evaluate for lipid- containing mature adipocytes (A and B) or lysed, mRNA extracted, and processed for quantitative PCR to evaluate expression changes on adipocyte differentiation genes (C and D). Graphs show fold changes vs. N Vis. N, control without BMPs or IC; BMP4 IC, BMP4 pretreatment followed by IC. Data are mean 6 SEM of n = 2 independent experiments. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. Gene expression analysis also indicated that the block in showed signiﬁcantly higher expression of genes involved differentiation was at an early step involved in the stimu- in positive regulation of adipocyte differentiation, including lation of PPARg. Thus, standard IC increased expression Pparg (3.2-fold), Cepba (1.3-fold), the dickkopf homolog of Cebpa by greater than twofold in APCs from both Vis DKK2 (9.1-fold), Stat5a (2.9-fold), and the BMPs associated and SC fat, and Cebpa was not further increased by BMP-4 with WAT differentiation, Bmp2 (1.5-fold) and Bmp4 (1.5- pretreatment (Fig. 2D). On the other hand, in SC APCs, fold) (all P , 0.05). By contrast, Vis APCs showed increased Pparg increased ﬁvefold after differentiation using standard expression of genes that can act as inhibitors of adipo- IC or BMP-4 plus IC. However, in Vis APCs, Pparg ex- cyte differentiation, including Gata2 (7.4-fold) and Tgfb2 pression was not changed by treatment with IC but was (1.8-fold) (Table 1 and Supplementary Table 1). It is in- increased sixfold when cells were pretreated by BMP-4 plus teresting that APCs from the Vis depot also expressed IC (Fig. 2D). Taken together, these data indicate that the higher levels of markers found in bone marrow mesen- block in differentiation in Vis APCs lies in the inability of chymal cells (17), including Lif, Igf1, Igfbp7, Ctgf, Mgp, Trib2 standard induction hormones to trigger the expression of and Pgn1/Bgn (Table 1). Pparg and induce the transcriptional events required for Depot-speciﬁc gene expression signatures. We (5,6) differentiation and that this block can be released by stim- and others (7,18,19) have previously shown that mature ulation of the BMP pathway. Since C/EBPa and PPARg can adipocytes from Vis and SC fat have different patterns of regulate expression of each other in a positive feedback gene expression, including genes involved in adipocyte manner (16) and since Cebpa is induced by standard IC in function and developmental patterning. Comparing the Vis fat, the blockade appears to be in the ability of C/EBPa results from APCs in the current study with our previous to stimulate a normal increase in PPARg. results in mature adipocytes from mice, we could identify Proadipogenic gene expression signature in SC APCs. depot-speciﬁc signatures. These consisted of 19 genes that To identify possible differences between adipocyte pre- were much more highly expressed in the Vis depot and cursors isolated from SC and Vis depots that might con- 32 genes that were much more highly expressed in the SC tribute to altered differentiation and other properties of depot in both preadipocytes and adipocytes (Table 2). the adipocytes, we performed microarray analysis on Gene set enrichment analysis of the microarray data freshly isolated SC and Vis APCs from 7- to 8-week-old showed that SC APCs have increased expression in three mice. Consistent with their increased ability to differenti- major gene sets when compared with Vis APCs: 1)che- ate, when compared with Vis preadipocytes, SC APCs mokines, 2)peptidases, and 3) proteins involved in the diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1693 ADIPOCYTE PRECURSOR CELL SUBTYPES TABLE 1 as the percentage of total SVF cells in each depot, young Pro- and anti-adipogenic gene expression signatures in Vis and mice had a signiﬁcantly higher percentage of APCs than old SC APCs mice in Vis fat (22 vs. 16%) but a smaller percentage of APCs in SC fat than old mice (3 vs. 13%) (Fig. 3A). Fold P value It is interesting that obesity-prone C57BL/6 mice already Proadipogenic genes higher had almost twofold higher numbers of APCs per gram of in SC APCs fat than 129 mice in both depots by age 3 months. C57BL/6 Cepba 1.3 0.03 mice also had ninefold more other SVF cells per gram of Pparg 3.2 0.0001 fat in the SC depot than 129 mice, whereas the number of Dkk2 9.1 0.0004 other SVF cells in the Vis depot was similar. However, the Stat5a 2.9 0.0001 percentage of APCs did not change between genotypes in Bmp2 1.5 0.001 Vis fat but was higher in the SC fat from 129 mice than in Bmp4 1.5 0.02 the same depot from C57BL/6 mice (17 vs. 4%) (Fig. 3B). Anti-adipogenic genes higher Between males and females, APCs and other SVF cells in Vis APCs per gram of fat or per depot did not differ signiﬁcantly. Gata2 7.4 0.0005 However, male mice did have a twofold higher number of Tgfb2 1.8 0.01 other SVF cells in the Vis depot than female mice. As a re- Bone marrow mesenchymal cell sult, the calculated percentage of APCs in the Vis depot markers higher in Vis APCs (but not in the SC depot) from female mice was almost Lif 2.46 0.039 twice as high as that in males (29 6 3vs. 15 6 2%) (Fig. 3C). Igf1 2.2 0.00008 Effect of HFD on adipocyte precursor frequency in Igfbp7 2.07 0.00004 young versus adult mice. Previous studies in humans Ctgf 4.29 0.003 show that early onset obesity is associated with adipocyte Mgp 11.88 0.003 hyperplasia, whereas late onset of obesity is the result of Trib2 1.75 0.047 adipocyte hypertrophy and hyperplasia; studies in rodents Pgn1/Bgn 1.77 0.000003 show hyperplasia can occur in both young and old animals (20–22). To determine if HFD-induced obesity might alter the number and percentage of APCs and other cells in the vesicular fraction or microsomes (Table 2). In the Vis APCs, the most enriched gene sets fell into four major SVF at different ages, we used two experimental paradigms: one in which the HFD was started just after weaning categories: 1) muscle related, with three related gene sets myoﬁbril, sarcomere, and contractile ﬁber genes (P , 0.02); (3 weeks old) and a second in which mice were started on the HFD as young adults (8–10 weeks old). As shown in 2) monooxygenase activity; 3) developmental; and 4) negative Fig. 4A and B, APC number increased after 2 and 4 weeks regulation of cytokine biosynthesis (Table 3). Genes most differentially expressed between Vis and on HFD in Vis and SC depots in both young and adult mice; however, the increase in APCs was larger when the SC APCs. The most differentially expressed gene, which was highly expressed in Vis APCs, was the transcription mice were started on the HFD at a young age. Thus, Vis APC numbers increased by 80 and 60% after 2 and 4 weeks factor Tcf21, whereas in SC APCs, the most differentially expressed gene was the transcription factor Lhx8 (LIM on HFD in young mice compared with 60 and 30% increases in adult mice. This difference was more marked in SC APCs, homeobox 8). Another interesting gene with signiﬁcantly higher expression in SC versus Vis APCs was Cd24. This with 80 and 270% increases in cell numbers after 2 and 4 weeks on HFD in young mice compared with only 30 membrane protein was previously used as a surface marker (10) to isolate the APCs from a mixture of Vis and SC white and 40% increases in adult mice. By contrast, the change in other SVF cells was greatest in the Vis depot but similar fat depots. It is clear that this would result in preferential isolation of the SC APCs from this mixed fat cell pool. A in young and adult mice, with increases of 210–270% in both young and adult mice on the HFD, whereas the number heat map listing the 100 most differentially expressed genes of other SVF cells in the SC depot increased modestly is shown in Supplementary Fig. 2. Effect of sex, strain, and age on adipocyte precursor (60 and 70% for young and adult mice, respectively). Thus, HFD increases the number of other SVF cells only in the Vis frequency. To evaluate how the number of APCs and other SVF cells change between different genetic and environ- depot, consistent with the increase in inﬂammatory cells in this compartment (23). When the percentage of APCs in the mental conditions, we analyzed APC frequency in young (2-month-old) versus old (1-year-old) mice, in obesity-prone depot was calculated, the percentage of APCs increased by 250 and 180% after 2 and 4 weeks on HFD in young animals (C57BL/6) versus obesity-resistant (129Sv/J) mice, and in male versus female mice. Aging altered dramatically the but was unchanged in adult animals. Thus, young pre- pubertal mice have an increase in APCs on HFD that is number of APCs. When expressed per gram of adipose tis- sue, APC numbers were twofold higher in young than in old greater than that seen in adult animals, especially in the SC depot (Fig. 4). mice in both depots (Fig. 3A). However, because fat mass increases with age, the total number of APCs per depot was increased by more than threefold in Vis and more than DISCUSSION fourfold in SC fat in old versus young mice. The number of non-APCs (i.e., other SVF cells) per gram of fat was eight- Adipose tissue serves as an important regulator of metabolic fold higher in SC fat of young versus old mice but similar in homeostasis, and in this regard, not all fat is functionally both age-groups in the Vis depot (Fig. 3A). However, the equivalent. Thus, accumulation of Vis fat is associated with total number of other SVF cells per fat pad was almost insulin resistance and a high risk for type 2 diabetes and sixfold higher in Vis fat of old versus young mice, while it metabolic syndrome, whereas accumulation of SC fat has remained similar in the SC depot between young and old been shown to have a possible protective value against mice. Therefore, when the number of APCs was calculated these metabolic abnormalities. This appears to relate to 1694 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES TABLE 2 Depot-speciﬁc signature of APCs and adipocytes from Vis and SC fat Fold difference Gene name Gene symbol Adipo Preads Localization Depot-speciﬁc signature—Vis Angiotensinogen Agt 7.5 5.8 Secreted Aldo-keto reductase family 1, member C12 Akr1c12 2.6 6.9 Cytoplasm AMP deaminase 3 Ampd3 2.5 2.8 Cytoplasm Anillin, actin binding protein (scraps homolog, Drosophila) Anln 5.3 2.8 Cytoplasm/Nucleus Cysteine and glycine-rich protein 1 Csrp1 2.6 6.2 Nucleus Cytochrome P450, family 1, subfamily b, polypeptide 1 Cyp1b1 11.0 3.2 ER/Microsomes Hydroxyprostaglandin dehydrogenase 15 (NAD) Hpgd 2.2 2.0 Cytoplasm Nuclear receptor subfamily 2, group F, member 1 Nr2f1 5.5 3.0 TF Nuclear receptor subfamily 4, group A, member 1 Nr4a1 3.2 2.3 TF Pterin 4 a carbinolamine dehydratase (TCF1) 1 Pcbd1 2.5 2.6 TF Protocadherin 7 Pcdh7 2.6 2.1 PM Plakophilin 2 Pkp2 2.9 2.8 PM Prolactin receptor Prlr 3.8 2.9 PM Serum amyloid A3 pseudogene Saa3p 2.6 5.2 Secreted Solute carrier family 8, member 1 Slc8a1 2.3 4.2 PM Secretory leukocyte peptidase inhibitor Slp1 3.1 18.7 Secreted Serine/arginine-rich protein speciﬁc kinase 2 Srpk2 2.2 2.2 Cytoplasm/Nucleus Thrombospondin 1 Thbs1 5.8 3.6 Secreted Transcription factor 21 Tcf21 3.0 216.2 TF Depot-speciﬁc signature—SC Angiopoietin-like 1 Angptl1 3.5 2.1 Secreted Apolipoprotein D Apod 3.8 2.0 Secreted Chemokine (C-C motif) ligand 8 Ccl8 3.4 4.2 Secreted Cadherin 2 Cdh2 2.9 2.5 PM Cadherin 9 Cdh9 2.9 17.1 PM Procollagen, type XII, a 1 Col12a1 3.4 3.4 Secreted Procollagen, type I, a 1 Col1a1 4.7 3.3 Secreted Early B-cell factor 3 Ebf3 2.0 4.1 TF Engrailed 1 En1 8.0 4.5 TF Estrogen receptor 1 (a) Esr1 2.5 3.0 TF Fibroblast growth factor 13 Fgf13 2.1 3.8 Secreted Fibronectin type III domain containing 1 Fndc1 2.9 2.0 Secreted Histocompatibility 2, Q region locus 10 H2q10 4.2 10.8 Membrane Interferon-g–inducible protein 47 Iﬁ47 4.0 2.8 ER/Membrane Interferon-g–induced GTPase Igtp 2.0 2.4 Cytoplasm/Membrane Interleukin-18 receptor 1 Il18r1 16.5 25.0 PM Myelin basic protein Mbp 3.9 9.1 Nucleus/Cytoplasm/IntMem Matrix metallopeptidase 3 Mmp3 3.5 21.5 Secreted Major urinary protein 1 /// major urinary protein 2 Mup1/Mup2 375.1 3.6 Secreted Major urinary protein 3 Mup3 63.5 10.3 Secreted Osteomodulin Omd 3.8 2.4 Secreted Procollagen C-endopeptidase enhancer protein Pcolce 2.8 2.9 Secreted Protein disulﬁde isomerase associated 5 Pdia5 5.9 2.1 ER Serine (or cysteine) peptidase inhibitor, clade F, member 1 Serpinf1 2.9 2.0 Secreted Secreted frizzled-related protein 2 Sfrp2 5.4 2.6 Secreted Short stature homeobox 2 Shox2 8.7 95.4 TF Sine oculis-related homeobox 4 homolog (Drosophila) Six4 2.2 2.1 TF ST6-N-acetylgalactosaminide a-2,6-sialyltransferase 5 St6galnac5 2.3 2.8 Golgi T-box 15 Tbx15 12.2 145.4 TF Transm prot with EGF-like and 2 follistatin-like domains 2 Tmeff2 3.0 2.0 PM Ubiquitin D Ubd 5.7 2.3 Cytoplasm Ubiquitin speciﬁc peptidase 18 Usp18 3.7 5.0 Cytoplasm ER, endoplasmic reticulum; TF, transcription factor; PM, plasma membrane; IntMem, internal membrane. intrinsic differences in the adipocytes in these fat depots With regard to differentiation, previous studies show that (2,5,24–26). In the current study, we demonstrate that APCs SVF cells taken from SC fat of either rodents or humans from SC and Vis fat have important differences in terms of differentiate more readily in culture than those from Vis fat their gene expression signatures, their ability to differenti- (26–28). We show that the same is true for isolated APCs ate, their response to growth factors, and their regulation in from these depots but ﬁnd that this is because the APCs normal physiology and disease. from SC and Vis depots have different differentiation diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1695 ADIPOCYTE PRECURSOR CELL SUBTYPES TABLE 3 Gene set enrichment analysis Gene set Gene symbol Gene name Fold change P value Enriched in Vis APCs Muscle related Des Desmin 5.5 0.000 Myoﬁbril Tpm2 Tropomyosin 2 (b) 3.6 0.020 Sarcomere Tpm1 Tropomyosin 1 (a) 2.5 0.000 Contractile ﬁber Myoz2 Myozenin 2 4.8 0.005 Tnnt2 Troponin T type 2 4.5 0.019 Tnni3 Troponin I type 3 2.4 0.019 Monooxygenase activity Pah Phenylalanine hydrogenase 18.8 0.000 Fmo2 Flavin containing monooxygenase 2 4.7 0.000 Cyp1b1 Cytochrome P450 family 1 subfamily b 3.2 0.000 Cyp27a1 Cytochrome P450 family 27 subfamily a 2.4 0.000 Fmo1 Flavin containing monooxygenase 1 2.1 0.000 Cyp7b1 Cytochrome P450 family 7 subfamily b 1.9 0.008 Developmental Mesoderm development Tcf21 Transcription factor 21 216.2 0.000 Eya2 Eyes absent homolog 2 4.9 0.003 Slit2 Slit homolog 2 5.5 0.000 Mest Mesoderm speciﬁc transcript hom 1.6 0.010 Developmental maturation Rnd1 r family GTPase 1 2.7 0.001 Ereg Epiregulin 1.9 0.015 Pick1 Protein interacting with PRKCA1 1.4 0.006 Tgfb2 Transforming growth factor, b 2 1.8 0.010 Myh11 Myosin, heavy chain 11, s 1.9 0.040 Negative regulation of cytokine biology Inhbb Inhibin, b B 8.6 0.001 Inhba Inhibin, b A 3.9 0.000 Ela2 Elastase 2 2.5 0.001 Il6 Interleukin 6 1.8 0.001 Enriched in SC APCs Chemokines Cxcl9 Chemokine (C-C motif) ligand 9 5.3 0.001 Chemokine activity Cxcl14 Chemokine (C-X-C motif) ligand 14 4.2 0.002 Chemokine receptor binding Ccl8 Chemokine (C-C motif) ligand 8 4.2 0.000 Cxcl13 Chemokine (C-X-C motif) ligand 13 5.4 0.007 Cxcl10 Chemokine (C-X-C motif) ligand 10 1.9 0.000 Ccl22 Chemokine (C-C motif) ligand 22 5 0.007 Peptidase activity Cysteine type endopeptidase activity Lgmn Legumain 2.8 0.001 Casp9 Caspase 9 2.8 0.003 Ctsk Cathepsin K 1.7 0.000 Casp1 Caspase 1 1.9 0.009 Usp7 Ubiquitin speciﬁc peptidase 1.7 0.001 Uchl1 Ubiquitin carboxyl-terminal 1.3 0.010 Ctsb Cathepsin B 1.2 0.002 Aminopeptidase activity Trhde TRH-degrading enzyme 10.2 0.000 Lnpep Leucyl/cystinyl aminopeptidase 1.5 0.018 Anpep Alanyl (membrane) aminopeptidase 1.8 0.020 Vesicular fraction Stx16 Syntaxin 16 1.9 0.007 Microsome Mgst3 Microsomal gluthation S-transferase 3 1.7 0.001 Oas2 29-59-oligoadenylate synthetase 2 2.2 0.03 Ltc4s Leukotriene C4 synthase 4.5 0.04 Ero1lL ERO1-like 1.4 0.02 Slc35b1 Solute carrier family 35, member b1 1.3 0.02 Sts Steroid sulfatase 1.3 0.01 Tmed2 Transmembrane emp24 domain trafﬁcking protein 2 1.4 0.01 Copa Coatomer protein complex subunit a 1.5 0.001 requirements. While SC APCs differentiate well using the C3H10T1/2 to an adipocyte lineage (29), whereas com- standard IC, Vis APCs differentiate very poorly under the mitted cell lines, such as 3T3-L1, do not have this re- same conditions. However, pretreatment of Vis APCs with quirement. Indeed, Vis APCs are more like mesenchymal BMP-4 or BMP-2 allows these cells to undergo full differ- stem cells and less committed to adipocyte differentiation entiation. BMP-2 and BMP-4 have been shown to be re- than cells bearing the same surface markers from SC fat. quired for commitment of the mesenchymal stem cell line Vis APCs also show higher levels of expression of Gata2 1696 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES FIG. 3. Genetic and environmental impact on number (#) and percentage (%) of APCs and other SVF cells from Vis and SC fat depots. FACS sorting 2 2 analysis of Vis and SC APCs and other SVF cells. Data were calculated using only live cells. Double positive CD34, SCA1 (and CD45 , CD31 ,and 2 + + + Ter119 ) cells were considered APCs, whereas CD45 ,CD31 ,and Ter119 cells were considered as other SVF cells. A: 8-week-old vs. 1-year-old C57BL/6 mice. m, month. B: C57BL/6 (B6) vs. 129 mice (3 months old). C: C57BL/6 male (M) vs. female (F) mice (6 months old). Five mice per group were used in two independent experiments. Data are mean 6 SEM. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. and Tgfb2, factors known to inhibit adipogenesis (23,30), indicating the isolated APCs from both depots do represent as well as higher levels of expression of mesenchymal the preadipocyte pool. This resistance to differentiation may stem cell markers (Lif, Ctgf, and Mgp), whereas SC APCs explain why Vis fat expansion during adulthood occurs show higher expression of proadipogenic genes (Pparg, mainly by hypertrophy, whereas SC fat expansion is due + + Cebpa, Bmp2, Bmp4, and Dkk2). Thus, Sca1 and CD34 primarily to hyperplasia (32). SC APCs are more committed to the adipocyte lineage Although others have analyzed differences in gene ex- than Vis APCs expressing the same markers and have pression between Vis and SC adipose tissue (5,19,33), ours fewer requirements for becoming mature adipocytes. is the ﬁrst study to identify genes that are differentially At a cellular level, this difference in differentiation is due expressed between APCs in different depots. Tcf21 is the to a blockade in the ability of the IC to stimulate expres- most differentially expressed gene between Vis and SC sion of Pparg. Thus, after treatment with IC, including the adipocyte precursors, with the higher levels in Vis cells. PPARg agonist rosiglitazone, Vis APCs show increased This transcription factor also shows higher expression in Cebpa but a failure of induction of Pparg and downstream mature adipocytes of Vis fat (34). While Tcf21 has been adipogenic markers. BMP-2 and BMP-4 signals overcome described (34) as a marker for white preadipocytes com- the block and induce differentiation in Vis APCs. This may pared with brown preadipocytes and muscle, in this study, be in part because SC APCs already have enough endoge- preadipocytes were isolated only from epididymal fat, nous BMP to trigger cell commitment, or it may depend which have much higher expression than SC preadipocytes. on other differences between the two adipocyte precursor The levels of Tcf21 in SC preadipocytes are similar to those types. BMPs have been shown to induce Pparg through in brown preadipocytes and muscle. activation of schnurri-2, which forms a transcriptional com- The most differentially expressed gene in SC APCs is the plex with C/EBPa and SMAD1/4 (31). However, we ﬁnd no LIM homeobox gene Lhx8. This gene had been previously difference in the level of schnurri-2 (Hivep2) between Vis and described as a marker for brown preadipocytes when ep- SC fat. In any case, in the presence of BMP-2, BMP-4, and IC, ididymal fat was the only source of WAT (34). However, 2 + + almost all of the Lin ,CD34 , and SCA1 cells isolated from Lhx8 is not unique to brown preadipocytes but also a strong both Vis and SC depots are able to become adipocytes, marker for SC preadipocytes (162-fold higher than in Vis diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1697 ADIPOCYTE PRECURSOR CELL SUBTYPES FIG. 4. Effect of HFD on number (#) and percentage (%) of APCs and other SVF cells from Vis and SC fat depots. FACS sorting analysis of Vis and 2 2 2 SC APCs and other SVF cells. Data were calculated using only live cells. Double positive CD34, SCA1 (and CD45 , CD31 , and Ter119 ) cells were + + + considered APCs, whereas CD45 , CD31 , and Ter119 cells were considered as other SVF cells. FACS sorted cells from mice placed on chow diet (CD) or HFD for 1, 2, and 4 weeks starting at age 4 weeks (A) or 8 weeks (B). Three to ﬁve mice per group were used in two independent experiments. Data are mean 6 SEM. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. APCs). Thus, the pattern of gene expression in SC white especially in Vis fat. While sex has no effect on number of fat has several similarities with that of brown fat, as com- APCs in the SC depot, females show a trend to increased pared with Vis white fat. Another gene more highly ex- APCs and lower other SVF cells per gram of fat in Vis fat. pressed in SC APCs than in Vis precursors is Cd24. This This could reﬂect a reduced number of macrophages in the membrane protein was previously used as a marker (10) to Vis depot of female mice and account for the higher insulin purify the APCs but should probably not be used unless sensitivity of adipocytes from Vis fat of females compared one is attempting to separate different populations from with those from males (35). a mixture of white fat depots. Finally, the potential for new adipocytes also parallels Our study also shows the dynamic nature of the adipo- the genetic risk for obesity. Thus, the obesity-prone cyte precursor pool. We ﬁnd that HFD increases the num- C57BL/6 mouse has approximately twofold more APCs per ber of APCs in both Vis and SC depots by as much as 270%. gram of fat than the obesity-resistant 129 mouse by age The increase is bigger when animals are started on HFD at 3 months. In adult animals, this is accompanied by in- a young age compared with when challenged as adults. Joe creased content of lymphocytes and macrophages and et al. (32) reported a similar increase in bromodeoxyuridine expression of proinﬂammatory genes in Vis fat of C57BL/6 labeling of APCs in SC fat but a much smaller increase in mice, contributing further to the tendency of C57BL/6 mice labeling of Vis fat after 60 days of HFD. Whether this reﬂects to develop insulin resistance upon aging or HFD-induced differences in methodology, age of the mice, and a longer obesity (36). period of HFD is unknown. In conclusion, APCs from Vis and SC depots differ dra- Another difference between depots after challenge with matically in terms of their differentiation ability, gene ex- HFD is that the number of non-APCs in the Vis depot pression signatures, growth factor requirements, and increases dramatically after 2–4 weeks on HFD, whereas capacity to expand upon genetic and environmental stim- the number of other SVF cells in the SC depot remains uli. SC APCs are more committed to differentiate, whereas constant. This causes a dramatic change in the relative those from Vis depots have an anti-adipogenic proﬁle and proportion of cell types in these depots. Thus, the per- require growth factors, such as BMP-2 or BMP-4, to un- centage of APCs in Vis fat decreases in mice on HFD dergo differentiation. This helps explain why SC fat ex- compared with those on chow diet, whereas the percent- pands more by hyperplasia, whereas Vis fat expands by age of APCs in the SC depot either increases or remains hypertrophy. These depot-speciﬁc gene signatures can constant. This could explain why SC fat stays metaboli- serve as a powerful tool to identify and manipulate spe- cally ﬁt in obesity, whereas Vis fat develops inﬂammation, ciﬁc characteristics in the differentiation and functional contributing to insulin resistance. properties of adipocytes and ultimately should help us un- Fat mass increases with age, as does the number of derstand how these two fat depots have opposing contri- APCs per depot, by three- to fourfold or more. As with butions to the development of diabetes and metabolic HFD, however, the number of other SVF cells also increases, syndrome. 1698 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES 12. Schulz TJ, Huang TL, Tran TT, et al. Identiﬁcation of inducible brown ACKNOWLEDGMENTS adipocyte progenitors residing in skeletal muscle and white fat. Proc Natl This work was supported by National Institutes of Health Acad Sci U S A 2011;108:143–148 Grant R01-DK-082659 and the Mary K. Iacocca Professor- 13. Ahrens M, Ankenbauer T, Schröder D, Hollnagel A, Mayer H, Gross G. ship. The study was also facilitated by support from the Expression of human bone morphogenetic proteins-2 or -4 in murine mes- Joslin Diabetes and Endocrinology Research Center Core enchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 1993;12:871–880 Laboratories (DK-036836). Y.M. was supported by the Pew 14. Bowers RR, Lane MD. A role for bone morphogenetic protein-4 in adipo- Latin American Fellows Program in the Biological Sciences, cyte development. Cell Cycle 2007;6:385–389 the Mentor-Based American Diabetes Association Grant 15. Schulz TJ, Tseng YH. Emerging role of bone morphogenetic proteins in Award, and the Mexican Council of Science (CONACyT) adipogenesis and energy metabolism. Cytokine Growth Factor Rev 2009; Postdoctoral Fellowship. C.R.K. was supported by a grant 20:523–531 from the Loveman Foundation. 16. Rosen ED, Hsu CH, Wang X, et al. C/EBPalpha induces adipogenesis through PPARgamma: a uniﬁed pathway. Genes Dev 2002;16:22–26 No potential conﬂicts of interest relevant to this article 17. Igarashi A, Segoshi K, Sakai Y, et al. Selection of common markers for were reported. bone marrow stromal cells from various bones using real-time RT-PCR: Y.M. conceived and designed the experiments, researched effects of passage number and donor age. Tissue Eng 2007;13:2405–2417 data, and wrote the manuscript. B.E. researched data, 18. Vohl MC, Sladek R, Robitaille J, et al. A survey of genes differentially contributed to discussion, and reviewed and edited the expressed in subcutaneous and visceral adipose tissue in men. Obes Res 2004;12:1217–1222 manuscript. M.A.M. and S.G. researched data and re- 19. Tchkonia T, Lenburg M, Thomou T, et al. Identiﬁcation of depot-speciﬁc viewed and edited the manuscript. T.J.S. set up the meth- human fat cell progenitors through distinct expression proﬁles and de- odology, contributed to discussion, and reviewed and velopmental gene patterns. Am J Physiol Endocrinol Metab 2007;292: edited the manuscript. Y.-H.T. contributed to discussion E298–E307 and reviewed and edited the manuscript. C.R.K. conceived 20. Salans LB, Cushman SW, Weismann RE. Studies of human adipose tissue. and designed the experiments, reviewed the data, and Adipose cell size and number in nonobese and obese patients. J Clin Invest wrote the manuscript. C.R.K. is the guarantor of this work 1973;52:929–941 21. Hirsch J, Batchelor B. Adipose tissue cellularity in human obesity. Clin and, as such, had full access to all the data in the study and Endocrinol Metab 1976;5:299–311 takes responsibility for the integrity of the data and the 22. Lemonnier D. Effect of age, sex, and site on the cellularity of the adipose accuracy of the data analysis. tissue in mice and rats rendered obese by a high-fat diet. J Clin Invest 1972; Parts of this study were presented in abstract form at the 51:2907–2915 70th Scientiﬁc Sessions of the American Diabetes Associ- 23. Wu H, Ghosh S, Perrard XD, et al. T-cell accumulation and regulated on ation, Orlando, Florida, 25–29 June 2010. activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity. Circulation 2007;115:1029–1038 The authors thank Joyce LaVecchio, Giri Buruzula, and 24. Hauner H, Entenmann G. Regional variation of adipose differentiation in the Flow Cytometry core for help with cytometry; Joshua cultured stromal-vascular cells from the abdominal and femoral adipose Schroeder and the Genomics core for microarray process- tissue of obese women. Int J Obes 1991;15:121–126 ing; Michael Rourk and Graham Smyth for their expertise 25. Lafontan M, Girard J. Impact of visceral adipose tissue on liver metabo- in animal care; and Madelana Basile and Jen Davison for lism. Part I: heterogeneity of adipose tissue and functional properties of editing the manuscript (all from Joslin Diabetes Center). visceral adipose tissue. Diabetes Metab 2008;34:317–327 26. Tchkonia T, Giorgadze N, Pirtskhalava T, et al. Fat depot-speciﬁc char- acteristics are retained in strains derived from single human pre- REFERENCES adipocytes. Diabetes 2006;55:2571–2578 1. Misra A, Garg A, Abate N, Peshock RM, Stray-Gundersen J, Grundy SM. 27. Prunet-Marcassus B, Cousin B, Caton D, André M, Pénicaud L, Casteilla L. Relationship of anterior and posterior subcutaneous abdominal fat to in- From heterogeneity to plasticity in adipose tissues: site-speciﬁc differ- sulin sensitivity in nondiabetic men. Obes Res 1997;5:93–99 ences. Exp Cell Res 2006;312:727–736 2. Snijder MB, Dekker JM, Visser M, et al. Associations of hip and thigh 28. Adams M, Montague CT, Prins JB, et al. Activators of peroxisome pro- circumferences independent of waist circumference with the incidence of liferator-activated receptor g have depot-speciﬁc effects on human pre- type 2 diabetes: the Hoorn Study. Am J Clin Nutr 2003;77:1192–1197 adipocyte differentiation. J Clin Invest 1997;100:3149–3153 3. Tran TT, Yamamoto Y, Gesta S, Kahn CR. Beneﬁcial effects of sub- 29. Bowers RR, Kim JW, Otto TC, Lane MD. Stable stem cell commitment to cutaneous fat transplantation on metabolism. Cell Metab 2008;7:410–420 the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 4. Wajchenberg BL, Giannella-Neto D, da Silva ME, Santos RF. Depot-speciﬁc gene. Proc Natl Acad Sci U S A 2006;103:13022–13027 hormonal characteristics of subcutaneous and visceral adipose tissue and 30. Tong Q, Dalgin G, Xu H, Ting CN, Leiden JM, Hotamisligil GS. Function of their relation to the metabolic syndrome. Horm Metab Res 2002;34:616–621 GATA transcription factors in preadipocyte-adipocyte transition. Science 5. Gesta S, Blüher M, Yamamoto Y, et al. Evidence for a role of developmental 2000;290:134–138 genes in the origin of obesity and body fat distribution. Proc Natl Acad Sci 31. Jin W, Takagi T, Kanesashi SN, et al. Schnurri-2 controls BMP-dependent U S A 2006;103:6676–6681 adipogenesis via interaction with Smad proteins. Dev Cell 2006;10:461–471 6. Yamamoto Y, Gesta S, Lee KY, Tran TT, Saadatirad P, Kahn CR. Adipose 32. Joe AW, Yi L, Even Y, Vogl AW, Rossi FM. Depot-speciﬁc differences in depots possess unique developmental gene signatures. Obesity (Silver adipogenic progenitor abundance and proliferative response to high-fat Spring) 2010;18:872–878 diet. Stem Cells 2009;27:2563–2570 7. Cantile M, Procino A, D’Armiento M, Cindolo L, Cillo C. HOX gene network 33. Atzmon G, Yang XM, Muzumdar R, Ma XH, Gabriely I, Barzilai N. Differ- is involved in the transcriptional regulation of in vivo human adipogenesis. ential gene expression between visceral and subcutaneous fat depots. J Cell Physiol 2003;194:225–236 Horm Metab Res 2002;34:622–628 8. Tchoukalova YD, Koutsari C, Votruba SB, et al. Sex- and depot-dependent 34. Timmons JA, Wennmalm K, Larsson O, et al. Myogenic gene expression differences in adipogenesis in normal-weight humans. Obesity (Silver signature establishes that brown and white adipocytes originate from Spring) 2010;18:1875–1880 distinct cell lineages. Proc Natl Acad Sci U S A 2007;104:4401–4406 9. Spalding KL, Arner E, Westermark PO, et al. Dynamics of fat cell turnover 35. Macotela Y, Boucher J, Tran TT, Kahn CR. Sex and depot differences in in humans. Nature 2008;453:783–787 adipocyte insulin sensitivity and glucose metabolism. Diabetes 2009;58: 10. Rodeheffer MS, Birsoy K, Friedman JM. Identiﬁcation of white adipocyte 803–812 progenitor cells in vivo. Cell 2008;135:240–249 36. Mori MA, Liu M, Bezy O, et al. A systems biology approach identiﬁes in- 11. Tang W, Zeve D, Suh JM, et al. White fat progenitor cells reside in the ad- ﬂammatory abnormalities between mouse strains prior to development of ipose vasculature. Science 2008;322:583–586 metabolic disease. Diabetes 2010;59:2960–2971 diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1699 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diabetes Pubmed Central http://www.deepdyve.com/lp/pubmed-central/intrinsic-differences-in-adipocyte-precursor-cells-from-different-21fQpBR0za

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Publisher: Pubmed Central
Copyright: © 2012 by the American Diabetes Association.
ISSN: 0012-1797
eISSN: 1939-327X
DOI: 10.2337/db11-1753
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Abstract

ORIGINAL ARTICLE Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots Yazmín Macotela, Brice Emanuelli, Marcelo A. Mori, Stephane Gesta, Tim J. Schulz, Yu-Hua Tseng, and C. Ronald Kahn expression have also been observed in human pre- Obesity and body fat distribution are important risk factors for adipocytes from different depots (8), but exactly how this the development of type 2 diabetes and metabolic syndrome. relates to intrinsic differences in adipose tissues and the Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. In propensity for obesity and insulin resistance is unclear. the current study, we demonstrate that adipocyte precursor cells Spalding et al. (9) recently demonstrated that in humans, (APCs) isolated from visceral and subcutaneous white adipose ;10% of the adipocyte pool turns over annually and that depots of mice have distinct patterns of gene expression, the absolute number of adipocytes turning over in indi- differentiation potential, and response to environmental and viduals with obesity is approximately double from that in genetic inﬂuences. APCs derived from subcutaneous fat differ- lean individuals. Exactly how adipocytes in different de- entiate well in the presence of classical induction cocktail, pots turn over and to what extent this is inﬂuenced by whereas those from visceral fat differentiate poorly but can be genetic or environmental factors is unknown. However, induced to differentiate by addition of bone morphogenetic using lineage tracing and ﬂuorescence-activated cell protein (BMP)-2 or BMP-4. This difference correlates with major differences in gene expression signature between subcutaneous sorting (FACS) strategies, two groups (10,11) have devel- and visceral APCs. The number of APCs is higher in obesity- oped techniques to identify and isolate the adipocyte pre- prone C57BL/6 mice than obesity-resistant 129 mice, and the cursor cells (APCs) from the stromovascular fraction number in both depots is increased by up to 270% by exposure of (SVF) of white fat. In the current study, we show that APCs mice to high-fat diet. Thus, APCs from visceral and subcutaneous are dynamic and respond to different environmental and depots are dynamic populations, which have intrinsic differences genetic factors. We show that APCs from Vis versus SC fat in gene expression, differentiation properties, and responses to differ in their speciﬁc gene expression signatures and that environmental/genetic factors. Regulation of these populations may SC APCs are more adipogenic and require fewer growth provide a new target for the treatment and prevention of obesity factors, whereas Vis APCs have anti-adipogenic charac- and its metabolic complications. Diabetes 61:1691–1699, 2012 teristics and require additional growth factor stimulation to differentiate. These results show that there are intrinsic differences between preadipocytes from different depots here are two main types of white adipose tissue and that these differences contribute to the differential (WAT) in humans and rodents—subcutaneous properties and turnover of adipocytes in different depots. (SC) fat and visceral (intra-abdominal [Vis]) fat. TThese contribute differentially to disease risk. RESEARCH DESIGN AND METHODS Accumulation of Vis adipose tissue is associated with ad- Mice. C57BL/6 mice and 129 mice were obtained from The Jackson Laboratory verse metabolic outcomes, whereas increased amounts of and kept under a normal diurnal cycle in a temperature-controlled room. Mice SC fat has been viewed as neutral or even beneﬁcial in its were fed with standard chow containing 22% of calories from fat (Mouse Diet 9F metabolic effects (1–3). While part of this difference may 5020; PharmaServ) or a 60% high-fat diet (HFD) (OpenSource Diet D12492; Research Diets). For the HFD versus chow diet study, two cohorts of mice were be related to anatomical location with different patterns of used starting at aged 3 and 8 weeks. Animal care and study protocols were venous drainage, over the past few years, it has become approved by the animal care committee of Joslin Diabetes Center and were in clear that these two types of WAT differ in their intrinsic accordance with National Institutes of Health guidelines. characteristics, including levels of adipokine secretion, Adipocyte precursor isolation and ﬂow cytometry. Epididymal and ingui- insulin sensitivity, lipolysis rate, and tendency to develop nal fat pads, representing Vis and SC fat, respectively, were cut in small pieces and inﬂammation (4). We and others (5–7) recently have shown incubated with 1 mg/mL collagenase I for 30 min. The cell suspension was ﬁltered through a 150-mmol/L nylon mesh, and the SVF was isolated by low-speed that this correlates with differences in gene expression be- centrifugation. For FACS analysis, erythrocyte-free SVF cells were incubated tween adipocytes in different depots, including the expres- with a mix of antibodies against different surface markers as described previ- sion of fundamental development and patterning genes. ously (10) and sorted using an Aria ﬂow cytometer (BD Biosciences). Dead cells Differences in differentiation and developmental gene were removed using propidium iodide staining. Cells negative for Ter119, CD45, and CD31 and positive for both SCA1 and CD34 were considered as APCs. Cell culture and differentiation of APCs. Cells were grown as described previously (12) with some modiﬁcations. Medium containing 60% Dulbecco’s From the Joslin Diabetes Center, Harvard Medical School, Boston, Massachu- modiﬁed Eagle’s medium–low glucose (Invitrogen) and 40% MCDB201 (Sigma- setts. Corresponding author: C. Ronald Kahn, [email protected]. Aldrich) and supplemented with Normocin 0.1 mg/mL (InvivoGen), 10% FBS, Received 19 December 2011 and accepted 5 March 2012. 13 insulin-transferrin-selenium mix, 13 linoleic acid conjugated to BSA, DOI: 10.2337/db11-1753 1 nmol/L dexamethasone, and 0.1 mmol/L L-ascorbic acid 2-phosphate (Sigma- This article contains Supplementary Data online at http://diabetes Aldrich) was used as growth medium. This medium was further supplemented .diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1753/-/DC1. with 10 ng/mL epidermal growth factor (PeproTech), 10 ng/mL leukemia inhibitory Y.M. is currently afﬁliated with the Instituto de Neurobiología, Universidad factor (Millipore), 10 ng/mL platelet-derived growth factor BB (PeproTech), Nacional Autónoma de México, Querétaro, Mexico. and 5 ng/mL basic ﬁbroblast growth factor (Sigma-Aldrich). Once sorted, cells 2012 by the American Diabetes Association. Readers may use this article as were rinsed with medium and plated at 50,000 cells per well in 24-well plates. long as the work is properly cited, the use is educational and not for proﬁt, After 6–7 days, cells reached ;80% conﬂuence. Medium was changed every and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. other day. For differentiation, cells were seeded at 20,000 cells per well in diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1691 ADIPOCYTE PRECURSOR CELL SUBTYPES 48-well plates. After cells reached 80% conﬂuence, they were treated or not for including rosiglitazone, and differentiation was assessed 2 days with 3.3 nmol/L BMP-2 or BMP-4 (R&D Systems) in growth medium by lipid accumulation and expression of markers of ter- with 2% FBS and without growth factors. Medium was then replaced by dif- minal differentiation, including Slc2a4 (Glut-4), Adipoq ferentiation medium (growth medium with no growth factors but with 2% FBS, 1 (adiponectin), Lep (leptin), and Fabp4 (fatty acid binding mmol/L dexamethasone, 0.5 mmol/L isobutylmethylxanthine, 100 nmol/L in- protein or aP2). Using this approach, APCs from the SC fat sulin, and 1 mmol/L rosiglitazone) for 3 days, after which the medium was replaced with growth medium containing 2% FBS and 100 nmol/L insulin for 2 differentiated well within 7 days after induction, with .90% more days; for the last 2–3 days of differentiation, cells were incubated with of the cells containing large lipid droplets (Fig. 2A). In fact, growth medium with 2% FBS alone. ;10% of SC APCs differentiated in the absence of any RNA extraction and PCR. RNA was extracted from cells using RNeasy hormonal induction (Fig. 2A). By contrast, with the same (QIAGEN). Reverse transcription was performed with 0.3 mg RNA by using High protocol, ,20% of APCs from the Vis depot could be in- Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). duced to differentiate (Fig. 2A). This raised two possibili- Real-time PCR was performed using Maxima SYBR Green (Fermentas, Glen Burnie, MD) in duplicate using the ABI Prism 7900 System (Applied Biosystems) ties: one was that the Vis APCs were not really adipocyte under the following conditions: 50°C 22min, 95°C 210 min, and 40 cycles of 95°C precursors; the other was that the APCs were missing some 215 s, 60°C 220 s, and 72°C 230 s. Dissociation protocols were conducted after signals required for differentiation. every run to check for primer speciﬁcity. To obtain relative expression values, BMPs are known to induce commitment of mesenchymal 2DCt we calculated the 2 parameter for each individual sample using cycle stem cells into adipocytes, muscle, and bone (13–15). We threshold values of TATA-box-binding protein as an endogenous control. hypothesized that perhaps BMP signaling was lacking in the Microarray. Approximately 1 3 10 sorted cells per sample were used to isolate RNA for the microarray analysis. Five independent RNA samples each APCs from the Vis depot. We therefore pretreated subcon- from a pool of isolated APCs from 10 different mice from each depot were ﬂuent Vis and SC APCs with 3.3 nmol/L BMP-4 for 2 days analyzed on Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). All prior to the addition of the induction cocktail (IC) and then data were subjected to global normalization to an intensity of 1,500 using the assessed differentiation. With the SC APCs, BMP-4 pre- Gene Chip Software MAS V. 5.0. All differences reported had a P value ,0.05. treatment did not further enhance the almost complete dif- ferentiation achieved by the IC alone (Fig. 2A). However, RESULTS with the Vis APCs, BMP-4 pretreatment markedly enhanced Isolation of APCs from different WAT depots. APCs differentiation, resulting in almost 90% of cells showing typ- were isolated by FACS from SVFs of Vis and SC fat from ical lipid droplets (Fig. 2A). Pretreatment with BMP-2 in- mice as described above and in Supplementary Fig. 1. duced Vis APCs to differentiate at a similar extent as BMP-4 Cells negative for CD45, CD31, and Ter119 and positive for (Fig. 2B) but had no further effect on SC APCs (not shown). CD34 and SCA1 were considered APCs. Cells positive for These differences in differentiation potential were con- CD45, CD31, and/or Ter119 (endothelial cells, platelets, ﬁrmed by quantitative PCR analysis for expression of ter- macrophages, white blood cells, osteoclasts, and eryth- minal adipocyte differentiation genes. SC APCs 7–8 days rocytes) were designated as “other SVF.” Only live cells were after treatment with standard IC with or without BMP-4 counted. In a typical sorting, using male C57BL/6 mice aged treatment showed marked increases in expression of Fabp4 8 weeks, Vis fat contained 0.9 6 0.1 3 10 APCs and 3.5 6 (730-fold), Slc2a4 (300-fold), Adipoq (730-fold), and Lep 0.8 3 10 other SVF cells per fat pad, whereas SC fat con- (20-fold) (Fig. 2C, right three bars, each panel). On the other 5 5 tained 0.65 6 0.08 3 10 APCs and 7.1 6 0.7 3 10 other SVF hand, Vis APCs treated with standard IC showed little ac- cells per pad (Fig. 1A). Thus, the percentage of stromovas- cumulation of lipid and very small increases in expression cular cells, which are APCs, was almost three times higher in of these differentiation markers (Fig. 2C, left two bars, each Vis fat than in SC fat (21 6 3.7 vs. 7.8 6 1.7%) (Fig. 1B). panel). However, when the Vis APCs were pretreated with APCs from different fat depots differ in their BMP-4 followed by IC, the increases in gene expression differentiation requirements. To conﬁrm that the iso- were similar to those seen in SC APCs after differentiation, lated APCs were preadipocytes, we analyzed the differ- with large increases in Fabp4 (480-fold), Slc2a4 (160-fold), entiation ability of both Vis and SC APCs. Conﬂuent APC Adipoq (660-fold), and Lep (21-fold) (Fig. 2C, third bar, each cultures were treated with the typical induction protocol, panel). FIG. 1. Frequency of APCs and other SVF cells in SC vs. Vis depots. Typical FACS sorting analysis of APCs and other SVF cells from SC and Vis 2 2 depots isolated from male mice aged 7–9 weeks. Data were calculated using only live cells. Double positive CD34, SCA1 (and CD45 , CD31 , and 2 + + + Ter119 ) cells were considered APCs, whereas CD45 , CD31 , and Ter119 cells were considered as other SVF cells. A: Number of cells per depot. B: Percent of APCs in both depots relative to other SVF cells. Data are mean 6 SEM of n = 4 independent experiments. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. 1692 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES FIG. 2. Higher differentiation capacity in APCs from SC than from Vis depots. After FACS sorting, APCs from SC and Vis depots were placed in culture; upon 80% conﬂuence, they were treated with or without BMP2 or BMP4 and 2 days later, were induced to differentiate (insulin, iso- butylmethylxanthine, dexamethasone, and rosiglitazone). Seven days after induction of differentiation, they were photographed to evaluate for lipid- containing mature adipocytes (A and B) or lysed, mRNA extracted, and processed for quantitative PCR to evaluate expression changes on adipocyte differentiation genes (C and D). Graphs show fold changes vs. N Vis. N, control without BMPs or IC; BMP4 IC, BMP4 pretreatment followed by IC. Data are mean 6 SEM of n = 2 independent experiments. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. Gene expression analysis also indicated that the block in showed signiﬁcantly higher expression of genes involved differentiation was at an early step involved in the stimu- in positive regulation of adipocyte differentiation, including lation of PPARg. Thus, standard IC increased expression Pparg (3.2-fold), Cepba (1.3-fold), the dickkopf homolog of Cebpa by greater than twofold in APCs from both Vis DKK2 (9.1-fold), Stat5a (2.9-fold), and the BMPs associated and SC fat, and Cebpa was not further increased by BMP-4 with WAT differentiation, Bmp2 (1.5-fold) and Bmp4 (1.5- pretreatment (Fig. 2D). On the other hand, in SC APCs, fold) (all P , 0.05). By contrast, Vis APCs showed increased Pparg increased ﬁvefold after differentiation using standard expression of genes that can act as inhibitors of adipo- IC or BMP-4 plus IC. However, in Vis APCs, Pparg ex- cyte differentiation, including Gata2 (7.4-fold) and Tgfb2 pression was not changed by treatment with IC but was (1.8-fold) (Table 1 and Supplementary Table 1). It is in- increased sixfold when cells were pretreated by BMP-4 plus teresting that APCs from the Vis depot also expressed IC (Fig. 2D). Taken together, these data indicate that the higher levels of markers found in bone marrow mesen- block in differentiation in Vis APCs lies in the inability of chymal cells (17), including Lif, Igf1, Igfbp7, Ctgf, Mgp, Trib2 standard induction hormones to trigger the expression of and Pgn1/Bgn (Table 1). Pparg and induce the transcriptional events required for Depot-speciﬁc gene expression signatures. We (5,6) differentiation and that this block can be released by stim- and others (7,18,19) have previously shown that mature ulation of the BMP pathway. Since C/EBPa and PPARg can adipocytes from Vis and SC fat have different patterns of regulate expression of each other in a positive feedback gene expression, including genes involved in adipocyte manner (16) and since Cebpa is induced by standard IC in function and developmental patterning. Comparing the Vis fat, the blockade appears to be in the ability of C/EBPa results from APCs in the current study with our previous to stimulate a normal increase in PPARg. results in mature adipocytes from mice, we could identify Proadipogenic gene expression signature in SC APCs. depot-speciﬁc signatures. These consisted of 19 genes that To identify possible differences between adipocyte pre- were much more highly expressed in the Vis depot and cursors isolated from SC and Vis depots that might con- 32 genes that were much more highly expressed in the SC tribute to altered differentiation and other properties of depot in both preadipocytes and adipocytes (Table 2). the adipocytes, we performed microarray analysis on Gene set enrichment analysis of the microarray data freshly isolated SC and Vis APCs from 7- to 8-week-old showed that SC APCs have increased expression in three mice. Consistent with their increased ability to differenti- major gene sets when compared with Vis APCs: 1)che- ate, when compared with Vis preadipocytes, SC APCs mokines, 2)peptidases, and 3) proteins involved in the diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1693 ADIPOCYTE PRECURSOR CELL SUBTYPES TABLE 1 as the percentage of total SVF cells in each depot, young Pro- and anti-adipogenic gene expression signatures in Vis and mice had a signiﬁcantly higher percentage of APCs than old SC APCs mice in Vis fat (22 vs. 16%) but a smaller percentage of APCs in SC fat than old mice (3 vs. 13%) (Fig. 3A). Fold P value It is interesting that obesity-prone C57BL/6 mice already Proadipogenic genes higher had almost twofold higher numbers of APCs per gram of in SC APCs fat than 129 mice in both depots by age 3 months. C57BL/6 Cepba 1.3 0.03 mice also had ninefold more other SVF cells per gram of Pparg 3.2 0.0001 fat in the SC depot than 129 mice, whereas the number of Dkk2 9.1 0.0004 other SVF cells in the Vis depot was similar. However, the Stat5a 2.9 0.0001 percentage of APCs did not change between genotypes in Bmp2 1.5 0.001 Vis fat but was higher in the SC fat from 129 mice than in Bmp4 1.5 0.02 the same depot from C57BL/6 mice (17 vs. 4%) (Fig. 3B). Anti-adipogenic genes higher Between males and females, APCs and other SVF cells in Vis APCs per gram of fat or per depot did not differ signiﬁcantly. Gata2 7.4 0.0005 However, male mice did have a twofold higher number of Tgfb2 1.8 0.01 other SVF cells in the Vis depot than female mice. As a re- Bone marrow mesenchymal cell sult, the calculated percentage of APCs in the Vis depot markers higher in Vis APCs (but not in the SC depot) from female mice was almost Lif 2.46 0.039 twice as high as that in males (29 6 3vs. 15 6 2%) (Fig. 3C). Igf1 2.2 0.00008 Effect of HFD on adipocyte precursor frequency in Igfbp7 2.07 0.00004 young versus adult mice. Previous studies in humans Ctgf 4.29 0.003 show that early onset obesity is associated with adipocyte Mgp 11.88 0.003 hyperplasia, whereas late onset of obesity is the result of Trib2 1.75 0.047 adipocyte hypertrophy and hyperplasia; studies in rodents Pgn1/Bgn 1.77 0.000003 show hyperplasia can occur in both young and old animals (20–22). To determine if HFD-induced obesity might alter the number and percentage of APCs and other cells in the vesicular fraction or microsomes (Table 2). In the Vis APCs, the most enriched gene sets fell into four major SVF at different ages, we used two experimental paradigms: one in which the HFD was started just after weaning categories: 1) muscle related, with three related gene sets myoﬁbril, sarcomere, and contractile ﬁber genes (P , 0.02); (3 weeks old) and a second in which mice were started on the HFD as young adults (8–10 weeks old). As shown in 2) monooxygenase activity; 3) developmental; and 4) negative Fig. 4A and B, APC number increased after 2 and 4 weeks regulation of cytokine biosynthesis (Table 3). Genes most differentially expressed between Vis and on HFD in Vis and SC depots in both young and adult mice; however, the increase in APCs was larger when the SC APCs. The most differentially expressed gene, which was highly expressed in Vis APCs, was the transcription mice were started on the HFD at a young age. Thus, Vis APC numbers increased by 80 and 60% after 2 and 4 weeks factor Tcf21, whereas in SC APCs, the most differentially expressed gene was the transcription factor Lhx8 (LIM on HFD in young mice compared with 60 and 30% increases in adult mice. This difference was more marked in SC APCs, homeobox 8). Another interesting gene with signiﬁcantly higher expression in SC versus Vis APCs was Cd24. This with 80 and 270% increases in cell numbers after 2 and 4 weeks on HFD in young mice compared with only 30 membrane protein was previously used as a surface marker (10) to isolate the APCs from a mixture of Vis and SC white and 40% increases in adult mice. By contrast, the change in other SVF cells was greatest in the Vis depot but similar fat depots. It is clear that this would result in preferential isolation of the SC APCs from this mixed fat cell pool. A in young and adult mice, with increases of 210–270% in both young and adult mice on the HFD, whereas the number heat map listing the 100 most differentially expressed genes of other SVF cells in the SC depot increased modestly is shown in Supplementary Fig. 2. Effect of sex, strain, and age on adipocyte precursor (60 and 70% for young and adult mice, respectively). Thus, HFD increases the number of other SVF cells only in the Vis frequency. To evaluate how the number of APCs and other SVF cells change between different genetic and environ- depot, consistent with the increase in inﬂammatory cells in this compartment (23). When the percentage of APCs in the mental conditions, we analyzed APC frequency in young (2-month-old) versus old (1-year-old) mice, in obesity-prone depot was calculated, the percentage of APCs increased by 250 and 180% after 2 and 4 weeks on HFD in young animals (C57BL/6) versus obesity-resistant (129Sv/J) mice, and in male versus female mice. Aging altered dramatically the but was unchanged in adult animals. Thus, young pre- pubertal mice have an increase in APCs on HFD that is number of APCs. When expressed per gram of adipose tis- sue, APC numbers were twofold higher in young than in old greater than that seen in adult animals, especially in the SC depot (Fig. 4). mice in both depots (Fig. 3A). However, because fat mass increases with age, the total number of APCs per depot was increased by more than threefold in Vis and more than DISCUSSION fourfold in SC fat in old versus young mice. The number of non-APCs (i.e., other SVF cells) per gram of fat was eight- Adipose tissue serves as an important regulator of metabolic fold higher in SC fat of young versus old mice but similar in homeostasis, and in this regard, not all fat is functionally both age-groups in the Vis depot (Fig. 3A). However, the equivalent. Thus, accumulation of Vis fat is associated with total number of other SVF cells per fat pad was almost insulin resistance and a high risk for type 2 diabetes and sixfold higher in Vis fat of old versus young mice, while it metabolic syndrome, whereas accumulation of SC fat has remained similar in the SC depot between young and old been shown to have a possible protective value against mice. Therefore, when the number of APCs was calculated these metabolic abnormalities. This appears to relate to 1694 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES TABLE 2 Depot-speciﬁc signature of APCs and adipocytes from Vis and SC fat Fold difference Gene name Gene symbol Adipo Preads Localization Depot-speciﬁc signature—Vis Angiotensinogen Agt 7.5 5.8 Secreted Aldo-keto reductase family 1, member C12 Akr1c12 2.6 6.9 Cytoplasm AMP deaminase 3 Ampd3 2.5 2.8 Cytoplasm Anillin, actin binding protein (scraps homolog, Drosophila) Anln 5.3 2.8 Cytoplasm/Nucleus Cysteine and glycine-rich protein 1 Csrp1 2.6 6.2 Nucleus Cytochrome P450, family 1, subfamily b, polypeptide 1 Cyp1b1 11.0 3.2 ER/Microsomes Hydroxyprostaglandin dehydrogenase 15 (NAD) Hpgd 2.2 2.0 Cytoplasm Nuclear receptor subfamily 2, group F, member 1 Nr2f1 5.5 3.0 TF Nuclear receptor subfamily 4, group A, member 1 Nr4a1 3.2 2.3 TF Pterin 4 a carbinolamine dehydratase (TCF1) 1 Pcbd1 2.5 2.6 TF Protocadherin 7 Pcdh7 2.6 2.1 PM Plakophilin 2 Pkp2 2.9 2.8 PM Prolactin receptor Prlr 3.8 2.9 PM Serum amyloid A3 pseudogene Saa3p 2.6 5.2 Secreted Solute carrier family 8, member 1 Slc8a1 2.3 4.2 PM Secretory leukocyte peptidase inhibitor Slp1 3.1 18.7 Secreted Serine/arginine-rich protein speciﬁc kinase 2 Srpk2 2.2 2.2 Cytoplasm/Nucleus Thrombospondin 1 Thbs1 5.8 3.6 Secreted Transcription factor 21 Tcf21 3.0 216.2 TF Depot-speciﬁc signature—SC Angiopoietin-like 1 Angptl1 3.5 2.1 Secreted Apolipoprotein D Apod 3.8 2.0 Secreted Chemokine (C-C motif) ligand 8 Ccl8 3.4 4.2 Secreted Cadherin 2 Cdh2 2.9 2.5 PM Cadherin 9 Cdh9 2.9 17.1 PM Procollagen, type XII, a 1 Col12a1 3.4 3.4 Secreted Procollagen, type I, a 1 Col1a1 4.7 3.3 Secreted Early B-cell factor 3 Ebf3 2.0 4.1 TF Engrailed 1 En1 8.0 4.5 TF Estrogen receptor 1 (a) Esr1 2.5 3.0 TF Fibroblast growth factor 13 Fgf13 2.1 3.8 Secreted Fibronectin type III domain containing 1 Fndc1 2.9 2.0 Secreted Histocompatibility 2, Q region locus 10 H2q10 4.2 10.8 Membrane Interferon-g–inducible protein 47 Iﬁ47 4.0 2.8 ER/Membrane Interferon-g–induced GTPase Igtp 2.0 2.4 Cytoplasm/Membrane Interleukin-18 receptor 1 Il18r1 16.5 25.0 PM Myelin basic protein Mbp 3.9 9.1 Nucleus/Cytoplasm/IntMem Matrix metallopeptidase 3 Mmp3 3.5 21.5 Secreted Major urinary protein 1 /// major urinary protein 2 Mup1/Mup2 375.1 3.6 Secreted Major urinary protein 3 Mup3 63.5 10.3 Secreted Osteomodulin Omd 3.8 2.4 Secreted Procollagen C-endopeptidase enhancer protein Pcolce 2.8 2.9 Secreted Protein disulﬁde isomerase associated 5 Pdia5 5.9 2.1 ER Serine (or cysteine) peptidase inhibitor, clade F, member 1 Serpinf1 2.9 2.0 Secreted Secreted frizzled-related protein 2 Sfrp2 5.4 2.6 Secreted Short stature homeobox 2 Shox2 8.7 95.4 TF Sine oculis-related homeobox 4 homolog (Drosophila) Six4 2.2 2.1 TF ST6-N-acetylgalactosaminide a-2,6-sialyltransferase 5 St6galnac5 2.3 2.8 Golgi T-box 15 Tbx15 12.2 145.4 TF Transm prot with EGF-like and 2 follistatin-like domains 2 Tmeff2 3.0 2.0 PM Ubiquitin D Ubd 5.7 2.3 Cytoplasm Ubiquitin speciﬁc peptidase 18 Usp18 3.7 5.0 Cytoplasm ER, endoplasmic reticulum; TF, transcription factor; PM, plasma membrane; IntMem, internal membrane. intrinsic differences in the adipocytes in these fat depots With regard to differentiation, previous studies show that (2,5,24–26). In the current study, we demonstrate that APCs SVF cells taken from SC fat of either rodents or humans from SC and Vis fat have important differences in terms of differentiate more readily in culture than those from Vis fat their gene expression signatures, their ability to differenti- (26–28). We show that the same is true for isolated APCs ate, their response to growth factors, and their regulation in from these depots but ﬁnd that this is because the APCs normal physiology and disease. from SC and Vis depots have different differentiation diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1695 ADIPOCYTE PRECURSOR CELL SUBTYPES TABLE 3 Gene set enrichment analysis Gene set Gene symbol Gene name Fold change P value Enriched in Vis APCs Muscle related Des Desmin 5.5 0.000 Myoﬁbril Tpm2 Tropomyosin 2 (b) 3.6 0.020 Sarcomere Tpm1 Tropomyosin 1 (a) 2.5 0.000 Contractile ﬁber Myoz2 Myozenin 2 4.8 0.005 Tnnt2 Troponin T type 2 4.5 0.019 Tnni3 Troponin I type 3 2.4 0.019 Monooxygenase activity Pah Phenylalanine hydrogenase 18.8 0.000 Fmo2 Flavin containing monooxygenase 2 4.7 0.000 Cyp1b1 Cytochrome P450 family 1 subfamily b 3.2 0.000 Cyp27a1 Cytochrome P450 family 27 subfamily a 2.4 0.000 Fmo1 Flavin containing monooxygenase 1 2.1 0.000 Cyp7b1 Cytochrome P450 family 7 subfamily b 1.9 0.008 Developmental Mesoderm development Tcf21 Transcription factor 21 216.2 0.000 Eya2 Eyes absent homolog 2 4.9 0.003 Slit2 Slit homolog 2 5.5 0.000 Mest Mesoderm speciﬁc transcript hom 1.6 0.010 Developmental maturation Rnd1 r family GTPase 1 2.7 0.001 Ereg Epiregulin 1.9 0.015 Pick1 Protein interacting with PRKCA1 1.4 0.006 Tgfb2 Transforming growth factor, b 2 1.8 0.010 Myh11 Myosin, heavy chain 11, s 1.9 0.040 Negative regulation of cytokine biology Inhbb Inhibin, b B 8.6 0.001 Inhba Inhibin, b A 3.9 0.000 Ela2 Elastase 2 2.5 0.001 Il6 Interleukin 6 1.8 0.001 Enriched in SC APCs Chemokines Cxcl9 Chemokine (C-C motif) ligand 9 5.3 0.001 Chemokine activity Cxcl14 Chemokine (C-X-C motif) ligand 14 4.2 0.002 Chemokine receptor binding Ccl8 Chemokine (C-C motif) ligand 8 4.2 0.000 Cxcl13 Chemokine (C-X-C motif) ligand 13 5.4 0.007 Cxcl10 Chemokine (C-X-C motif) ligand 10 1.9 0.000 Ccl22 Chemokine (C-C motif) ligand 22 5 0.007 Peptidase activity Cysteine type endopeptidase activity Lgmn Legumain 2.8 0.001 Casp9 Caspase 9 2.8 0.003 Ctsk Cathepsin K 1.7 0.000 Casp1 Caspase 1 1.9 0.009 Usp7 Ubiquitin speciﬁc peptidase 1.7 0.001 Uchl1 Ubiquitin carboxyl-terminal 1.3 0.010 Ctsb Cathepsin B 1.2 0.002 Aminopeptidase activity Trhde TRH-degrading enzyme 10.2 0.000 Lnpep Leucyl/cystinyl aminopeptidase 1.5 0.018 Anpep Alanyl (membrane) aminopeptidase 1.8 0.020 Vesicular fraction Stx16 Syntaxin 16 1.9 0.007 Microsome Mgst3 Microsomal gluthation S-transferase 3 1.7 0.001 Oas2 29-59-oligoadenylate synthetase 2 2.2 0.03 Ltc4s Leukotriene C4 synthase 4.5 0.04 Ero1lL ERO1-like 1.4 0.02 Slc35b1 Solute carrier family 35, member b1 1.3 0.02 Sts Steroid sulfatase 1.3 0.01 Tmed2 Transmembrane emp24 domain trafﬁcking protein 2 1.4 0.01 Copa Coatomer protein complex subunit a 1.5 0.001 requirements. While SC APCs differentiate well using the C3H10T1/2 to an adipocyte lineage (29), whereas com- standard IC, Vis APCs differentiate very poorly under the mitted cell lines, such as 3T3-L1, do not have this re- same conditions. However, pretreatment of Vis APCs with quirement. Indeed, Vis APCs are more like mesenchymal BMP-4 or BMP-2 allows these cells to undergo full differ- stem cells and less committed to adipocyte differentiation entiation. BMP-2 and BMP-4 have been shown to be re- than cells bearing the same surface markers from SC fat. quired for commitment of the mesenchymal stem cell line Vis APCs also show higher levels of expression of Gata2 1696 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES FIG. 3. Genetic and environmental impact on number (#) and percentage (%) of APCs and other SVF cells from Vis and SC fat depots. FACS sorting 2 2 analysis of Vis and SC APCs and other SVF cells. Data were calculated using only live cells. Double positive CD34, SCA1 (and CD45 , CD31 ,and 2 + + + Ter119 ) cells were considered APCs, whereas CD45 ,CD31 ,and Ter119 cells were considered as other SVF cells. A: 8-week-old vs. 1-year-old C57BL/6 mice. m, month. B: C57BL/6 (B6) vs. 129 mice (3 months old). C: C57BL/6 male (M) vs. female (F) mice (6 months old). Five mice per group were used in two independent experiments. Data are mean 6 SEM. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. and Tgfb2, factors known to inhibit adipogenesis (23,30), indicating the isolated APCs from both depots do represent as well as higher levels of expression of mesenchymal the preadipocyte pool. This resistance to differentiation may stem cell markers (Lif, Ctgf, and Mgp), whereas SC APCs explain why Vis fat expansion during adulthood occurs show higher expression of proadipogenic genes (Pparg, mainly by hypertrophy, whereas SC fat expansion is due + + Cebpa, Bmp2, Bmp4, and Dkk2). Thus, Sca1 and CD34 primarily to hyperplasia (32). SC APCs are more committed to the adipocyte lineage Although others have analyzed differences in gene ex- than Vis APCs expressing the same markers and have pression between Vis and SC adipose tissue (5,19,33), ours fewer requirements for becoming mature adipocytes. is the ﬁrst study to identify genes that are differentially At a cellular level, this difference in differentiation is due expressed between APCs in different depots. Tcf21 is the to a blockade in the ability of the IC to stimulate expres- most differentially expressed gene between Vis and SC sion of Pparg. Thus, after treatment with IC, including the adipocyte precursors, with the higher levels in Vis cells. PPARg agonist rosiglitazone, Vis APCs show increased This transcription factor also shows higher expression in Cebpa but a failure of induction of Pparg and downstream mature adipocytes of Vis fat (34). While Tcf21 has been adipogenic markers. BMP-2 and BMP-4 signals overcome described (34) as a marker for white preadipocytes com- the block and induce differentiation in Vis APCs. This may pared with brown preadipocytes and muscle, in this study, be in part because SC APCs already have enough endoge- preadipocytes were isolated only from epididymal fat, nous BMP to trigger cell commitment, or it may depend which have much higher expression than SC preadipocytes. on other differences between the two adipocyte precursor The levels of Tcf21 in SC preadipocytes are similar to those types. BMPs have been shown to induce Pparg through in brown preadipocytes and muscle. activation of schnurri-2, which forms a transcriptional com- The most differentially expressed gene in SC APCs is the plex with C/EBPa and SMAD1/4 (31). However, we ﬁnd no LIM homeobox gene Lhx8. This gene had been previously difference in the level of schnurri-2 (Hivep2) between Vis and described as a marker for brown preadipocytes when ep- SC fat. In any case, in the presence of BMP-2, BMP-4, and IC, ididymal fat was the only source of WAT (34). However, 2 + + almost all of the Lin ,CD34 , and SCA1 cells isolated from Lhx8 is not unique to brown preadipocytes but also a strong both Vis and SC depots are able to become adipocytes, marker for SC preadipocytes (162-fold higher than in Vis diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1697 ADIPOCYTE PRECURSOR CELL SUBTYPES FIG. 4. Effect of HFD on number (#) and percentage (%) of APCs and other SVF cells from Vis and SC fat depots. FACS sorting analysis of Vis and 2 2 2 SC APCs and other SVF cells. Data were calculated using only live cells. Double positive CD34, SCA1 (and CD45 , CD31 , and Ter119 ) cells were + + + considered APCs, whereas CD45 , CD31 , and Ter119 cells were considered as other SVF cells. FACS sorted cells from mice placed on chow diet (CD) or HFD for 1, 2, and 4 weeks starting at age 4 weeks (A) or 8 weeks (B). Three to ﬁve mice per group were used in two independent experiments. Data are mean 6 SEM. Statistics were analyzed by Student t test (two-way, unequal variance). *P < 0.05. APCs). Thus, the pattern of gene expression in SC white especially in Vis fat. While sex has no effect on number of fat has several similarities with that of brown fat, as com- APCs in the SC depot, females show a trend to increased pared with Vis white fat. Another gene more highly ex- APCs and lower other SVF cells per gram of fat in Vis fat. pressed in SC APCs than in Vis precursors is Cd24. This This could reﬂect a reduced number of macrophages in the membrane protein was previously used as a marker (10) to Vis depot of female mice and account for the higher insulin purify the APCs but should probably not be used unless sensitivity of adipocytes from Vis fat of females compared one is attempting to separate different populations from with those from males (35). a mixture of white fat depots. Finally, the potential for new adipocytes also parallels Our study also shows the dynamic nature of the adipo- the genetic risk for obesity. Thus, the obesity-prone cyte precursor pool. We ﬁnd that HFD increases the num- C57BL/6 mouse has approximately twofold more APCs per ber of APCs in both Vis and SC depots by as much as 270%. gram of fat than the obesity-resistant 129 mouse by age The increase is bigger when animals are started on HFD at 3 months. In adult animals, this is accompanied by in- a young age compared with when challenged as adults. Joe creased content of lymphocytes and macrophages and et al. (32) reported a similar increase in bromodeoxyuridine expression of proinﬂammatory genes in Vis fat of C57BL/6 labeling of APCs in SC fat but a much smaller increase in mice, contributing further to the tendency of C57BL/6 mice labeling of Vis fat after 60 days of HFD. Whether this reﬂects to develop insulin resistance upon aging or HFD-induced differences in methodology, age of the mice, and a longer obesity (36). period of HFD is unknown. In conclusion, APCs from Vis and SC depots differ dra- Another difference between depots after challenge with matically in terms of their differentiation ability, gene ex- HFD is that the number of non-APCs in the Vis depot pression signatures, growth factor requirements, and increases dramatically after 2–4 weeks on HFD, whereas capacity to expand upon genetic and environmental stim- the number of other SVF cells in the SC depot remains uli. SC APCs are more committed to differentiate, whereas constant. This causes a dramatic change in the relative those from Vis depots have an anti-adipogenic proﬁle and proportion of cell types in these depots. Thus, the per- require growth factors, such as BMP-2 or BMP-4, to un- centage of APCs in Vis fat decreases in mice on HFD dergo differentiation. This helps explain why SC fat ex- compared with those on chow diet, whereas the percent- pands more by hyperplasia, whereas Vis fat expands by age of APCs in the SC depot either increases or remains hypertrophy. These depot-speciﬁc gene signatures can constant. This could explain why SC fat stays metaboli- serve as a powerful tool to identify and manipulate spe- cally ﬁt in obesity, whereas Vis fat develops inﬂammation, ciﬁc characteristics in the differentiation and functional contributing to insulin resistance. properties of adipocytes and ultimately should help us un- Fat mass increases with age, as does the number of derstand how these two fat depots have opposing contri- APCs per depot, by three- to fourfold or more. As with butions to the development of diabetes and metabolic HFD, however, the number of other SVF cells also increases, syndrome. 1698 DIABETES, VOL. 61, JULY 2012 diabetes.diabetesjournals.org Y. MACOTELA AND ASSOCIATES 12. Schulz TJ, Huang TL, Tran TT, et al. Identiﬁcation of inducible brown ACKNOWLEDGMENTS adipocyte progenitors residing in skeletal muscle and white fat. Proc Natl This work was supported by National Institutes of Health Acad Sci U S A 2011;108:143–148 Grant R01-DK-082659 and the Mary K. Iacocca Professor- 13. Ahrens M, Ankenbauer T, Schröder D, Hollnagel A, Mayer H, Gross G. ship. The study was also facilitated by support from the Expression of human bone morphogenetic proteins-2 or -4 in murine mes- Joslin Diabetes and Endocrinology Research Center Core enchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 1993;12:871–880 Laboratories (DK-036836). Y.M. was supported by the Pew 14. Bowers RR, Lane MD. A role for bone morphogenetic protein-4 in adipo- Latin American Fellows Program in the Biological Sciences, cyte development. Cell Cycle 2007;6:385–389 the Mentor-Based American Diabetes Association Grant 15. Schulz TJ, Tseng YH. Emerging role of bone morphogenetic proteins in Award, and the Mexican Council of Science (CONACyT) adipogenesis and energy metabolism. Cytokine Growth Factor Rev 2009; Postdoctoral Fellowship. C.R.K. was supported by a grant 20:523–531 from the Loveman Foundation. 16. Rosen ED, Hsu CH, Wang X, et al. C/EBPalpha induces adipogenesis through PPARgamma: a uniﬁed pathway. Genes Dev 2002;16:22–26 No potential conﬂicts of interest relevant to this article 17. Igarashi A, Segoshi K, Sakai Y, et al. Selection of common markers for were reported. bone marrow stromal cells from various bones using real-time RT-PCR: Y.M. conceived and designed the experiments, researched effects of passage number and donor age. Tissue Eng 2007;13:2405–2417 data, and wrote the manuscript. B.E. researched data, 18. Vohl MC, Sladek R, Robitaille J, et al. A survey of genes differentially contributed to discussion, and reviewed and edited the expressed in subcutaneous and visceral adipose tissue in men. Obes Res 2004;12:1217–1222 manuscript. M.A.M. and S.G. researched data and re- 19. Tchkonia T, Lenburg M, Thomou T, et al. Identiﬁcation of depot-speciﬁc viewed and edited the manuscript. T.J.S. set up the meth- human fat cell progenitors through distinct expression proﬁles and de- odology, contributed to discussion, and reviewed and velopmental gene patterns. Am J Physiol Endocrinol Metab 2007;292: edited the manuscript. Y.-H.T. contributed to discussion E298–E307 and reviewed and edited the manuscript. C.R.K. conceived 20. Salans LB, Cushman SW, Weismann RE. Studies of human adipose tissue. and designed the experiments, reviewed the data, and Adipose cell size and number in nonobese and obese patients. J Clin Invest wrote the manuscript. C.R.K. is the guarantor of this work 1973;52:929–941 21. Hirsch J, Batchelor B. Adipose tissue cellularity in human obesity. Clin and, as such, had full access to all the data in the study and Endocrinol Metab 1976;5:299–311 takes responsibility for the integrity of the data and the 22. Lemonnier D. Effect of age, sex, and site on the cellularity of the adipose accuracy of the data analysis. tissue in mice and rats rendered obese by a high-fat diet. J Clin Invest 1972; Parts of this study were presented in abstract form at the 51:2907–2915 70th Scientiﬁc Sessions of the American Diabetes Associ- 23. Wu H, Ghosh S, Perrard XD, et al. T-cell accumulation and regulated on ation, Orlando, Florida, 25–29 June 2010. activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity. Circulation 2007;115:1029–1038 The authors thank Joyce LaVecchio, Giri Buruzula, and 24. Hauner H, Entenmann G. Regional variation of adipose differentiation in the Flow Cytometry core for help with cytometry; Joshua cultured stromal-vascular cells from the abdominal and femoral adipose Schroeder and the Genomics core for microarray process- tissue of obese women. Int J Obes 1991;15:121–126 ing; Michael Rourk and Graham Smyth for their expertise 25. Lafontan M, Girard J. Impact of visceral adipose tissue on liver metabo- in animal care; and Madelana Basile and Jen Davison for lism. Part I: heterogeneity of adipose tissue and functional properties of editing the manuscript (all from Joslin Diabetes Center). visceral adipose tissue. Diabetes Metab 2008;34:317–327 26. Tchkonia T, Giorgadze N, Pirtskhalava T, et al. Fat depot-speciﬁc char- acteristics are retained in strains derived from single human pre- REFERENCES adipocytes. Diabetes 2006;55:2571–2578 1. Misra A, Garg A, Abate N, Peshock RM, Stray-Gundersen J, Grundy SM. 27. Prunet-Marcassus B, Cousin B, Caton D, André M, Pénicaud L, Casteilla L. Relationship of anterior and posterior subcutaneous abdominal fat to in- From heterogeneity to plasticity in adipose tissues: site-speciﬁc differ- sulin sensitivity in nondiabetic men. Obes Res 1997;5:93–99 ences. Exp Cell Res 2006;312:727–736 2. Snijder MB, Dekker JM, Visser M, et al. Associations of hip and thigh 28. Adams M, Montague CT, Prins JB, et al. Activators of peroxisome pro- circumferences independent of waist circumference with the incidence of liferator-activated receptor g have depot-speciﬁc effects on human pre- type 2 diabetes: the Hoorn Study. Am J Clin Nutr 2003;77:1192–1197 adipocyte differentiation. J Clin Invest 1997;100:3149–3153 3. Tran TT, Yamamoto Y, Gesta S, Kahn CR. Beneﬁcial effects of sub- 29. Bowers RR, Kim JW, Otto TC, Lane MD. Stable stem cell commitment to cutaneous fat transplantation on metabolism. Cell Metab 2008;7:410–420 the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 4. Wajchenberg BL, Giannella-Neto D, da Silva ME, Santos RF. Depot-speciﬁc gene. Proc Natl Acad Sci U S A 2006;103:13022–13027 hormonal characteristics of subcutaneous and visceral adipose tissue and 30. Tong Q, Dalgin G, Xu H, Ting CN, Leiden JM, Hotamisligil GS. Function of their relation to the metabolic syndrome. Horm Metab Res 2002;34:616–621 GATA transcription factors in preadipocyte-adipocyte transition. Science 5. Gesta S, Blüher M, Yamamoto Y, et al. Evidence for a role of developmental 2000;290:134–138 genes in the origin of obesity and body fat distribution. Proc Natl Acad Sci 31. Jin W, Takagi T, Kanesashi SN, et al. Schnurri-2 controls BMP-dependent U S A 2006;103:6676–6681 adipogenesis via interaction with Smad proteins. Dev Cell 2006;10:461–471 6. Yamamoto Y, Gesta S, Lee KY, Tran TT, Saadatirad P, Kahn CR. Adipose 32. Joe AW, Yi L, Even Y, Vogl AW, Rossi FM. Depot-speciﬁc differences in depots possess unique developmental gene signatures. Obesity (Silver adipogenic progenitor abundance and proliferative response to high-fat Spring) 2010;18:872–878 diet. Stem Cells 2009;27:2563–2570 7. Cantile M, Procino A, D’Armiento M, Cindolo L, Cillo C. HOX gene network 33. Atzmon G, Yang XM, Muzumdar R, Ma XH, Gabriely I, Barzilai N. Differ- is involved in the transcriptional regulation of in vivo human adipogenesis. ential gene expression between visceral and subcutaneous fat depots. J Cell Physiol 2003;194:225–236 Horm Metab Res 2002;34:622–628 8. Tchoukalova YD, Koutsari C, Votruba SB, et al. Sex- and depot-dependent 34. Timmons JA, Wennmalm K, Larsson O, et al. Myogenic gene expression differences in adipogenesis in normal-weight humans. Obesity (Silver signature establishes that brown and white adipocytes originate from Spring) 2010;18:1875–1880 distinct cell lineages. Proc Natl Acad Sci U S A 2007;104:4401–4406 9. Spalding KL, Arner E, Westermark PO, et al. Dynamics of fat cell turnover 35. Macotela Y, Boucher J, Tran TT, Kahn CR. Sex and depot differences in in humans. Nature 2008;453:783–787 adipocyte insulin sensitivity and glucose metabolism. Diabetes 2009;58: 10. Rodeheffer MS, Birsoy K, Friedman JM. Identiﬁcation of white adipocyte 803–812 progenitor cells in vivo. Cell 2008;135:240–249 36. Mori MA, Liu M, Bezy O, et al. A systems biology approach identiﬁes in- 11. Tang W, Zeve D, Suh JM, et al. White fat progenitor cells reside in the ad- ﬂammatory abnormalities between mouse strains prior to development of ipose vasculature. Science 2008;322:583–586 metabolic disease. Diabetes 2010;59:2960–2971 diabetes.diabetesjournals.org DIABETES, VOL. 61, JULY 2012 1699

Journal

Diabetes – Pubmed Central

Published: Jun 15, 2012

References

MisraAGargAAbateNPeshockRMStray-GundersenJGrundySM Relationship of anterior and posterior subcutaneous abdominal fat to insulin sensitivity in nondiabetic men. Obes Res 1997;5:93–999112243
SnijderMBDekkerJMVisserM Associations of hip and thigh circumferences independent of waist circumference with the incidence of type 2 diabetes: the Hoorn Study. Am J Clin Nutr 2003;77:1192–119712716671
TranTTYamamotoYGestaSKahnCR Beneficial effects of subcutaneous fat transplantation on metabolism. Cell Metab 2008;7:410–42018460332
WajchenbergBLGiannella-NetoDda SilvaMESantosRF Depot-specific hormonal characteristics of subcutaneous and visceral adipose tissue and their relation to the metabolic syndrome. Horm Metab Res 2002;34:616–62112660870
GestaSBlüherMYamamotoY Evidence for a role of developmental genes in the origin of obesity and body fat distribution. Proc Natl Acad Sci U S A 2006;103:6676–668116617105
YamamotoYGestaSLeeKYTranTTSaadatiradPKahnCR Adipose depots possess unique developmental gene signatures. Obesity (Silver Spring) 2010;18:872–87820111017
CantileMProcinoAD’ArmientoMCindoloLCilloC HOX gene network is involved in the transcriptional regulation of in vivo human adipogenesis. J Cell Physiol 2003;194:225–23612494461
TchoukalovaYDKoutsariCVotrubaSB Sex- and depot-dependent differences in adipogenesis in normal-weight humans. Obesity (Silver Spring) 2010;18:1875–188020300084
SpaldingKLArnerEWestermarkPO Dynamics of fat cell turnover in humans. Nature 2008;453:783–78718454136
RodehefferMSBirsoyKFriedmanJM Identification of white adipocyte progenitor cells in vivo. Cell 2008;135:240–24918835024
TangWZeveDSuhJM White fat progenitor cells reside in the adipose vasculature. Science 2008;322:583–58618801968
SchulzTJHuangTLTranTT Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proc Natl Acad Sci U S A 2011;108:143–14821173238
AhrensMAnkenbauerTSchröderDHollnagelAMayerHGrossG Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 1993;12:871–8808274220
BowersRRLaneMD A role for bone morphogenetic protein-4 in adipocyte development. Cell Cycle 2007;6:385–38917314508
SchulzTJTsengYH Emerging role of bone morphogenetic proteins in adipogenesis and energy metabolism. Cytokine Growth Factor Rev 2009;20:523–53119896888
RosenEDHsuCHWangX C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev 2002;16:22–2611782441
IgarashiASegoshiKSakaiY Selection of common markers for bone marrow stromal cells from various bones using real-time RT-PCR: effects of passage number and donor age. Tissue Eng 2007;13:2405–241717596118
VohlMCSladekRRobitailleJ A survey of genes differentially expressed in subcutaneous and visceral adipose tissue in men. Obes Res 2004;12:1217–122215340102
TchkoniaTLenburgMThomouT Identification of depot-specific human fat cell progenitors through distinct expression profiles and developmental gene patterns. Am J Physiol Endocrinol Metab 2007;292:E298–E30716985259
SalansLBCushmanSWWeismannRE Studies of human adipose tissue. Adipose cell size and number in nonobese and obese patients. J Clin Invest 1973;52:929–9414693656
HirschJBatchelorB Adipose tissue cellularity in human obesity. Clin Endocrinol Metab 1976;5:299–3111085232
LemonnierD Effect of age, sex, and site on the cellularity of the adipose tissue in mice and rats rendered obese by a high-fat diet. J Clin Invest 1972;51:2907–29155080416
WuHGhoshSPerrardXD T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity. Circulation 2007;115:1029–103817296858
HaunerHEntenmannG Regional variation of adipose differentiation in cultured stromal-vascular cells from the abdominal and femoral adipose tissue of obese women. Int J Obes 1991;15:121–1262040549
LafontanMGirardJ Impact of visceral adipose tissue on liver metabolism. Part I: heterogeneity of adipose tissue and functional properties of visceral adipose tissue. Diabetes Metab 2008;34:317–32718550411
TchkoniaTGiorgadzeNPirtskhalavaT Fat depot-specific characteristics are retained in strains derived from single human preadipocytes. Diabetes 2006;55:2571–257816936206
Prunet-MarcassusBCousinBCatonDAndréMPénicaudLCasteillaL From heterogeneity to plasticity in adipose tissues: site-specific differences. Exp Cell Res 2006;312:727–73616386732
AdamsMMontagueCTPrinsJB Activators of peroxisome proliferator-activated receptor γ have depot-specific effects on human preadipocyte differentiation. J Clin Invest 1997;100:3149–31539399962
BowersRRKimJWOttoTCLaneMD Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 gene. Proc Natl Acad Sci U S A 2006;103:13022–1302716916928
TongQDalginGXuHTingCNLeidenJMHotamisligilGS Function of GATA transcription factors in preadipocyte-adipocyte transition. Science 2000;290:134–13811021798
JinWTakagiTKanesashiSN Schnurri-2 controls BMP-dependent adipogenesis via interaction with Smad proteins. Dev Cell 2006;10:461–47116580992
JoeAWYiLEvenYVoglAWRossiFM Depot-specific differences in adipogenic progenitor abundance and proliferative response to high-fat diet. Stem Cells 2009;27:2563–257019658193
AtzmonGYangXMMuzumdarRMaXHGabrielyIBarzilaiN Differential gene expression between visceral and subcutaneous fat depots. Horm Metab Res 2002;34:622–62812660871
TimmonsJAWennmalmKLarssonO Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages. Proc Natl Acad Sci U S A 2007;104:4401–440617360536
MacotelaYBoucherJTranTTKahnCR Sex and depot differences in adipocyte insulin sensitivity and glucose metabolism. Diabetes 2009;58:803–81219136652
MoriMALiuMBezyO A systems biology approach identifies inflammatory abnormalities between mouse strains prior to development of metabolic disease. Diabetes 2010;59:2960–297120713682

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Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots

Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots

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Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots

Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots

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