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Separation of human adipocytes by size: hypertrophic fat cells display distinct gene expression

Margareta Jernås^*, Jenny Palming^*, Kajsa Sjöholm^*, Eva Jennische, Per-Arne Svensson^*, Britt G. Gabrielsson^*, Max Levin, Anders Sjögren, Mats Rudemo, Theodore C. Lystig^*, Björn Carlsson^*, Lena M. S. Carlsson^* and Malin Lönn^*,1

^* Research Centre for Endocrinology and Metabolism, Division of Body Composition and Metabolism, Department of Internal Medicine,

Institute of Anatomy and Cell Biology, and

Cardiovascular Institute and Wallenberg Laboratory, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden; and

Department of Mathematical Statistics, Chalmers University of Technology, Göteborg, Sweden

¹ Correspondence: RCEM/Division of Body Composition and Metabolism, Department of Internal Medicine, Vita Stråket 15, SE 413 45 Göteborg, Sweden. E-mail: malin.lonn{at}medic.gu.se

SPECIFIC AIMS

Enlargement of subcutaneous (s.c.) abdominal adipocytes is associated with insulin resistance and is an independent predictor of type 2 diabetes. The aim of the present study was to detect factors linking human adipocyte hypertrophy to insulin resistance/type 2 diabetes.

PRINCIPAL FINDINGS

1. Isolated human adipocytes, from a single adipose tissue sample, can be separated into populations of small cells and large cells
Most previous studies of the impact of adipocyte size, have studied fat cells or biopsies with different mean adipocyte diameters obtained from different tissue locations or even from different donors. Therefore, differences in environmental conditions or genetic factors that affect adipocyte gene expression and metabolism could not be excluded. Thus, it is not clear whether the functions of the fat cell vary with adipocyte size per se.

In the present study, a technique for separation of human adipocytes by size was developed. The technique, based on cell buoyancy and mesh filtration, separated isolated adipocytes from an adipose tissue sample into populations of small cells (mean 57.6±3.54 µm) and large cells (mean 100.1±3.94 µm). The mean size and the size distribution of the small and large populations, determined by computerized image analysis, differed significantly (P<0.005 and P<0.001, respectively) (Fig. 1 ).

View larger version (83K): [in this window] [in a new window]   Figure 1. Separation of human adipocytes by size. A) Representative images of human adipocytes from one adipose tissue sample before separation (All) and after separation (Small and Large). Microspheres (98.00 µm in diameter) were used for reference in the computerized image analysis. B) Size distributions, determined by computerized image analysis, and mean diameters in random samples of all adipocytes (n=742), small adipocytes (n=1965), and large adipocytes (n=2382) from one adipose tissue sample. Patient characteristics; man, age: 41 yr, body mass index (BMI): 26.0 kg/m2. C) Mean diameter of small and large adipocytes from 12 adipose tissue biopsies. Error bars indicate SEM.

2. Microarray analysis of the cell populations identified genes with markedly higher mRNA expression in large cells than small cells
Gene expression profiling (Affymetrix GeneChip HG-U133A arrays composed of 22,283 probe sets) of the cell populations separated from adipose tissue from three subjects identified 14 genes with more than 4-fold higher expression in large cells than small cells (P< 0.01) (Table 1 ). Classification by cellular or organism function based on Gene Ontology definitions revealed that five of those genes were immune-related. The remaining nine genes were referred to structure (four), unknown function (three), growth (one) and transport (one) (Table 1) . Differences in sample preparation or hybridization were excluded since there was no difference between small and large adipocytes in the expression of LRP10, CLN3, or COBRA1, suitable reference genes for studies of human adipose tissue.

3. SAA and TM4SF1 were 20-fold higher expressed in the large adipocytes as determined by real-time RT-PCR, and adipocyte size correlated with the expression of both SAA and TM4SF1
One immune-related gene, serum amyloid A (SAA), and one gene with unknown function, transmembrane 4 L six family member 1 (TM4SF1), were selected among the genes with >4-fold higher expression in large vs. small cells for further analysis (Table 1) . Leptin, being 3-fold higher expressed in the large adipocytes (data not shown), and previously suggested to be higher expressed in large compared with small adipocytes, was also included in the following studies.

The up-regulation of SAA, TM4SF1 and leptin expression in large cells was confirmed by real-time RT-PCR chain reaction analysis of small (mean 59.3±4.47 µm) and large (mean 97.1±5.69 µm) adipocytes from seven different adipose tissue samples. In all cases, SAA, TM4SF1 and leptin were expressed at higher levels in large cells (P=0.018). The mean fold increase in expression was 18.7 ± 15.1 for SAA, 22.3 ± 6.4 for TM4SF1, and 3.9 ± 1.4 for leptin. In addition, adipocyte size correlated with the expression of SAA (P=0.015), TM4SF1 (P=0.012), and leptin (P=0.0009).

4. In comparison with 17 other human tissues/cell types by microarray, large adipocytes displayed by far the highest SAA and TM4SF1 expression
GeneChip HG-U133A expression profiles from 17 different tissues were downloaded from the SymAtlas dataset (http://symatlas.gnf.org/Symatlas/). For comparison of gene expression in different tissues, the signal value for each gene was normalized by dividing the signal by the average signal of the entire array for each tissue. In addition, our own expression profiles, originating from small and large adipocytes, were included and normalized as outlined above. SAA, TM4SF1 and leptin expression levels in large adipocytes were compared to the levels in other human tissues and small adipocytes. The three genes were expressed at markedly higher levels in large adipocytes than in all other tissues/cell types.

5. The higher mRNA expression of SAA and TM4SF1 in large compared to small adipocytes was reflected also at the protein level
Expression of SAA and TM4SF1 in adipocytes was also demonstrated immunohistochemically. Although SAA immunoreactivity varied between cells of the same size, it was generally greater in large than in small adipocytes in the same sample. TM4SF1 immunoreactivity was demonstrated mainly in large but to some extent also in medium-sized adipocytes. Positive TM4SF1 signal appeared in a dot-like pattern in the cell membrane. Small adipocytes were completely without TM4SF1 immunoreactivity.

CONCLUSIONS AND SIGNIFICANCE

In this study, we developed a new technique to separate populations of small and large human adipocytes from a single adipose tissue sample. The two populations of cells obtained with our technique differed significantly in size, as determined with a computer-based image-analysis method that allows rapid analysis of 10-fold more cells than conventional methods. DNA microarray analysis of the two populations showed that several genes were expressed at markedly higher levels in the large cells, demonstrating that hypertrophy per se can significantly alter gene expression and thereby presumably adipocyte function (Fig. 2 ).

View larger version (47K): [in this window] [in a new window]   Figure 2. Schematic diagram. Flow chart and summary of the principal findings (A). Potential mechanisms by which serum amyloid A (SAA) and transmembrane 4 L six family member 1 (TM4SF1), expressed at the highest levels by large adipocytes, may mediate a link between hypertrophic obesity and insulin resistance/type 2 diabetes (B).

Previous studies of the metabolic activity of small and large adipocytes have indicated that adipocyte size influences various adipocyte metabolic functions. However, the cells of different sizes were obtained from different adipose tissue locations or from different donors. Thus, it was not possible to exclude environmental influences, such as nutritional/hormonal conditions, or genetic factors that might have affected gene expression and thus adipocyte metabolism. Our technique avoids these problems and will facilitate metabolic studies of fat cells of different sizes. For example, a positive correlation between human adipocyte size and leptin expression/secretion has previously been suggested. In the present study of human adipocytes from a single adipose tissue sample, the previous findings were confirmed since leptin was indeed expressed at higher levels in the large cells in all cases. Moreover, we identified several genes that, compared with leptin, showed a more pronounced differential expression in large vs. small adipocytes (Fig. 2) .

Among the fourteen genes with markedly higher expression in large compared with small adipocytes, five were classified as immune-related; E-selectin, interleukin-8, SAA, C1q receptor 1, and CXCL2 also known as MIP-2 or macrophage inflammatory protein-2. Components of the metabolic syndrome, such as obesity and type 2 diabetes, are associated with a systemic increase in inflammatory markers. The acute-phase proteins SAA and C-reactive protein have attracted particular attention because they are independent risk factors for coronary artery disease. We, and others, have previously shown that adipose tissue is a major site of SAA production and is likely to be a major source of circulating SAA in obese patients. The current study extends these findings by demonstrating that SAA is expressed at the highest concentration by the large adipocytes. SAA has been implicated in inflammation, insulin resistance and impairment of reverse cholesterol transport. Our data may therefore suggest that adipocyte-derived SAA, likely having both local effects and endocrine functions, is a potential mediator of the link between hypertrophic adipocytes and type 2 diabetes (Fig. 2) .

To summarize, we have developed a technique to separate human adipocytes, from a single adipose tissue sample, by size. The resulting populations of small and large adipocytes have significantly different cell size distributions. Gene expression profiling of the small and large adipocytes identified genes, many of them immune-related, with markedly higher mRNA expression in the large cells. Two of those genes, SAA (an acute-phase protein implicated in inflammation, insulin resistance and impairment of reverse cholesterol transport) and TM4SF1 (a membrane protein with unknown function), were 20-fold higher expressed in the large cells, a difference reflected also at the protein level. Moreover, in comparison with several other human tissues, large adipocytes displayed by far the highest SAA and TM4SF1 expression. In the light of previous studies reporting that adipocyte hypertrophy is associated with insulin resistance and is an independent predictor of type 2 diabetes, the findings in the current work provide novel insights into the molecular connection between hypertrophic obesity and insulin resistance/type 2 diabetes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5678fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5678fjev1 20/9/1540    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jernås, M. Articles by Lönn, M. Search for Related Content PubMed PubMed Citation Articles by Jernås, M. Articles by Lönn, M.