Bioscience Reports, Vol. 12, No. 2, 1992
Amino Acid Uptake Regulation by Cell Growth in Cultured Hepatocytes Isolated from Fetal and Adult Rats S. Leoni, L2 S. Spagnuolo, 2 M. Massimi, 2 F. Terenzi 2 and L. Conti Devirgifiis 2 Received March 23, 1991 Amino acid uptake mediated by system A was studied in cultured fetal and adult hepatocytes, subjected to growth stimulation by EGF and insulin, or to growth inhibition by high cell density. The mitogenic stimulation induced a strong transport increase only in fetal cells, while the cell density-dependent growth inhibition, probably mediated by molecules present on adult hepatocyte membranes, provoked the decrease of amino acid uptake only in the adult cells. The results indicate that the different modulation of amino acid transport by cell growth is dependent on the age and the differentiation stage of hepatocytes. KEY WORDS: amino acid transport; cell growth; rat hepatocytes.
INTRODUCTION Amino acid uptake mediated by system A appears to be an important parameter controlling cellular growth. This transport system is modulated by hormones, growth factors and substrates [Kilberg et al., 1985; Saier et al., 1988]. Data concerning rat hepatocytes during fetal life [Leoni et al., 1987] and liver regeneration [Le Cam et al., 1979] indicate that proliferating cells have increased amino acid transport with respect to quiescent hepatocytes, suggesting a close relationship between cellular growth and amino acid uptake [Nakamura et al., 1984; Handlogten and Kilberg, 1988]. However it is difficult to obtain univoca! responses analyzing an "in v i v o " system, that is affected by several different controls. Cultured liver cells represent a suitable model for studying the correlation between amino acid transport and cell growth, since they can be induced to enter in S phase "in vitro" by several hormones and growth factors [Cruise et al., 1985; 1Dept. of Cellular and Developmental Biology, University "La Sapienza", p. le A. Moro, 5; 00185 Rome, Italy. 2 To whom correspondence should be addressed. 135 0144-8463/92/0400-0135506.50/0O 1992 Plenum Publishing Corporation
136
Leoni, Spagnuolo, Massimi,Terenziand Conti Devirgiliis
Sand and Christoffersen, 1987; Ichihara A, 1991]. EGF [James and Bradshaw, 1984; Carpenter and Cohen, 1990], insulin and insulin-like factors, transforming growth factor 06 hepatopoietin and other similar substances act as stimulators of hepatocyte mitogenesis [Alison, 1986; Michalopoulos, 1990; Selden and Hodgson, 1991]. Other factors like transforming growth factor /3 act negatively on hepatocyte proliferation by means of growth inhibition [Mashima et al., 1988; Jakowlew et al., 1991; Tsao et al., 1991]. Growth inhibition is also achieved by high cell density. Cell density and adhesion to a suitable substratum have particular influence on the DNA synthesis in hepatocyte cultures. As demonstrated for a variety of cells in culture, the activity of system A is also regulated by cell density. However the effect of density can be different in cells in active growth with respect to the quiescent cells [Handlogten and Kilberg, 1988]. Therefore, in this work we have used less differentiated cells, which were actively proliferating and which showed high AIB transport, such as fetal hepatocytes, and differentiated quiescent cells, with low AIB transport, such as adult hepatocytes, with the aim of understanding whether growth stimulation or growth inhibition could affect transport system A differently in these two different physiological conditions. In particular we have used primary hepatocyte cultures prepared from fetal or adult rats in which the proliferation was induced by EGF or inhibited by high cell density.
MATERIALS A N D METHODS
Adult (3-month-old) and fetal (18-20-day-old) Wistar rats were used to obtain isolated hepatocytes by collagenase (Boerhinger Mannheim) dissociation, as previously described [Moldeus et al., 1978; Conti Devirgiliis et at., 1981]. The isolated cells were suspended in RPMI medium, supplemented with 10% FCS, 0, 1 ~M dexametasone, 0.5 ~g/ml amphotericine B, 100 ~tg/ml gentamicine, and plated at various initial densities (0.6.106 - 3 9 106 cells/dish) on 60 mm diameter plastic dishes, coated with collagen. The medium was replaced after the first 3 h (adult) or 5 h (fetus) by fresh medium containing 5% FCS with the same supplements, and subsequently daily. 10-8M EGF and 10-7M insulin (Sigma Chemical, Co) were added after 24 h of culture when monolayers were obtained (0 time). Incubation was continued for further 24 or 48 h. DNA synthesis was measured by adding to cultures 1/~Ci/ml [3H-methyl]-thymidine (specific activity 84.2 Ci/mmol; NEN, Boston MA) at the appropriate time (0-24-48 h), according to Nakamura et al. [1983]. [1-14C]-2-Aminoisobutyric acid (AIB) (0.15/tCi/ml, specific activity 4060 mCi/mmol, NEN, Boston, MA) was used to examine amino acid transport. The assay was performed both in the sodium-containing and in the sodium-free media as described elsewhere [Leoni et at., 1987]. Before the AIB uptake assay, the culture medium was replaced with Krebs-Henseleit solution (KH) for at least 1 h, to obtain amino acid starvation, which enhances the system A transport activity, as previously demonstrated [Leoni et al., 1987].
A m i n o acid uptake in cultured hepatocytes
137
Table
1. [3H-methyl]-thymidine incorporation into D N A in hepatocytes isolated from fetal and adult rats with ( + H o r ) or without (C) h o r m o n e addition at 24 h or 48 h or culture 24 h
Fetal Adult
48 h
C
+Hor
C
+Hor
71.0 b 5.9
276.0 ~ 33.5 a
88.0 b 18,7
211.0 a 75.U
D a t a are reported as d p m / 2 h / / ~ g protein and are the m e a n of at least five experiments. SD is less than 15%o P < 0,001 with respect to the control. b p < 0,001 with respect to the adult.
Plasma membranes from rat liver (adult and 18-20-day-old fetus) were prepared according to the method of Hubbard et al. (1983). The membranes, purified about 15 fold from the homogenate, as judged by their 5'-nucleotidase activity, were stored at -70~ until use. Aliquots were added after plating, replaced after medium change and maintained for a further 24 h. After washing and KH incubation of the cultures as described above, AIB uptake and thymidine incorporation were assayed. Proteins were measured by the method of Lowry et al. [1951].
RESULTS
The effect of EGF addition on D N A sythesis and on AIB transport in hepatocyte cultures is shown in Tables 1 and 2. In cells from adult animals thymidine incorporation was low in the control plates and increased following hormonal stimulation, mainly at 48h. AIB transport was not affected in these conditions. On the contrary, in hepatocytes from fetal livers EGF stimulation had an effect on both parameters. In fact as far as thymidine incorporation is concerned, a strong increase was present at 24 and 48 h; the control values were also higher with respect to the adult values. AIB Table 2. [14-C]AIB uptake in hepatocytes isolated from fetal and adult rats assayed in the absence (C) and in the presence of h o r m o n e s ( + H o r ) at 24 and 48 h of culture. 24 h
Fetal Adult
48 h
C
+Hor
C
+Hor
1104 c 269
1705 a 295
1260 c 285
2284 b 359
D a t a are reported as d p m / m g prot./15' and are the m e a n of at least five experiments. SD is less t h a n t 5 % . ~P
Amino Acid Uptake Regulation by Cell Growth in Cultured Hepatocytes Isolated from Fetal and Adult Rats S. Leoni, L2 S. Spagnuolo, 2 M. Massimi, 2 F. Terenzi 2 and L. Conti Devirgifiis 2 Received March 23, 1991 Amino acid uptake mediated by system A was studied in cultured fetal and adult hepatocytes, subjected to growth stimulation by EGF and insulin, or to growth inhibition by high cell density. The mitogenic stimulation induced a strong transport increase only in fetal cells, while the cell density-dependent growth inhibition, probably mediated by molecules present on adult hepatocyte membranes, provoked the decrease of amino acid uptake only in the adult cells. The results indicate that the different modulation of amino acid transport by cell growth is dependent on the age and the differentiation stage of hepatocytes. KEY WORDS: amino acid transport; cell growth; rat hepatocytes.
INTRODUCTION Amino acid uptake mediated by system A appears to be an important parameter controlling cellular growth. This transport system is modulated by hormones, growth factors and substrates [Kilberg et al., 1985; Saier et al., 1988]. Data concerning rat hepatocytes during fetal life [Leoni et al., 1987] and liver regeneration [Le Cam et al., 1979] indicate that proliferating cells have increased amino acid transport with respect to quiescent hepatocytes, suggesting a close relationship between cellular growth and amino acid uptake [Nakamura et al., 1984; Handlogten and Kilberg, 1988]. However it is difficult to obtain univoca! responses analyzing an "in v i v o " system, that is affected by several different controls. Cultured liver cells represent a suitable model for studying the correlation between amino acid transport and cell growth, since they can be induced to enter in S phase "in vitro" by several hormones and growth factors [Cruise et al., 1985; 1Dept. of Cellular and Developmental Biology, University "La Sapienza", p. le A. Moro, 5; 00185 Rome, Italy. 2 To whom correspondence should be addressed. 135 0144-8463/92/0400-0135506.50/0O 1992 Plenum Publishing Corporation
136
Leoni, Spagnuolo, Massimi,Terenziand Conti Devirgiliis
Sand and Christoffersen, 1987; Ichihara A, 1991]. EGF [James and Bradshaw, 1984; Carpenter and Cohen, 1990], insulin and insulin-like factors, transforming growth factor 06 hepatopoietin and other similar substances act as stimulators of hepatocyte mitogenesis [Alison, 1986; Michalopoulos, 1990; Selden and Hodgson, 1991]. Other factors like transforming growth factor /3 act negatively on hepatocyte proliferation by means of growth inhibition [Mashima et al., 1988; Jakowlew et al., 1991; Tsao et al., 1991]. Growth inhibition is also achieved by high cell density. Cell density and adhesion to a suitable substratum have particular influence on the DNA synthesis in hepatocyte cultures. As demonstrated for a variety of cells in culture, the activity of system A is also regulated by cell density. However the effect of density can be different in cells in active growth with respect to the quiescent cells [Handlogten and Kilberg, 1988]. Therefore, in this work we have used less differentiated cells, which were actively proliferating and which showed high AIB transport, such as fetal hepatocytes, and differentiated quiescent cells, with low AIB transport, such as adult hepatocytes, with the aim of understanding whether growth stimulation or growth inhibition could affect transport system A differently in these two different physiological conditions. In particular we have used primary hepatocyte cultures prepared from fetal or adult rats in which the proliferation was induced by EGF or inhibited by high cell density.
MATERIALS A N D METHODS
Adult (3-month-old) and fetal (18-20-day-old) Wistar rats were used to obtain isolated hepatocytes by collagenase (Boerhinger Mannheim) dissociation, as previously described [Moldeus et al., 1978; Conti Devirgiliis et at., 1981]. The isolated cells were suspended in RPMI medium, supplemented with 10% FCS, 0, 1 ~M dexametasone, 0.5 ~g/ml amphotericine B, 100 ~tg/ml gentamicine, and plated at various initial densities (0.6.106 - 3 9 106 cells/dish) on 60 mm diameter plastic dishes, coated with collagen. The medium was replaced after the first 3 h (adult) or 5 h (fetus) by fresh medium containing 5% FCS with the same supplements, and subsequently daily. 10-8M EGF and 10-7M insulin (Sigma Chemical, Co) were added after 24 h of culture when monolayers were obtained (0 time). Incubation was continued for further 24 or 48 h. DNA synthesis was measured by adding to cultures 1/~Ci/ml [3H-methyl]-thymidine (specific activity 84.2 Ci/mmol; NEN, Boston MA) at the appropriate time (0-24-48 h), according to Nakamura et al. [1983]. [1-14C]-2-Aminoisobutyric acid (AIB) (0.15/tCi/ml, specific activity 4060 mCi/mmol, NEN, Boston, MA) was used to examine amino acid transport. The assay was performed both in the sodium-containing and in the sodium-free media as described elsewhere [Leoni et at., 1987]. Before the AIB uptake assay, the culture medium was replaced with Krebs-Henseleit solution (KH) for at least 1 h, to obtain amino acid starvation, which enhances the system A transport activity, as previously demonstrated [Leoni et al., 1987].
A m i n o acid uptake in cultured hepatocytes
137
Table
1. [3H-methyl]-thymidine incorporation into D N A in hepatocytes isolated from fetal and adult rats with ( + H o r ) or without (C) h o r m o n e addition at 24 h or 48 h or culture 24 h
Fetal Adult
48 h
C
+Hor
C
+Hor
71.0 b 5.9
276.0 ~ 33.5 a
88.0 b 18,7
211.0 a 75.U
D a t a are reported as d p m / 2 h / / ~ g protein and are the m e a n of at least five experiments. SD is less than 15%o P < 0,001 with respect to the control. b p < 0,001 with respect to the adult.
Plasma membranes from rat liver (adult and 18-20-day-old fetus) were prepared according to the method of Hubbard et al. (1983). The membranes, purified about 15 fold from the homogenate, as judged by their 5'-nucleotidase activity, were stored at -70~ until use. Aliquots were added after plating, replaced after medium change and maintained for a further 24 h. After washing and KH incubation of the cultures as described above, AIB uptake and thymidine incorporation were assayed. Proteins were measured by the method of Lowry et al. [1951].
RESULTS
The effect of EGF addition on D N A sythesis and on AIB transport in hepatocyte cultures is shown in Tables 1 and 2. In cells from adult animals thymidine incorporation was low in the control plates and increased following hormonal stimulation, mainly at 48h. AIB transport was not affected in these conditions. On the contrary, in hepatocytes from fetal livers EGF stimulation had an effect on both parameters. In fact as far as thymidine incorporation is concerned, a strong increase was present at 24 and 48 h; the control values were also higher with respect to the adult values. AIB Table 2. [14-C]AIB uptake in hepatocytes isolated from fetal and adult rats assayed in the absence (C) and in the presence of h o r m o n e s ( + H o r ) at 24 and 48 h of culture. 24 h
Fetal Adult
48 h
C
+Hor
C
+Hor
1104 c 269
1705 a 295
1260 c 285
2284 b 359
D a t a are reported as d p m / m g prot./15' and are the m e a n of at least five experiments. SD is less t h a n t 5 % . ~P
