Glucose stimulates transcription of fatty acid synthase and malic enzyme in avian hepatocytes

F. Bradley Hillgartner and Tina Charron

Department of Biochemistry, School of Medicine, West Virginia University, Morgantown, West Virginia 26506

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transcription of fatty acid synthase (FAS) and malic enzyme (ME) in avian liver is low during starvation or feeding a low-carbohydrate, high-fat diet and high during feeding a high-carbohydrate, low-fat diet. The role of glucose in the nutritional control of FAS and ME was investigated by determining the effects of this metabolic fuel on expression of FAS and ME in primary cultures of chick embryo hepatocytes. In the presence of triiodothyronine, glucose (25 mM) stimulated an increase in the activity and mRNA abundance of FAS and ME. These effects required the phosphorylation of glucose to glucose 6-phosphate but not further metabolism downstream of the aldolase step of the glycolytic pathway. Xylitol mimicked the effects of glucose on FAS and ME expression, suggesting that an intermediate of the pentose phosphate pathway may be involved in mediating this response. The effects of glucose on the mRNA abundance of FAS and ME were accompanied by similar changes in transcription of FAS and ME. These data support the hypothesis that glucose plays a role in mediating the effects of nutritional manipulation on transcription of FAS and ME in liver.

fatty acid synthesis; nutritional regulation; glucose signal; liver; thyroid hormone

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IN BIRDS AND MAMMALS, one of the functions of the de novo synthesis of long-chain fatty acids is the provision of energy reserves during periods when dietary carbohydrate intake exceeds the immediate energy needs of the animal. Accordingly, rates of long-chain fatty acid synthesis in liver and adipose tissue are rapid during feeding a high-carbohydrate, low-fat diet, and slow during starvation or feeding a low-carbohydrate, high-fat diet (22, 49). Two enzymes that are involved in the synthesis of long-chain fatty acids are malic enzyme (ME, EC 1.1.1.40) and fatty acid synthase (FAS, EC 2.3.1.85). ME catalyzes the oxidative decarboxylation of malate to pyruvate and carbon dioxide, simultaneously generating NADPH from NADP+. FAS is a multifunctional polypeptide that catalyzes the synthesis of a molecule of palmitate from 1 molecule of acetyl- CoA, 7 molecules of malonyl-CoA, and 14 molecules of NADPH. The ME reaction supplies about 50% of the NADPH required for palmitate synthesis in rat liver and adipose tissue and nearly all of the NADPH needed for palmitate synthesis in chicken liver (9, 26, 36). The concentrations of FAS and ME in liver and adipose tissue are correlated with rates of fatty acid synthesis during different nutritional conditions. Thus the amount of FAS and ME is increased during feeding a high-carbohydrate, low-fat diet and decreased during starvation or feeding a low-carbohydrate, high-fat diet (6, 13, 17, 19, 27). Diet-induced changes in the concentrations of FAS and ME are mediated by alterations in the transcription rate of the respective genes (2, 5, 20, 31, 37). An understanding of the mechanisms controlling the transcription of FAS and ME is important because altered regulation of fatty acid synthesis is associated with several disease states such as obesity, diabetes, and atherosclerosis (22, 43).

Insulin, 3,5,3'-triiodothyronine (T3), and glucagon are widely considered to be humoral factors that communicate changes in nutritional status to the liver and other organs, thereby mediating the nutritional regulation of FAS and ME. This deduction is based on the observation that stimulation of transcription of FAS and ME caused by feeding a high-carbohydrate, low-fat diet is preceded or paralleled by increases in the molar ratio of insulin/glucagon and the levels of T3 in the blood (18, 22). A major role for insulin, T3, and glucagon is also indicated by the observation that these hormones regulate expression of lipogenic enzymes in various cell culture systems. For example, in primary cultures of chick embryo hepatocytes, T3 stimulates a 10- and 60-fold increase in transcription of FAS and ME, respectively (40, 47). Insulin accelerates the increase in transcription of FAS and ME caused by T3, whereas glucagon completely blocks the stimulatory effect of T3 on ME transcription.

Hormones may not be the only agents that participate in the nutritional regulation of FAS and ME. The level of glucose in the blood is also modulated by alterations in nutritional status. For example, the concentration of glucose in the blood is low in starved animals and high in animals fed a high-carbohydrate diet (22). Previous studies have shown that glucose stimulates an increase in the synthesis and mRNA abundance of FAS and ME in rat hepatocyte cultures (12, 32, 33, 38). Glucose also has been reported to cause an increase in FAS mRNA levels in rat white adipose tissue pieces (11) and the human hepatoma cell line Hep G2 (44). In experimental models of non-insulin-dependent diabetes mellitus, increased glucose flux across the liver is associated with elevated rates of fatty acid synthesis and increased expression of FAS (3, 45). Collectively, these findings suggest that glucose may serve as an extracellular signal in the control of lipogenic enzyme expression. In the present study, we investigated this possibility further by determining the effects of glucose on transcription of FAS and ME in chick embryo hepatocytes. We show that glucose increases the transcription of FAS and ME and that this effect is dependent on the presence of T3. We also provide data suggesting that an intermediate of the nonoxidative branch of the pentose phosphate pathway is involved in mediating the effects of glucose on expression of FAS and ME. Finally, based on comparison of the glucose response in chick embryo hepatocytes with that reported in rat hepatocytes and Hep G2 cells, we propose that fundamental differences exist between these cell culture systems in the mechanism by which glucose regulates expression of FAS and ME.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. Nucleotides (Pharmacia Biotechnology), proteinase K (Boehringer Mannheim), [alpha -32P]UTP, and [alpha -32P]dCTP (ICN Biochemicals) were purchased from the indicated sources. Crystalline bovine insulin was a gift from Lilly. All other chemicals were from Sigma or of the highest purity commercially available.

Preparation and maintenance of isolated hepatocytes. Unincubated embryonated eggs from white Leghorn chickens were obtained from Truslow Farms (Chestertown, MD) and incubated in an electric forced draft incubator at 37.5 ± 0.5°C and 80% relative humidity. Embryos (19 days of age) were killed by decapitation, the livers were removed, and hepatocytes were isolated (15). The isolated hepatocytes were incubated in Waymouth's medium MD705/1 (GIBCO) containing penicillin (60 µg/ml) and streptomycin (100 µg/ml) on untreated plastic petri dishes (Fisher) at 40°C in a humidified atmosphere of 5% CO2 and 95% air. One 90-mm petri dish contained 1 ml of cell suspension (2-3 mg total protein, ~1 × 107 cells) and 9 ml of medium. The medium on all plates was changed after about 18 h of incubation. Hormone and other additions were as described in the legends to the figures and table. Because T3 is rapidly degraded in serum-free Waymouth's medium (19), high concentrations of T3 (1.6 µM) were employed in long-term incubations to ensure that optimal levels of T3 were present throughout the incubation. FAS (15), ME (50), and protein (42) were assayed by the indicated methods.

Isolation of RNA and quantitation of mRNA levels. Medium was removed and RNA was extracted from cells by the guanidinium thiocyanate-phenol-chloroform method (4). Total RNA (15 µg) was separated by size in 0.9% agarose, 0.7 M formaldehyde gels, and then transferred to a Nytran membrane (Schleicher & Schuell) using a vacuum blotting apparatus (Pharmacia Biotechnology). The RNA was crosslinked to the membrane by ultraviolet and baked at 80°C for 30-60 min. RNA blots were hybridized with 32P-labeled DNA probes labeled by random priming (8). Hybridization and washes were as described (1). Membranes were subjected to storage phosphor autoradiography. Hybridization signals were quantified using ImageQuant software (Molecular Dynamics).

Nuclear run-on assay of transcription rates. Nuclei were isolated from the pooled cells of fifteen 90-mm plates (40, 41). Approximately 5 × 107 nuclei were stored at -80°C in 100-µl aliquots containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, 75 mM NaCl, 0.1 mM EDTA, 5 mM dithiothreitol, 0.125 mM phenylmethylsulfonyl fluoride, and 50% glycerol. The in vitro elongation reactions were carried out as described (14, 20). 32P-labeled RNA transcripts were purified by the method of Linial et al. (29), using NICK columns (Pharmacia Biotechnology) as described in the manufacturer's instructions. Denaturation of DNA probes, application of DNA to GeneScreen membranes, hybridization of 32P-labeled RNA transcripts to membrane-bound DNA, and posthybridization washes were carried out as described (20). Hybridization signals were quantified as previously described.

DNA probes. Chicken cDNAs for FAS, glyceraldehyde-3-phosphate dehydrogenase, and beta -actin were generously provided by Drs. Gordon G. Hammes (Duke University), Robert Schwartz (Baylor College of Medicine), and Don W. Cleveland (Johns Hopkins University), respectively. These DNAs were used as probes in both Northern and nuclear run-on analyses. The chicken cDNA for ME was provided by Dr. Alan G. Goodridge (Ohio State University) and was used as a probe in Northern analysis. The chicken ME genomic DNA, ME-4.8-5', has been described (31, 40) and was used as a probe in nuclear run-on assays.

Statistics. Data were subjected to analysis of variance, and statistical comparisons were made with Dunnett's test or Student's t-test. Statistical significance is defined as P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Glucose stimulates expression of FAS and ME in chick embryo hepatocytes. The ability of glucose to regulate the expression of FAS and ME in avian liver was investigated by determining the effects of different concentrations of glucose on the activities and mRNA levels for FAS and ME in primary cultures of chick embryo hepatocytes incubated in the absence or presence of insulin, T3, or insulin plus T3. Incubation of hepatocytes with T3 in the presence of 2.5 mM glucose stimulated a 2.3- and 42.6-fold increase in the activities of FAS and ME, respectively (Table 1). An increase in the glucose concentration in the culture medium to 25 mM amplified the T3-induced increase in FAS and ME activity by 3.1- and 1.5-fold, respectively. Glucose (25 mM) caused a similar stimulation in the activities of FAS and ME in hepatocytes incubated with insulin plus T3. Addition of glucose by itself or in combination with insulin had no effect on the activities of FAS or ME. Thus glucose increases the activities of ME and FAS in avian hepatocytes, and this effect requires the presence of T3.

                              
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Table 1.   Glucose amplifies T3-induced increase in activities of FAS and ME in chick embryo hepatocytes

Next, we determined the effects of varying the glucose concentration of the culture medium on the mRNA abundance of FAS and ME in hepatocytes incubated with insulin or insulin plus T3. In the absence of glucose, addition of insulin plus T3 stimulated a 3.4- and 26-fold increase in the amount of mRNA for FAS and ME, respectively (Fig. 1). An increase in the glucose concentration of the culture medium enhanced the T3-induced accumulation of mRNA for FAS and ME. Maximal levels of FAS mRNA were observed at a glucose concentration of 15 mM and were 5.1 and 4.2 times the levels observed at 0 and 2.5 mM glucose, respectively. Maximal levels of ME mRNA were observed at a lower glucose concentration (11 mM) and were 2.2 and 1.5 times the levels observed at 0 and 2.5 mM glucose, respectively. The levels of mRNA for FAS and ME remained high as the glucose concentration of the culture medium was increased from 15 to 30 mM and from 11 to 30 mM, respectively. A similar dose-response relationship between glucose concentration and mRNA levels for FAS and ME was observed in hepatocytes incubated with T3 alone (data not shown). In the absence of T3, addition of glucose with or without insulin had no effect on the abundance of the mRNAs for FAS and ME (Fig. 1 and data not shown). These results indicate that the effects of glucose on expression of FAS and ME are concentration dependent and that glucose acts at a pretranslational step to increase the activities of FAS and ME. The effects of glucose on the abundance of mRNAs for FAS and ME were specific because beta -actin mRNA levels were not affected by alterations in the concentration of glucose in the culture medium (data not shown).


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Fig. 1.   Concentration dependence of effects of glucose on mRNA levels for fatty acid synthase (FAS, A) and malic enzyme (ME, B). square  and black-square, insulin plus 3,5,3'-triiodothyronine (T3); open circle  and bullet , insulin. Hepatocytes were isolated and incubated in Waymouth's medium lacking glucose. At about 18 h of incubation medium was changed to one containing glucose at concentrations indicated. Insulin or insulin plus T3 was added at this time. Medium was changed to one of same composition after 24 h of treatment. After 48 h of treatment total RNA was isolated and levels of mRNA for FAS and ME were measured by Northern analysis as described in MATERIALS AND METHODS. Hybridization signals from representative experiment are shown in top panel of each section. In bottom panel of each section abundance of mRNA for FAS or ME is quantitated. FAS and ME mRNA levels in hepatocytes treated with 0 mM glucose and insulin were set at 1. Values are means ± SE of 4 experiments. * Significantly different (P < 0.05) from cells incubated with insulin and T3 and 0 mM glucose.

Role of intermediates of glycolytic pathway and pentose phosphate pathway in mediating effects of glucose on expression of FAS and ME. To assess the specificity of the action of glucose and to determine whether the effects of glucose on expression of FAS and ME are mediated by glucose per se or by specific intermediates of the glycolytic pathway, we investigated the ability of various metabolic fuels and nonmetabolizable analogs of glucose to stimulate the accumulation of mRNA for FAS and ME in the presence of T3. In hepatocytes incubated with 5 mM dihydroxyacetone or 10 mM lactate and 1 mM pyruvate, the accumulation of mRNA for FAS and ME was similar to that of cells incubated with 0 mM glucose (Fig. 2). Addition of dihydroxyacetone or lactate and pyruvate had no effect on the abundance of mRNA for FAS and ME in the absence of T3 (data not shown). These results indicate that metabolism downstream of the aldolase step in the glycolytic pathway is not sufficient for the glucose-induced increase in the abundance of mRNA for FAS and ME.


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Fig. 2.   Effect of metabolic fuels and nonmetabolizable glucose analogs on abundance of mRNA for FAS (A) and ME (B). Hepatocytes were isolated and incubated in Waymouth's medium lacking glucose. At 18 h of incubation medium was changed to one containing insulin plus T3 and the following metabolites were added: glucose (20 mM), dihydroxyacetone (5 mM), lactate (Lact, 10 mM), pyruvate (Pyr, 1 mM), 3-O-methylglucose (3-O-MG, 20 mM), fructose (20 mM), and mannose (20 mM). Medium was changed to one of same composition after 24 h of treatment. After 48 h of treatment total RNA was isolated and levels of mRNA for FAS and ME were measured by Northern analysis as described in MATERIALS AND METHODS. 2-Deoxyglucose (2-DOG, 20 mM) was added during last 24 h of 48-h treatment with 10 mM lactate and 1 mM pyruvate. Hybridization signals from representative experiment are shown in top panel of each section. In bottom panel of each section, the abundance of mRNA for FAS or ME is quantitated. FAS and ME mRNA levels in hepatocytes treated with 0 mM glucose were set at 1. Values are means ± SE (n = 4). * Significantly different (P < 0.05) from cells incubated with 0 mM glucose. ** Significantly different (P < 0.05) from cells incubated with Lact/Pyr.

To determine whether the effects of glucose on expression of FAS and ME require the phosphorylation of glucose to glucose 6-phosphate, we examined the ability of 3-O-methylglucose to enhance the accumulation of mRNA for these genes in the presence of T3. 3-O-Methylglucose is a glucose analog that is transported into hepatocytes but is not phosphorylated (39). In these experiments, the culture medium was supplemented with 10 mM lactate and 1 mM pyruvate to provide an additional energy source for the cells. Incubation of hepatocytes with 20 mM glucose in the presence of lactate and pyruvate increased the mRNA abundance for FAS and ME by 4.2- and 1.9-fold, respectively, whereas addition of 20 mM 3-O-methylglucose in the presence of lactate and pyruvate had no effect on the levels of mRNA for FAS and ME (Fig. 2). These results indicate that glucose must be phosphorylated for it to be effective in stimulating the expression of FAS and ME.

We next investigated the role of glucose 6-phosphate in mediating the effects of glucose on expression of FAS and ME. 2-Deoxyglucose is a glucose analog that is transported into hepatocytes and phosphorylated to 2-deoxyglucose 6-phosphate but is not further metabolized (24). Hepatocytes were incubated with 2-deoxyglucose for only 24 h to minimize the possible effects of intracellular ATP depletion on expression of FAS and ME. Addition of 2-deoxyglucose (20 mM) during the last 24 h of a 48-h treatment with insulin, T3, 10 mM lactate, and 1 mM pyruvate had no effect on accumulation of mRNA for FAS and ME (Fig. 2), whereas addition of glucose during the last 24 h of this treatment period caused a 3.7- and 1.8-fold increase in mRNA levels for FAS and ME, respectively (data not shown). These data suggest that glucose 6-phosphate is not involved in signaling glucose-induced changes in FAS and ME expression.

Stimulation of FAS and ME expression by glucose may be mediated by intermediates of the pentose phosphate pathway. To examine this possibility, we tested the effects of xylitol on the abundance of mRNA for FAS and ME in chick embryo hepatocytes incubated in the presence of T3. Xylitol is metabolized to xylulose 5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway (51). Addition of xylitol in the presence of 10 mM lactate and 1 mM pyruvate increased the abundance of mRNA for FAS and ME in a dose-dependent manner (Fig. 3). Maximal mRNA levels for FAS and ME were observed at a xylitol concentration of 8 mM and were increased by 3.5- and 2.1-fold, respectively, relative to levels observed at 0 mM xylitol. Fructose and mannose were also effective in stimulating an increase in mRNA abundance for FAS and ME in the presence of T3 (Fig. 2). Xylitol, fructose, and mannose had no effect on FAS and ME mRNA levels in the absence of T3 (data not shown). The effects of xylitol, fructose, and mannose on the abundance of mRNA for FAS and ME were specific because beta -actin mRNA levels were not affected by the presence of these sugars in the medium (data not shown).


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Fig. 3.   Xylitol stimulates increase in abundance of mRNA for FAS (A) and ME (B). Closed bars, xylitol; stippled bars, glucose. Hepatocytes were isolated and incubated in Waymouth's medium lacking glucose. At 18 h of incubation medium was changed to one containing insulin plus T3 plus 10 mM lactate and 1 mM pyruvate. Xylitol or glucose was added at this time at concentrations indicated. Medium was changed to one of same composition after 24 h of treatment. After 48 h of treatment total RNA was isolated and levels of mRNA for FAS and ME were measured by Northern analysis as described in MATERIALS AND METHODS. Hybridization signals from representative experiment are shown in top panel of each section. In bottom panel of each section abundance of mRNA for FAS or ME is quantitated. FAS and ME mRNA levels in hepatocytes incubated without xylitol and glucose were set at 1. Values are means ± SE (n = 4). * Significantly different from cells incubated with 0 mM xylitol and glucose (P < 0.05).

Role of transcription in mediating effects of glucose on abundance of mRNA for FAS and ME. We next determined whether the effects of glucose on the abundance of mRNA for FAS and ME were due to changes in gene transcription. Nuclear run-on assays were performed using nuclei prepared from hepatocytes treated with or without T3 in the presence of 2.5 mM glucose or 25 mM glucose. In hepatocytes incubated with T3 and 25 mM glucose, transcription of FAS and ME was increased by 3.8- and 1.8-fold, respectively, relative to cells incubated with T3 and 2.5 mM glucose (Fig. 4). Glucose had no effect on transcription of FAS and ME in hepatocytes incubated without T3. These changes in the transcription of FAS and ME were comparable to alterations in accumulation of the respective mRNAs for these genes (Fig. 1), indicating that the effects of glucose are mediated primarily by a transcriptional mechanism. The effects of glucose on transcription of FAS and ME were specific because transcription of the genes for glyceraldehyde-3-phosphate dehydrogenase and beta -actin were not affected by alterations in the concentration of glucose in the culture medium (Fig. 4).


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Fig. 4.   Glucose stimulates transcription of FAS and ME. Closed bars, +T3; stippled bars, -T3. Hepatocytes were isolated and incubated in Waymouth's medium containing 2.5 mM glucose. At 18 h of incubation medium was changed to one of same composition or to one containing 25 mM glucose. Insulin or insulin plus T3 was added at this time. After 24 h of treatment nuclei were isolated and transcription of FAS and ME was measured as described in MATERIALS AND METHODS. Hybridization signals of representative experiment are shown in A. Transcription of FAS and ME is quantitated in B and C, respectively. Transcription of FAS and ME in cells incubated with insulin and 2.5 mM glucose was set at 1. Each point represents mean ± SE of 3 experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * Significantly different from cells incubated with T3 and 2.5 mM glucose (P < 0.05). ** Significantly different from cells incubated with T3 (P < 0.05).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous work investigating the regulation of FAS and ME in chick embryo hepatocytes has focused on the ability of hormones to control the expression of these enzymes. In this report, we demonstrate that the important dietary substrate glucose also regulates expression of FAS and ME in avian hepatocytes. Our results indicate that glucose stimulates an increase in expression of FAS and ME and that this effect is manifested only in the presence of T3 (Table 1 and Figs. 1 and 4). The finding that T3 is required for glucose responsiveness in chick embryo hepatocytes contrasts with the results of previous studies examining the effects of glucose on lipogenic enzyme expression in rat hepatocytes and white adipose tissue pieces and in the human hepatoma cell line Hep G2. In rat hepatocytes, glucose is effective in stimulating expression of FAS and ME in the absence of T3 (12, 32, 33, 38). Stimulation of FAS expression by glucose in Hep G2 cells and rat white adipose tissue pieces is also not dependent on the presence of T3 (11, 44). Thus, in avian hepatocytes, glucose regulates the expression of FAS and ME by modulating the activity of the T3 signaling pathway, whereas in Hep G2 cells and rat hepatocytes and adipocytes, a different mechanism appears to be involved in mediating the effects of glucose on expression of lipogenic enzymes. The reason for these differences in the mechanism of action of glucose is presently unclear.

Another interesting characteristic of the glucose-induced increase in expression of FAS and ME in chick embryo hepatocytes is that it does not require the presence of insulin (Table 1). This observation contrasts with the results of previous studies in rat hepatocytes demonstrating that the stimulatory effects of glucose on expression of FAS and ME require the presence of insulin (12, 32, 33, 38). Differences between chick hepatocytes and rat hepatocytes in the requirement for insulin for glucose responsiveness may be due to differences in the properties of hexokinases that catalyze the phosphorylation of glucose to glucose 6-phosphate. In rat hepatocytes, hexokinase activity is dependent on the presence of insulin (23), whereas, in chicken hepatocytes, hexokinase activity is not dependent on the presence of insulin (28, 46). As in chick embryo hepatocytes, the effect of glucose on FAS expression in Hep G2 cells does not require the presence of insulin (44). Thus hexokinase activity in Hep G2 cells is probably insulin independent.

Two different signaling pathways have been suggested to play a role in mediating the effects of glucose on lipogenic enzyme expression in rat hepatocytes. Girard and co-workers (10, 13) have postulated that the proximal signal mediating the effects of glucose on FAS expression is glucose 6-phosphate. This proposal is based on the observations that 2-deoxyglucose is effective in mimicking the effects of glucose on FAS mRNA levels and that glucose-induced alterations in FAS mRNA abundance are positively correlated with changes in intracellular glucose 6-phosphate levels. Mariash and Oppenheimer (32) have suggested that another signaling pathway is involved in mediating the effects of glucose on ME expression in rat hepatocytes. They have proposed that the proximal signal mediating the glucose-induced increase in ME expression arises at the level of mitochondrial oxidation of pyruvate. This proposal is based on the observations that any carbohydrate intermediate entering the glycolytic pathway at or above pyruvate is effective in stimulating ME activity and that dichloroacetic acid, a stimulator of pyruvate dehydrogenase, is able to increase ME activity in the absence of insulin or the presence of glucagon. Interestingly, our results in chick embryo hepatocytes suggest that neither glucose 6-phosphate nor a metabolite distal of the aldolase step of the glycolytic pathway is involved in mediating the effects of glucose on expression of FAS and ME. 2-Deoxyglucose, dihydroxyacetone, and lactate plus pyruvate were not effective in stimulating FAS and ME mRNA levels in chick embryo hepatocytes (Fig. 2). A lack of a role for glucose 6-phosphate in mediating glucose-induced changes in lipogenic enzyme expression in chick embryo hepatocytes is supported by the observation that expression of acetyl-CoA carboxylase in these cells is not closely correlated with intracellular levels of glucose 6-phosphate (21). Thus our findings provide further support for the proposal that the mechanism mediating the effects of glucose on expression of FAS and ME in chick hepatocytes is fundamentally different from that in rat hepatocytes.

Intermediates of the nonoxidative branch of the pentose phosphate pathway have also been proposed to mediate the effects of glucose on gene expression. This hypothesis is based on the observation that glucose treatment increases xylulose 5-phosphate levels in perfused liver (30), and that low concentrations of xylitol are effective in mimicking the stimulatory effects of glucose on L-pyruvate kinase transcription in mhAT3-F hepatoma cells without altering intracellular glucose 6-phosphate levels (7). Our finding that xylitol is effective in stimulating the accumulation of mRNA for FAS and ME in chick embryo hepatocytes (Fig. 3) is consistent with xylulose 5-phosphate or another intermediate of the pentose phosphate pathway playing a role in regulating the expression of these enzymes.

Recently, Mourrieras et al. (34) have reported that stimulation of FAS and S14 mRNA levels by glucose and xylitol in rat hepatocyte cultures is more closely correlated with changes in intracellular glucose 6-phosphate levels than xylulose 5-phosphate levels, supporting the hypothesis that glucose 6-phosphate is involved in the regulation of lipogenic enzyme expression. These observations contrast with the findings of Doiron et al. (7) demonstrating that stimulation of L-pyruvate kinase transcription by xylitol in mhAT3-F cells is not associated with changes in intracellular glucose 6-phosphate levels and data from our laboratory (21) demonstrating that stimulation of acetyl-CoA carboxylase expression by glucose in chick embryo hepatocytes is not closely correlated with glucose 6-phosphate levels. One explanation for the contradictory observations is that the effects of glucose and xylitol on lipogenic enzyme expression are mediated by different metabolic intermediates in different cell types. Another possibility is that neither glucose 6-phosphate nor xylulose 5-phosphate is involved in mediating these responses and that another unidentified metabolic intermediate functions in the transduction of the glucose signal. Resolution of these discrepant findings will require additional analyses that directly define the metabolites involved in the regulation of lipogenic enzyme expression by glucose. One such approach is to trace the intracellular pathway for glucose from the target sequences in lipogenic genes to the active metabolite. Experiments are in progress aimed at characterizing the cis-acting elements that mediate the effects of glucose on lipogenic gene transcription in chick embryo hepatocytes.

Another significant finding of the present study is that transcription is the primary process through which glucose increases expression of FAS and ME in chick embryo hepatocytes (Fig. 4). This finding supports the hypothesis that glucose plays a role in mediating the effects of nutritional manipulation on transcription of FAS and ME in liver. Our data demonstrating that glucose acts through a transcriptional mechanism to regulate FAS expression in chick embryo hepatocytes contrast with the results of a previous report (44) examining the effects of glucose on FAS expression in Hep G2 cells. In the latter cell line, the stimulatory effect of glucose on abundance of FAS mRNA is due largely to changes in the stability of this mRNA. The reason for the discrepant results between the chicken and human systems is unclear. One possibility is that chickens and humans have evolved different mechanisms for the regulation of expression of lipogenic enzymes by glucose. Alternatively, differences in the mechanism of glucose action between chick embryo hepatocytes and Hep G2 cells may be due to the transformed nature of the latter cell line.

The effects of starvation and refeeding a high-carbohydrate, low-fat diet on transcription of FAS and ME are mimicked quantitatively in primary cultures of chick embryo hepatocytes by manipulating the concentrations of T3, insulin, glucagon, and glucose in the culture medium (Fig. 4) (40, 47). Thus this cell culture system appears to be a good model for the regulation of FAS and ME in livers of intact chickens. For similar reasons, primary cultures of rodent hepatocytes also appear to be a good model for the regulation of lipogenic enzymes in livers of intact rats (12, 32, 33, 38). Because the mechanism by which glucose regulates FAS and ME in chick embryo hepatocytes appears to be different from that in rat hepatocytes, class-specific differences may exist in the mechanism by which dietary carbohydrate regulates lipogenic enzyme transcription in vivo.

Because the effects of glucose on transcription of FAS and ME in chick embryo hepatocytes are dependent on the presence of T3, we postulate that a metabolite(s) of glucose enhances the ability of the liganded form of the nuclear T3 receptor to stimulate the transcription of these genes. How can this metabolite increase the transcriptional activity of the nuclear T3 receptor? One possibility is that the metabolite directly binds to the nuclear T3 receptor and regulates its function or interacts with a nuclear protein that modulates the transcriptional activity of the nuclear T3 receptor. For example, a metabolite of glucose may be a ligand for an as yet to be described orphan receptor that dimerizes with the nuclear T3 receptor. Another possibility is that a metabolite of glucose triggers a phosphorylation-dephosphorylation cascade, resulting in an alteration in the phosphorylation state of the nuclear T3 receptor or a protein that modulates the activity of the nuclear T3 receptor. Protein phosphorylation is thought to play a role in regulating T3 action because protein kinase inhibitors inhibit and protein phosphatase inhibitors enhance the effects of T3 on gene transcription (25, 48). Elucidation of the molecular mechanisms by which glucose regulates transcription of FAS and ME in hepatocytes may lead to new insights on how metabolic intermediates regulate gene expression in higher eucaryotes.

    ACKNOWLEDGEMENTS

This work was supported by the American Heart Association, West Virginia Affiliate.

    FOOTNOTES

Address for reprint requests: F. B. Hillgartner, Dept. of Biochemistry, PO Box 9142, West Virginia Univ., Morgantown, WV 26506-9142.

Received 12 September 1997; accepted in final form 25 November 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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