¹Department of Pediatrics, University of Ulm, Ulm, Germany

²Department of Orthopaedics, University of Ulm, Ulm, Germany

³Department of Pathology, University of Ulm, Ulm, Germany

⁴Diabetes Research Institute at the University of Duesseldorf, Duesseldorf, Germany

Correspondence to: M Wabitsch, Department of Pediatrics, University of Ulm, Prittwitzstr. 43, 89075 Ulm, Germany. E-mail: martin.wabitsch@medizin.uni-ulm.de

OBJECTIVE: To develop and to characterize a human preadipocyte cell strain with high capacity for adipose differentiation serving as a model for studying human adipocyte development and metabolism in vitro.

METHODS: Cells were derived from the stromal cells fraction of subcutaneous adipose tissue of an infant with Simpson-Golabi-Behmel syndrome (SGBS). Adipose differentiation was induced under serum-free culture conditions by exposure to 10 nM insulin, 200 pM triiodothyronine, 1 µM cortisol and 2 µM BRL 49653, a PPAR agonist.

RESULTS: During the differentiation process SGBS cells developed a gene expression pattern similar to that found in differentiating human preadipocytes with a characteristic increase in fat cell-specific mRNAs encoding lipoprotein lipase (LPL), glycero-3-phosphate dehydrogenase (GPDH), GLUT4, leptin and others. Differentiated SGBS cells exhibited an increase in glucose uptake upon insulin stimulation and in glycerol release upon catecholamine exposure. SGBS adipocytes were morphologically, biochemically and functionally identical to in vitro differentiated adipocytes from healthy subjects. However, while preadipocytes from healthy control infants rapidly lost their capacity to differentiate after a few cell divisions in culture, SGBS cells maintained their differentiation capacity over many generations: upon appropriate stimulation 95% of SGBS cells of generation 30 developed into adipocytes. A mutation in the glypican 3 gene was not detected in the patient. Thus, it remains unclear whether the molecular alteration in SGBS cells is also responsible for the high differentiation capacity and further investigations are required.

CONCLUSION: The human cell strain described here provides an almost unlimited source of human preadipocytes with high capacity for adipose differentiation and may, therefore, represent a unique tool for studying human fat cell development and metabolism.

International Journal of Obesity (2001) 25, 8-15

human adipocyte; human preadipocyte; differentiation; peroxisome proliferator activated receptor

Introduction

The study of human fat cell development and metabolism has become an important issue in the last decade.¹ Adipocyte differentiation in humans is known to be a multi-step process that is regulated by a variety of factors in a complex manner.² Today, primary cultures of human preadipocytes are widely used to study adipose differentiation and metabolism since it has emerged that substantial differences exist in the developmental pattern and function of adipocytes among species.² However, the technique of primary culture has its limitations. A particular disadvantage is that adipose tissue samples from different donors and possibly from different anatomical sites may lead to some unpredictable variation in the characteristics of the cells. In addition, the amount of available tissue is usually rather limited, which may complicate experimental studies.

Such problems prompted efforts to establish clonal, human-derived preadipocte cell lines which should not only exhibit stable cellular characteristics but should also provide an unlimited source of homogenous cell material. However, previous attemps to establish a human preadipocyte cell line were not convincingly successful. To our knowledge, there have only been two human preadipocyte cell lines described so far: one cell line was derived from brown adipose tissue³ and another one derived from white adipose tissue with only low differentiation rates.⁴ In a recent study, Tontonoz et al reported that human liposarcoma cells can be induced to undergo adipose differentiation in the presence of thiazolidinediones.⁵ These cells may have an undefined differentiation block that can be overcome by maximal activation of the peroxisome proliferator-activated receptor (PPAR) pathway.

In the present study, we describe a human preadipocyte cell strain derived from subcutaneous white adipose tissue of a patient with Simpson-Golabi-Behmel syndrome (SGBS), a rare X-linked disorder characterized by pre- and postnatal overgrowth (MIM 312870).^6,7,8 The molecular defect causing this syndrome is not yet known, although mutations in the Glypican 3 gene, a gene involved in the control of organ growth, has been identified as associated with the syndrome in some of the reported patients.^9,10 The cell strain established exhibits a high capacity for adipose differentiation, resulting in mature fat cells which are biochemically and functionally similar to human adipocytes.

Methods

Materials

Culture media were obtained from Life Technologies (Gaithersburg, MD, USA). Radio-labelled chemicals were purchased from Amersham (Buckinghamshire, UK). Other chemicals and reagents were purchased from Sigma (Munich, Germany). BRL 49653 was a gift from SmithKline Beecham (London, UK). Recombinant human insulin and human growth hormone were kindly provided by Novo Nordisk (Gentofte, Denmark), and recombinant human IGF-I by Eli Lilly (Bad Homburg, Germany).

Patients characteristics and adipose tissue collection

Patient with Simpson - Golabi - Behmel syndrome. The male infant was born after 35 weeks of gestation by Caesarean section due to prenatal overgrowth. The patient had a birth weight of 3.840 kg, a birth length of 50 cm, a head circumference of 39.5 cm, and showed the clinical signs of Simpson-Golabi-Behmel-syndrome (MIM 312870). The main features were expanded subcutaneous fat depots, a disproportionately large head with prominent forehead, a coarse facies, a wide nasal bridge and upturned nasal tip, a large mouth, a central cleft of the lower lip, postaxial hexadactyly of all four limbs and an additional lumbal vertebra. The patient died at the age of 3 months due to a malignant tumor with angioblastic and mainly trophoblastic components. A sample of subcutaneous adipose tissue (500 mg) was obtained post-mortem with informed consent from the parents.

Control subjects. To compare the differentiation capacity of SGBS cells with preadipocytes from normal infants subcutaneous adipose tissue samples (100-300 mg) were obtained from two male and one female healthy infants at the age of 1, 6 and 7 months undergoing herniotomia. The study was approved by the Ethical Committee of the University of Ulm.

Cell culture

Preadipocytes were prepared from adipose tissue samples by collagenase digestion and cultured according to an established protocol.¹¹ Pre-confluent cells were repeatedly subcultured in DMEM/Ham's F12 (1:1) medium containing 10% FCS and antibiotics. The preadipocytes from the infant with Simpson-Golabi-Behmel syndrome (SGBS) were called SGBS cells. The SGBS cultures have been repeatedly shown to be mycoplasma-free. In the experiments performed either serum-containing medium or serum-free basal medium (DMEM/F12 (1:1) supplemented with 10 µg/ml transferrin, 15 mM NaHCO₃, 15 mM HEPES, 33 mM biotin, 17 mM pantothenate, and antibiotics) was used.

Cell proliferation

Cell proliferation experiments were performed by seeding cells at low density (4000/cm²). To determine the proliferation rate, cells were detached at the time points indicated with Hanks' balanced salt solution (HBSS) containing 0.05% trypsin and 0.02% EDTA and counted in a hemocytometer. The doubling time of the cells was estimated from growth curves during the exponential growth phase. In addition to the direct cell counts [³H]thymidine incorporation (0.2 mCi/well) was used as a measure of DNA synthesis as described previously.¹²

Induction of differentiation

Preadipocytes were grown in serum-containing medium until reaching confluence. To induce adipose differentiation cells were repeatedly washed with PBS buffer and cultured thereafter in serum-free, basal medium supplemented with 10 nM insulin, 200 pM triiodothyronine, and 1 µM cortisol (adipogenic medium) with or without addition of 2 µM BRL 49653 for the first 10 days. In some cultures 500 µmol/l 1-methyl-3-isobutylxanthine (IBMX) and 0.25 µmol/L dexamethasone was added to increase the differentiation rate. The medium was changed twice a week. The percentage of morphologically differentiated adipocytes was determined after 20 days of culture by microscopic counting of total cell number and the number of differentiated cells in defined areas in random order.

RT-reaction and duplex PCR

Reverse transcripase (RT)-reaction and duplex-polymerase chain reaction (PCR) were performed as described previously with some modifications.^13,14 To study the time-course of expression of adipocyte-specific genes during differentiation of SGBS cells total RNA was extracted by the method of Chomczynski and Sacchi¹⁵ at different time points during differentiation (day 0: undifferentiated cells, day 10: during adipose differentiation; day 20: after differentiation). RNA-specific primer pairs (GC-content about 50%, PCR product length 200-500 bp) were selected from human cDNA libraries (Wisconsin Package Version 9.1, Genetic Computer Group, Madison, WI) and were used for semiquantitative duplex PCR with the transcription factor Sp1 as internal standard. Electrophoretically separated PCR products were stained with ethidium bromide and the fluorescence intensities were measured using the Image Master VDS system (Pharmacia Biotec, San Francisco, CA). Results are expressed as ratios between the specific cDNA transcript and the internal marker.

Determination of GPDH activity glucose transport, lipogenesis and lipolysis

The activity of GPDH in cell extracts was measured as shown earlier.¹⁶ Enzyme activity was expressed in milliunits per mg of cellular protein, 1 mU being equal to the oxidation of 1 nmol NADH/min. Transmembraneous glucose transport was determined using non-metabolizable [³H]2-deoxy-D-glucose and lipogenesis was assessed by incubation with 3-D-[¹⁴C]glucose as described earlier.¹² Glycerol release was determined as a measure of lipolysis and was measured by a luminometric kinetic assay.¹⁷

Leptin radioimmunoassay

Leptin concentrations in the media from SGBS cultures were measured at different time points of the adipose differentiation process and under defined culture conditions using a highly sensitive radioimmunoassay described recently.¹⁸

Cytogenetics and comparative genome hybridization (CGH) analysis

Metaphase chromosomes were prepared from peripheral blood cells of the patient and from SGBS cells of the 30th generation and G-banded according to conventional procedures and karyotyped. DNA-hybridization was performed according to an established method.¹⁹

Glypican 3 mutation analysis

DNA was isolated from peripheral blood cells by the standard salt extraction method.²⁰ Genomic DNA from the patient and unaffected controls were amplified for individual exons and flanking intronic sequences of the GPC3 gene by PCR using oligonucleotide primers designed according to the genomic sequences deposited in the gene bank. Single-stranded conformational polymorphism analysis was performed according to Bunge et al.²¹

Results

Cytogenetics and mutation analysis

The karyotype of the patient with SGBS obtained from peripheral blood cells was normal, with 46,XY with a structurally abnormal chromosome 22 (enlarged section of the short arm), probably without clinical significance since the father of the patient showed the same abnormality and had a normal phenotype. The karyotype of SGBS cells of the 30th generation was identical to the one seen in peripheral blood cells of the patient. No mutation in the gene for Glypican 3 could be detected by single-stranded conformational polymorphism analysis.

Proliferation of SGBS cells

SGBS cells were morphologically fibroblast-like, like the stromal cells from adipose tissue samples from the healthy control infants. To assess growth characteristics cells were inoculated at a low density and cultured in a medium containing 10% fotal calf serum. Under these conditions, SGBS cells had a doubling time of 38.4±1.0 h, slightly higher than the doubling time of stromal cells from the three healthy infants (31.3±2.6 h). Both, SGBS cells and preadipocytes from the control subjects reached confluence at a density of approximately 25 000/cm².

Differentiation of SGBS cells into adipocytes

When SGBS cells, after having reached confluency, were cultured in serum-free medium without adipogenic factors, none of the cells underwent adipose differentiation. However, the vast majority of SGBS cells cultured in serum-free medium supplemented with insulin, triiodothyronine and cortisol developed into mature adipocytes within 20 days. During this period, most cells accumulated lipids and finally showed the typical morphological criteria of in vitro-differentiated adipose cells, such as round shape and a cytoplasma filled with lipid droplets (Figure 1). When differentiated cells were cultured for another 5-10 days in the adipogenic medium many cells developed a monolocular appearance. Insulin and cortisol had a similar dose-dependent stimulatory effect on adipose differentiation of SGBS cells as on stromal cells from healthy controls.

Subculture of SGBS cells in serum-containing medium resulted in a continuous decline of the potential for differentiation in adipogenic medium. However, addition of the PPAR agonist BRL 49653 to the adipogenic medium stimulated adipose differentiation in subcultures in a dose-dependent fashion, with a maximal effect at 2 µM/l, thus inhibiting the fast decline of differentiation and preserving a high capacity for differentiation over many generations (Figure 2). At the 10th generation 24% of the SGBS cells differentiated into adipocytes in adipogenic medium without BRL 49653, whereas 92% of the cells differentiated in adipogenic medium supplemented with 2 µM BRL 49653. In the 50th generation, none of the cells underwent adipose differentiation in the adipogenic medium alone, but still more than 50% converted into fat cells in adipogenic medium supplemented with 2 µM BRL 49653. The percentage of differentiated cells could be significantly stimulated by addition of 500 µmol/l IBMX and 0.25 µmol/l dexamethasone for the first 3 days to adipogenic medium supplemented with BRL49653, resulting in differentiation rates of 95% at generation 30 and 78% at generation 50 (data not shown).

Comparison of the capacity for adipose differentiation between SGBS cells and primary preadipocytes from control infants

Between 75 and 80% of the preadipocytes derived from healthy infants differentiated into adipocytes in normal adipogenic medium after first inoculation. When preadipocytes from the three control infants were allowed to proliferate in serum-containing medium and were subsequently subcultured, their capacity for adipose differentiation decreased rapidly, even in the presence of BRL 49653. In the absence of BRL 49653, adipose differentiation was observed for not more than eight generations (data not shown), whereas in the presence of 2 µM BRL 49653 the differentiation rate of cells in generation 8 was between 20% and 53%.

However, also under these conditions, preadipocytes from control infants completely lost their capacity for adipose differentiation until generation 16 (Table 1). The addition of IBMX and dexamethasone for the first 3 days could not prevent the fast decline in the differentiation capacity of control cells (data not shown).

Gene expression pattern during the differentiation of SGBS cells

The phenotypical changes during adipose differention of SGBS cells were associated with marked changes in the gene expression pattern. Figure 3 shows the results of a representative semi-quantitative RT-PCR experiment with RNA extracted before (day 0), during (day 10), and after differentiation (day 20) as well as from SGBS cells cultured in basal medium for 20 days. The time-course of specific mRNA expression showed early increases of specific mRNAs for LPL and LBP and late increases of mRNAs encoding for GLUT 4, leptin and GPDH (Figure 3). This pattern was not different to that found in differentiating human preadipocytes from normal subjects (data not shown).

Functional characteristics of in vitro differentiated SGBS adipocytes

In accordance with the expression of GPDH mRNA, the activity of this enzyme, which is essential for triglyceride synthesis, increased during adipose differentiation from undetectable levels on day 1 to activities between 1100 and 1600 U/mg protein on day 20 in a time-course similar to that observed in human adipocyte precursor cells in primary culture.

Another feature of mature adipocytes is the development of a insulin-responsive glucose transport system. Figure 4 shows the effect of insulin on glucose uptake in differentiated SGBS cells. Insulin was found to exert a dose-dependent stimulation on 2-deoxy-D-glucose uptake (Figure 4A). Maximal glucose uptake was more than 10-fold higher in differentiated SGBS adipocytes as compared to preadipocytes. A similar pattern was seen when incorporation of [¹⁴C]-labelled D-glucose into cellular lipids was determined. Again, there was a dose-dependent stimulation of glucose uptake by increasing concentrations of insulin in differentiated SGBS adipocytes, whereas no detectable incorporation of glucose was observed in undifferentiated SGBS (Figure 4B), indicating that lipid accumulation in differentiated SGBS cells was due to lipogenesis from glucose. For comparison we determined insulin-stimulated glucose-transport in in vitro differentiated adipocytes from adult control patients (n=3), which showed a comparable ED₅₀ but a slightly lower maximal effect (76.0±8.9 vs 87.5±7.5 cpm/(10⁶ cells 10 min).

In vitro differentiated SGBS adipocytes not only expressed HSLm RNA but also became sensitive to lipolytic -adrenergic agents. Figure 5 shows the dose-dependent stimulation of glycerol release into the culture medium after exposure to isoprenaline and adrenaline. The effects of the two adrenergic compounds were almost superimposible, with maximal effects at 1 µM.

Release of leptin by in vitro differentiated SGBS adipocytes

It is now well established that human adipocytes produce and release leptin in proportion to the state of development and fat cell size.²² Using a highly specific and sensitive radioimmunoassay the release of leptin into the culture medium was measured in SGBS cells before and after adipose differentiation. While leptin was not detectable in undifferentiated SGBS cells, there was a significant increase of leptin release into the culture medium of up to 3.3 ng/24 h per 10⁶ adipocytes (data not shown).

Prerequisites for utilization of SGBS cells as a model for studying human fat cell differentiation and metabolism with an almost unlimited source of homogeneous cells

Repeated freezing and thawing of SGBS cells did not change their proliferation behavior nor their differentiation capacity. Together with the findings described above, this indicates that there is an almost infinitive number of SGBS cells with high capacity for adipose differentiation, eg 10⁶ cells at generation 10 will provide 2²⁰´10⁶=1´10¹³ cells at generation 30. Ninety-five percent of SGBS cells at generation 30 differentiate into adipocytes when they are cultured in adipogenic medium supplemented with 2 µM BRL 49653, and with 0.25 mmol/l IBMX and dexamethasone for the first 3 days. Differentiated SGBS cells could be maintained for more than 3 weeks in a chemically defined medium without losing their viability, allowing the performance of long-term incubation experiments.

Discussion

The results of these experiments clearly show that SGBS cells represent a human cell strain with a high capacity for adipose differentiation. In contrast to preadipocytes from control patients, SGBS cells retain their potential to develop into adipocytes over at least 50 generations. In addition, in vitro differentiated SGBS adipocytes demonstrate stable expression of adipocyte-specific genes and functional characteristics of adipose cells over this period, making these cells a valuable tool for studying fat cell development and function in a human model. SGBS cells do not fullfill the criteria for a cell line since they are not immortalized and after approximately 70 generations they lose their capacity to proliferate, and finally die (data not shown).

This cell strain was established from a patient with Simpson-Golabi-Behmel syndrome. To date, the molecular defect of most cases with SGBS is unknown. Since there is no biochemical test for reliable identification available, diagnosis is based upon typical clinical signs (MIM 312870).^6,7,8 The exaggerated growth is probably due to an imbalance between growth-promoting and growth-inhibitory factors at specific early developmental stages, resulting in defective growth inhibition or apoptosis in certain cell types.^23,24 Recently, a mutation in the gene encoding for glypican 3 was detected in a minority of patients with SGBS.¹⁰ Glypican-3 is a cell-surface proteoglycan which is involved in the control of organ growth.^25,26 However, the exact role of glypican-3 in the control of organ growth is not known. Like the majority of patients with SGBS, our patient did not exhibit the mutation of the glypican-3 gene. The mutation screening, however, does not completely exclude a point mutation or a mutation affecting the expression of glypican-3.

SGBS cells kept a high capacity to develop into adipocytes under serum-free culture conditions in the presence of insulin, triiodothyronine and cortisol over several generations, which is not seen in preadipocytes from control infants as shown here or in preadipocytes from adults as reported earlier.¹¹ A high capacity for adipose differentiation could be further maintained over many generations by addition of the PPAR agonist BRL 49653. A quantitatively comparable effect of BRL 49653 was not found in subcultures of preadipocytes from control infants.

The adipogenic effect of thiazolidinediones is well known. They are ligands of PPAR and upon binding to this nuclear receptor they are able to activate the PPAR/RXR complex. Activation of PPAR was found to be a critical event for the induction of adipose differentiation.²⁷ The activated PPAR/RXR heterodimer binds to DNA and regulates the transcription of adipocyte-specific genes.²⁸ The high sensitivity of SGBS cells for the action of PPAR agonists is obviously a characteristic of the SGBS cell strain. If this is a specific feature of preadipocytes from patients with this syndrome, it could help to explain the clinical finding of an enlarged fat mass. However, this finding has to be confirmed in cultures from other patients with SGBS. It also remains an interesting challenge to clarify the mechanism responsible for the high sensitivity to PPAR agonists and the subsequent high differentiation capacity. Further studies are required to address these issues.

Apart from their high capacity for adipose differentiation, SGBS cells behave similarly to human preadipocytes in primary culture. Doubling time and density at confluence were comparable between the two models. The pattern and time-course of gene expression in SGBS cells during differentiation was comparable to the findings in human preadipocytes in primary culture and also resembled the characteristics of preadipocyte cell lines derived from rodents like 3T3-L1, 3T3-F442A or ob17 cells.² In addition, our investigations of glucose transport, lipogenesis and lipolysis showed that in vitro differentiated SGBS adipocytes were functionally not distinguishable from human adipocytes. This similarity allows SGBS cells to be used as a model system of human adipocytes which may offer some advantages, such as fewer limitations in terms of cell resources and high homogeneity between passages. The almost unlimited number of, for example generation 30 SGBS cells in our laboratory, possessing a differentiation rate of 95%, allows us to also distribute these cells to other scientists interested in studying human fat cell development and metabolism.

In conclusion, we decribe here for the first time a human preadipocyte cell strain which demonstrates a high capacity for adipose differentiation over many generations, providing an almost unlimited source of identical human preadipocytes and in vitro differentiated adipocytes. Differentiated SGBS cells behave biochemically and functionally like human adipocytes differentiated in primary culture. Therefore, this cell strain represents a new useful tool for the study of human fat cell development and metabolism in vitro.

Acknowledgements

Cytogenetics and CGH analysis were performed by Dr M. Djalali, Department of Medical Genetics, University of Ulm, and GPC3 mutation analysis by Ulrike Orth, Institute of Human Genetics, University of Hamburg, Germany. The authors wish to thank Simone Schmid, Uta Späth and Silvia Röderer for their excellent technical assistance. The authors gratefully acknowledge the helpful discussions and technical advices of Dr Hans Tornqvist, Department of Pediatrics, University of Lund, Sweden, and Dr Claude Marcus, Department of Pediatrics, Huddinge University Hospital, Sweden. This work was partly supported by a grant from the Deutsche Forschungsgemeinschaft (WA 1096/1-1).

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Figure 1 Photomicrographs of SGBS cells before and after adipose differentiation (32nd generation). (A) Undifferentiated cells during the proliferation phase (magnification 200-fold). (B) Undifferentiated cells in confluent state (magnification 100-fold). (C) Differentiated cells on day 20 (magnification 100-fold). (D) Differentiated cells on day 25, Oil Red Staining (magnification 100-fold).

Figure 2 Adipose differentiation of SGBS-preadipocytes in dependency of cell generation. After confluence SGBS cells were cultured in serum-free, chemically defined adipogenic medium with and without addition of the PPAR-agonist BRL 49653. The percentage of differentiated cells was determined on day 20. Results are presented as means of a representative experiment (s.d.<10%).

Figure 3 Time-course of gene-expression during differentiation of SGBS cells. RNA was extracted from SGBS cells grown in adipogenic medium (+) on day 0, 10, and 20 corresponding to undifferentiated cells (day 0+), cells during differentiation (day 10+), and differentiated cells (day 20+), and from SGBS cells grown in non-adipogenic medium (-) on day 20 as control of undifferentiated cells (day 20-). Specific mRNA expression was measured by RT-PCR and expressed as ratio of specific mRNA to Sp1 mRNA.

Figure 4 Glucose uptake and lipogenesis in SGBS-cells. (A) Cellular uptake of 2-deoxy-D-glucose was determined in differentiated (circles) and undifferentiated (triangles) SGBS cells in response to increasing insulin concentrations as described in the Methods. Means±s.d. of three independent experiments are given in duplicate. (B) Lipogenesis was measured in differentiated (circles) and undifferentiated (triangles) SGBS cells in response to increasing insulin concentrations as described in the Methods. The results are expressed as radioactivity (cpm) incorporated into cellular lipids. Means±s.d. of three independent experiments are given in duplicate.

Figure 5 Lipolysis in SGBS cells. SGBS adipocytes were stimulated with increasing concentrations of adrenaline (triangles) or isoprenaline (circles) and glycerol release into the culture medium was measured as described in the Methods. Results are given as means±s.d. of three independent experiments in triplicate.

Table 1 Capacity for adipose differentiation of preadipocytes derived from healthy control infants and of SGBS cells