Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Experimental Cell Research 168 (1987) 15-30

Development of a Chemically Defined for Differentiation of Rat Adipose SYLVIANE

DESLEX, RAYMOND

Serum-free Medium Precursor Cells

NEGREL and GERARD AILHAUD*

Cenire de Biochimie du CNRS (LP 7300), Laboratoire Biologie du Developpement du Tissu Adipeux, Facultt! des Sciences, 06034 Nice Ctdex, France

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transfenin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [“?Zlglucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose COnVCrSiOn process. @ 1987 Academic Press, Inc.

Over the last 10 years, numerous studies performed on preadipocyte cell lines and cell strains have allowed the delineation of the main features of the adipose conversion process [l]. After a period of growth arrest at confluence, a limited proliferation of susceptible cells occurring thereafter leads to the formation of fat cell clusters in which adipose-like cells have acquired both the morphological and functional characteristics of mature adipocytes [2-4]. In Ob17 cells, early markers of adipose conversion, such as lipoprotein lipase (LPL), are detected before any visible triacylglycerol accumulation, whereas the full expression of late markers, such as glycerol-3-phosphate dehydrogenase (GPDH), takes place after the post-confluent mitoses [2, 5-81. In order to gain a better understanding of the factors involved in the control of growth and differentiation of adipose precursor cells, serum-free media have been defined [9-121. In most cases, serum-free media were able to support cell proliferation but unable to support cell differentiation. Therefore, addition of a mixture of dexamethasone and isobutylmethylxanthine (IBMX) for a few days, serum at low concentrations, other hormones, or serum-derived components, were required to promote adipose cell differentiation. For instance, cells of the * To whom offprint requests should be addressed. 2-878331

Copyricht @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827187 $03.00

16

Deslex, Negrel und Ailhuud

teratoma-derived 12.46clonal line grown in a mixture of Dulbecco’s modified Eagle (DME) and Ham’s FI2 media supplemented with insulin, transferrin, fibroblast growth factor (FGF) and fibronectin, undergo adipose conversion upon addition of dexamethasone and IBMX for a few days [ 131,whereas sheep preadipose tibroblasts in secondary culture require insulin, dexamethasone, fibroblast growth factor (FGF) and a lipid mixture [12]. In addition, Ob17 cells established in our laboratory [4] are able to convert to adipose-like cells in a medium supplemented with insulin, transferrin, triiodothyronine, growth hormone, fetuin and a serum fraction enriched in some still unidentified adipogenic factor(s) [ 141. The present report shows that cells of the stromal-vascular fraction, obtained from various rat adipose deposits, are able to differentiate in primary culture when maintained in a simple chemically defined medium containing insulin, transferrin and triiodothyronine.

MATERIALS Cell Preparation

AND METHODS

and Cell Culture

Male Wistar rats used in this study were fed ad libitum with ordinary chow diet and killed by cervical dislocation and diethyl-ether anesthesia. Unless otherwise stated, 4-week-old animals were used. Adipose deposits (periepididymal, retroperitoneal, subcutaneous of the inguinal region) were immediately resected under sterile conditions, freed as much as possible from blood capillaries and then bathed in isotonic saline supplemented with antibiotics. The mammary tissue was carefully removed in the case of the inguinal subcutaneous fat tissue. Stromal-vascular cells were obtained by collagenase digestion of the minced tissues. The digestion was performed during 40 min at 37°C under controlled agitation in DME medium containing 2 mg/ml collagenase and 20 mg/ml bovine serum albumin. Cells and tissue remnants were then poured at room temperature into DME medium containing (unless otherwise stated) 10% fetal bovine serum (FBS) and filtrated through a 25 urn nylon screen. The filtrate was centrifugated at 600 g for 5 min in order to collect a first pellet of stromal-vascular cells. The floating fat layer was resuspended under the same conditions as above, washed and centrifuged. Both pellets were pooled and resuspended in DME medium supplemented with 10% FBS, 62 mg/l penicillin, 50 mg/l streptomycin, 33 uM biotin and 17 uM pantothenate. Aiiquots of the suspension of stromal-vascular cells were counted with a Couher counter, under conditions which exclude the counting of blood cells. Stromal-vascular cells were plated into 35mm culture dishes at a mean cell density of lo4 cells/cm2 and maintained in 2 ml of this serumsupplemented medium at 37°C for 12-24 h in an atmosphere of 96.5% sir/3.5% COz. After attachment, cells were then thoroughly washed twice for at least 1 h with a mixture of Dulbecco’s modified Eagles medium (DME)/Ham’s F12 medium (1 : 1; v/v) containing 15 mM NaHCO,, 15 mM HEPES buffer pH 7.4, biotin, pantothenate and antibiotics as above. Cells were subsequently maintained in this standard serum-free culture medium supplemented with insulin, transferrin and triiodothyronine. Unless otherwise stated, this medium is referred to as ITT medium. Culture media were changed every 3 days. Cells were counted as above after trypsin treatment in phosphate-buffered saline (PBS) pH 7.4 containing 5 mM EDTA. Eight independent control experiments showed that the plating efficiency was 100+15 % (blood cells excluded). In some experiments (see table 1 and fig. I), FBS was omitted and replaced by some physiologically-relevant glycoproteins which are known to mediate cell attachment and spreading in culture [15]. In that case, (i) cells and tissue remnants were poured into DME medium alone; (ii) culture dishes were pre-coated for 1 h at 37°C in the presence of 1 ml of ITT medium containing 5 ug/ml vitronectin, 10 &ml fibronectin or 10 t&ml laminin, or a combination of laminin and vitronectin (or Iibronectin) at the same concentrations. Then stromal-vascular cells were plated on each type of pre-coated dishes in the presence of ITT medium alone or supplemented with the same corresponding adhesive protein(s). No medium change occurred in these experiments by contrast to those performed in the presence of FBS. Exp CellRes 168(1987)

Adipose conversion in serum-free medium Fluorescence

17

Microscopy

Immunofluorescent labelling of LPL was performed on permeabilized cells as previously described [16], using an immunoserum raised in goat against the homogeneous rat adipose tissue enzyme (obtained through the courtesy of Professor J. Etienne, Paris) and a rabbit anti-goat IgG fraction labelled with rhodamine isothiocyanate.

Chemical and Enzymatic

Assays

Glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and lactate dehydrogenase (LDH; EC 1.1.1.27) were assayed spectrophotometrically as previously described [17, 181 using either cell homogenates or Triton X-l 14 cell lysates [16]. Lipoprotein lipase (LPL; EC 3.1.1.34) was assayed on Triton X-l 14 lysates after removing the detergent according to Bordier [16-191. All specific activities were expressed in mU/mg protein, i.e. in nmol/min/mg of protein [20]. DNA and triacylglycerol cellular content were estimated according to Labarca & Paigen [21] and to Wahlefeld [22], using DNA from Ob17 cells and triolein as standards, respectively.

Incorporation

of Radioactive

Precursors

into Cellular Lipids and DNA

Labelling of cellular lipids was performed by adding 5 pCi/dish of [U-‘4C]glucose (8.5 GBq/mmol) to 2 ml of culture medium 24 h before cell collection. Pre-labelling intended to lipolysis experiments was performed by adding twice 0.5 @i/dish of [l-‘4C]acetate (2 GBq/mmol) at 48 h time intervals before the beginning of experiments. Lipids from cells and culture medium were extracted according to Bligh & Dyer [23] and analysed on silica-gel plates using a mixture of hexane/diethyl-ether/formic acid (80 : 20 : 1) as developing solvent. Areas corresponding to lipid standards run in parallel were scraped and counted by liquid scintillation. Experiments of [‘Hlthymidine incorporation into DNA were performed on freshly prepared stromal-vascular cells suspended in DME medium supplemented with 10% FBS, labelled thymidine (1 FM; 111 kBq/pmol) and additives as above. After 24 or 96 h, attached cells (mean cell density of lo4 cells/cm2) were thoroughly washed three times at 37°C with PBS, treated at room temperature with 3 % formaldehyde for 40 min, then with 50 mM ammonium chloride and at last washed three times with PBS and twice with 90 % ethanol. Autoradiographs of labelled cells were obtained as previously described [2].

Materials Culture media and FBS were obtained from Gibco (Cergy-Pontoise, France) and collagenase from Boehringer (Mannheim, F.R.G.). [l-‘4C]Acetate was a product from Commissariat B 1’Energie Atomique (France). [U-14C]Glucose, glycerol tri[9,10-3H]oleate and recombinant Thr-59 IGF-I were purchased from Amersham Corp. (Buckinghamshire, UK). Bacterially produced mIGF-I was kindly provided by Dr A. Skottner-Lundin (KabiVitrum AB, Stockholm, Sweden). Fibroblast growth factor (FGF) was a product of Collaborative Research Inc. (Lexington, Mass.). Bovine growth hormone (bGH) was obtained through the National Hormone and Pituitary Program (NIADDK, Baltimore, Md.). Human fibronectin, human vitronectin, mouse laminin and bovine dermal collagen were from Institut J. Boy (Reims, France), Calbiochem. Brand Biochemicals (La Jolla, Calif.), Gibco-BRL (CergyPontoise, France) and Collagen Corporation (Palo Alto, Calif.), respectively. Crystalline bovine insulin, human transfetin, triiodothyronine, bovine serum albumin, isoproterenol, reagents for the enzymatic determination of triacylglycerol, as well as other chemicals were from Sigma Chemical Co. (St. Louis, MO.).

RESULTS As previously reported [IO], after plating in the presence of 10% FBS, stromalvascular cells isolated from rat periepididymal adipose tissue were subsequently able to proliferate in a serum-free hormone-supplemented medium (4F medium) containing insulin, transferrin, FGF and a rat submaxillary gland extract. Initial attempts to promote differentiation of rat stromal-vascular cells in primary culExp Cell Res 168 (1987)

18

Deslex, Negrel und Ailhaud

ture under these serum-free conditions led to a low frequency of adipose conversion despite a supplementation with growth hormone and triiodothyronine. The well-documented property of growth factors and other mitogens to behave as antagonists of adipose conversion [14, 24-261 prompted us to remove both FGF and the submaxillary gland extract routinely present in the 4F medium. As shown below, extensive adipose conversion of stromal-vascular cells became detectable upon exposure to a mixture of DME/Ham’s F12 medium (1 : I ; v/v) supplemented with insulin (5 yg/ml), transferrin (10 ug/ml) and triiodothyronine (200 PM). This medium has been referred to a ITT medium. Most of our experiments were undertaken with 4-week-old rats, since earlier reports [27, 281, as well as our own morphological observations, showed that periepididymal (fig. 1A, B) and subcutaneous stromal-vascular cells (not shown) obtained from animals at that age displayed, when grown and maintained in serum-supplemented medium containing 17 nM insulin and 2 nM triiodothyronine, a high frequency of adipose conversion. As shown in fig. 1A for the periepididymal stromal-vascular cells, a large number of lipid-filled adipose-like cells was routinely observed under these conditions, 10 days post-inoculation. In contrast, stromal-vascular cells originating from older animals (lgweek-old) retained a fibroblastic appearance in serum-supplemented medium (fig. 1B) and did not differentiate significantly. A different picture emerged in serum-free medium. As illustrated in fig. 1 C, a very low proportion of periepididymal stromal-vascular cells, if any, contained cytoplasmic lipid droplets 48 h after plating, i.e. 24 h after shifting the cells in ITT medium. However, a visible lipid accumulation began usually at day 4, increased rapidly thereafter and culminated around day 7-10. At that time, a very large proportion (90-95 %) of periepididyma1 stromal-vascular cells accumulated lipid droplets (tig. 1D), providing the presence of insulin or IGF-I (fig. 1E, G and uide infra). It is of interest to note that periepididymal stromal-vascular cells isolated from older rats (16-20 weekold) were also able to differentiate and to accumulate lipid droplets (6&70% of the cell population) when maintained for a few days in ITT medium (fig. 1F) Fig. I. Morphological appearance of stromal-vascular cells maintained under various culture conditions and immunostaining of LPL in differentiating cells. After plating and maintenance overnight in serum-supplemented medium, periepididymal stromal-vascular cells isolated from (A, C, D, E, G, H) 4- or (B, F) Is-week-old rats were washed and fed with serum-supplemented medium containing (A, B) 17 nM insulin; and 2 nM triiodothyronine; (C, D, F, If) serum-free ITT medium and insulin-deprived ITT medium (G) supplemented or (E) not with 10 nM IGF-I. Micrographs of representative fields under each condition were taken 2 days (C), 4 days (H), 7 days (D-G) or 10 days (A, B) after plating. (A-G) Phase-contrast; (H) immunofluorescence staining of lipoprotein lipase; (0 autoradiograph of stromal-vascular cells from 4-week-old rats maintained in the presence of [3H]thymidine as described in Materials and Methods for 24 h (la) or 96 h (Zb) (see Results); (J) micrograph of stromal-vascular cells from 4-week-old rats plated on dishes pre-coated with laminin and maintained for 5 days in ITT medium supplemented with 10 ug/ml laminin (see Materials and Methods for details). Similar pictures were obtained with cells plated on laminin-pre-coated dishes in the presence of ITT medium alone as well as with cells plated on dishes pre-coated with vitronectin or tibronectin, or various combinations of adhesive proteins (see text), in the presence of ITT medium alone or supplemented with the same corresponding protein(s). Bar, 100 urn. Exp Cell Res 168 (1987)

Adipose conversion

in serum-free medium

19

20

Deslex, Negrel and Ailhaud

Table 1. Acquisition maintained

of adipocyte in ITT medium

phenotypes

in periepididymal

stromal-vascular

cell:

Days relative to inoculation 1 Triacylglycerol content (nmole/yg DNA) Incorporation of [W]glucose into (dpm/dish and (%)) Cellular lipids Polar lipids Triacylglycerol Specific activity (mU/mg) Lipoprotein lipase Glycerol-3-phosphate dehydrogenase Lactate dehydrogenase

und.

und. und. 1 100-2 500

5

1

9.5

50

19 900 (100) 6 350 (32) 10 550 (53)

221 850 (100) 21 520 (9.7) 173 930 (78.4)

8-20 480-l 350 [950”-2 5806] 1 820-3 610 [3 370’-6 670b]

l&30 950-l 870 1 900-3 250

Cells were plated in serum-supplemented medium (day 0) and then shifted to ITT medium. Triacylglycerol content incorporation of [‘4C]glucose into cellular lipids and enzyme activities were determined at the times indicated a described in Materials and Methods. The values of triacylglycerol content and [i4C]glucose incorporation are th means of duplicate determinations on two separate dishes from the same series of cells; they did not differ by mor than 7 %. Owing to some variations observed for the onset and extent of differentiation as a function of time afte plating, enzyme activities are reported as minimal and maximal values determined at the times indicated fror different series of cells. The reported values for day 1 are representative of two independent experiments; thos reported for days 5 and 7 are representative of four independent experiments for LDH and at least eight independer experiments for LPL and GPDH. The numbers in parentheses correspond to values obtained when stromal-vascuh cells were plated, in the absence of FBS, on dishes pre-coated (a) with fibronectin and maintained in ITT mediui containing fibronectin; (b) same as in (a) plus laminin; (c) same as in (a) but laminin alone instead of fibronectin. Th values obtained under the other conditions (see Materials and Methods) were found to tit between the reporte values. Und., Undetectable.

despite their rather low ability to differentiate in serum-supplemented medium (fig. 1B). The accumulation of lipid droplets in periepididymal stromal-vascular cells maintained in ITT medium was shown to correspond to a true increase in the intracellular triacylglycerol content, reaching a value of 50 nmole/ug DNA at day 7 (table 1). It is noteworthy that, since exogenous lipids were absent from the culture medium, the triacylglycerol accumulation should rely exclusively on endogenous fatty acid synthesis and esterification. Thus, it was no surprise to observe a potent increase in the rate of endogenous fatty acid synthesis from glucose. A marked rise (11-fold) in the total incorporation of [‘4C]glucose into cellular lipids was observed between day 5 and day 7 (table 1). This rise was mainly due to triacylglycerol synthesis, since lipid analysis by thin-layer chromatography showed that the incorporation of labeled glucose into polar lipids and triacylglycerol was enhanced approx. 3- and 16-fold, respectively. This enhancement was therefore due to an increase in the rate of both fatty acid synthesis and esterification, the latter being directed preferentially toward triacylglycerol synthesis. Since stromal-vascular cells were able to convert into adipose-like cells, the occurrence of lipoprotein lipase as enzyme marker of adipose conversion was Exp CellRes I~%(19871

Adipose conversion in serum-free medium

21

next investigated. This enzyme could be clearly detected in differentiating cells by indirect immunofluorescence microscopy using specific antibodies (fig. 1ZZ): most cells, if not all, contained LPL. In agreement with our previous studies performed on Ob17 preadipose cells [29], immunostaining of LPL showed that this secreted enzyme was concentrated mainly in the Golgi region. LPL and GPDH activities were also directly assayed in cell extracts (table 1). They were undetectable early after plating but became rapidly expressed at high levels, reaching specific activity values similar to those found in mature rodent adipocytes as well as in differentiated Ob17 and 3T3F442A cells [3,4]. In most experiments the maximal activity of LPL was attained at a time when the GDPH activity was still increasing. It is of interest to note that the activity of LDH, which is not directly related to lipogenesis, remained essentially unchanged as a function of time in culture (table 1). In the experiments so far described, it is important to note that cell plating was performed in the presence of 10% FBS. The role of the latter, which contains like human serum both tibronectin and vitronectin [30], was likely to provide some adhesive proteins known to mediate the attachment of various cell types to the substratum

WI. Clearly, as shown in table 1 and fig. 1, exposure of the cells to FBS after collagenase digestion and during the 24-h period of cell attachment proved to be unnecessary for their subsequent adipose conversion in ITT medium. When cell attachment was taking place in the presence of FBS for 24 h, the nuclear labelling index was low (~20 %), indicating no significant cell growth (fig. 1la), in contrast to cells exposed for 96 h where the nuclear labelling index was close to 100% (fig. 1Zb). Furthermore stromal-vascular cells, isolated from rat periepididymal adipose tissue, were plated on dishes pre-coated with vitronectin, tibronectin or laminin, or a combination of these adhesive proteins, and then maintained in ITT medium containing or not the same corresponding protein(s). The results of table 1 show that adipose precursor cells were able within 5 days to express high activity levels of GPDH (table 1) and to accumulate lipid droplets (fig. 1.Z). Precoating of the dishes proved to be critical, since similar results, if not identical, were obtained when the cells were subsequently maintained in ITT medium containing (or not) the adhesive proteins. The ability of ITT medium to support adipose conversion of stromal-vascular cells from periepididymal fat pad was extended to other adipose deposits. As the results of table 2 show, stromal-vascular cells isolated from retroperitoneal and subcutaneous inguinal fat tissues were able to express significant levels of LPL and GPDH activities and to accumulate lipid droplets (not shown) when maintained in ITT medium. Among the best characteristic phenotypes of adipose cells is their ability to mobilize intracellular triacylglycerol in response to lipolytic hormones. This point was further investigated on stromal-vascular cells differentiated in ITT medium and in which triacylglycerol molecules were prelabelled on their fatty acid Exp Cell Res 168 (1987)

22

Deslex, Negrel and Ailhaud

Table 2. Development medium ofstromal-vascular adipose tissues

of

enzyme markers during adipose conuersion in ITT cells isolatedfrom subcutaneous and retroperitoneal Specific activity

(mU/mg) Days after inoculation

Lipoprotein lipase

Glycerol-3-phosphate dehydrogenase

Lactate dehydrogenase

Subcutaneous stromal-vascular cells 2 8 11

und. 12.5 18.7

und. 400 840

2 450 2 580

6

720 1 550

3 130 3 220

Retroperitoneal stromal-vascular cells 6 12

The reported values are the mean determined on duplicate dishes from one series of cells for both adipose sites; they did not differ by more than 5 %. See Materials and Methods for details. Und., Undetectable.

moiety after incorporation of [14C]acetate, as previously described [31]. The curves in fig. 2 demonstrate clearly that, after differentiation in ITT medium, periepididymal and subcutaneous stromal-vascular cells were able to release very significant amounts of radioactivity in response to isoproterenol used as a typical /?-agonist. This lipolytic response occurred at at P-agonist concentration in the range of that reported in numerous studies performed on adipocytes [32]. Some SO-90% of the released radioactivity co-chromatographed with unesterified (free) fatty acids, The specific mobilization of radioactive endogenous triglycerides was assessed by their net cellular decrease (see caption to fig. 2). Taken together, these results demonstrate that the morphological differentiation of stromal-vascular cells isolated from three different adipose deposits was accompanied by the acquisition of functional properties of mature adipocytes. Therefore, lipid-filled stromal-vascular cells which were able to differentiate in ITT medium can be considered as typical adipose cells. The relative importance of each additive to the DMEIHam’s F12 medium for the maximal expression of enzyme markers of adipose conversion was estimated by individual removal. The results of fig. 3 indicate no obvious requirement for transferrin. Nevertheless, this iron-binding protein was routinely kept in the culture medium because it binds also heavy metals which might be present as contaminants and might have cytotoxic effects under serum-free conditions [34]. The absence of triiodothyronine led to a halving of the activity levels of LPL and GPDH. A more dramatic decrease (5- to lo-fold) was observed in the absence of insulin. Therefore, dose-response relationships of insulin to the activities of enzyme markers were determined. The experiments reported in fig. 4 were performed on a long-term basis after exposure of the stromal-vascular cells from Exp Cell Res 168 (1987)

Adipose conversion in serum-free medium

0

30

120

00

0

30

00

23

120

TIME (mid

Fig. 2. Lipolytic response of periepididymal and subcutaneous stromal-vascular cells after differentiation in ITT medium. (A) Periepididymal; or (B) subcutaneous stromal-vascular cells were prelabelled during the course of their differentiation in ITT medium containing [‘4C]acetate as described in Materials and Methods. Lipolysis experiments were performed 7 days after plating. Before assays, in order to remove insulin bound to cells and thus to exclude any anti-lipolytic effect brought by this hormone 1331,cells were washed four times for 15 min at 37°C in a mixture of DME/Ham’s F12 medium containing 1% BSA and then incubated at 37°C in the same medium supplemented (u) or not (0 --0) with 2.5 nM isoproterenol. 200 ul samples were taken and counted at time 30,60 and 120 min. Each point is representative of the mean of triplicate dishes from the same series of cells. Their values did not differ by more than 6 %. The mobilization of endogenous triacylglycerol was assessed by determining at time 120 min the percentages of radioactivity recovered in the different intracellular lipid fractions. These percentages were for cells exposed or not to isoproterenol: Triacylglycerol (A) 57 and 68%; (B) 45 and 54%. Unesterified fatty acids (A) 4.5 and 7.2%; (B) 8 and 8.5%. Polar lipids (A) 33 and 16%; (B) 39.5 and 27.5 %. In both experiments more than 80 % of the radioactivity released from the cells into the medium co-chromatographed with unesterified fatty acids.

-TrF

-73

L -TrF -TS

-Ins

El -T,F

-73

-TrF -T3

4”‘

Fig. 3. Effect of removal of individual factors on the activity levels of enzyme markers of adipose conversion. Specific activities of (A) LPL; (B) GPDH were determined on duplicate dishes of

periepididymal stromal-vascular cells after 4 days in W, ITT medium, or 0, in ITT medium deprived of -TrF, transferrin; -23, triiodothyronine; -Trf-T3, transferrin and triiodothyronine; -ins, insulin. The values, which did not exceed +5% from the mean, are reported in percent of the mean values obtained for cells maintained in ITT medium and taken as 100% (6 and 1260 mU/mg for LPL and GPDH); they are representative of two independent experiments. Exp Cdl Res 168 (1987)

24

Deslex, Negrel and Ailhaud

0

1.7

17

INSULIN

850

170

CONCENTRATION

(nM)

Fig. 4. Dose-response relationship of insulin to the activities of enzyme markers of adipose conversion. After plating and maintenance overnight in serum-supplemented medium, stromal-vascular cells from inguinal subcutaneous (A, l ) or periepididymal adipose tissue (A, 0) were shifted to a serumfree medium containing 10 ug/ml transferrin and 200 pM triiodothyronine, in the presence or absence of insulin as indicated. A, A, LPL; 0, 0, GPDH activities were determined at day 8 postinoculation. The mean values from duplicate dishes are reported (+5% from the mean); they are representative of two independent experiments. 100% correspond for LPL activity to 20 and 16 mU/mg for stromal-vascular cells from inguinal subcutaneous and periepididymal fat tissue, respectively, and for GPDH activity to 1200 mU/mg.

periepididymal and inguinal subcutaneous adipose tissues to the hormone. The activities of LPL and GPDH were determined as a function of insulin concentration. The half-maximally and maximally effective concentrations on the activity levels of LPL were found to be 4 and 170 nM for periepididymal stromal-vascular cells and 1 and 17 nM for inguinal subcutaneous stromal-vascular cells, respectively. The corresponding values on the activity levels of GPDH were found to be 170 and 850 nM as a minimal estimate for periepididymal stromal-vascular cells and 40 and 850 nM for inguinal subcutaneous stromal-vascular cells, respectively. Thus the data show that the insulin concentrations required to observe an effect on the activity levels of LPL are within the physiological range. However, for stromal-vascular cells originating from any given site, the half-maximally and maximally effective concentrations of insulin on the activity levels of GPDH were at least lo-fold higher than those determined on the activity levels of LPL. Insulin-like growth factors IGF-I and IGF-II, which are polypeptides sharing a high degree of homology with insulin, are known to regulate growth of many tissues [35] and that of Ob17 cells in vitro [36], to promote differentiation of cultured myoblasts and erythroblasts [37, 381 as well as cultured oligodendrocytes [39] and to mimic the effect of insulin on the metabolism of cultured hepatocytes [40]. Therefore, as a comparison with insulin, the effects of IGF-I on adipose conversion were next investigated. As shown in fig. 5, the addition of IGF-I to triiodothyronine- and transferrin-containing medium produced a dramatic increase in the activity levels of LPL (fig. 5A) and GPDH (fig. 5B), in agreement with the micrograph of fig. 1 G. These increases occurred at IGF-I Exp Cell Res 168 (1987)

Adipose conversion in serum-free medium

0

2

10

20

0

2

0

IGF I CONCENTRATION

10

20

30

40

25

50

(nM)

Fig. 5. Dose-response relationship of IGF-I to the activities of enzyme markers of adipose conversion. After plating and maintenance overnight in serum-supplemented medium, stromal-vascular cells from inguinal subcutaneous adipose tissue were shifted to a serum-free medium containing 10 pg/ml transfertin and 200 pM triiodothyronine, in the presence or absence of IGF-I, with or without insulin as indicated. (A) LPL activities were determined at the times indicated in the absence (R) or presence (0) of 1.7 nM insulin. (B) GPDH activities were determined at day 6 post-inoculation in the absence (0) or presence (0) of 1.7 nM insulin. The reported values are the mean determined from duplicate dishes of a single series of cells; they did not differ by more than 5 %. The curves are representative of two independent experiments.

concentrations ranging from 2 to 50 nM. The simultaneous addition of 1.7 nM insulin and various concentrations of IGF-I did elicit higher activity levels of LPL and GPDH as compared with those obtained by addition of IGF-I alone. This increase was observed for LPL on both day 6 and day 8 (fig. 5A) and for GPDH at day 6 post-inoculation (fig. 5B). In order to establishwhether IGF-I and insulin promote differentiation independently from their stimulation of growth, the mitogenie effects of IGF-I and insulin were further investigated by determining the increase in cell number (determined by the increase in DNA content per dish) as a function of hormone concentration. The curves of fig. 6 indicate that the mitogenie potency of insulin, if any, is low at physiological concentrations and becomes significant only at concentrations above 17 nM; the small but significantly greater number of cells observed after a 6-day exposure of the cells to 1.7 nM insulin, as compared with cells not exposed to insulin (or IGF-I), was probably due to a visible loss of cells detaching from the substratum in the absence of either hormone. In contrast to insulin, IGF-I behaved as a potent mitogen at low concentrations and even more so in the presence of 1.7 nM insulin; this observation might be related to insulin activating the appearance of IGF-II and/or IGF-I receptors at the adipocyte cell surface 141-441.The increased order of potency, insulin < IGF-I < insulin plus IGF-I, remained similar on day 6 and day 8 post-inoculation (not shown). It is of interest to note that, under the conditions used for adipose conversion (i.e., 850 nM insulin; see figs l-4 and Exp Cell Res 168 (1987)

26

Deslex, Negrel and Ailhaud

01 +t 0

' 2

5

10

20

POLYPEPTIDE

50

100

CONCENTRATION

850

(nM)

Fig. 6. Effects of insulin and IGF-I on cell proliferation of stromal-vascular cells. After plating and maintenance overnight in serum-supplemented medium, stromal-vascular cells from inguinal subcutaneous adipose tissue were shifted to a serum-free medium containing 10 )&ml transferrin, 200 pM triiodothyronine and increasing concentrations of insulin (0) or IGF-I in the presence (A) or absence (A) of 1.7 nM insulin. DNA content was determined on duplicate dishes at day 6 post-inoculation as indicated in Materials and Methods; the mean values are reported (3% from the mean). The results are expressed by taking as 100% the maximal value obtained; it corresponds to 4 ug DNA per dish for cells grown in the presence of 850 nM insulin.

tables 1 and 2), the mitogenic potency of ITT medium remains quite low, as only about one to two cell doublings could be observed within 6-8 days after inoculation. DISCUSSION Growth and differentiation of preadipose cells from established clonal lines have been reported previously to take place in serum-free medium [ 13, 141.In the case of diploid adipose precursor cells, stromal-vascular cells isolated from sheep perirenal and subcutaneous adipose tissues were shown to differentiate at a low frequency into adipose-like cells in a medium which was serum-free but not fully defined in chemical terms [12]. In numerous studies performed on adipose precursor cells isolated from rodent adipose tissues, the formation of new fat cells has been shown to occur in the presence of serum or plasma 127, 28, 451. In that respect, considerable variations in the potential to promote adipose conversion of sera and plasma from different species have been reported [46]. Moreover, under a given defined culture condition in serum-supplemented medium, the frequency of adipose precursor cells to convert into adipose-like cells varied as a function of the animal’s age and the adipose site [28, 471, as well as between cell clones from a single site 1481.Our results demonstrate that no serum or plasma is indeed required for differentiation of adipose precursor cells isolated from rat adipose Exp Cell Res 168 (1987)

Adipose conversion in serum-free medium

27

tissues, providing the cells are exposed to insulin or IGF-I, triiodothyronine and transferrin. The low mitogenic potency of this serum-free medium has allowed us to obtain, at a very high frequency, differentiated cells present as monolayers (fig. 1). By contrast, in a serum-supplemented medium of higher mitogenic potency, the same cells grew as multilayers with time in culture and showed a lower frequency of adipose conversion. This lower frequency could be due either to a decrease in the frequency of cell commitment and/or to a medium favouring the growth of fibroblast-like non-differentiating cells. Clearly, in serum-free medium, these events did not take place: after a few days in ITT medium, the proportion of adipose cells was >90 %. This observation is in agreement with the fact that most differentiated cells, if not all, contained LPL. Both observations are of interest since they indicate, under these optimized culture conditions, that primary cultures of stromal-vascular cells were largely devoid of contaminating cells. In some other experiments performed in ITT medium and in which the proportion of adipose-like cells was actually lower (60-70 %), endothelial cells were detected as contaminants and could be recognized by their characteristic cobblestone morphology (not shown). In any event, it is clear that ITT medium is not a selective medium unique to stromal-vascular cells originating from the periepididymal adipose tissue (fig. 1, table I), since this medium was also able to support differentiation of stromal-vascular cells originating from retroperitoneal and inguinal subcutaneous adipose tissues (table 2, fig. 2). The results presented in figs 1 and 2, as well as in tables 1 and 2, indicate also that differentiated stromal-vascular cells are indeed converted to adipose cells. These cells were able in the first place to synthesize and accumulate triacylglycerol to levels reported for cells maintained in serum-supplemented medium 141. Incidently, the triacylglycerol accumulation indicates that no ‘induction’ by exogenous substrate is required for the acquisition of enzyme markers of adipose conversion, as previously demonstrated for 3T3-F442A [49] and Ob17 cells [4]. These cells were able in the second place to mobilize triacylglycerol stores, and differentiated stromal-vascular cells did release unesterified fatty acids in response to a lipolytic drug. Regarding the critical role of insulin in promoting the adipose conversion of stromal-vascular cells, the results appear different at first sight from those reported for the Ob17 preadipocyte cell line. Ob17 cells were shown in insulindeprived serum to express LPL at activity levels only half those determined in cells exposed to insulin-supplemented serum [50]. In contrast to the expression of this early marker, the expression of late markers of adipose conversion, GPDH and acid: CoA ligase, was more dependent upon the presence of insulin. The opposite observations made on Ob17 and stromal-vascular cells with respect to insulin requirement can be reconciled if one recalls that FCS contains high levels of insulin-like growth factors able to substitute for insulin [35]. In agreement with this hypothesis, in stromal-vascular cells, LPL was significantly expressed in Exp Cell Res 168 (1987)

28

Deslex, Negrel and Ailhaud

IGF-I-supplemented serum-free medium and the addition of insulin to IGF-Icontaining medium led to a 1.2- to 3-fold increase in the activity level of this enzyme (fig. 5A). However, in the case of stromal-vascular cells (but not in Ob17 cells) IGF-I was also able to replace insulin for the expression of GPDH (fig. 5 B). The results in figs 4 and 5B indicate no major differences between insulin and IGF-I in promoting the expression of GPDH, since the ECso values were found to be 40 and 8 nM, respectively. As already mentioned, it is of interest to note that the half-maximally effective concentrations of insulin are different for the expression of LPL and GPDH, only the former taking place within a physiological range of concentrations. This might be due to the requirement of a mitogenic stimulus for the expression of GPDH, but not for that of LPL, as already observed in Ob17 cells [2, 5, 81. Consistent with this hypothesis, the enhancement in the expression of GPDH activity in the presence of 1.7 nM insulin and various concentrations of IGF-I (fig. 5B) was correlated with the stimulation of cell proliferation observed under the same conditions (fig. 6). Thus these results indicate that, when stromal-vascular cells are exposed to a physiological concentration of insulin, IGF-I present at low concentrations plays a permissive role for adipose cell differentiation. Regarding the role of triiodothyronine, stromal-vascular cells showed only a partial requirement for the expression of LPL in serum-free medium (fig. 3). This result is at variance with data obtained on Ob17 cells in serum-supplemented medium ([51] and A. Doglio et al., unpublished work). The lack of growth hormone requirement for adipose conversion of stromal-vascular cells in serumfree medium is also at variance with data reported on the differentiation of cells from preadipocyte clonal lines in serum-supplemented medium 152, 531. It is unlikely that exposure of stromal-vascular cells to FBS (containing high levels of growth hormone) is responsible for this lack of dependence, since plating in the presence of FBS (depleted of growth hormone) led to an identical differentiation in ITT medium (not shown). Among other possibilities to explain the lack of growth hormone requirement could be the fact that stromal-vascular cells have been exposed to triiodothyronine and growth hormone in vivo before inoculation in vitro and thus could keep a ‘memory’ of hormone exposure. It could be envisaged that, in preadipocyte clonal lines, a ‘remodelling’ of the chromatin structure could take place, allowing some promoter regions to regain a part or all of their hormone dependence. This phenomenon could be due to the very large number of cell doubling which occurred during the establishment of the cell lines and/or to the significant number of mitoses occurring during the growth phase of preadipose cells. If this were so, since stromal-vascular cells do not divide to any great extent in ITT medium, this phenomenon would be negligible or incomplete with respect to growth hormone or triiodothyronine dependence, respectively. The aim of this study was primarily to examine whether adipose precursor cells can differentiate in a serum-free, chemically defined medium. The results demonstrate that the hormones which are required for differentiation in vitro should Exp Cell Res 168 (1987)

Adipose conversion in serum-free medium

29

circulate in abundance in vivo. Hence it is tempting to postulate that the regulatory step for the formation of new fat cells might be found in the stimulation by specific mitogens of the proliferation of growth-arrested, dormant adipose precursor cells. Possibly this stimulation would take place in response to increased concentrations of either insulin or more likely insulin-like growth factors present in the circulation, as a function of diets [54], and/or in response to mitogenic factors produced locally by hypertrophied fat cells. Clearly, the development of a simple chemically defined medium for the differentiation of diploid adipose precursor cells opens the possibility to characterize inhibitors or activators of the adipose conversion process. The authors wish to thank Professor J. Etienne (Paris) for the kind gift of anti-lipoprotein lipase antibodies and Dr C. Vannier (Nice, France) for help in performing immunofluorescence staining of LPL. We are indebted to Mrs G. Oillaux for expert secretarial assistance and to Miss M. Cazales for the preparation of culture media. This work was supported by the Centre National de la Recherche Scientifique (LP 7300), the Institut National de la Recherche Agronomique (grant no. 4388) and the Fondation pour la Recherche Medicale Francaise.

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Received April 1, 1986 Revised version received July 14, 1986

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