Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots

By Virginia Duncan,2014-12-30 19:27
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    Intrinsic Differences in Adipocyte

    Precursor Cells From Different

    White Fat Depots

    1. Yazmín Macotela,

    2. Brice Emanuelli,

    3. Marcelo A. Mori,

    4. Stephane Gesta,

    5. Tim J. Schulz,

    6. Yu-Hua Tseng and

    7. C. Ronald Kahn?

    1. Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 1. Corresponding author: C. Ronald Kahn, c.ronald.kahn{at} Next Section


    Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. In the current study, we demonstrate that adipocyte precursor cells (APCs) isolated from visceral and subcutaneous white adipose depots of mice have distinct patterns of gene expression, differentiation potential, and response to environmental and genetic influences. APCs derived from subcutaneous fat differentiate well in the presence of classical induction cocktail, whereas those from visceral fat differentiate poorly but can be induced to differentiate by addition of bone morphogenetic protein (BMP)-2 or BMP-4. This difference correlates with major differences in gene expression signature between subcutaneous and visceral APCs. The number of APCs is higher in obesity-prone C57BL/6 mice than obesity-resistant 129 mice, and the number in both depots is increased by up to 270% by exposure of mice to high-fat diet. Thus, APCs from visceral and subcutaneous depots are dynamic populations, which have intrinsic differences in gene expression, differentiation properties, and responses to environmental/genetic factors. Regulation of these populations may provide a new target for the treatment and prevention of obesity and its metabolic complications. There are two main types of white adipose tissue (WAT) in humans and rodents

    subcutaneous (SC) fat and visceral (intra-abdominal [Vis]) fat. These contribute differentially to disease risk. Accumulation of Vis adipose tissue is associated with adverse metabolic outcomes, whereas increased amounts of SC fat has been viewed as neutral or even beneficial in its metabolic effects (13). While part of this

    difference may be related to anatomical location with different patterns of venous drainage, over the past few years, it has become clear that these two types of WAT differ in their intrinsic characteristics, including levels of adipokine secretion, insulin sensitivity, lipolysis rate, and tendency to develop inflammation (4). We and others

    (57) recently have shown that this correlates with differences in gene expression between adipocytes in different depots, including the expression of fundamental development and patterning genes. Differences in differentiation and developmental gene expression have also been observed in human preadipocytes from different depots (8), but exactly how this relates to intrinsic differences in adipose tissues and the propensity for obesity and insulin resistance is unclear.

Spalding et al. (9) recently demonstrated that in humans, ?10% of the adipocyte pool

    turns over annually and that the absolute number of adipocytes turning over in individuals with obesity is approximately double from that in lean individuals. Exactly how adipocytes in different depots turn over and to what extent this is influenced by genetic or environmental factors is unknown. However, using lineage tracing and fluorescence-activated cell sorting (FACS) strategies, two groups (10,11) have

    developed techniques to identify and isolate the adipocyte precursor cells (APCs) from the stromovascular fraction (SVF) of white fat. In the current study, we show that APCs are dynamic and respond to different environmental and genetic factors. We show that APCs from Vis versus SC fat differ in their specific gene expression signatures and that SC APCs are more adipogenic and require fewer growth factors, whereas Vis APCs have anti-adipogenic characteristics and require additional growth factor stimulation to differentiate. These results show that there are intrinsic differences between preadipocytes from different depots and that these differences contribute to the differential properties and turnover of adipocytes in different depots. Previous SectionNext Section



    C57BL/6 mice and 129 mice were obtained from The Jackson Laboratory and kept under a normal diurnal cycle in a temperature-controlled room. Mice were fed with standard chow containing 22% of calories from fat (Mouse Diet 9F 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 used starting at aged 3 and 8 weeks. Animal care and study protocols were approved by the animal care committee of Joslin Diabetes Center and were in accordance with National Institutes of Health guidelines.

    Adipocyte precursor isolation and flow cytometry.

    Epididymal and inguinal fat pads, representing Vis and SC fat, respectively, were cut in small pieces and incubated with 1 mg/mL collagenase I for 30 min. The cell suspension was filtered through a 150-μmol/L nylon mesh, and the SVF was isolated

    by low-speed centrifugation. For FACS analysis, erythrocyte-free SVF cells were incubated with a mix of antibodies against different surface markers as described previously (10) and sorted using an Aria flow cytometer (BD Biosciences). Dead cells 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 modified Eagle’s medium–low glucose (Invitrogen) and

    40% MCDB201 (Sigma-Aldrich) and supplemented with Normocin 0.1 mg/mL (InvivoGen), 10% FBS, 1× insulin-transferrin-selenium mix, 1× linoleic acid conjugated to BSA, 1 nmol/L dexamethasone, and 0.1 mmol/L L-ascorbic acid 2-

    phosphate (Sigma-Aldrich) was used as growth medium. This medium was further supplemented with 10 ng/mL epidermal growth factor (PeproTech), 10 ng/mL leukemia inhibitory factor (Millipore), 10 ng/mL platelet-derived growth factor BB (PeproTech), and 5 ng/mL basic fibroblast growth factor (Sigma-Aldrich). Once sorted, cells were rinsed with medium and plated at 50,000 cells per well in 24-well plates. After 67 days, cells reached ?80% confluence. Medium was changed every other day. For differentiation, cells were seeded at 20,000 cells per well in 48-well plates. After cells reached 80% confluence, they were treated or not for 2 days with 3.3 nmol/L BMP-2 or BMP-4 (R&D Systems) in growth medium with 2% FBS and without growth factors. Medium was then replaced by differentiation medium (growth medium with no growth factors but with 2% FBS, 1 μmol/L dexamethasone, 0.5 μmol/L

    isobutylmethylxanthine, 100 nmol/L insulin, and 1 μmol/L rosiglitazone) for 3 days,

    after which the medium was replaced with growth medium containing 2% FBS and 100 nmol/L insulin for 2 more days; for the last 23 days of differentiation, cells were

    incubated with growth medium with 2% FBS alone.

    RNA extraction and PCR.

    RNA was extracted from cells using RNeasy (QIAGEN). Reverse transcription was performed with 0.3 μg RNA by using High Capacity cDNA Reverse Transcription Kit

    (Applied Biosystems, Foster City, CA). Real-time PCR was performed using Maxima SYBR Green (Fermentas, Glen Burnie, MD) in duplicate using the ABI Prism 7900 System (Applied Biosystems) under the following conditions: 50?C ?2 min, 95?C ?10 min, and 40 cycles of 95?C ?15 s, 60?C ?20 s, and 72?C ?30 s. Dissociation protocols were conducted after every run to check for primer specificity. To obtain ?ΔCtrelative expression values, we calculated the 2 parameter for each individual

    sample using cycle threshold values of TATA-box-binding protein as an endogenous control.


    6Approximately 1 × 10 sorted cells per sample were used to isolate RNA for the microarray analysis. Five independent RNA samples each from a pool of isolated APCs from 10 different mice from each depot were analyzed on Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). All data were subjected to global normalization to an intensity of 1,500 using the Gene Chip Software MAS V. 5.0. All differences reported had a P value <0.05.

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    Isolation of APCs from different WAT depots.

    APCs were isolated by FACS from SVFs of Vis and SC fat from mice as described above and in Supplementary Fig. 1. Cells negative for CD45, CD31, and Ter119 and positive for CD34 and SCA1 were considered APCs. Cells positive for CD45, CD31, and/or Ter119 (endothelial cells, platelets, macrophages, white blood cells, osteoclasts, and erythrocytes) were designated as ―other SVF.‖ Only live cells were

    counted. In a typical sorting, using male C57BL/6 mice aged 8 weeks, Vis fat 55contained 0.9 ? 0.1 × 10 APCs and 3.5 ? 0.8 × 10 other SVF cells per fat pad, 55whereas SC fat contained 0.65 ? 0.08 × 10 APCs and 7.1 ? 0.7 × 10 other SVF cells

    per pad (Fig. 1A). Thus, the percentage of stromovascular cells, which are APCs, was almost three times higher in Vis fat than in SC fat (21 ? 3.7 vs. 7.8 ? 1.7%) (Fig. 1B).

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    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 depots isolated from male mice aged 79 weeks. 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. A: Number of cells per

    depot. B: Percent of APCs in both depots relative to other SVF cells. Data are mean ? SEM of n = 4 independent experiments. Statistics were analyzed by Student t test

    (two-way, unequal variance). *P < 0.05.

    APCs from different fat depots differ in their differentiation requirements.

    To confirm that the isolated APCs were preadipocytes, we analyzed the differentiation ability of both Vis and SC APCs. Confluent APC cultures were treated with the typical induction protocol, including rosiglitazone, and differentiation was assessed by lipid accumulation and expression of markers of terminal differentiation, including Slc2a4

    (Glut-4), Adipoq (adiponectin), Lep (leptin), and Fabp4 (fatty acid binding protein or

    aP2). Using this approach, APCs from the SC fat differentiated well within 7 days after induction, with >90% of the cells containing large lipid droplets (Fig. 2A). In fact,

    ?10% of SC APCs differentiated in the absence of any hormonal induction (Fig. 2A).

    By contrast, with the same protocol, <20% of APCs from the Vis depot could be induced to differentiate (Fig. 2A). This raised two possibilities: one was that the Vis APCs were not really adipocyte precursors; the other was that the APCs were missing some signals required for differentiation.