JOURNAL OF CELLULAR PHYSIOLOGY 149339-346 (1991i

Ad ipogenic Activity Produced by Hepatocyte-Derived Cell lines and by Normal Hepatocytes in Primary Culture HIROKAZU ZAITSU AND GINETTE SERRERO* W . Alton /ones Cell Science Center Inc., Lake Placid, New York 12946 Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLCIPRFI5, and SKHep-1 do not contain adipogenic activity. Conditioned medium from HepGZ cells also stimulated differentiation of 3T3-L, cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, i s destroyed by treatment at lOO"C, with protease, with 2-mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cells also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.

Preadipocyte cell lines are useful model systems to study adipose differentiation (Serrero, 1984). Most adipogenic cell lines require the presence of fetal calf serum or fetuin (a fraction of fetal calf serum) to optimally undergo differentiation, although the degree of this serum dependency is variable (Serrero, 1984; Gaillard et al., 1985). This serum requirement has allowed investigators to demonstrate the existence of adipogenic factors in fetal bovine serum (Kuri-Harcuch and Green, 1979; Serrero e t al., 1979; Gaillard e t al., 1984). Although growth hormone and glucocorticoids have been shown to have a potent adipogenic activity (Morikawa et al., 1982; Schiwek and Loffler 19861, their presence accounts only partially for the adipogenic activity of fetal bovine serum (Nixon and Green, 1984; Schiwek and Loffler 1986). This suggests that other serum factor(s) not yet identified have adipogenic activity. Reports in the literature have indicated the presence of adipogenic activity in the circulation that is at higher levels in obese patients as compared with normal subjects (Sypnieska et al., 1986) and in obese rodents (Loffler et al., 1983), although this difference of level in obese patients could be due to dietary control (Hauner et al., 1989). It is possible to assume that the serum requirement observed for adipogenic cell lines in vitro corresponds to these circulating factors in vivo. Therefore, characterizing the serum adipogenic factor is very important. Biochemical characterization of adipogenic activity has been undertaken using fetuin a s a source of the adipogenic factor (Zaitsu and Serrero, 1990).However, the complex nature of fetuin has made the study of the adipogenic factor difficult. Moreover, one important question that the use of fetuin as a 0 1991 WILEY-LISS, INC.

source of adipogenic factor does not allow to be answered is the identity of the site of synthesis of the adipogenic factorb). Exploring such a question is very important if one wishes to purify the factor, clone the gene coding for it, and study the regulation of its synthesis. Since liver is the organ where several serum proteins are synthesized and since normal hepatocytes in culture have been shown to secrete these proteins (Kaighn and Prince, 1971; Leffert and Sell, 1974; Jeejeebhoy et al., 1975), we have investigated in the present paper whether or not hepatocytes synthesize adipogenic factor. For this purpose, experiments were performed to determine if culture medium conditioned by either normal primary hepatocytes or by hepatocytederived cell lines could stimulate the differentiation of adipogenic cell lines and adipocyte precursors in primary culture.

MATERIALS AND METHODS Cell culture 1246 cells, derived from C3H mouse teratoma, were cultivated in tissue culture plasticware (Costar, Cambridge, MA) in Dulbecco's modified Eagle's medium/ Ham's F12 nutrient mixture (1:l mixture) (Gibco, Grand Island, NY) (referred to as DMEIF12) supple-

Received March 13, 1991; accepted June 6 , 1991.

"To whom reprint requestsicorrespondence should be addressed. Hirokazu Zaitsu is now a t Kyushu University, Department of Pediatrics, Faculty of Medicine, Fukuoka, Japan.

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mented with 1.2 gil sodium bicarbonate (Sigma, St. Louis, MO), 15 mM HEPES, pH 7.4, (Research Organics, Cleveland, OH) and 10% fetal calf serum (FCS) (HyClone, Logan, UT) in humidified atmosphere of 95% air-5% COz a t 37°C. All hepatoma derived cell lines were cultivated in RITC 80-7 medium (Kyokuto Pharmaceuticals, Tokyo, Japan) containing 2% FCS. Adipose differentiation assay Subconfluent 1246 cells were inoculated at a density of 1.5 x lo4 cellsiwell (4.5 cm2) in 12-well plates (Costar) with DMEiFl2 supplemented with 2% FCS at day 0. At day 1, the medium was replaced by defined medium consisting of DMEiFl2 supplemented with insulin (10 pgiml) (Sigma, St. Louis, MO), transferrin (10 pgiml) (Sigma), and FGF (5 ngiml) (Collaborative Research, Waltham, MA). Cells Cultivated in this defined medium were exposed to dexamethasone (2 x M) (Sigma),isobutylmethylxanthine (2 x lo-, M) (Aldrich Chemical Co., Milwaukee, WI), and indomethacin (3 x M) (Sigma) from day 4 to day 6. Cells were further incubated in DMEiFl2 containing insulin and transferrin and were harvested a t day 11. Adipose differentiation was examined by measurement of glycerol 3 phosphate dehydrogenase specific activity (G3PDH) as described previously (Serrero, 1985). Conditioned medium from either hepatocytes or from hepatoma-derived cell lines and fractions to assay were added a t day 1, day 4, and day 6. When cells were harvested, duplicate wells were pooled and used for the assay. Control plates corresponding to cells cultivated in defined medium alone were used as negative controls. Cells cultivated in the defined medium supplemented with 250 pgiml of crude Pedersen fetuin (Sigma) were used as positive controls. Unless otherwise specified in the figure legends and tables, duplicate dishes were used per condition in the experiments described in this paper. Values given for G3PDH specific activity correspond to the average value for duplicate dishes, the standard deviation being less than 10%. 3T3-Ll cells (lo5 cellsi35 mm dish) were cultivated in the same conditions as 1246 cells. Collection of conditioned medium from primary cultures of rat hepatocytes Rat primary hepatocytes were derived from 2-10day-old New Zealand Brown (NBR) rats (Trudeau Institute, Saranac Lake, NY). In experiment A of Figure 7, the liver was minced and digested with 0.1% collagenase in Hepes-buffered saline (pH 7.0) a t 37°C according to the method of Hoshi and Kan (1986). In this way, hepatocytes are co-cultivated with nonhepatocytes. In experiments B and C in Figure 7, hepatocytes and nonhepatocytes were separated by the method of Shimaoka et al. (1987) using differential centrifugation a t 50g for 1 min. Cells collected in this way were resuspended in RITC 80-7 medium containing 5 pM FeSO, [7HzOl (Sigma) after a rinse. The medium was collected 24 h later and assayed for the presence of adipose differentiation stimulating activity, as described in the previous paragraph. Collection of conditioned media from hepatoma derived cell lines All cell lines were cultivated in RITC 80-7 medium containing 2% FCS a t 37°C. When cells became sub-

confluent, medium was replaced with RITC 80-7 containing 5 pM FeS0, [7HzO] again. The medium was collected 48 h later and assayed for its ability to stimulate adipose differentiation. For biochemical characterization, HepG2 cells were cultivated in Minimum Essential Medium (MEM) supplemented with 2% FCS until they reached confluency. The cells were then washed with factor-free serum-free MEM and were maintained in factor-free serum-free MEM. Medium was collected every 4 days.

Biochemical characterization of HepG2-derived adipogenic factor A semi-purified sample obtained by gel filtration on Sephacryl S-300 was used. Acid and alkali sensitivity was tested by incubating the samples at pH 2.5 or pH 11.0 for 24 h a t 4°C followed by dialysis against 20 mM phosphate buffer a t pH 7.0. A control untreated sample was dialyzed against 20 mM phosphate buffer at pH 7.0. Heat stability was examined by boiling samples for 10 min. Sensitivity to a disulfide-reducing agent was tested by incubating the samples with 0.2 M 2-mercaptoethanol a t room temperature for 6 h then dialyzing them against 20 mM phosphate buffer (pH 7.0) before assaying them. Protease sensitivity was examined by incubating samples with immobilized pronase conjugated to agarose beads (Streptomycesgriseus, Sigma) a t 37°C for 6 h. Pronase was removed by centrifugation before use.

RESULTS AND DISCUSSION Conditioned medium from hepatocyte derived cell line contains adipogenic activity Adipogenic activity of conditioned media from hepatocyte derived cell lines was determined by following their ability to stimulate the differentiation of the C3H mouse teratoma-derived adipogenic cell line 1246. Differentiated 1246 cells present all the characteristics of mature adipocytes (Serrero and Khoo, 1982). They can proliferate and differentiate in a defined medium which can also sustain differentiation of primary culture of newborn rodent adipocyte precursors (Serrero and Mills, 1987). Although the differentiation of 1246 cells can occur in defined medium, i t is stimulated by the addition of serum or fetuin (Serrero and Khoo, 1982; Zaitsu and Serrero, 1990). As 1246 cells have a simpler growth requirement than other adipogenic cell lines (Serrero and Khoo 1982; Serrero et al., 1979; Gaillard et al., 1984) and as they differentiate maximally in 11 days, they represent a n easy and suitable bioassay system for measuring adipogenic activity of serum fractions or conditioned media. Routinely, differentiation of 1246 cells was determined by following morphological changes characteristic of adipocytes by measuring accumulation of triglycerides and by determining the increase in the level of glycerol-3-phosphate dehydrogenase (G3PDH) specific activity, which is a late marker of adipose differentiation. It is well known that several established cell lines derived from liver maintain, in culture, some or all of the differentiated characteristics of functional hepatocytes. In particular, these cell lines are able to synthesize and secrete several types of serum proteins and constitute excellent systems for studying liver func-

HEPATOCYTE-DERIVED ADIPOGENIC ACTIVITY

tions (McNab et al., 1976; Knowles et al., 1980; Nakabayashi et al., 1982; Reid and Jefferson 1984; Doyle et al., 1985).We determined whether or not hepatocytederived cell lines secreted adipogenic activity. Conditioned media from 5 different liver-derived cell lines were examined for the presence of adipogenic activity. They ranged from the differentiated cell lines HepG2, Hep3B (Knowles et al., 19801, and HUH-7 (Nakabayashi et al., 1982) to the semi-differentiated cell line PLCIPRFI5 (McNab e t al., 19761, to the undifferentiated hepato-adenocarcinoma cell line SK-Hep-1 (Doyle et al., 1985). As shown in Figure 1, only the media collected from the well differentiated cell lines HepG2 and Hep3B were able to stimulate 1246 cell differentiation by 3.6-fold (HepG2) and 2.6-fold (Hep3B), respectively. The addition of the same concentration of media from the undifferentiated cell lines PLC/PRF/5 and Sk-Hepl resulted in a decrease of G3PDH specific activity, instead of a n increase. These results could suggest that only cell lines which maintained functional properties of differentiated hepatocytes could secrete the adipogenic factor. However, the addition of culture medium from the semi-differentiated HUH-7 cell line also inhibited the increase in G3PDH specific activity in 1246 cells as observed for the semi- and nondifferentiated cell lines PLC/PRF/5 and SK Hepl. This could indicate that the lack of stimulation and even inhibition of 1246 cell differentiation resulting from the addition of the conditioned media could also be due to the fact that the three cell types produced inhibitors of differentiation rather than to the fact that they do not have the characteristics of functional hepatocytes. The fact that HepG2 cells synthesize and secrete the adipose inducing factor indicate that in human subjects, the liver may be a site of production for adipogenic activity. In addition to stimulating increases in G3PDH specific activity, the addition of HepG2 CM to the culture medium of 1246 cells resulted in a higher percentage of differentiated cells (Fig. 2) and a higher level of triglyceride accumulated. After 11 days in culture cells cultivated in 4F medium alone accumulated 165 pg triglycerides/106 cells, whereas cells cultivated in the presence of HepG2 CM contained 380 pg of triglycerides/lQ6cells. We examined the effect of increasing amounts of concentrated HepG2 CM on the proliferation and differentiation of 1246 cells maintained for 11 days in culture. As shown in Figure 3a and Figure 3b, the addition of HepG2 CM to the defined medium resulted in a moderate stimulation of cell proliferation (1.25fold) and a 3.5-fold increase in G3PDH specific activity. A 50% stimulation of G3PDH specific activity was observed in the presence of 6 pg protein from HepG2 CM/ml of culture medium. It should be noted that the addition of a concentration of a t least 100 pg/ml of crude Pedersen fetuin was required to stimulate the same increase of G3PDH specific activity in the 1246 cells (Zaitsu and Serrero 1990). Maximal stimulation of G3PDH specific activity by fetuin required a protein concentration of 250 pgiml or higher, as compared with a protein concentration of 50-60 pglml for HepG2 CM. For 1246 cells, the addition of 2% fetal bovine serum would be required to provide a similar stimulation of adipose differentiation. Higher concentrations of serum

341

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PRFI5

Fig. 1. Adipose differentiation-inducing activity in cell culture fluid collected from human hepatocyte derived cell lines. Conditioned media were collected a s described in the Methods section. Test cultures received 30% of each conditioned medium, in triplicate manner. 1246 cells cultivated i n the presence of fetuin (250 Fgiml) were used a s positive control. G3PDH specific activity in negative control culture (cultivated in defined medium only a s described in the Materials and Methods section) was 94 mUimg protein.

were found inhibitory (data not shown). When comparing Figure 3a and Figure 3b, one could observe that the addition of 20 pl/ml of HepG2 CM (120 pgiml of protein) resulted in a further stimulation of cell proliferation, but in a slight inhibition in the level of G3PDH specific activity. The nature of this difference is not clear and could be due to the presence in the crude conditioned medium of inhibitors of differentiation. Interestingly, when determining the effect of the same HepG2 CM on the short term growth of 1246 cells, a 2.1-fold increase in cell number was observed (Fig. 4). After 11 days in culture (Fig. 3a) the effect was only 1.25 fold.

HepG2 CM stimulates differentiation of 3T3-Ll cells and of primary culture of adipocyte precursors in defined medium We investigated whether or not HepG2 CM could stimulate the differentiation of other adipogenic cell lines, such a s 3T3-Ll cells, or primary culture of adipocyte precursors. As indicated in Figure 5a, HepG2 conditioned medium could replace fetuin to stimulate the differentiation of 3T3-Ll cells. In addition, HepG2 conditioned medium was also able to stimulate the differentiation of adipocyte precursors isolated from epididymal fat pads of 21 day old rats in primary culture (Fig. 5b). These latter results suggest that the adipogenic factor produced by liver-derived cell lines may represent a physiological regulator of adipose differentiation.

ZAITSU AND SERRERO

342

A

HepG2 CM

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Fig. 2. Photomicrograph of 1246 cells cultivated defined medium: (A) in the absence or (B) in the presence of HepGX CM (protein concentration 55 kgiml). Magnification x 100.

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Fig. 3. Effect of increasing concentrations of concentrated HepG2 conditioned medium on (a) the proliferation and (b)the G3PDH specific activity of 1246 cells cultivated for 11days as described in the Materials and Methods section. The values correspond to the average of quadruplicate dishes and are given with their standard deviation. HepG2 CM was concentrated 25-fold by ultrafiltration on Filtron cassette having a molecular weight cut off of 10,000 dalton. The final protein concentration of concentrated HepG2 CM was 6 mgiml.

Biochemical characterization of HepG2-derived adipogenic factor

The fraction precipitated by 30-50% ammonium sulfate precipitation was used for biochemical characterization of the adipogenic factor as described in the Conditioned medium collected from HepG2 cells (pro- Methods section and in the legend to Figure 6. Molectein concentration of 250 kg/ml) was concentrated ular sieve fractionation on Sephacryl S300 of concen40-fold by ultrafiltration through a 10,000 molecular trated HepG2 medium indicated that the adipogenic weight cut off membrane and then precipitated be- activity eluted in one single broad peak with a n appartween 30% and 50% ammonium sulfate. We ensured ent molecular weight between 445 and 232 kDa (Fig. 6). that material having a molecular weight less than We have shown previously that when crude Pedersen 10,000 which went through the ultrafiltration mem- fetuin was chromatographed on a column of Sephacryl brane and the fractions which were precipitated by S300 in the same conditions a s the ones used for HepG2 0-30% ammonium sulfate or which remained in solu- CM, three distinct peaks of adipogenic activity were tion after the treatment with 30-50% ammonium sul- obtained (Zaitsu and Serrero, 1990). They had a molecfate did not contain any significant adipogenic activity. ular weight greater than 669 kDa, between 445 and 232

343

HEPATOCYTE-DERIVED ADIPOGENIC ACTIVITY 2

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Fig. 4. Effect of increasing concentrations of concentrated HepG2 CM on the short term proliferation of 1246 cells. Cells were plated at a density of 7,000 cellsiwell in 24-well dishes in 1 ml of defined medium consisting of DME-F12 medium supplemented with fibronectin (2 Fgirnl), insulin (10 pgiml), transferrin (10 pg/ml), and FGF (25 ngiml) alone or in the presence of increasing amounts of concentrated

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Fig. 5. (a)Adipogenic activity of HepG2 conditioned media for 3T3 L1 cells. 3T3-Ll cells were inoculated at a n initial cell density of lo7 cellsidish (35 mm dish) in DME-F12 medium supplemented with 2% FBS and then switched to defined medium consisting of DME-F12 medium supplemented with insulin (10 pgiml), transferrin (10 Fgiml), and FGF (25 ngiml) alone (Cont) or containing either 250 ugiml of crude Pedersen fetuin or 55 ugiml of HepG2 conditioned medium (HepG2 CM). At day 4 cells were treated with dexamethasone (2 x lo7 M) and isobutylmethylxanthine (2 X M) for 48 h as described in the Methods section for 1246 cells. Cells were harvested at day 14. The value for G3PDH activity for the control was 200 mUimg protein; (b) Adipogenic activity of HepG2 CM for rat epididymal adipocytes precursors in primary culture. Adipocyte precursors were isolated by collagenase digestion of epididymal fat pads of 21-day-old New Zealand Brown rats and cultivated in defined medium as described previously (Serrero and Mills, 1987). The value of G3PDH specific activity for the control was 50 mUimg protein.

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HepG2 CM prepared as described in the legend to Figure 3. Cells were detached with a solution of 1 mgiml trypsin containing 1 mM EDTA and counted with a Coulter counter. The values for cell number correspond to the average of quadruplicate determinations and are given with their standard deviation.

kDa, and around 69 kDa, respectively (Zaitsu and Serrero, 1990). These results would suggest that HepG2 CM contains only one adipogenic factor possibly similar to the second peak of activity found in Pedersen fetuin. Biochemical characterization of the HepG2-derived adipogenic factor (summarized in Table 1) indicated that the activity was completely lost after being treated with acid (pH 2.5 for 24 h at 4"C), heat (lOO"C, 10 m i d , protease (1.8 mUimg protein, 6 h at 37"C), or 2-mercaptoethanol (0.2 M, 6 h a t room temperature). The factor was relatively stable after being exposed to alkaline conditions (pH 11.0 for 24 h at 4°C). When treated a t pH 9.4, HepG2 fraction retained the totality of its adipogenic activity. These results would suggest that HepG2 derived adipogenic factor shares biochemical similarities with the second major adipogenic fraction, defined as FII found in Pedersen fetuin, but not with the first adipogenic factor, defined a s FI, as found in Pedersen fetuin (Zaitsu and Serrero 1990). Based on these results one could assume that one of the adipogenic factors found in fetuin could be synthesized and secreted by the liver. Our previous studies on fetuin derived adipogenic factors have shown t h a t the factors are different from known large molecular weight proteins found in serum such a s a2-macroglobulin and lipoproteins. The fact that HepG2 derived adipogenic factor may be similar to FII fraction of fetuin would indicate that this is also the case for the hepatocyte-derived factor. Further characterization of adipogenic factors contained in fetuin and in HepG2 CM is required to investigate these possibilities in detail.

Conditioned medium from rat hepatocytes in primary culture stimulates adipose differentiation of 1246 cells Hepatocytes and non-hepatocyte cells were isolated by collagenase digestion of NBR rat livers and were cultivated in primary culture. Hepatocytes and nonhepatocyte cells were prepared using two different

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Fig. 6. Molecular sieve fractionation of adipose differentiation-inducing factor secreted by HepG2 cells on Sephacryl S300 column. HepGZ conditioned medium was concentrated 40-fold by ultrafiltration through a 10,000 molecular weight cut off membrane and then was precipitated between 3 0 6 and 50% of ammonium sulfate. The resulting pellet was dissolved with water and loaded on a Sephacryl S300 column (2.6 cm x 90 cm) equilibrated in 20 mM phosphate buffer

pH 7.0 containing 0.1 M, NaCl. Protein concentration of each fraction was measured with BioRad protein assay. Every two fractions were pooled and an identical volume (40 Fliml) was assayed for adipogenic activity. Specific activity of G3PDH in control culture was 65 mUimg protein. Thyroglobulin (669 kD), ferritin (445 kD), catalase (232 kD), and bovine serum albumin (67 kD) were used as molecular weight markers.

experimental procedures as described in the Method section. Figure 7 shows the results of three separate experiments with duplicate dishes using culture media collected from three distinct primary cultures. The variation for G3PDH specific activity within each experimental point was less than 10%. Different dilutions of medium conditioned by NBR r a t primary hepatocytes stimulated 1246 cell differentiation in a dose-dependent fashion: 1.4-fold for a 10% dilution of hepatocyte derived medium and 3-fold for a 30% dilution of hepatocyte medium. In contrast, medium obtained by cultivating the nonhepatocyte cellular fraction obtained from liver did not have any stimulating effect on 1246 cell differentiation; it even inhibited differentiation (Fig. 7C). These results indicate that normal hepatocytes synthesize and secrete a n adipose differentiation stimulating activity. Biochemical characterization of the liver-derived adipogenic medium indicates that it is found in HepG2 conditioned medium a s a large molecular weight form. It is not known whether or not this is also the case in physiological conditions or if this is due to a n artefact of preparation of the HepG2 conditioned medium. Fetuin derived adipogenic factors are also found as large molecular weight proteins (Zaitsu and Serrero, 1990).

It is known that in serum several large molecular weight proteins, such as a2-macroglobulin, can bind a wide variety of smaller proteins, particularly growth factors (Huang e t al., 1984). Whether or not the large molecular weight determined for adipogenic factors is due to their association with these binding proteins is currently under study in our laboratory. In vivo and in vitro studies have led to the identification of some of the hormones and growth factors controlling adipose tissue development (Serrero, 1984). These studies have also shown that serum contains a potent adipogenic activity which cannot be totally replaced by any of the known adipogenic hormones, including growth hormone and glucocorticoids, and which plays a n important role in the regulation of adipose tissue development in vivo (Nixon and Green, 1984; Schiwek and Loffler, 1986). In fact, although glucocorticoids and growth hormone have been shown to accelerate 1246 cell differentiation (Serrero 19841, the addition of fetuin or HepG2 CM could further stimulate adipose differentiation even in their presence. Studies using different adipogenic cell lines have shown that fetal bovine serum could be totally replaced by the addition of fetuin to stimulate differentiation (Gaillard et al., 1985; Zaitsu and Serrero, 1990),

345

HEPATOCYTE-DERIVED ADIPOGENIC ACTIVITY TABLE 1. Biochemical characteristics of adipogenic factor secreted by HepG2 cells' Conditions No treatment pH 2.5 pH 11.0 Heat 2-ME Protease

G3PDH W control

100 11

47 0 18 5

'Sample prepared by gel filtration of concentrated HepGZ CM on Sephacryl S-300 was treated as described in the Materials and Methods section. Remaining activity was shown in the percentage of the value determinedin nontreated sample taken as 100%.

thereby suggesting that the adipogenic factors found in serum are also found in fetuin. In fact, the addition of 250 Fgiml fetuin has the same adipogenic activity as fetal bovine serum for 3T3-Ll cells and for 1246 cells (data not shown). The data presented here show that crude HepG2 CM also has a high adipogenic activity for 1246 cells for 3T3-Ll cells and for primary culture of adipocyte precursors and can replace fetuin to stimulate their differentiation. We have shown previously that fetuin contained two adipogenic factors (Zaistu and Serrero, 1990). The results presented here suggest that HepG2 has only one adipogenic factor, HepG2 CM probably being similar to the second factor present in fetuin. Further purification of these factors is required in order to investigate in detail the contribution of these various factors in stimulating adipose differentiation. Factors stimulating either differentiation or proliferation of preadipocytes have been purified from human plasma (Wier and Scott, 1986) or identified in rat serum (Li et al., 1989). The site of production of these factors has not yet been identified. Our preliminary results indicate that the HepG2 derived factor has different biochemical characteristics from either of these two serum-derived factors. In spite of these two examples, characterization or purification of adipogenic factorb) from serum has remained difficult because of the variability in adipogenic activity among various lots of serum, the complex nature of serum, and the presence of a large variety of contaminating proteins. The data presented here suggest for the first time that hepatocytes may be the site of production of a t least one serum-derived adipogenic activity. Moreover, HepG2 CM may represent a simpler starting material to purify this factor. Further studies are required to identify the biochemical nature of this adipogenic factor and to investigate if liver is also the physiological site of synthesis of circulating adipogenic factor in vivo. Solving such a question may be vary useful for furthering our understanding of the physiological regulation of adipose tissue development.

ACKNOWLEDGMENTS The authors wish to thank Dr. Mikio Kan for his help and advice, Dr. Shinichi Yamada for the gift of RITC80-7 medium, Nancy Lepak for helping with the triglyceride assays, Marilyn Hauer for the preparation of the manuscript, and Marina LaDuke for the illustra-

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Fig. 7. Adipose differentiation-inducing activity in cell culture fluid from rat primary hepatocytes. Duplicate dishes of 1246 cells were cultivated in defined medium alone (Cont), in the presence of a 30% dilution of unconditioned RITC 80-7 medium (NCM)or in the presence of a 30% dilution of RITC 80-7 medium conditioned by the liverderived primary culture (Hep CM). In experiments B and C, conditioned media from hepatocyte cultures (Hep CM) and from nonhepatocyte cells derived from liver (NHepCM) were assayed. Specific activity of G3PDH in control culture were 27 m U h g protein (A),24 mUimg protein (B), and 72 mUimg protein (0,respectively.

tions. This work was supported by grants PO1 DK38639 from the National Institute of Health and 2003 from the Council for Tobacco Research.

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Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture.

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipoge...
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