Biol Cell (1992) 76, 131-138 © Elsevier, Paris

131

Original article

Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes Ana Rosa Rinc6n-Sdnchez, Ascenci6n Hern~mdez, Ma de Lourdes L6pez, Tomds Mendoza-Figueroa* Department o f Pharmacology and Toxicology, Centro de Investigaci6n y de Estudios A vanzados del IPN, A P 14-740, Mexico City 07000, DF Mexico (Received 10 August 1992; accepted 19 November 1992) Summary - The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from

female rats and explore the potential use of this culture system to study the effect of hormones, drugs and toxic chemicals on it. Hepatocytes, seeded on a feeder layer of 3T3 cells, maintained for 2 weeks their typical morphology. The cultures were able to take up [~4C]acetic and [t4C]oleic acid from the culture medium and incorporate them into lipids. The synthesis and secretion of lipids by [t4C]acetic acid-labeled cultures had a maximum value after 11 and 13 days in culture. Triacylglycerols were the main lipidic species synthesized and secreted by hepatocytes (up to 67% of the total lipids); they also synthesized and secreted phospholipids, cholesterol and cholesterol esters from [~4C]acetic acid. Similarly, [J4C]oleic acid-labeled cultures synthesized and secreted mostly triacylglycerols (up to 60-70% of the total lipids), but. they were also able to incorporate the labeled precursor into both cellular and secreted phospholipids and cholesterol esters. The activity of glycerol-phosphate-dehydrogenase, marker enzyme of glycerolipid synthesis, decreased slightly during the culture time whereas the activity of malic enzyme, marker of fatty acid synthesis, increased. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially triacylglycerols, from both [J4C]acetic and [14C]oleic acid for at least 2 weeks and that they maintain enzyme activities related with the synthetic pathways of glycerolipids and fatty acids. They also suggest that these cultures could be a useful model system to study lipid metabolism and the effect of hormones, drugs and toxic chemicals on it. lipid metabolism / female rat hepatocytes

Introduction

The liver is known to carry out central metabolic functions in various aspects of lipid and lipoprotein metabolism, such as uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol and phospholipids and formation and secretion of specific classes of lipoproteins. The liver also participates in other essential aspects of plasma lipoprotein metabolism [10]. Lipid metabolism is influenced by genetic, dietetic and hormonal factors. Because of the different metabolic needs for male and female reproduction the hepatic tissue shows considerable sexual dimorphism. Although the origin of sexual dimorphism is genetic in nature, the liver phenotypic expression is mediated via the sex hormones [8]. Gonadal steroids influence the concentration of fatty acid binding protein in the rat liver [1]. Fatty acids are taken up and metabolized at significantly higher rates by the female than by the male rat liver [26, 30]. Hepatocyte plasma membranes from female rats have a greater avidity for fatty acids as compared with membranes from male rats. This allows the female liver to metabolize fatty adids from

* Correspondence and reprints Abbreviations: C, cholesterol; CE, cholesterol esters; dpm, disintegrations per minute; FA, fatty acid; GPD, L-glycerol 3-phosphate dehydrogenase; HDL, high density lipoprotein; ME, malic enzyme; PL, phospholipids; TLC, thin layer chromatography; TG, triacylglycerols; VLDL, very low density lipoprotein.

blood at higher rates than the male liver [27]. Isolated perfused livers and hepatocytes from male animals secrete less triacylglycerols and incorporate less fatty acids into them than livers from female rats [25, 26, 30, 32]. These genderrelated differences in triacylglycerol metabolism by the liver include altered lipid and lipoprotein composition of the very low density lipoprotein particle of the male compared to that of the female [30, 32]. Recently, primary cultures of hepatocytes have been used extensively in metabolic studies [9]. These cultures allow study of the metabolic expression of liver cells in a defined environment, independently of hormonal or nutritional variations. However, the utility of these cultures is limited due to their short survival and the rapid loss of their differentiated functions. We have described a system for the culture of adult rat hepatocytes from male rats seeded on a feeder layer of 3T3 cells, which maintain for several weeks their typical morphology and differentiated functions like albumin secretion, basal and inducible levels of cytochrome-P450, synthesis and secretion of several lipids to the culture medium [6, 16]. These cultures were used to study the effect of drugs and toxic chemicals on lipid metabolism [7, 20, 22]. The aim of this work was to characterize the lipid metabolism in female rat hepatocytes cultivated on a feeder layer of 3T3 cells and explore their possible use to study the effect of hormones, drugs and toxic chemicals on it. The hepatocytes were cultivated for 2 weeks and we measured the synthesis and secretion of lipids (triacylglycerols and other complex lipids).from [14C]acetic and [14C]oleic

132

AR Rinc6n-S~nchez et al ture medium. [i~C]oleic acid labeled cultures were rinsed twice with 2 ml of serum-free medium and refed with 0.5 ml of fresh non-radioactive medium. After 24 h incubation the medium was removed from the [t4C]acetic and p~C]oleic acid labeled cultures, the dishes were rinsed and the cells were scraped in cold phosphate-buffer (0.1 M pH 7.4) and disrupted by sonication. Lipids from culture medium (secreted lipids) and cells (cellular lipids) were extracted as described by Cham and Knowles [3] and analyzed by thin layer chromatography.

acid. Besides, we d e t e r m i n e d the activity o f two lipogenic e n z y m e s , malic e n z y m e a n d g l y c e r o p h o s p h a t e deh y d r o g e n a s e , m a r k e r s o f fatty acid a n d glycerolipid synthesis, respectively.

Materials and methods Materials

Thin layer chromatography Collagenase type IV, hydrocortisone, insulin and d-biotin were purchased from Sigma Chemical Co (St Louis, MO). Mitomycin c was a gift from Bristol-Myers Co (Syracuse, NY). Calf serum was obtained from Hy Clone Laboratories (Logan, UT). [l,2-14C]acetate (55 mCi/mmol), and (l-t4C)oleic aicd (59 mCi/mmol) were obtained from New England Nuclear (Boston, MA). All other reagents were of analytical grade and were purchased from JT Baker (Mexico City, Mexico).

The extracted lipids were evaporated under a nitrogen stream and dissolved in 30 ill of chloroform containing lipid extract from female rat serum as a carrier. Samples of 15 t~l were analyzed by TLC on plates of silica gel 60 (0.22 mm thickness, Merck, Darmstadt, Germany) using heptane:di-isopropyl ether:acetic acid (60:40:4) as mobil phase [12]. The radioactivity that comigrated with triacylglycerol, phospholipid, cholesterol, cholesterol ester and fatty acid purified standards was determined by liquid scintillation counting in a Packard spectrophotometer model Tri-Carb 460 using Instagel (Packard Instruments Co, Inc, Downers Grove, IL). Disintegrations per minute (dpm) were determined automatically by the external standard method after correction for quenching.

Cell cultures Female Wistar rats (150-200 g) were fed ad libitum and maintained with day-night cycles of 12 h. Hepatocytes were isolated by the collagenase-perfusion method [2] with some modifications [20] and seeded and cultivated as previously described [16, 20].

Enzyme assays Cell trlorphology Extracts from cell cultures were prepared and assayed for Lglycerol 3-phosphate dehydrogenase (EC l . l , l . 8 ) (GPD) and malic enzyme (L-malate:NADP oxidoreductase (descarboxylating), EC. I. I. 1.40) (ME) activities as described [13-15, 33). Protein content was determined by the method of Lowry et al [18].

Cultured hepatocytes were maintained for 2 weeks and cell morphology was examined and photographed with an Olympus inverted photomicroscope model IMT equipped with a photographic system model PM-10AD.

Statistical analysis

Uptake and incorporation o f [#4C]acetic and [~C]oleic acid into cellular and secreted lipids

The results are expressed as the mean _+ standard deviation of duplicate determinations from four culture dishes in a typical experiment. Similar results were obtained in 2 - 3 independent experiments. Statistical analysis of the results was made with the Student's t-test with a significance level of P < 0.05.

At the indicated culture times 0.2 ~Ci of [14C]acetic acid or p4C]oleic acid were added per ml of culture medium. After 2 h incubation, the uptake of the radioactive precursor was determined by measuring the radioactivity that remained in the cul-

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Lipid metabolism in female rat hepatocytes

133

Results

Uptake of [J4C]acetic and [14]oleic acid

Morphology and protein content of cultured hepatocytes from female rat

C u l t u r e d h e p a t o c y t e s r e m o v e d f r o m the culture m e d i u m r a d i o a c t i v e p r e c u r s o r s n e e d e d for lipid synthesis. A b o u t 650/o o f the a d d e d [14C]oleic acid was r e m o v e d f r o m the culture m e d i u m d u r i n g a 2-h p e r i o d b y 24-h cultures, whereas only 220/0 o f the a d d e d [14C]acetic acid was rem o v e d d u r i n g the s a m e p e r i o d (table II). T h e u p t a k e o f []4C]oleic acid decreased up to 28°/0 with respect to the initial value after 2 weeks in culture (table II). O n the other h a n d , the u p t a k e o f [14C]acetic acid increased 42070 a f t e r 6 days in culture but decreased d u r i n g the second week. Cultures o f lethally t r e a t e d 3T3 cells were a b l e to r e m o v e b o t h r a d i o a c t i v e p r e c u r s o r s f r o m culture m e d i u m b u t to a lower extent than the 3T3-hepatocyte cultures (table II).

F r e s h l y isolated h e p a t o c y t e s a d h e r e d to the 3T3 feeder layer with high efficiency (80-90070). 24 h a f t e r seeding, hepatocytes f o r m e d the typical hepatic cords showing their c h a r a c t e r i s t i c p o l y g o n a l m o r p h o l o g y , with g r a n u l o u s cytoplasm, intercellular spaces resembling biliary canaliculi a n d central nucleus (fig l a ) . A f t e r 8 a n d 15 days in culture, h e p a t o c y t e s f o r m e d cell m o n o l a y e r s d u e to the ass o c i a t i o n o f small g r o u p s (fig lb). A t these culture times h e p a t o c y t e s m a i n t a i n e d their typical c u b o i d a l m o r p h o l o gy with s o m e i n t r a c y t o p l a s m i c lipid d r o p l e t s (fig lc). T h e p r o t e i n c o n t e n t o f h e p a t o c y t e s c u l t u r e d for 24 h was 1.26 ___ 0.06 mg per culture dish ( m e a n _+ SD, n = 4). P r o t e i n c o n t e n t increased 13 a n d 20070 with respect to the initial value after 8 a n d 15 d a y s in culture, respectively (table I). A b o u t 25070 o f the total p r o t e i n c o n t e n t per dish was due to 3T3 cells (table I). Since the p r o t e i n c o n t e n t was p r a c tically u n c h a n g e d d u r i n g the culture time a n d differences between culture dishes were low (table I), the results d e s c r i b e d b e l o w were expressed per culture dish a n d not per mg o f p r o t e i n .

Incorporation of [t4C]acetic acid into lipids by cultured hepatocytes W e d e t e r m i n e d the synthesis a n d secretion o f lipids by cult u r e d h e p a t o c y t e s by m e a s u r i n g the i n c o r p o r a t i o n o f

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Table I1. Uptake of [14C]acetic and [~4C]oleic acid. At the indicated culture times (days) cells were incubated for 2 h with 0.2 #Ci/ml of [14C]acetic or [14C]oleic acid. The uptake of the radioactive precursors was determined by the difference between initial and residual radioactivity in the culture'medium after 2 h of incubation.

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134

AR Rinc6n-S~inchezet al this culture time TG represented 67% of the labeled secreted lipids. 39 and 22% of labeled cellular and released lipids, respectively, corresponded to PL in 24-h cultures (fig 3). Incorporation of the precursor into total PL increased also during the second week in culture with a maximum value at the 13th day that was 1.5-fold higher than that observed in 24-h cultures. The increase was due mostly to incorporation into secreted P L that was 4.3-fold higher than that seen in 24-h cultures (fig 3). 11 and 5% of the label incorporated by the 24-h cultures corresponded to C and CE, respectively (fig 3). The incorporation into total C and CE increased during the second week in culture with maximum values at the 13th day that were 60 and 72% higher, respectively, than those observed in 24-h cultures. These increases were due to in-

[14C]acetic acid into cellular, secreted and total (cellular plus secreted) lipids. The incorporation of radioactive precursor into total lipids decreased about 4 0 % during the first week in culture, but at the 1lth and 13th day increased about two-fold due mostly to the higher incorporation into secreted lipids (fig 2). The T L C analysis of the secreted and cellular lipids showed that T G were the main lipidic species. After 24 h of cell seeding about 52 and 36% of labeled cellular and secreted lipids, respectively, were T G (fig 3). The incorporation of []4C]acetic acid into total T G during the culture time was very similar to that observed for total lipids, with a maximum value at the 13th day that was 1.9-fold higher than the 24-h value. This increase was due mostly to incorporation into secreted TG that was eightfold higher than that observed in 24-h cultures (fig 3). At

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Lipid metabolism in female rat hepatocytes corporation into cellular C and secreted CE and the observed values were 2.9- and 3.5-fold higher, respectively, than those measured in 24-h cultures (fig 3).

Incorporation of [14C]oleic acid by cultured hepatocytes Although the incorporation of [14C]oleic acid into total lipids decreased slightly during the first week of culture, the observed values between 8 and 15 days were unchanged with respect to the initial values (fig 4). While the incorporation of the labeled precursor into cellular lipids decreased about 40-50°70 in hepatocytes cultured for 4-15o70 days, the observed values for secreted lipids increased more than two-fold (2.1-2.8) during the same period (fig 4). As in experiments with [~4C]acetic acid, the T L C analysis of lipids showed that T G were the main lipidic species. In 24-h cultures 45 and 50°7o o f the labeled cellular and secreted lipids, respectively, were TG and this value reached 60-70o7o during the culture time (fig 5). As seen for total lipids the incorporation of [14C]oleic acid into cellular T G decreased 2.8-3.8-fold during the same period. The incorporation of [14C]oleic acid into total PL was unchanged during the culture time. 27 and 33% of the labeled cellular and secreted lipids, respectively, corresponded to PL in 24-h cultures. While cellular PL showed slight changes during culture time, secreted P L increased 1 . 5 - 2 . l - f o l d with respect to the initial values (fig 5). Labeled diglycerides were also found in cellular lipids but were not secreted into the culture medium. 15 and 5°7o of the labeled cellular lipids were 1,3- and 1,2-diglycerides, respectively, in 24-h cultures. These values showed slight modification during the culture time (fig 5).

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4% of the labeled cellular and secreted lipids corresponded to CE in 24-h cultures. While the incorporation of the labeled precursor into cellular CE decreased 2.7-4.7-fold during the culture time, the incorporation into secreted CE increased 2 6 - 7 4 % during the same period, producing a reduction of 38-50°7o in the incorporation into total CE. Less than 1% of label incorporated corresponded to cellular and secreted FA (fig 5). These results show that while the synthesis of different lipids by cultured hepatocytes showed slight changes during 2 weeks in culture, the secretion of these lipids, mostly TG, increased as seen in experiments with [14C]acetic acid (fig 3).

Activity of two lipogenic enzymes in cultured hepatocytes To determine the levels of the lipid synthesis pathways in cultured hepatocytes, we measured the activities of malic enzyme and glycerol-phosphate-dehydrogenase, marker enzymes of fatty acid and glycerolipid synthesis, respectively [33]. The activity of GPD in 24-h cultures was 95.9 _+ 13.2 nmol/min per culture dish (mean _+ SD, n = 4). This value decreased during the first week in culture, with a maximum decrease of 380/o at the 8th day (fig 6). The activity of GPD increased slightly during the second week and after 15 days in culture it was 750/o of the initial value (fig 6). On the other hand, the activity of malic enzyme in 24-h cultures was 14.72 _+ 1.40 nmol/min per culture dish (mean _+ SD, n = 4) and increased during the culture time with maximum values at the 1lth and 13th day that were 68 and 58% higher than the 24-h value (fig 7). These enzyme activities were due mostly to hepatocytes, since cultures of lethally treated 3T3 cells showed 5-10°70 of the enzyme activities measured in the 3T3-hepatocyte cultures (figs 6, 7). These results show that 3T3-hepatocyte cultures maintain for at least 2 weeks enzyme activities related with the synthetic pathways of glycerolipids and fatty acids, in agreement with the experiments with labeled precursors.

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Our results show that female rat hepatocytes cultured on a feeder layer of 3T3 cells survive for long periods maintaining their typical morphology and resembling the organization of the normal liver cords. The hepatocyte viability was also maintained during the culture time. The protein content, a rough estimate of cell viability, increased lightly during the 15 days. Since hepatocytes in these conditions do not proliferate, the increase could be due to proliferation of non-parenchymatous cells like epithelial and Ito cells [21]. Since these cells were seen occasionally in the culture and hepatocytes secrete extracellular matrix, specially between contact areas with the 3T3 cells [16], and lethally treated feeder layers of 3T3 cells secrete some products into the culture medium like collagen [11], the increase in cell protein could be due to extracellular matrix production. The hepatocytes in vivo have the capacity of taking up large quantities o f fatty acids from the blood [23]. The culture of hepatocytes on 3T3 cells allowed the expression of this differentiated function for 2 weeks. Up to 68.50/o o f the added [14C]oleic acid was taken up by the hepatocytes after a pulse of 2 h and this uptake was about twice that of [14C]acetic acid (ta.ble II). The entry of fatty acids

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into liver cells is a physical-chemical and not a biologically facilitated process [4, 5, 24, 25]. Female hepatocytes cultivated on a feeder layer of 3T3 cells carried out for 2 weeks the synthesis and secretion o f several lipids into the culture medium from both [14C]acetic and [14C]oleic acid (figs 2 - 5 ) . The incorporation of the labeled precursors into cellular lipids was higher during the first day in culture (figs 2, 4) due probably to increased synthesis of several lipids necessary to repair the

membrane injury produced during the isolation by the collagenase digestion. After that the incorporation of both labeled precursors into cellular lipids decreased and remained at this level during the culture time. The incorporation of both precursors into secreted lipids was mostly into T G (60-65%0), followed by PL (25%0), C (15%) and CE ( 3 - 6 % ) (figs 3, 5). 1, 3-diglycerides are intermediate species of T G and PL synthesis and these lipids are not secreted. The incorporation into fatty acids was lower than

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Fig 7. Malic enzyme activity of cultured hepatocytes. 3T3 cells (O) and 3T3-hepatocytes (O) were maintained in culture for 15 days. At the indicated culture times, the cells were scraped, disrupted by sonication and centrifuged at l0 000 rpm for 3 rain. Enzymatic activity was determined spectrophotometrically at 340 nm in the postmitochondrial supernatant as described in Materials and methods. Asterisks represent significantly different values from the 24-h cultures by the Student's t-test (P < 0.05).

1% and it was maintained at this level during the culture time. The incorporation of the radioactive precursors into lipids by 3T3 cells was 65°7o and 38% lower than that of [14C]acetic and [14C]oleic acid, respectively, by the 3T3-hepatocyte cultures (figs 2, 4). The percentage of the lipids secreted into the culture medium was similar to that of these lipids in VLDL [10] and these results suggest that the lipid secretion by the 3T3-hepatocyte cultures could be in the form of lipoproteins (VLDL and HDL) [29, 31]. Further work is necessary in order to determine the secretion of lipoproteins by these cultures and their lipid composition. In the fasting state the liver accounts for most of lipoprotein production and the hepatocyte cultures resemble more closely the fasting liver due to the limited supply of fatty acids. The time course of incorporation of radioactive precursors into secreted lipids shows an increase after 6 days in culture (figs 2, 4). It has been shown that cultures of hepatocytes seeded on plastic and maintained with supplemented medium synthesize and secrete apoproteins corresponding to all classes of lipoproteins [28]. The synthesis increases during the culture time, reaching a maximum at the fifth day after isolation [28]. The 3T3-hepatocyte cultures could require several days to have the optgmal levels of apoproteins necessary for lipid secretion. Preliminary results have shown that cultures of 3T3-hepatocytes from male rats have stable or increasing levels of mRNAs of apoproteins E and A-1 during 4 weeks in culture (unpublished data). The enzymes involved in the synthesis o f glycerolipids and fatty acids were maintained in the hepatocyte cultures for 2 weeks. The levels of GPD and ME correlated with

the results obtained with the radioactive precursors. These studies show that enzymatically isolated hepatocytes cultivated on 3T3 cells are a useful experimental system for studies on lipid metabolism. The content of the estrogen receptor and its distribut i o n between cytosol and nucleus does not vary during estrous cycle in the rat [19]. Therefore we used female rats without considering the estrous cycle. The concentration of 17 fl-estradiol in the culture medium was 16.7 pmol/l due to the added serum while the physiologic level of circulating steroids in women is 150-370 pmol/1 [17]. Although the results obtained in this study and those obtained previously with male rat hepatocytes [6, 7, 20, 22] are not strictly comparable, it seems that lipid metabolism is similar in both cultures, including the uptake of fatty acids, the percentage of TG in the secreted lipids and the GPD and malic enzyme activities. It is not surprising since the ctllture conditions, the medium composition and mostly the content of sex hormones are the same in cultured hepatocytes isolated from both male and female rats. However, in the studies with oleic acid it was found that the proportion of the total TG secreted into the culture medium by female hepatocytes was higher than that secreted by male hepatocyte cultures [6, 7, 20]. This difference could suggest a higher secretion of TG by female rat hepatocytes. Further work is necessary in order to determine if at physiologic estrogen levels the female hepatocytes maintain their sexual dimorphism in vitro with respect to lipid metabolism and if there are sex-related differences in response to different agents. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially TG, from both [14C]acetic and [14C]oleic acid

138

AR Rinc6n-S~inchezet a/

and that they maintain for at least 2 weeks enzyme activities related with the synthetic pathways of glycerolipids and fatty acids. They also suggest that these cultures could be a useful model system to study lipid metabolism and the effect of hormones, drugs and toxic chemicals on it.

Acknowledgments The authors acknowledge the art work by Mr Alfredo Padilla. AR Rinc6n-S~mchez was supported by a scholarship from CONACYT, Mexico.

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Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes.

The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from female rats and explore the potent...
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