Effects of transforming precursor cells in vitro
growth factor-\g=a\on chicken adipocyte
S. C. Butterwith, C. D. Peddie and C. Goddard Department of Cellular and Molecular Biology, AFRC Institute of Animal Physiology Genetics Research, Edinburgh Research Station, Roslin, Midlothian eh25 9ps, U.K. received
and
23 December 1991
ABSTRACT
TGF-\g=a\ in chicken adipocyte precursor cells in vitro. Both TGF-\g=a\and EGF stimulated incorporation
The hyperplastic capacity of adipose tissue resides in a group of fibroblast-like adipocyte precursor cells. There is evidence to suggest that their proliferation and differentiation is regulated by insulin-like growth factor-I (IGF-I) and transforming growth factor-\g=b\ (TGF-\g=b\)but there is less information about other growth factors which may also participate in adipocyte precursor cell hyperplasia. Transforming growth factor-\g=a\ (TGF-\g=a\) is a 50 amino acid polypeptide which has been shown to stimulate proliferation in both neoplastic and normal cell types acting through the epidermal growth factor (EGF) receptor. We have studied the regulation of DNA synthesis and the activity of lipoprotein lipase
by
INTRODUCTION
in 3T3-L1 preadipocytes (Weiner, Shah, Smith et al. 1989) is also consistent with an autocrine or paracrine role. The effect of other growth factors on preadipocytes is less well established. DNA synthesis is increased by platelet-derived growth factor (PDGF) in the 3T3-L1 and Ob 1771 cell lines (Aoki, Kawada, Umyema & Sugimoto, 1990) and in chicken adipocyte precursor cells (Butterwith & Goddard, 1991). Serrerò (1987) demonstrated that epidermal growth factor (EGF) and transforming growth factor- (TGF- ) stimula¬ ted the proliferation and inhibited the differentiation of rat adipocyte precursors in the presence of a high concentration of insulin. TGF- is a member of the EGF family which can induce transmembrane signalling as either soluble or membrane-anchored forms (Massague, 1990). The soluble form of TGF- is a 50 amino acid polypeptide which has marked structural homology to EGF (Marquardt, Hunkapiller, Hood et al. 1984). Its bio¬ logical effects are thought to be mediated through the EGF receptor and there is no evidence for a distinct TGF- receptor (Lyons & Moses, 1990). Although
tissue hyperplasia occurs by proliferation and differentiation of fibroblast-like adipocyte pre¬ cursor cells. Although conditions have been devel¬ oped for the culture of adipocyte precursor cells from
Adipose
a
number of species
(see Van, 1985), there is still much
to learn about the control of hyperplasia by growth factors at the cellular level. The use of a number of
different cell lines and preadipocytes in primary cul¬ ture has clearly established that insulin-like growth factor-I (IGF-I) stimulates both proliferation and differentiation of preadipocytes (Deslex, Negrel & Ailhaud, 1987; Smith, Wise, Berkowitz et al. 1988; Butterwith & Goddard, 1991), and the presence of IGF-I mRNA in differentiated Ob 1771 cells (Doglio, Dani, Fredrikson et al. 1987) and in primary porcine adipocyte precursors (Gaskins, Kim, Wright et al. 1990) suggests that the effect may be via an autocrine or paracrine mechanism. Similarly, the stimulation of DNA synthesis in adipocyte precursors by trans¬ forming growth factor-ßl (TGF-ßl) (Butterwith & Goddard, 1991) and the presence of TGF-ß mRNA
[3H]thymidine
into DNA in a dose-dependent TGF-\g=a\ was approximately 180-fold more potent than EGF. Addition of TGF-\g=a\in combination with IGF-I, TGF-\g=b\1 or platelet-derived growth factor produced a synergistic increase in DNA synthesis. Short-term incubation with TGF-\g=a\reduced lipoprotein lipase activity by 23%. These results show that TGF-\g=a\is a potent mitogen in these adipocyte precursor cells and can inhibit their differentiation in vitro and may participate in the regulation of adipose tissue development in vivo. Journal of Endocrinology (1992) 134, 163\p=n-\168 of
manner.
TGF- was initially found in tumours, a number of studies have shown that it is produced by normal cells (see Derynck, 1990) and it should be considered as a normal physiological ligand for the EGF receptor. Its production and function in normal cells is not well understood, and its role in adipocyte development is restricted to a single report (Serrerò, 1987). The pre¬ sent study was designed to investigate the potential role of TGF- in the regulation of adipocyte pre¬ cursor proliferation and differentiation.
MATERIALS AND METHODS was a gift from Dr R. Derynck (Genentech, South San Francisco, CA, U.S.A.). Recombinant cDNA-derived IGF-I, PDGF-AB and TGF- were from Bachern, Saffron Waiden, Essex, U.K. EGF (receptor grade) and bovine serum albumin (radioim¬ munoassay grade) were purchased from Sigma, Poole, Dorset, U.K. Gentamicin and Ultroser were from Life Technologies, Paisley, Strathclyde, U.K. Tissue cul¬ ture plates (Costar; 24-multiwell plates) and medium 199 (M199) were from Northumbria Biologicals, Cramlington, Northumberland, U.K. [6-3H] Thymidine (25 Ci/mmol) was from Amersham Inter¬ national pic, Amersham, Bucks, U.K.
TGF-ßl
Animals Female broiler chickens of a commercial line were obtained from D. B. Marshall, Newbridge, Mid¬ lothian, U.K. They were housed in brooders with a lighting pattern of 14 h light: 10 h darkness and fed on standard broiler diets. Birds were killed at 2-3 weeks of age by cervical dislocation, and abdominal adipose tissue was removed for the preparation of adipocyte precursor cells.
Wilkie & Goddard (1991). Each experiment was repeated three times with different batches of cells. We have previously indicated that there is a good correlation between DNA synthesis and cell number (Butterwith & Goddard, 1991). Differentiation of adipocyte precursor cells was investigated in conditions described by Butterwith & Griffin (1989). After 7 days in culture the cells form a confluent monolayer, develop lipoprotein lipase activity, and after 14 days they resemble lipid-rich mature adipocytes. To assess short-term effects on differentiation, cells were incubated in the presence or absence of TGF- added for 18 h, 8 days after reach¬ ing confluence. Lipoprotein lipase activity was used as a marker of differentiation and measured as previ¬ ously described (Butterwith & Griffin, 1989). Statistics
The ALLFIT program (De Lean, Munson & Rodbard, 1978) was used to analyse dose-response curves and calculate half-maximum effective concen¬ trations (ED50) for [3H]thymidine incorporation, and a two-way analysis of variance using the MINITAB statistical package (Minitab Statistical Software, State College, PA, U.S.A.) to test for synergistic interactions.
RESULTS
Incorporation
of
[3H]thymidine into
chicken
adipo¬
cyte precursor cell DNA in the absence of added
Ultroser or exogenous growth factor varied between batches of cells but was a mean of 1936± 1054 d.p.m./well (s.e.m. 3) in these experi¬ ments. The addition of M199 containing 4% Ultroser increased [3H]thymidine incorporation to 122 880±67 421d.p.m./well (n = 2), a 43-fold increase. =
Effect of EGF
Adipocyte precursor cells were prepared and cultured in supplemented M199 as previously described (Butterwith & Griffin, 1989). They were seeded in multiwell plates at a density of 32 000 cells/well (2 cm2) in M199 (1 ml) containing 4% (v/v) Ultroser and incubated for 66 h at 37 °C in a humidified atmos¬ phere containing 5% C02. In experiments designed to measure DNA synthesis, the cells were washed and incubated in M199 without supplements for a further 24 h before the addition of growth factors, together with [3H]thymidine (0-25 pCi/well). Incorporation of [3H]thymidine into DNA was determined after a further 18 h incubation as described by Duelos,
The effect of EGF or TGF- on [3H]thymidine incorporation into DNA in chicken adipocyte pre¬ cursor cells in the absence of other factors is shown in Fig. 1. These structurally related peptides stimulated the incorporation of [3H]thymidine in a dose-depen¬ dent manner. The maximum response to TGF- (6-9fold) occurred at a concentration of about 1 pg/1 with an ED50 of 0- 323 pg/1. The response to EGF (8-2-fold) did not reach a maximum at a concentration of 100 pg/1 and did not allow accurate calculation of the ED50. We estimate the ED50 to be greater than 58pg/l showing that TGF- was more potent than EGF (approximately 180-fold).
or
TGF-
DNA
Cell culture
on
synthesis
'S ff
•
4000-
3000
2000
-
1000
S
-
j=
11
.
'
001
0 0001
01
1
100
10
1000
Peptide concentration (µg/\) figure 1. Effect of increasing concentrations of transforming growth factor- (TGF- ; ·) and epidermal growth factor (EGF; O) on the stimulation of [3H]thymidine incorporation into
DNA in chicken adipocyte precursor cells in culture. Values are the mean ± s.e.m. (n 4) in one representative experiment out of three. =
Effect on DNA synthesis of TGF- in combination with IGF-I, TGF-ßl or PDGF
The effect of incubating adipocyte precursor cells with increasing concentrations of TGF- in the presence
of IGF-I (5 pg/1) is shown in Fig. 2. This concentra¬ tion caused maximum DNA synthesis in an IGF-I dose-response curve when added alone to adipocyte precursors (Butterwith & Goddard, 1991). DNA syn¬ thesis was increased by the presence of both factors together and it was greater than the additive effect in incubations with each factor alone. The stimulation was synergistic at TGF- concentrations of 0-1 pg/1 (F=5-35, P
growth factor-\g=a\on chicken adipocyte
S. C. Butterwith, C. D. Peddie and C. Goddard Department of Cellular and Molecular Biology, AFRC Institute of Animal Physiology Genetics Research, Edinburgh Research Station, Roslin, Midlothian eh25 9ps, U.K. received
and
23 December 1991
ABSTRACT
TGF-\g=a\ in chicken adipocyte precursor cells in vitro. Both TGF-\g=a\and EGF stimulated incorporation
The hyperplastic capacity of adipose tissue resides in a group of fibroblast-like adipocyte precursor cells. There is evidence to suggest that their proliferation and differentiation is regulated by insulin-like growth factor-I (IGF-I) and transforming growth factor-\g=b\ (TGF-\g=b\)but there is less information about other growth factors which may also participate in adipocyte precursor cell hyperplasia. Transforming growth factor-\g=a\ (TGF-\g=a\) is a 50 amino acid polypeptide which has been shown to stimulate proliferation in both neoplastic and normal cell types acting through the epidermal growth factor (EGF) receptor. We have studied the regulation of DNA synthesis and the activity of lipoprotein lipase
by
INTRODUCTION
in 3T3-L1 preadipocytes (Weiner, Shah, Smith et al. 1989) is also consistent with an autocrine or paracrine role. The effect of other growth factors on preadipocytes is less well established. DNA synthesis is increased by platelet-derived growth factor (PDGF) in the 3T3-L1 and Ob 1771 cell lines (Aoki, Kawada, Umyema & Sugimoto, 1990) and in chicken adipocyte precursor cells (Butterwith & Goddard, 1991). Serrerò (1987) demonstrated that epidermal growth factor (EGF) and transforming growth factor- (TGF- ) stimula¬ ted the proliferation and inhibited the differentiation of rat adipocyte precursors in the presence of a high concentration of insulin. TGF- is a member of the EGF family which can induce transmembrane signalling as either soluble or membrane-anchored forms (Massague, 1990). The soluble form of TGF- is a 50 amino acid polypeptide which has marked structural homology to EGF (Marquardt, Hunkapiller, Hood et al. 1984). Its bio¬ logical effects are thought to be mediated through the EGF receptor and there is no evidence for a distinct TGF- receptor (Lyons & Moses, 1990). Although
tissue hyperplasia occurs by proliferation and differentiation of fibroblast-like adipocyte pre¬ cursor cells. Although conditions have been devel¬ oped for the culture of adipocyte precursor cells from
Adipose
a
number of species
(see Van, 1985), there is still much
to learn about the control of hyperplasia by growth factors at the cellular level. The use of a number of
different cell lines and preadipocytes in primary cul¬ ture has clearly established that insulin-like growth factor-I (IGF-I) stimulates both proliferation and differentiation of preadipocytes (Deslex, Negrel & Ailhaud, 1987; Smith, Wise, Berkowitz et al. 1988; Butterwith & Goddard, 1991), and the presence of IGF-I mRNA in differentiated Ob 1771 cells (Doglio, Dani, Fredrikson et al. 1987) and in primary porcine adipocyte precursors (Gaskins, Kim, Wright et al. 1990) suggests that the effect may be via an autocrine or paracrine mechanism. Similarly, the stimulation of DNA synthesis in adipocyte precursors by trans¬ forming growth factor-ßl (TGF-ßl) (Butterwith & Goddard, 1991) and the presence of TGF-ß mRNA
[3H]thymidine
into DNA in a dose-dependent TGF-\g=a\ was approximately 180-fold more potent than EGF. Addition of TGF-\g=a\in combination with IGF-I, TGF-\g=b\1 or platelet-derived growth factor produced a synergistic increase in DNA synthesis. Short-term incubation with TGF-\g=a\reduced lipoprotein lipase activity by 23%. These results show that TGF-\g=a\is a potent mitogen in these adipocyte precursor cells and can inhibit their differentiation in vitro and may participate in the regulation of adipose tissue development in vivo. Journal of Endocrinology (1992) 134, 163\p=n-\168 of
manner.
TGF- was initially found in tumours, a number of studies have shown that it is produced by normal cells (see Derynck, 1990) and it should be considered as a normal physiological ligand for the EGF receptor. Its production and function in normal cells is not well understood, and its role in adipocyte development is restricted to a single report (Serrerò, 1987). The pre¬ sent study was designed to investigate the potential role of TGF- in the regulation of adipocyte pre¬ cursor proliferation and differentiation.
MATERIALS AND METHODS was a gift from Dr R. Derynck (Genentech, South San Francisco, CA, U.S.A.). Recombinant cDNA-derived IGF-I, PDGF-AB and TGF- were from Bachern, Saffron Waiden, Essex, U.K. EGF (receptor grade) and bovine serum albumin (radioim¬ munoassay grade) were purchased from Sigma, Poole, Dorset, U.K. Gentamicin and Ultroser were from Life Technologies, Paisley, Strathclyde, U.K. Tissue cul¬ ture plates (Costar; 24-multiwell plates) and medium 199 (M199) were from Northumbria Biologicals, Cramlington, Northumberland, U.K. [6-3H] Thymidine (25 Ci/mmol) was from Amersham Inter¬ national pic, Amersham, Bucks, U.K.
TGF-ßl
Animals Female broiler chickens of a commercial line were obtained from D. B. Marshall, Newbridge, Mid¬ lothian, U.K. They were housed in brooders with a lighting pattern of 14 h light: 10 h darkness and fed on standard broiler diets. Birds were killed at 2-3 weeks of age by cervical dislocation, and abdominal adipose tissue was removed for the preparation of adipocyte precursor cells.
Wilkie & Goddard (1991). Each experiment was repeated three times with different batches of cells. We have previously indicated that there is a good correlation between DNA synthesis and cell number (Butterwith & Goddard, 1991). Differentiation of adipocyte precursor cells was investigated in conditions described by Butterwith & Griffin (1989). After 7 days in culture the cells form a confluent monolayer, develop lipoprotein lipase activity, and after 14 days they resemble lipid-rich mature adipocytes. To assess short-term effects on differentiation, cells were incubated in the presence or absence of TGF- added for 18 h, 8 days after reach¬ ing confluence. Lipoprotein lipase activity was used as a marker of differentiation and measured as previ¬ ously described (Butterwith & Griffin, 1989). Statistics
The ALLFIT program (De Lean, Munson & Rodbard, 1978) was used to analyse dose-response curves and calculate half-maximum effective concen¬ trations (ED50) for [3H]thymidine incorporation, and a two-way analysis of variance using the MINITAB statistical package (Minitab Statistical Software, State College, PA, U.S.A.) to test for synergistic interactions.
RESULTS
Incorporation
of
[3H]thymidine into
chicken
adipo¬
cyte precursor cell DNA in the absence of added
Ultroser or exogenous growth factor varied between batches of cells but was a mean of 1936± 1054 d.p.m./well (s.e.m. 3) in these experi¬ ments. The addition of M199 containing 4% Ultroser increased [3H]thymidine incorporation to 122 880±67 421d.p.m./well (n = 2), a 43-fold increase. =
Effect of EGF
Adipocyte precursor cells were prepared and cultured in supplemented M199 as previously described (Butterwith & Griffin, 1989). They were seeded in multiwell plates at a density of 32 000 cells/well (2 cm2) in M199 (1 ml) containing 4% (v/v) Ultroser and incubated for 66 h at 37 °C in a humidified atmos¬ phere containing 5% C02. In experiments designed to measure DNA synthesis, the cells were washed and incubated in M199 without supplements for a further 24 h before the addition of growth factors, together with [3H]thymidine (0-25 pCi/well). Incorporation of [3H]thymidine into DNA was determined after a further 18 h incubation as described by Duelos,
The effect of EGF or TGF- on [3H]thymidine incorporation into DNA in chicken adipocyte pre¬ cursor cells in the absence of other factors is shown in Fig. 1. These structurally related peptides stimulated the incorporation of [3H]thymidine in a dose-depen¬ dent manner. The maximum response to TGF- (6-9fold) occurred at a concentration of about 1 pg/1 with an ED50 of 0- 323 pg/1. The response to EGF (8-2-fold) did not reach a maximum at a concentration of 100 pg/1 and did not allow accurate calculation of the ED50. We estimate the ED50 to be greater than 58pg/l showing that TGF- was more potent than EGF (approximately 180-fold).
or
TGF-
DNA
Cell culture
on
synthesis
'S ff
•
4000-
3000
2000
-
1000
S
-
j=
11
.
'
001
0 0001
01
1
100
10
1000
Peptide concentration (µg/\) figure 1. Effect of increasing concentrations of transforming growth factor- (TGF- ; ·) and epidermal growth factor (EGF; O) on the stimulation of [3H]thymidine incorporation into
DNA in chicken adipocyte precursor cells in culture. Values are the mean ± s.e.m. (n 4) in one representative experiment out of three. =
Effect on DNA synthesis of TGF- in combination with IGF-I, TGF-ßl or PDGF
The effect of incubating adipocyte precursor cells with increasing concentrations of TGF- in the presence
of IGF-I (5 pg/1) is shown in Fig. 2. This concentra¬ tion caused maximum DNA synthesis in an IGF-I dose-response curve when added alone to adipocyte precursors (Butterwith & Goddard, 1991). DNA syn¬ thesis was increased by the presence of both factors together and it was greater than the additive effect in incubations with each factor alone. The stimulation was synergistic at TGF- concentrations of 0-1 pg/1 (F=5-35, P
