J. Biochem. I l l , 25-30 (1992)
Nutritional and Hormonal Regulation of mRNA Levels of Lipogenic Enzymes in Primary Cultures of Rat Hepatocytes1 Hitomi Fukuda, Akihiko Katsurada, and Nobuko Iritani2 Tezukayama Gakuin College, 4-2-2, Harumidai, Sakai, Osaka 590-01 Received for publication, July 29, 1991
and protein were required to induce fatty acid synthase and glucose 6-phosphate dehydrogenase, primarily at the transcription step (2, 3). Glucose 6-phosphate dehydrogenase induction was rather protein- than carbohydrate-dependent (3). Polyunsaturated fat feeding reduced the transcriptional rates for these lipogenic enzymes (1-4). Moreover, we found that insulin is importantly involved in the transcription of Lipogenic enzymes and also in their translation (1-3, 6, 7). Triiodothyronine-treatment markedly increased malic enzyme synthesis primarily at the transcription step in both normal and diabetic rats (4, 6). The effect of triiodothyronine on the other lipogenic enzymes was observed in the diabetic state. The triiodothyronine and insulin actions appeared to be synergistic. However, the results in whole animals are influenced by so many factors that it is hard to draw definite conclusions as to the mechanisms of actions of hormones and nutrients. In particular, it is diflicult to analyze the interactions of hormones and nutrients. Thus, it is advantageous to use hepatocytes in primary culture for further studies on the mechanisms regulating the gene expression of lipogenic enzymes. We previously investigated the effects of amino acids and various kinds of fatty acid species on lipogenic enzyme induction at the enzyme level in cultured hepatocytes (20). Goodridge et aL (9, 10) reported hormonal regulation of gene expression of malic enzyme and fatty acid synthase in chick embryo hepatocytes in culture. However, the nutritional and hormonal regulation of gene expression of acetyl-CoA carboxylase and glucose 6-phosphate dehydrogenase in cultured hepatocytes has not been
The first step in the pathway of long-chain fatty acid biosynthesis is mediated by acetyl-CoA carboxylase [EC 6.4.1.2], which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. The second step is the conversion of acetyl-CoA and malonyl-CoA to long-chain fatty acids by the multifunctional enzyme, fatty acid synthase [EC 2.3.1.85], in the presence of NADPH. Malic enzyme [EC 1.1.1.40] and glucose 6-phosphate dehydrogenase [EC 1.1.1.49] are donors of NADPH. It is well known that the cellular contents of these lipogenic enzymes in liver vary with different nutritional, hormonal, and genetic conditions. However, only limited information on the regulatory mechanism for gene expression of the enzymes is available at present (1-19). We have cloned the cDNAs of all four lipogenic enzymes, and investigated the nutritional and hormonal regulation of their gene expression in rat liver (1-5). On the feeding of a carbohydrate diet (without protein) to fasted rats, the transcriptional rate, mRNA concentration and enzyme induction of acetyl-CoA carboxylase similarly increased to the levels with a carbohydrate/protein diet (2). In these animals, the transcriptional rate and mRNA concentration of malic enzyme increased to the levels with the carbohydrate/protein diet, whereas the enzyme induction increased only to 60% (4). It appears that protein-feeding is not necessary to induce the acetyl-CoA carboxylase nor to induce malic enzyme mRNA. However, both carbohydrate 1
This work was supported in part by Japanese private school promotion funds. 1 To whom correspondence should be addressed. Vol. I l l , No. 1, 1992
25
Downloaded from https://academic.oup.com/jb/article-abstract/111/1/25/808969 by university of winnipeg user on 23 January 2019
The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.
26
H. Fukuda et al.
reported. The mechanisms of nutritional and hormonal regulation of gene expression of lipogenic enzymes have not been sufficiently elucidated. Thus, in the present work, we have investigated the effects of glucose, insulin, and triiodothyronine on the mRNA induction of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase in cultured rat hepatocytes. EXPERIMENTAL PROCEDURES
Preparation of RNA and Dot Blot Hybridization Assay—Total cellular RNA was isolated from cultured cells by the guanidine thiocyanate/LiCl procedure (23). The cDNAs cloned by us (1-4) were labeled with a multiprimer DNA labeling system kit (Amersham) using [33P]dCTP. To measure the mRNA concentration, the RNA (10 ^g) was denatured with formamide at 65'C for 15 min, spotted on a nylon filter, and then irradiated with ultraviolet light for 5 min The filter was prehybridized for at least 4 h at 50"C in a reaction mixture comprising 50 mM Tris-HCl (pH 7.5), 0.09 M sodium citrate, 0.9 M NaCl, 5 x Denhardt's solution, 1% SDS, 50% formamide, and 100 //g/ml of salmon sperm DNA (medium A). The hybridization was carried out for 36 h at 42*C in medium A with "P-labeled cDNA. The filter was then washed with 1.5 mM sodium citrate, 15 mM
RESULTS
Time Courses of Induction of Lipogenic Enzyme mRNAs by Glucose and/or Insulin—The time courses of induction of lipogenic enzyme mRNAs by insulin and/or glucose in cultured hepatocytes of rats are compared in Fig. 1. The mRNA concentrations become maximum after 16-24 h. Therefore, to investigate the effects of additions on the mRNA concentrations in the following experiments, the mRNA concentrations were measured after 24 h incubation of hepatocytes in medium containing test hormones and/or carbohydrates. With insulin and glucose together, the mRNA concentrations increased to about 8-10-fold the control values for hepatocytes cultured in the absence of insulin and glucose. The mRNA induction was very slightly increased by either glucose or insulin alone. The half-lives of the mRNAs can be roughly calculated from the time courses, as done by Goodridge et al. (18, 19), according to Schimke (25) and Haining (26). The values were about 8 h for acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme, and 9-10 h for glucose 6-phosphate dehydrogenase in the presence of glucose and insulin. Effects of Glucose on Induction of Lipogenic Enzyme mRNAs—The addition of glucose to the medium caused marked dose-dependent increases in the induction of the mRNAs by insulin, as shown in Fig. 2. The mRNA concentrations of acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme reached the maximum levels, 4-5-fold increases, at the glucose concentrations of 5, 20, and 10 mM, respectively. The acetyl-CoA carboxylase mRNA induction appeared to be the most sensitive to glucose among the lipogenic enzymes. On the contrary, the mRNA concentration of glucose 6-phosphate dehydrogenase reached a low mayimiim level and rather decreased at higher concentrations of glucose. Furthermore, fructose or glycerol could substitute for glucose considerably well (Table I). Effect of Insulin on Induction of Lipogenic Enzyme mRNAs—Insulin caused dose-dependent increases in the mRNA concentrations of lipogenic enzymes in the presence of glucose (Fig. 3). The maximum concentrations were 35-fold those for hepatocytes cultured without insulin. The insulin levels required to induce the maximum mRNA concentrations were below 10~9, but 10~ 7 ,10~\ and 10~8 M, respectively, for acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase. Thus, acetyl-CoA carboxylase mRNA induction was the most sensitive to insulin as well as to glucose (Fig. 2). Effects of Hormones on Induction of Lipogenic Enzyme mRNAs—The effects of hormones on the induction of J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/1/25/808969 by university of winnipeg user on 23 January 2019
Materials—Collagenase (type I), bovine serum albumin (essentially fat-free), insulin (bovine pancreas), 3,3',5-triiodo-L-thyronine, and dexamethasone were obtained from Sigma Chemical. William's medium E was obtained from Flow Laboratories and fetal bovine serum from Gibco. Tissue culture dishes were purchased from Corning, [a32 P]dCTP (lllTBq/mmol) was purchased from ICN Biochemical, nylon filters (Hybond N) from Amersham, guanidine thiocyanate from Fluka, and /3-actin DNA probe from Wako Chemical. Other chemicals were mostly purchased from Sigma. Cell Culture—Male Wistar rats (200-300 g, Japan SLC) fed on a laboratory chow were used, except for in experiments involving fatty acid addition. For experiments involving fatty acid addition, rats adapted to a fat-free diet were used (21). The hepatocytes were isolated by a modification of the method of Seglen (22), and were suspended at 5X10 6 cells/ml in William's medium E containing 5% fetal bovine serum, 1 ^M dexamethasone, and 0.1//M insulin, supplemented with glucose at the concentration of 10 mM, in addition to penicillin at 100 units/ml and streptomycin at 100 /xg/ml. The cell viability in all experiments was above 95%, as judged from 0.4% Trypan Blue exclusion. After 4 h, the medium was replaced by serum-free and insulin-free medium, and incubation was continued for 16 h for plating. Then the medium was replaced by the medium containing the test hormones and/ or carbohydrates, as indicated in the tables and figures. Fatty acids were added to the medium as their albumin complexes at a final concentration of 0.1 mM. The complexes were prepared by sonicating a mixture of 1 mM fatty acid and 3 mg/ml bovine serum albumin in William's medium E. The hepatocytes were cultured in a humidified chamber at 37'C under 5% CO2 in air. The medium was changed every 12 h. The cells on individual plates were harvested after 24 h incubation unless otherwise indicated. The medium was aspirated from the dishes and cells were washed three times with 10 ml of 0.15 M NaCl solution.
NaCl, and 0.1% SDS at room temperature for 20 rnin, and then three times at 55*C for 20 min. The filters were exposed for varying lengths of time at — 70'C to Kodak X-Omat AR film with Dupont Lighting Plus intensifying screens. The relative densities of the hybridization signals were determined by scanning the autoradiograms at 525 nm (Shimadzu, Model CS-9000, Kyoto). Analyses—Statistical evaluation of the results was carried out by one-way analysis of variance followed by inspection of all differences between pairs of means by means of the least significant differences test (24). The half-life of mRNA was calculated by the methods of Schimke (25) and Haining (26).
Regulation of Lipogenic Enzyme mRNA Levels lipogenic enzyme mRNAs are shown in Table II. The medium contained 10 mM glucose throughout the experiments on hormone effects. Insulin addition increased the mRNA concentrations of lipogenic enzymes by 4-6-fold above the control values for hepatocytes cultured in the presence of glucose without any hormones. Moreover,
27
Fig. 2. Dose-dependent effect of glucose on induction of lipogenic enzyme mRNAs. Hepatocytea were cultured for 24 h in the presence of the indicated concentrations of glucose, 0.1//M insulin and 1//M dexamethasone. The mRNA concentrations of acetylCoA carboxylase (A), fatty acid synthase (B), malic enzyme (C), glucose 6-phosphate dehydrogenase (D), and /?-actin (E) were determined by dot blot hybridization assay. Each point is the mean of four determinations and expressed relative to the mRNA concentration in hepatocytes cultured without glucose. Means± SD.
6 -
-c
10
8
16
24
48
Time after addition (h) Fig. 1. Time courses of induction of lipogenic enzyme mRNAs by insulin and/or glucose. Hepatocytes were cultured with 0.1 //M insulin (•), 10 mM glucose (O), and 0.1 ^M insulin+ 10mM glucose (A) in the presence of 1 ^M dexamethasone for the indicated times. The mRNA concentrations of acetyl-CoA carboxylase (A), fatty acid synthase (B), malic enzyme (C), glucose 6-phosphate dehydrogenase (D), and £-actin (E) were determined by dot blot hybridization assay. Each point is the mean offivedeterminations and expressed relative to the mRNA concentration in hepatocytes cultured in the absence of glucose and inmilin for the indicated time. Means±SD.
10
20
30
Glucose concentration
TABLE I. Effects of carbohydrates on mRNA concentrations in cultured hepatocytes. After 16 h preculture without serum and hormones, hepatocytes were incubated with glucose, fructose, or glycerol at 10 mM for 24 h in the presence of 1 ^M dexamethasone and 0.1 ft M insulin. The mRNA concentrations were measured by dot blot hybridization assay. Values are means of 4-7 determinations and expressed relative to the control value for glucose addition. Means± SD. Means with different superscript letters for each enzyme are significantly different at p< 0.05 at least. mRNA concentration Treatment
Acetyl-CoA carboxylase
Fatty arid synthase
Malic enzyme
Glucose Fructose Glycerol
1.00±0.15" 0.78±0.10* 0.76±0.27"
1.00±0.13* 1.13±0.24* 0.71±0.15b
1.00±0.14' 0.73±0.14b 0.57±0.15"
Glucose 6-phosphate dehydrogenase
£-Actin
l.OOiO.161 0.65±0.19b 0.59±0.19b
1.00±0.13* 1.00±0.21* 1.26±0.20*
fold
Vol. I l l , No. 1, 1992
Downloaded from https://academic.oup.com/jb/article-abstract/111/1/25/808969 by university of winnipeg user on 23 January 2019
dexamethasone addition greatly increased the mRNA induction by insulin. Although dexamethasone alone only slightly increased the mRNA concentrations, dexamethasone appeared to amplify the effects of insulin. On the other hand, triiodothyronine addition markedly increased the mRNA concentrations of acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme. However, dexamethasone did not increase the mRNA induction by triiodothyronine and even tended to decrease it. Triiodothyronine had only a little effect on the mRNA induction of glucose 6-phosphate dehydrogenase. Triiodothyronine addition failed to enhance acetyl-CoA carboxylase, fatty acid synthase, and glucose 6-phosphate dehydrogenase mRNA induction by insulin together with dexamethasone, but enhanced additively the malic enzyme mRNA induction.
28
H. Fukuda et aL
TABLE II. Effects of hormones on mRNA concentrations in cultured hepatocytes. After 16 h preculture without serum and hormones, hepatocytes were incubated for 24 h with various hormones at the concentrations given in parentheses. The medium contained 10 mM glucose throughout the experiment. The mRNA concentrations for lipogenic eniymes and /J-actin were measured by dot blot hybridization assay. Values are means of 4-10 determinations and expressed relative to the control value for hepatocytes incubated in the presence of glucose without any hormones. Means+SD. Means with different superscript letters for each enzyme are significantly different at p
Nutritional and Hormonal Regulation of mRNA Levels of Lipogenic Enzymes in Primary Cultures of Rat Hepatocytes1 Hitomi Fukuda, Akihiko Katsurada, and Nobuko Iritani2 Tezukayama Gakuin College, 4-2-2, Harumidai, Sakai, Osaka 590-01 Received for publication, July 29, 1991
and protein were required to induce fatty acid synthase and glucose 6-phosphate dehydrogenase, primarily at the transcription step (2, 3). Glucose 6-phosphate dehydrogenase induction was rather protein- than carbohydrate-dependent (3). Polyunsaturated fat feeding reduced the transcriptional rates for these lipogenic enzymes (1-4). Moreover, we found that insulin is importantly involved in the transcription of Lipogenic enzymes and also in their translation (1-3, 6, 7). Triiodothyronine-treatment markedly increased malic enzyme synthesis primarily at the transcription step in both normal and diabetic rats (4, 6). The effect of triiodothyronine on the other lipogenic enzymes was observed in the diabetic state. The triiodothyronine and insulin actions appeared to be synergistic. However, the results in whole animals are influenced by so many factors that it is hard to draw definite conclusions as to the mechanisms of actions of hormones and nutrients. In particular, it is diflicult to analyze the interactions of hormones and nutrients. Thus, it is advantageous to use hepatocytes in primary culture for further studies on the mechanisms regulating the gene expression of lipogenic enzymes. We previously investigated the effects of amino acids and various kinds of fatty acid species on lipogenic enzyme induction at the enzyme level in cultured hepatocytes (20). Goodridge et aL (9, 10) reported hormonal regulation of gene expression of malic enzyme and fatty acid synthase in chick embryo hepatocytes in culture. However, the nutritional and hormonal regulation of gene expression of acetyl-CoA carboxylase and glucose 6-phosphate dehydrogenase in cultured hepatocytes has not been
The first step in the pathway of long-chain fatty acid biosynthesis is mediated by acetyl-CoA carboxylase [EC 6.4.1.2], which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. The second step is the conversion of acetyl-CoA and malonyl-CoA to long-chain fatty acids by the multifunctional enzyme, fatty acid synthase [EC 2.3.1.85], in the presence of NADPH. Malic enzyme [EC 1.1.1.40] and glucose 6-phosphate dehydrogenase [EC 1.1.1.49] are donors of NADPH. It is well known that the cellular contents of these lipogenic enzymes in liver vary with different nutritional, hormonal, and genetic conditions. However, only limited information on the regulatory mechanism for gene expression of the enzymes is available at present (1-19). We have cloned the cDNAs of all four lipogenic enzymes, and investigated the nutritional and hormonal regulation of their gene expression in rat liver (1-5). On the feeding of a carbohydrate diet (without protein) to fasted rats, the transcriptional rate, mRNA concentration and enzyme induction of acetyl-CoA carboxylase similarly increased to the levels with a carbohydrate/protein diet (2). In these animals, the transcriptional rate and mRNA concentration of malic enzyme increased to the levels with the carbohydrate/protein diet, whereas the enzyme induction increased only to 60% (4). It appears that protein-feeding is not necessary to induce the acetyl-CoA carboxylase nor to induce malic enzyme mRNA. However, both carbohydrate 1
This work was supported in part by Japanese private school promotion funds. 1 To whom correspondence should be addressed. Vol. I l l , No. 1, 1992
25
Downloaded from https://academic.oup.com/jb/article-abstract/111/1/25/808969 by university of winnipeg user on 23 January 2019
The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.
26
H. Fukuda et al.
reported. The mechanisms of nutritional and hormonal regulation of gene expression of lipogenic enzymes have not been sufficiently elucidated. Thus, in the present work, we have investigated the effects of glucose, insulin, and triiodothyronine on the mRNA induction of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase in cultured rat hepatocytes. EXPERIMENTAL PROCEDURES
Preparation of RNA and Dot Blot Hybridization Assay—Total cellular RNA was isolated from cultured cells by the guanidine thiocyanate/LiCl procedure (23). The cDNAs cloned by us (1-4) were labeled with a multiprimer DNA labeling system kit (Amersham) using [33P]dCTP. To measure the mRNA concentration, the RNA (10 ^g) was denatured with formamide at 65'C for 15 min, spotted on a nylon filter, and then irradiated with ultraviolet light for 5 min The filter was prehybridized for at least 4 h at 50"C in a reaction mixture comprising 50 mM Tris-HCl (pH 7.5), 0.09 M sodium citrate, 0.9 M NaCl, 5 x Denhardt's solution, 1% SDS, 50% formamide, and 100 //g/ml of salmon sperm DNA (medium A). The hybridization was carried out for 36 h at 42*C in medium A with "P-labeled cDNA. The filter was then washed with 1.5 mM sodium citrate, 15 mM
RESULTS
Time Courses of Induction of Lipogenic Enzyme mRNAs by Glucose and/or Insulin—The time courses of induction of lipogenic enzyme mRNAs by insulin and/or glucose in cultured hepatocytes of rats are compared in Fig. 1. The mRNA concentrations become maximum after 16-24 h. Therefore, to investigate the effects of additions on the mRNA concentrations in the following experiments, the mRNA concentrations were measured after 24 h incubation of hepatocytes in medium containing test hormones and/or carbohydrates. With insulin and glucose together, the mRNA concentrations increased to about 8-10-fold the control values for hepatocytes cultured in the absence of insulin and glucose. The mRNA induction was very slightly increased by either glucose or insulin alone. The half-lives of the mRNAs can be roughly calculated from the time courses, as done by Goodridge et al. (18, 19), according to Schimke (25) and Haining (26). The values were about 8 h for acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme, and 9-10 h for glucose 6-phosphate dehydrogenase in the presence of glucose and insulin. Effects of Glucose on Induction of Lipogenic Enzyme mRNAs—The addition of glucose to the medium caused marked dose-dependent increases in the induction of the mRNAs by insulin, as shown in Fig. 2. The mRNA concentrations of acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme reached the maximum levels, 4-5-fold increases, at the glucose concentrations of 5, 20, and 10 mM, respectively. The acetyl-CoA carboxylase mRNA induction appeared to be the most sensitive to glucose among the lipogenic enzymes. On the contrary, the mRNA concentration of glucose 6-phosphate dehydrogenase reached a low mayimiim level and rather decreased at higher concentrations of glucose. Furthermore, fructose or glycerol could substitute for glucose considerably well (Table I). Effect of Insulin on Induction of Lipogenic Enzyme mRNAs—Insulin caused dose-dependent increases in the mRNA concentrations of lipogenic enzymes in the presence of glucose (Fig. 3). The maximum concentrations were 35-fold those for hepatocytes cultured without insulin. The insulin levels required to induce the maximum mRNA concentrations were below 10~9, but 10~ 7 ,10~\ and 10~8 M, respectively, for acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase. Thus, acetyl-CoA carboxylase mRNA induction was the most sensitive to insulin as well as to glucose (Fig. 2). Effects of Hormones on Induction of Lipogenic Enzyme mRNAs—The effects of hormones on the induction of J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/111/1/25/808969 by university of winnipeg user on 23 January 2019
Materials—Collagenase (type I), bovine serum albumin (essentially fat-free), insulin (bovine pancreas), 3,3',5-triiodo-L-thyronine, and dexamethasone were obtained from Sigma Chemical. William's medium E was obtained from Flow Laboratories and fetal bovine serum from Gibco. Tissue culture dishes were purchased from Corning, [a32 P]dCTP (lllTBq/mmol) was purchased from ICN Biochemical, nylon filters (Hybond N) from Amersham, guanidine thiocyanate from Fluka, and /3-actin DNA probe from Wako Chemical. Other chemicals were mostly purchased from Sigma. Cell Culture—Male Wistar rats (200-300 g, Japan SLC) fed on a laboratory chow were used, except for in experiments involving fatty acid addition. For experiments involving fatty acid addition, rats adapted to a fat-free diet were used (21). The hepatocytes were isolated by a modification of the method of Seglen (22), and were suspended at 5X10 6 cells/ml in William's medium E containing 5% fetal bovine serum, 1 ^M dexamethasone, and 0.1//M insulin, supplemented with glucose at the concentration of 10 mM, in addition to penicillin at 100 units/ml and streptomycin at 100 /xg/ml. The cell viability in all experiments was above 95%, as judged from 0.4% Trypan Blue exclusion. After 4 h, the medium was replaced by serum-free and insulin-free medium, and incubation was continued for 16 h for plating. Then the medium was replaced by the medium containing the test hormones and/ or carbohydrates, as indicated in the tables and figures. Fatty acids were added to the medium as their albumin complexes at a final concentration of 0.1 mM. The complexes were prepared by sonicating a mixture of 1 mM fatty acid and 3 mg/ml bovine serum albumin in William's medium E. The hepatocytes were cultured in a humidified chamber at 37'C under 5% CO2 in air. The medium was changed every 12 h. The cells on individual plates were harvested after 24 h incubation unless otherwise indicated. The medium was aspirated from the dishes and cells were washed three times with 10 ml of 0.15 M NaCl solution.
NaCl, and 0.1% SDS at room temperature for 20 rnin, and then three times at 55*C for 20 min. The filters were exposed for varying lengths of time at — 70'C to Kodak X-Omat AR film with Dupont Lighting Plus intensifying screens. The relative densities of the hybridization signals were determined by scanning the autoradiograms at 525 nm (Shimadzu, Model CS-9000, Kyoto). Analyses—Statistical evaluation of the results was carried out by one-way analysis of variance followed by inspection of all differences between pairs of means by means of the least significant differences test (24). The half-life of mRNA was calculated by the methods of Schimke (25) and Haining (26).
Regulation of Lipogenic Enzyme mRNA Levels lipogenic enzyme mRNAs are shown in Table II. The medium contained 10 mM glucose throughout the experiments on hormone effects. Insulin addition increased the mRNA concentrations of lipogenic enzymes by 4-6-fold above the control values for hepatocytes cultured in the presence of glucose without any hormones. Moreover,
27
Fig. 2. Dose-dependent effect of glucose on induction of lipogenic enzyme mRNAs. Hepatocytea were cultured for 24 h in the presence of the indicated concentrations of glucose, 0.1//M insulin and 1//M dexamethasone. The mRNA concentrations of acetylCoA carboxylase (A), fatty acid synthase (B), malic enzyme (C), glucose 6-phosphate dehydrogenase (D), and /?-actin (E) were determined by dot blot hybridization assay. Each point is the mean of four determinations and expressed relative to the mRNA concentration in hepatocytes cultured without glucose. Means± SD.
6 -
-c
10
8
16
24
48
Time after addition (h) Fig. 1. Time courses of induction of lipogenic enzyme mRNAs by insulin and/or glucose. Hepatocytes were cultured with 0.1 //M insulin (•), 10 mM glucose (O), and 0.1 ^M insulin+ 10mM glucose (A) in the presence of 1 ^M dexamethasone for the indicated times. The mRNA concentrations of acetyl-CoA carboxylase (A), fatty acid synthase (B), malic enzyme (C), glucose 6-phosphate dehydrogenase (D), and £-actin (E) were determined by dot blot hybridization assay. Each point is the mean offivedeterminations and expressed relative to the mRNA concentration in hepatocytes cultured in the absence of glucose and inmilin for the indicated time. Means±SD.
10
20
30
Glucose concentration
TABLE I. Effects of carbohydrates on mRNA concentrations in cultured hepatocytes. After 16 h preculture without serum and hormones, hepatocytes were incubated with glucose, fructose, or glycerol at 10 mM for 24 h in the presence of 1 ^M dexamethasone and 0.1 ft M insulin. The mRNA concentrations were measured by dot blot hybridization assay. Values are means of 4-7 determinations and expressed relative to the control value for glucose addition. Means± SD. Means with different superscript letters for each enzyme are significantly different at p< 0.05 at least. mRNA concentration Treatment
Acetyl-CoA carboxylase
Fatty arid synthase
Malic enzyme
Glucose Fructose Glycerol
1.00±0.15" 0.78±0.10* 0.76±0.27"
1.00±0.13* 1.13±0.24* 0.71±0.15b
1.00±0.14' 0.73±0.14b 0.57±0.15"
Glucose 6-phosphate dehydrogenase
£-Actin
l.OOiO.161 0.65±0.19b 0.59±0.19b
1.00±0.13* 1.00±0.21* 1.26±0.20*
fold
Vol. I l l , No. 1, 1992
Downloaded from https://academic.oup.com/jb/article-abstract/111/1/25/808969 by university of winnipeg user on 23 January 2019
dexamethasone addition greatly increased the mRNA induction by insulin. Although dexamethasone alone only slightly increased the mRNA concentrations, dexamethasone appeared to amplify the effects of insulin. On the other hand, triiodothyronine addition markedly increased the mRNA concentrations of acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme. However, dexamethasone did not increase the mRNA induction by triiodothyronine and even tended to decrease it. Triiodothyronine had only a little effect on the mRNA induction of glucose 6-phosphate dehydrogenase. Triiodothyronine addition failed to enhance acetyl-CoA carboxylase, fatty acid synthase, and glucose 6-phosphate dehydrogenase mRNA induction by insulin together with dexamethasone, but enhanced additively the malic enzyme mRNA induction.
28
H. Fukuda et aL
TABLE II. Effects of hormones on mRNA concentrations in cultured hepatocytes. After 16 h preculture without serum and hormones, hepatocytes were incubated for 24 h with various hormones at the concentrations given in parentheses. The medium contained 10 mM glucose throughout the experiment. The mRNA concentrations for lipogenic eniymes and /J-actin were measured by dot blot hybridization assay. Values are means of 4-10 determinations and expressed relative to the control value for hepatocytes incubated in the presence of glucose without any hormones. Means+SD. Means with different superscript letters for each enzyme are significantly different at p
