High expression of leptin by human bone marrow adipocytes in

High expression of leptin by human bone marrow adipocytes in primary culture PATRICK LAHARRAGUE,*,1 DOMINIQUE LARROUY,* ANNE-MARIE FONTANILLES,* NATHALIE TRUEL,* ARTHUR CAMPFIELD,† RENATA TENENBAUM,† JEAN GALITZKY,‡ ¨ L X. CORBERAND,* LUC PE´NICAUD,* AND LOUIS CASTEILLA* JOE *UPRESA-CNRS 5018, UPS, IFR L. Bugnard, Faculte´ de Me´decine, Toulouse, France; †Hoffmann-La Roche, Nuttley, N.J. 07110–1199, USA; and ‡Unite´ INSERM 317, IFR L. Bugnard, Faculte´ de Me´decine Toulouse, France

Adipocytes participate in the microenvironment of the bone marrow (BM), but their exact role remains to be determined. It has recently been shown that leptin, a hormone secreted from extramedullary adipocytes, could be involved in hematopoiesis. Therefore we have developed a primary culture system of human BM adipocytes to characterize their differentiation and determine whether leptin is also secreted from these adipocytes. BM cells were cultured with fetal calf and horse sera. In the presence of dexamethasone, cells with vesicles containing lipids appeared within 15 days. They expressed glycerol phosphate dehydrogenase activity and a lipolytic activity in response to isoproterenol, but expressed neither the adrenergic b3 receptor nor the mitochondrial uncoupling protein UCP1. The addition of insulin alone to the culture media did not promote adipocyte differentiation. Leptin was expressed and secreted at high levels during adipocyte differentiation. Acute exposure of differentiated adipocytes to insulin had little effect on leptin expression whereas forskolin strongly inhibited it. These results show that although human BM adipocytes differ from extramedullary adipose tissues in their sensitivity to different effectors, they are a secondary source of leptin production. They suggest that BM adipocytes could contribute to hematopoiesis via the secretion of leptin in the vicinity of hematopoietic stem cells.—Laharrague, P., Larrouy, D., Fontanilles, A-M., Truel, N., Campfield, A., Tenenbaum, R., Galitzky, J., Corberand, J. X., Pe´nicaud, L., Casteilla, L. High expression of leptin by human bone marrow adipocytes in primary culture. FASEB J. 12, 747–752 (1998)

ABSTRACT

croenvironment (1). Indeed, it is well known that in healthy humans the number and size of adipocytes increase in the marrow cavity with advancing age when hematopoiesis decreases. The same phenomenon occurs in aplastic anemia (2). On the contrary, when hematopoiesis is stimulated by a peripheral cytopenia, the number of BM adipocytes decreases (3). Altogether, these results suggest an inverse relationship between BM adipocyte development and hematopoiesis potency. Extensive knowledge regarding adipose development in marrow has been obtained from permanent stromal cell lines derived from human BM (4), but primarily from rodent systems (5). A few studies have considered the model of primary culture. Results from these in vitro studies (5) suggest that 1) the process of adipogenesis may differ in bone marrow and extramedullary adipose tissues and could be unresponsive to insulin (6), and 2) BM adipocytes could contribute to hematopoiesis, as metabolic and energy reservoirs for developing blood cells, and through the production of extracellular matrix proteins favoring the expansion and differentiation of hematopoietic cells and the production of growth factors and cytokines (7). Among the products secreted from nonmedullary fat cells, leptin is a hormone whose main site of production and secretion is adipose tissue (8, 9). The recent identification of leptin receptor on hematopoietic stem cells pointed out the putative role of this hormone on hematopoiesis (10). This link was later confirmed by studies using different hematopoietic precursors (11, 12). Nevertheless, no data concerning the putative secretion of leptin from adipocytes in the vicinity of hematopoietic stem cells are available.

Key Words: adipose tissue · ob · corticosteroids · hematopoiesis 1

THE REGULATION OF proliferation and differentiation of hematopoietic precursors is performed through complex interactions with the cells that constitute the hematopoietic microenvironment. Bone marrow (BM)2 is the main hematopoietic site in adult mammals; among the various cell types present in BM, adipocytes could play an important role in this mi-

Correspondence: UPRESA-CNRS 5018, Hoˆpital ToulouseRangueil, 31403 Toulouse Cedex 4, France. 2 Abbrevations: ALPase, alkaline phosphatase; b3-AR, b3-adrenoceptor; BM, bone marrow; cAMP, cyclic AMP; G3PDH, glycerol-3-phosphate dehydrogenase; NE, norepinephrine; RPMI, Roswell Park Memorial Institute medium; RT-PCR, reverse transcription-polymerase chain reaction; TRAP, tartrateresistant acid phosphatase; UCP1, mitochondrial uncoupling protein-1.

0892-6638/98/0012-0747/$02.25 Q FASEB

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MATERIALS AND METHODS

Kaplow (14), a-naphthyl butyrate esterases according to Li et al. (15), myeloperoxidase according to Kaplow (16), and Sudan black according to Sheehan and Storey (17). The phenotype was established with alkaline phosphatase-anti-alkaline phosphatase technique and Mayer’s hematoxylin counterstain (18). The monoclonal antibody reacting with CD3 clusters was obtained from Becton-Dickinson (Mountain View, Calif.); the antibodies reacting with CD14, CD68, CD54, and HLA-DR were from Immunotech (Marseille, France); those reacting with CD19 and CD45 were from Dako (Trappes, France).

Subjects

Chemical and enzymatic assays

After we received informed consent, BM specimens were obtained from needle biopsies performed for diagnosis or periodic assessment from 40 patients (aged 44–84 years) with various nonmalignant hematological and nonhematological disorders, and from four healthy adult allogenic bone marrow donors (aged 23–47 years).

Assays were performed after washing the adherent cells twice with phosphate-buffered saline. Protein content was quantified by Bradford’s technique, using bovine serum albumin as a standard (Bio-Rad protein assay, Bio-Rad Laboratories, Hercules, Calif.). Cellular triglycerides were extracted with isopropanol and assayed by the colorimetric enzymatic method with glycerol-3-phosphate oxidase (Wako Chemicals, Neuss, Germany). For glycerol-3-phosphate dehydrogenase (G3PDH) determination, cells were homogenized in 20 mM Tris-HCl, pH 7.3, containing 1 mM EDTA and 1 mM b-mercaptoethanol, and the spectrophotometric assay was performed according to Wise and Green (19). Lipolysis was determined either by following the incorporation of [14C] acetate (Amersham, U.K.; 0.5 mCi/ml, 1 mCi/mmol) into the triglyceride fraction of adherent cells (20) or by enzymatic determination of the glycerol released directly by adherent cells in response to the nonselective b-adrenoceptor agonist isoproterenol (1006 M), the cAMP analog dibutyryl cAMP (1002 M), and the adenylate cyclase activator forskolin (1004 M), as previously described (21).

We have therefore developed a primary culture that successfully promotes a high rate of adipocyte differentiation from human BM stromal cells to determine whether leptin is expressed and secreted from these BM adipocytes.

Materials EDTA K3 vacutainers were obtained from Becton Dickinson (Meylan, France). NH4Cl was from Merck (Darmstadt, Germany); penicillin/streptomycin solution was obtained from Seromed (Biochrom, Berlin). RPMI-1640 (Roswell Park Memorial Institute medium) with Glutamax, minimum essential medium, and trypsin/EDTA were from Gibco BRL (Life Technologies, Cergy, France). Fetal calf serum and horse serum were purchased from DAP (Neuf-Brisach, France); hydrocortisone, dexamethasone, insulin, norepinephrine (NE), forskolin, dibutyryl cyclic AMP (cAMP), and isoproterenol were obtained from Sigma Chemical Co. (St. Louis, Mo.).

Preparation and analysis of RNA Cell culture EDTA-anti-coagulated BM samples of 2 ml were treated with 0.87% NH4Cl for erythrolysis and the mononuclear cells were resuspended immediately in RPMI supplemented with 10% heat-inactivated fetal calf serum, 10% horse serum, 1.6 mmol/ l L-glutamine, 100 mg/ml streptomycin, and 100 U/ml penicillin, with or without differentiating agents. They were layered either onto coverslips in 6-well culture plates or in 25 cm2 flasks at a concentration of 1–2 1 107 cells/ml and cultured at 377C in a humidified atmosphere of 5% CO2. The spent medium was replaced every 4 days with an equal amount of fresh medium. Myeloid and lymphoid cells either did not adhere or progressively detached and were removed. Adherent cells were examined and counted under an inverted microscope with phase contrast optics at 110 magnification. A confluent layer was observed after 2 to 5 wk of culture. Coverslip-adherent cells were processed for morphology and phenotype analysis. In culture flasks, the adherent cell layer was passaged either with 0.25% trypsin containing 0.02% EDTA or by scraping with a rubber policeman. For each system, the time for confluence of adherent cells and for the apparition of morphologically identifiable adipocytes (i.e., presence of more than two refractive cytoplasmic lipid droplets by phasecontrast microscopy) was determined, as well as the maximal number of clusters of adipocytic cells (reported per flask) observed during the primary culture.

Total RNA was extracted from trypsinized adherent stromal cells using the guanidine thiocyanate single-step technique of Chomczinski and Sacchi (22). Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) were performed using total RNA preparations with a 260/280 nm absorbance ratio between 1.9 and 2.1. cDNA synthesis and DNA amplification were performed as previously described (23). For UCP1, sense primer (5*-CTTCAGCGGCAAATCAGCT-3*) and antisense primer (5*-CACAGTCCATAGTCTGCCT-3*) matched with the human cDNA from positions 427 and 1056, respectively (24). The b3-adrenoceptor (b3-AR) sense primer (5*-ACCAACGTGTTCGTGACTTC-3*) matched with b3-AR cDNA at position 850–870 corresponding to the second transmembrane segment, and antisense primer (5*TAGATGAGCGGGTTGAAGGC-3*) matched at position 1654–1574 corresponding to the end of the seventh transmembrane segment (25). For the leptin gene ob, sense primer (5*-GATGACACCAAAACCCTCAT-3*) matched at position 85–104 (exon 2) and antisense primer (5*-CCACCACCTCTGTGGAGT-3*) matched at position 418–436 (exon 3) of human DNA. The PCR product (352 bp) was cloned into pBluescript, and sequence analysis revealed complete identity to the reported human leptin cDNA sequence (26). This PCR fragment was labeled by [g-32P]dCTP random priming for Northern analysis of total RNA (5 mg). Measurement of leptin protein concentration

Characterization of cultured cells The morphology of cells growing in culture was studied on May-Gru¨nwald-Giemsa stained coverslips. Staining for tartrateresistant acid phosphatase (TRAP) was performed according to Li et al. (13), alkaline phosphatase (ALPase) according to 748

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Leptin concentrations in culture medium were measured by enzyme-linked immunosorbent assay, using anti-human leptin antibodies obtained from Roche Gent. A monoclonal antibody raised in mice (2A5) immobilized on Nunc Maxisorb 96well plates was used in combination with rabbit polyclonal

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anti-serum (R46) and goat polyclonal anti-rabbit IgG conjugated to horseradish peroxidase (Boehringer-Mannheim, Mannheim, Germany). Leptin concentrations were calculated from standard curves based on recombinant human leptin protein. Minimum detection for the assay was 0.2 ng/ml of leptin. Colorimetric reaction was generated by addition of tetramethyl benzidine substrate. The lower level of detection using leptin antibodies obtained from Roche assay was 0.25 ng/ml of leptin.

RESULTS Characterization of BM fat cells in primary culture When cultured in the presence of fetal calf and horse sera only, initial adherent cells were mostly bipolar fibroblast-like cells, with macrophages appearing as a well-dispersed attached population. The entire culture became progressively overgrown by a relatively undifferentiated fibroblast-like population, with no evidence of differentiation toward blanket cells or adipocytes. These cells were nonphagocytic (opsonized zymosan) and did not stain with butyrate-esterase or TRAP, but were ALPase and CD54 positive; they were interspersed with some macrophages (CD14, CD68, CD45, and butyrate-esterase positive, phagocytosis of Candida albicans). BM cells cultured in RPMI supplemented with fetal calf and horse sera plus 1006 M dexamethasone gave rise to colonies of cells with multilocular vesicles evocative of lipid droplets. These cells appeared within 10–15 days of treatment among the fibroblastic adherent layer and could be maintained for up to 10– 12 wk. Staining with Sudan black B and oil red O confirmed the presence of lipid within the intracellular vesicles. These cells expressed neither fibroblast (ALPase, CD54) nor macrophage (CD14, CD68) markers, but exhibited a strong nonspecific butyrateesterase activity inhibited by sodium fluoride. The number of fat cell clusters in the flasks was (highly) significantly correlated (rÅ.671, Põ0.001) with the cellular triglyceride concentration. G3PDH activity could be detected only when fat cell clusters were present, and was significantly correlated either with the fat cell cluster number (rÅ.425, 0.01õPõ0.05) or the triglyceride concentration (r Å.428, 0.01õPõ0.05). According to these results, the fat cell cluster number, determined by phase-contrast microscopy, appeared to be the easiest and most reliable indicator of adipose conversion. Dibutyryl cAMP and forskolin significantly (Põ0.05) stimulated triglyceride hydrolysis as assessed both by [14C] and glycerol release in culture medium (Fig. 1). A similar effect was also observed with isoproterenol, indicating that these cells express at least one subtype of the b-adrenergic receptor (Fig. 1). However, the b3-AR could not be detected under basal conditions by RTPCR. Using the same technique, we were unable to

Figure 1. Lipolytic activity of BM adipocytes grown in the presence of 1006 M dexamethasone tested by enzymatic determination of the glycerol released directly in response to 1006 M isoproterenol, 1002 M dibutyryl cAMP, and 1004 M forskolin. The means {SE correspond to six separate experiments.

reveal the UCP1 mRNA, another marker of brown adipocyte phenotype (data not shown). No significant difference in the BM cultures and adipocytes was observed due to age, sex, or clinical status of the donor patients. Medication used by the patients also had no adverse effects on the cultures of stromal cells. Cultures with differentiation factors Hydrocortisone and dexamethasone significantly shortened the confluence time and were necessary for adipocytic differentiation in a dose-dependent fashion (1008 M hydrocortisone õ 1006 M hydrocortisone õ 1006 M dexamethasone) (Fig. 2A). NE (1007 M) modified neither the confluence time nor the differentiating effect of hydrocortisone or dexamethasone. Insulin (1008 M) alone had no effect on the proliferation or differentiation of BM stromal cells into committed fat cells (data not shown). Insulin did not improve the differentiating effect of dexamethasone (Fig. 2B). Leptin expression and secretion To investigate the expression of ob in BM adipocytes, we obtained a human ob probe from RT-PCR exper-

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ration, and lipolysis induced by b-adrenergic agonist. These adipose-like cells do not present the cytochemistry and immune phenotype corresponding to fibroblast and macrophage markers. It has been speculated that BM adipocytes could be more closely related to brown than to white adipocytes (1). In our culture system, NE treatment had no differentiating effect; fat cells expressed neither UCP1 nor the b3AR featuring the differentiated brown adipocytes in different mammal species. This suggests that adipocytes differentiated from human BM stroma cells are not true brown fat cells even if it cannot be excluded that, according to different conditions, BM adipocytes express UCP1 or assume a potential brown-like function. UCP1 has been detected in rodents by using RT-PCR in adipocytes differentiated from a rat bone marrow preadipocyte cell line (27). In some species, the percentage of BM adipocytes increases as the ambient temperature drops (28) and adipocytes are present mainly in the long bones where the internal temperature is relatively low (29). Cultivating BM stromal cells at 337C, as performed by some investigators (30), delayed the confluence time and

Figure 2. Influence of the culture medium on BM adipocyte differentiation: 1006 M hydrocortisone vs. 1006 M dexamethasone (A); 1006 M dexamethasone vs. 1006 M dexamethasone plus 1008 M insulin (B). The means { SE correspond to three separate experiments.

iments (see Materials and Methods). Even after washing in stringent conditions, this probe revealed a signal of the expected size. This signal could not be detected in the absence of multilocular cells in the primary culture (Fig. 3A). The expression of ob increased according to the differentiation state (Fig. 3A), reaching a high level compared to the expression observed in human subcutaneous adipose tissue (Fig. 3B). Together with ob mRNA detection, significant amounts of leptin could be detected in the culture supernatant (Fig. 3C). The ob mRNA expression was strongly decreased after forskolin treatment, whereas insulin had little inhibitory effect (Fig. 4).

DISCUSSION These data indicate that the fat cells obtained from human BM culture system were true adipocytes and that they expressed and secreted high levels of the adipocyte hormone leptin. The nature of lipid-laden cells appearing in the adherent layer as true adipocytes is confirmed by G3PDH activity, leptin expression, acetate incorpo750

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Figure 3. Northern blot analysis of ob mRNA from 5 mg of total RNA according to the time and number of adipocyte clusters in culture (A); comparison with extramedullary adipose tissue (B). Concentration of leptin in the culture supernatant (day 21) and in the absence (fibroblasts) or presence (adipocytes) of 1006 M dexamethasone. The means { SE correspond to three separate experiments (C).

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Figure 4. Regulation of ob mRNA by a 24 h treatment with 1008 M insulin (INS) and 1004 M forskolin (FK). Results are expressed as ob mRNA/actin mRNA ratio. The means { SE correspond to three separate experiments performed with 5 mg of total RNA *P õ 0.05; #P õ 0.001).

had no effect on the differentiation of BM fibroblast cells into adipocytes (data not shown). These results do not support the hypothesis of a temperature-inducible adipocyte differentiation. The positive effect of horse serum on adipose conversion could be related to a high content in cortisol. However, this beneficial effect could be restricted to human stromal cells, since horse serum was found to be ineffective for the adipose conversion of rabbit BM stromal cells (31). It was recently demonstrated that human serum also contains factors that are permissive for adipocyte formation in the presence of dexamethasone (32). We found that dexamethasone was more efficient than hydrocortisone for the proliferation and differentiation of human stromal preadipocytes as reported for primary culture from human subcutaneous adipose tissue (33). However, in the absence of horse serum, dexamethasone induces the differentiation of BM stromal cells into osteoblasts and mineralization of the matrix, with no adipocytes visible in the adherent layer (34). These results confirm that cultured BM stromal cells could differentiate into adipocytes and osteoblasts (32, 35), and suggest that a stromal stem cell could give rise to adipocytes and osteoblasts within bone. Our data also indicate that even if dexamethasone per se appears

unable to induce differentiation of BM stromal cells to adipocytes, glucocorticoid hormones are of great importance for the adipose conversion of fibroblast precursor cells in human species. Besides glucocorticoids, whose action is not clearly understood, insulin plays an important role in extramedullary adipocyte differentiation. Our results clearly indicate that insulin has no effect on the proliferative or differentiation capacities of nontransformed human BM stromal cells. This agrees with previous studies demonstrating that murine BM adipocytes in mixed cultures are unresponsive to insulin (1). Together, our results suggest that the sensitivity of human BM preadipocytes to adipogenic agents is different from that of extramedullary adipocytes. These data favor differences in development among adipose tissue according to their localization and, more specifically, their intra- or extramedullary localization. An additional argument in support of this hypothesis is that differentiation efficiency of BM stromal cells was not dependent on the sex or age of the donor. This is quite different from results obtained with extramedullary stromal-vascular cells of fat tissue (33). BM samples from patients presenting with primary hematologic diseases or hematologic manifestations secondary to other disorders underwent adipocyte differentiation with an efficiency that correlated with neither clinical status nor treatment. Moreover, no difference was observed between these samples and samples from healthy allogenic bone marrow donors. The most striking result in this study is the high expression and secretion of leptin from human medullary stromal cells in primary culture. Its detection when lipid-laden cells are present supports the adipocyte phenotype of BM cells differentiated in the presence of glucocorticoids. The significant level of expression we observed is in contrast to other culture systems where sensitive technique is needed (36). Since the normal adult skeleton contains about 1500 g of marrow, the amount of leptin secreted from BM adipocytes could contribute to leptin plasma levels. The very faint effect of insulin in our model is quite different from the results obtained with the 3T3 clonal line (37), whereas the strong inhibitory effect of forskolin on ob mRNA expression agrees with previous reports (38). The leptin receptor has been detected recently on human hematopoietic stem cells bearing the CD34 antigen (10). Some reports suggest a putative link between this hormone and hematopoiesis or inflammatory processes (11, 12, 39). Leptin secretion by the intramedullary adipocytes in the vicinity of stem cells could play a major role in the control of the expansion and differentiation of human primitive hematopoietic cells through paracrine interaction in BM microenvironment. The results described here demonstrate that our primary culture model is useful for investigating the function of human bone marrow adipocytes. We have

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begun this investigation by studying the microenvironment of the human bone marrow cells, the expression and secretion of leptin from bone marrow adipocytes, and would like to consider the relationship between adipogenesis and hematopoiesis in human species. We are grateful to J. S. Saulnier-Blache for assisting in chemical and enzymatic assays, C. Hirtz for preparating the manuscript, and N. Crowte for revising the English version.

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Received for publication January 9, 1998. Revised for publication January 29, 1998.

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