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Endocrinology Vol. 140, No. 7 2938-2947
Copyright © 1999 by The Endocrine Society

ARTICLES

Modulation of CCAAT/Enhancer-Binding Protein-{alpha} Gene Expression by Metabolic Signals in Rodent Adipocytes

Yihong Wang, Whaseon Lee-Kwon, Jennifer L. Martindale, Lisa Adams, Phillip Heller, Josephine M. Egan and Michel Bernier

Laboratory of Clinical Investigation (Y.W., W.L.-K., L.A., J.M.E., M.B.), Laboratory of Biological Chemistry (J.L.M.), and Laboratory of Cardiovascular Science (P.H.), National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825

Address all correspondence and requests for reprints to: Michel Bernier, Ph.D., Diabetes Section, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Box 23, Baltimore, Maryland 21224-6825. E-mail: bernierm{at}vax.grc.nia.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transcription factor CCAAT/enhancer-binding protein-{alpha} (C/EBP{alpha}) is a positive modulator of transcription for several adipocyte-specific genes that play a role in energy metabolism. However, there is little information available regarding the regulation of its expression by metabolic signals. Exposure to insulin for 5–24 h attenuated C/EBP{alpha} expression when 3T3-L1 adipocytes were incubated in 24 mM glucose, but not in 5.7 mM glucose. Nuclear run-on transcription assays indicated a transcriptional repression of C/EBP{alpha} gene, but not that of C/EBPß. Glucosamine, a product of the hexosamine pathway, in the presence of low glucose mimicked high glucose’s ability to reduce C/EBP{alpha} messenger RNA expression in insulin-treated cells. Similar results were obtained with xylitol, an activator of the pentose phosphate pathway. There was no correlation between the accumulation of hexosamine pathway metabolites (e.g. UDP-N-acetylhexosamines) and/or changes in intracellular protein glycosylation with the ability of high glucose, glucosamine, or xylitol to down-regulate C/EBP{alpha} gene expression. None of these treatments caused a reduction in intracellular ATP levels. Stable transfection of 3T3-L1 cells with the 5'-flanking 468-bp sequence of the mouse C/EBP{alpha} gene fused to luciferase demonstrated that promoter activity was also reduced by these nutrients. Of interest, treatment of rats with glucose or glucosamine led to a reduction in C/EBP{alpha} messenger RNA levels in epididymal, but not omental, fat. Taken together, these results suggest that metabolic signals serve to down-regulate C/EBP{alpha} expression both in vitro and in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CCAAT/ENHANCING-BINDING protein-{alpha} (C/EBP{alpha}) belongs to a well characterized family of transcription factors implicated in the establishment and maintenance of energy homeostasis in liver and adipocytes (1). These tissues are major sites for the storage and release of metabolic fuels. In addition to its critical role in preadipocyte differentiation, C/EBP{alpha} has been shown to bind to and trans-activate several metabolically important gene promoters in adipocytes, including the insulin-responsive glucose transporter GLUT4 (2), the adipocyte fatty acid-binding protein, aP2 (3), and the product of the ob gene (4, 5). C/EBP{alpha} is highly expressed in mature adipocytes both in cell culture and in vivo, which suggests that regulation of this protein may have a profound effect on adipocyte gene expression and metabolism. For example, it has been demonstrated that addition of tumor necrosis factor-{alpha} to adipocytes led to suppression of C/EBP{alpha} gene expression, concomitant with transcriptional repression of the GLUT4 gene (6). Similarly, glucocorticoids have been shown to regulate ob gene expression (7, 8), possibly as a result of transcriptional regulation of C/EBP{alpha} in 3T3-L1 adipocytes and in white adipose tissue in vivo (9). Other experiments examining the regulation of C/EBP{alpha} gene expression indicated that insulin and thiazolidinediones, a class of antidiabetic drugs, have opposite effects on its expression in 3T3-L1 adipocytes (10, 11). Despite its pivotal role in regulating the relative abundance of enzymes involved in carbohydrate and lipid metabolism in the liver and adipose tissue, little is known about the regulation of C/EBP{alpha} gene expression in response to metabolic signals.

It has been recently demonstrated that high concentrations of glucose enhance the expression of acetyl-coenzyme A carboxylase, fatty acid synthase, and L-pyruvate kinase in adipose tissue and hepatocytes as a result of transcriptional induction (12, 13) and extension of messenger RNA (mRNA) half-life (14, 15). Interestingly, insulin appears to play largely a permissive role in the transcriptional induction of these genes by promoting glucose phosphorylation into glucose-6-phosphate (12, 16). Doiron et al. (17) have proposed that high glucose induces the expression of L-pyruvate kinase (17) in hepatocytes through the formation of xylulose-5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway. Others have found that glucosamine, a product of the hexosamine pathway, mimics the effect of glucose in modulating growth factor expression (18). Furthermore, transient overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme in the hexosamine pathway, resulted in a 2-fold increase in glucose-mediated induction of transforming growth factor-{alpha} gene expression in vascular smooth muscle cells (19). Interestingly, a reduction in GLUT4 expression combined with insulin resistance were observed in mice overexpressing GFAT specifically in muscle and fat (20). It has been suggested that an increased flux through the hexosamine biosynthetic pathway acts as a cellular sensor of energy availability both in cultured cells and in vivo (21). It is not clear, however, whether glucose and its metabolites alter the expression of energy-related genes in part through modulation of C/EBP{alpha} gene expression. In this study, we examined the regulation of C/EBP{alpha} expression by metabolic signals, in particular glucose, glucosamine, and xylitol in fully differentiated 3T3-L1 adipocytes, and white adipose tissues of intact rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA), grown in 75-cm2 flasks, were cultured in DMEM (Paragon, Baltimore, MD) containing 24 mM D-glucose, 110 mg/liter pyruvate, 10% FCS, and penicillin-streptomycin (50 U/ml and 50 µg/ml, respectively) at 37 C in an atmosphere of air-CO2 (95:5). At confluence, monolayers of preadipocytes were induced to differentiate into adipocytes by switching to fresh medium (DMEM in the presence of 10% FBS; HyClone Laboratories, Inc., Logan, UT) supplemented with methylisobutylxanthine, dexamethasone, and insulin as previously described (22). Insulin was withdrawn from the medium 4 days after initiating differentiation. Cells were then maintained in DMEM and 10% FBS for an additional 4 days, with a medium change every 2 days. The incubation medium was composed of glucose-free or glutamine-free DMEM medium supplemented with 10% FBS and various substrates and insulin in various combinations according to experimental conditions.

Treatment of animals
Three-month-old Wistar rats from the Wistar colony at NIA (Baltimore, MD) were housed in a 12-h light, 12-h dark cycle, maintained on standard rat chow, and fed ad libitum. Rats were randomly placed into control and four treatment groups, with four to six animals in each group. Alzet microosmotic pumps (Alza Corp., Palo Alto, CA) were implanted in the interscapular region of each rat. Control animals received normal saline from their pumps, whereas treated rats were infused with either glucose (40%) at the rate of 1 µl/min or glucosamine at the rate of 30 µmol/kg·min alone and in the presence of insulin at 2 U/24 h (Humulin R, Eli Lilly & Co., Indianapolis, IN). During the last 10 h of a 48-h infusion, animals were fasted. After treatment, the rats were killed, and part of the epididymal and omental fats were quickly frozen in liquid nitrogen and stored at -80 C until analysis. Blood was collected at the time of killing for glucose and insulin determinations. This experimental protocol was approved by the animal care and use committee of the NIA.

RNA isolation and Northern blot analysis
Cellular RNA was extracted using guanidinium thiocyanate followed by ultracentrifugation through CsCl (23). Total RNA was denatured in formamide/formaldehyde for 2 min at 90 C before being electrophoresed (15 µg) on a 1.0% agarose gel containing 5% formaldehyde, blotted onto uncharged nylon membranes (Schleicher & Schuell, Inc., Keene, NH), and cross-linked with UV light (Stratalinker, Stratagene, La Jolla, CA). The membranes were prehybridized followed by hybridization with 32P-labeled complementary DNA (cDNA) probes as previously described (24). When possible, the membranes were hybridized with different cDNA probes without stripping, as long as the corresponding mRNAs were of different sizes. Blots were subjected to autoradiography at -70 C with Hyperfilm (Amersham, Arlington Heights, IL) and two intensifying screens. The relative amount of each mRNA was quantitated by electronic autoradiography using a Packard InstantImager (Meriden, CT). Subsequently, the membranes were reprobed with a ribosomal 18S probe to control for the amount of blotted RNA in each lane. The blots were hybridized with C/EBP{alpha} (1.2 kb) and C/EBPß (1.6 kb) cDNA probes (gifts from Dr. Steven L. McKnight, Tularik, Inc., San Francisco, CA) (25). In some cases, blots were also hybridized with a 2.8-kb rat GLUT1 cDNA probe (a gift from Dr. Morris J. Birnbaum, University of Pennsylvania, Philadelphia, PA) (26), a 1-kb mouse peroxisome proliferator-activated receptors (PPAR{gamma}2) cDNA probe (gift from Dr. Bruce M. Spiegelman, Harvard Medical School, Boston, MA) (27), or a 509-bp mouse leptin, 2.1-kb mouse GLUT4, or 0.422-kb mouse aP2 cDNA probes (gifts from Dr. M. Daniel Lane, Johns Hopkins Medical School, Baltimore, MD) (28, 29). All cDNAs were labeled to high specific activity with [32P]deoxy-CTP (Amersham) using random primer and Sequenase (U.S. Biochemical Corp., Cleveland, OH). The 18S oligonucleotide probe was end labeled using T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA) and [{gamma}-32P]ATP (~3000 Ci/mmol; Amersham).

Nuclear run-on transcription analysis
3T3-L1 adipocytes were cultured in DMEM supplemented with 10% FBS in the presence of 5.7 or 24 mM D-glucose plus 10 nM insulin for 6 h. The medium was replaced with fresh medium supplemented with the same mixture for an additional 6 h. For each nuclear preparation, cells from two flasks (75 cm2) were pooled. After culture medium removal, cells were broken in ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM NaCl, and 3 mM MgCl2. Nuclei isolation and run-on transcription assays were performed essentially as previously described (30). Signals were quantified by laser densitometry, and results were normalized to 3T3-L1 genomic DNA signals.

Reporter gene analysis
3T3-L1 preadipose cells stably transfected with a 468-bp 5'-segment of the C/EBP{alpha} gene (from nucleotides -343 to +125) fused to the luciferase reporter gene were provided by Dr. M. Daniel Lane (31). These cells were grown in 60-mm dishes and maintained in DMEM medium supplemented with 10% FCS and 10 µg/ml neomycin. The day of the experiment, cells were treated with 10 nM insulin in the presence of glucose or glucose metabolites for 5 h. Cells were washed twice with PBS, scraped, pelleted by centrifugation, and then resuspended in 100 mM potassium phosphate buffer (pH 7.8). Cells were lysed, and the lysates were centrifuged at 14,000 x g for 20 min at 4 C. Aliquots of the supernatants were taken for the luciferin/luciferase detection system (Promega Corp., Madison, WI) with an AutoLumat LB 953 luminometer (EG&G Berthold Analytical Instruments, Nashua, NH). The intraassay variation was less than 10%. Protein determination was carried out by the method of Bradford, using bovine {gamma}-globulin as standard.

ATP measurement
3T3-L1 adipocytes grown in 35-mm culture plates were incubated with glucose, glucosamine, or xylitol in the presence of 10 nM insulin for 8 h, and then aliquots of the culture medium were saved for glucose determination. Cells were washed in PBS and lysed with 0.25 ml ice-cold 0.6 N perchloric acid. The lysates were clarified by centrifugation at 14,000 x g for 15 min at 4 C and neutralized with 18 µl 5 M potassium carbonate. The salt precipitate was removed by centrifugation, after which 100 µl of the supernatants were diluted in 25 mM HEPES (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol. Aliquots were taken for measurement of ATP using the luciferin/luciferase detection system, and the values in the samples were corrected for protein content. An ATP standard curve (0–320 pmol/assay) was generated under the same conditions.

Assays
Glucose content in the plasma and culture medium was measured by the glucose oxidase method, whereas plasma insulin was assayed by RIA, as previously reported (32). Levels of UDP N-acetyl hexosamines (UDP-HexNAc), products of the hexosamine biosynthesis pathway and UDP-hexose (UDP-Hex) were measured in 3T3-L1 adipocytes as described by Buse et al. (33). Briefly, frozen cells were homogenized at 4 C in 1 ml 0.3 M perchloric acid, precipitates were pelleted by centrifugation, and the perchlorate was extracted from the supernatants with 2 vol trioctylamine:1,1,2-trichloro-trifluoroethane (1:4). The aqueous phase was stored at -70 C until analysis by HPLC. The extracts were filtered (0.45 µm), and HPLC was performed on a Capcell amino column (25 cm x 4.6 mm; Shiseido Co., Tokyo, Japan) eluted with a concaved gradient from 15 mM ammonium phosphate (pH 3.8) to 1 M ammonium phosphate (pH 4.5) over 45 min at a flow rate of 1 ml/min. The levels of UDP-HexNAc and UDP-Hex were quantified by UV absorption at 254 nm, compared with external standards, and corrected for protein content.

Western blot analysis
3T3-L1 adipocytes were cultured in DMEM supplemented with 10% FBS in the presence of the indicated sugars and 10 nM insulin for 8 h. The medium was replaced with fresh medium supplemented with the same mixture for an additional 16 h. Cells were then washed several times with cold PBS and lysed in Laemmli sample buffer (34) containing 7.5% 2-mercaptoethanol. After heating at 65 C for 20 min, insoluble material was pelleted by centrifugation. Equal amounts of protein from each sample were separated by SDS-PAGE under reducing conditions on 4–12% polyacrylamide gradient gel and then electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Novex, San Diego, CA). C/EBP{alpha}, TFIIH p89, and N-acetylglucosamine (GlcNAc)-containing proteins were detected by Western immunoblotting using the enhanced chemiluminescence detection system (Amersham). The polyclonal anti-C/EBP{alpha} and TFIIH p89 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), whereas monoclonal RL2 antibody was obtained from Affinity BioReagents, Inc. (Golden, CO).

Statistical analysis
Data are presented as the mean ± SEM. Comparison between groups were made by ANOVA coupled to Fisher’s protected least significant differences post-hoc test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glucose utilization in 3T3-L1 adipocytes
To determine the effects of insulin on glucose consumption, 3T3-L1 adipocytes were incubated with 10 nM insulin for various amounts of time in the presence of low (5.7 mM) or high (24 mM) glucose. Aliquots of the culture medium was then used for glucose measurement (Fig. 1AGo). Insulin rapidly increased the rate of glucose utilization, with initial velocities of 0.60 and 0.86 mM/h in the presence of 5.7 and 24 mM glucose, respectively. After stimulation with insulin for 11 h, there were less than 0.51 mM glucose remaining in the medium from cells incubated in low glucose, whereas more than 17.1 mM glucose remained in cells with high glucose (Fig. 1BGo). Under the same experimental conditions, the absence of insulin led to a minimal amount of glucose utilization with only 0.2 and 0.4 mM glucose consumed in cells maintained in low and high glucose, respectively.



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  		Figure 1. Insulin rapidly increases the utilization of glucose. A, 3T3-L1 adipocytes grown in monolayer culture were incubated in 5.7 mM (•) or 24 mM ({blacksquare}) glucose in the presence of 10 nM insulin for the indicated times. Aliquots of the medium were used for glucose determination. B, 3T3-L1 adipocytes were incubated in medium supplemented with 5.7 (filled bars) or 24 mM (open bars) glucose for 11 h with 0 and 10 nM insulin. Results are the means of four independent cultures with SEs less than 2%.

 
Effects of glucose and its metabolites on C/EBP{alpha} mRNA levels
To assess the effects of glucose and glucose metabolites on C/EBP{alpha} gene expression, 3T3-L1 adipocytes were incubated with low or high glucose for 8 h in the presence of 10 nM insulin. The medium was then replaced with fresh medium supplemented with the same experimental conditions, and the cells were incubated for a second 8-h period. This step was repeated a third time before extracting total RNA (Fig. 2AGo). This protocol was used to prevent glucose starvation to occur when insulin-treated 3T3-L1 adipocytes are maintained in low glucose for periods greater than 12 h (see above). Northern blot analysis indicated that cells exposed to high glucose for 24 h resulted in a significant 40–50% decrease in C/EBP{alpha} mRNA expression compared with that in cells in low glucose (P < 0.01). There was no difference in C/EBP{alpha} mRNA levels between cells incubated in low and high glucose in the absence of insulin (Fig. 2BGo). A reduction of C/EBP{alpha} mRNA was observed as early as 2 h after insulin addition and reached a plateau at around 5–6 h of treatment (Fig. 2CGo). It was previously reported that after a 24-h incubation with insulin, the level of C/EBPß mRNA was comparable with that of unstimulated 3T3-L1 adipocytes (10). Therefore, C/EBPß was chosen as a negative marker for C/EBP{alpha} mRNA expression. As shown in Fig. 2AGo, reprobing the blots with the C/EBPß cDNA probe revealed that expression of the C/EBPß gene was not altered by the ambient concentration of glucose. It is known that the expression of C/EBP{alpha} significantly influences trans-activation of the GLUT4 gene (2). As anticipated, the levels of GLUT4 mRNA followed a pattern of expression identical to that of C/EBP{alpha} mRNA, whereas GLUT1 mRNA levels remained unaffected whether cells were incubated in high or low glucose in the presence of insulin (Fig. 2AGo). The relative amount of immunoreactive C/EBP{alpha} protein was measured next by Western analysis of 3T3-L1 adipocyte extracts. At least two translation products of C/EBP{alpha} mRNA with molecular masses of 42 and 30 kDa have been described (10). As shown in Fig. 2DGo, the 42-kDa form of C/EBP{alpha} protein exhibited a significant decrease in cells treated with insulin and high glucose for 24 h. The 30-kDa C/EBP{alpha} isoform was not observed on Western analysis. Thus, these data represent the first demonstration of a selective reduction in C/EBP{alpha} mRNA and protein by glucose.



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  		Figure 2. Selective effect of glucose on insulin-stimulated expression of C/EBP{alpha}. A, Total RNA was extracted from insulin-treated 3T3-L1 adipocytes incubated in medium containing low (5.7 mM; {square}) or high (24 mM; {blacksquare}) levels of glucose for 24 h as described in Materials and Methods. Northern blot analysis was then performed using cDNA probes for C/EBP{alpha}, C/EBPß, GLUT4, and GLUT1. mRNA Levels were normalized to the 18S ribosomal RNA value in the same lane. Results are the mean ± SEM of three independent experiments. Insets show representative autoradiograms of Northern blots used for quantitation. **, P < 0.01. B, Effect of glucose on C/EBP{alpha} mRNA levels in the absence of insulin. C, Time course of glucose regulation on C/EBP{alpha} mRNA levels in insulin-treated cells. D, Western blot analysis of C/EBP{alpha} expression. Total cell extracts (30 µg) were prepared from 3T3-L1 adipocytes maintained in low (lane 1) or high (lane 2) glucose in the presence of 10 nM insulin for 24 h. Samples were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted using anti-C/EBP{alpha} antibody and the Amersham enhanced chemiluminescence detection system. The left margin indicates the Mr x 10-3. E, Lack of effect of L-glucose and 3-O-methylglucose.

 
When insulin-treated cells were incubated with 5.7 mM D-glucose in the presence of 19 mM L-glucose, which is unable to enter into the cells, or with 3-O-methylglucose, a nonmetabolizable glucose analog, no reduction in the accumulation of C/EBP{alpha} mRNA was observed (Fig. 2EGo). This indicates that hyperosmolarity was not responsible for the adverse effects of high ambient glucose on gene expression and that glucose metabolism has an essential role in these effects.

The transcription rate of C/EBP{alpha} gene was directly assessed by nuclear run-on assays in 3T3-L1 adipocyte nuclei isolated from cells treated with low or high glucose in the presence of insulin. Exposure of the cells to 24 mM D-glucose for 12 h resulted in a sharp decrease in the C/EBP{alpha} signals compared with that in 5.7 mM D-glucose (Fig. 3Go). This change in C/EBP{alpha} levels was accompanied by an attenuation of GLUT4 gene transcription, but not that of GLUT1 or C/EBPß. Taken together, these results indicate that a reduction in C/EBP{alpha} gene transcription is responsible at least in part for the observed effects of high glucose on C/EBP{alpha} mRNA content.



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  		Figure 3. Transcriptional inhibition of C/EBP{alpha} and GLUT4 gene expression by glucose. Nuclei were prepared from 3T3-L1 adipocytes treated with 10 nM insulin in the presence of 5.7 or 24 mM glucose for 12 h. A nuclear run-on experiment was then carried out. Newly synthesized RNA was hybridized to C/EBP{alpha}, GLUT4, GLUT1, and C/EBPß cDNAs or 3T3-L1 genomic DNA. Vector DNA (Bluescript) was present as a control. The data presented are from a representative experiment that was repeated twice with similar results.

 
Role of the hexosamine pathway in the regulation of C/EBP{alpha} mRNA levels by glucose
The metabolism of glucose through the hexosamine pathway may play a central role in the transcriptional repression of the C/EBP{alpha} gene by glucose. The rate-limiting step in this pathway is GFAT, which uses glutamine as an amide donor to convert fructose-6-phosphate to glucosamine-6-phosphate (35). Based on these observations, the determination of whether glucose flux through the hexosamine pathway is responsible for the glucose effect on C/EBP{alpha} expression was assessed in 3T3-L1 adipocytes transferred in glutamine-free medium and incubated in 24 mM glucose and insulin. The levels of C/EBP{alpha} mRNA were significantly increased by the withdrawal of glutamine (P < 0.01; Fig. 4AGo). Moreover, similar results were observed when the cells were pretreated with azaserine (Fig. 4AGo), an irreversible inhibitor of GFAT (35). These treatments were also performed in cells maintained in 5.7 mM glucose, and the results showed that both azaserine and glutamine depletion caused an increase in C/EBP{alpha} mRNA levels (Fig. 4BGo).



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  		Figure 4. Effect of glutamine depletion, azaserine, or glucosamine on C/EBP{alpha} mRNA levels. 3T3-L1 adipocytes maintained in high (A) or low (B) glucose were incubated in the absence or presence of 20 µM azaserine for 30 min before the addition of 10 nM insulin for 12 h. A second group of cells was exposed to glutamine-free culture medium in the presence of 10 nM insulin for 12 h. C, Cells in low glucose were treated or not with 2 mM glucosamine in the presence of 10 nM insulin for 12 h. In all experiments, the medium was replaced with fresh medium supplemented with the same mixture every 4 h for the next 12 h. After 24-h incubation, total RNA was isolated, and Northern blot analysis was performed. The relative expression of C/EBP{alpha} mRNA in the presence of high glucose and glutamine (A) or in the presence of 5.7 mM glucose without glucosamine (C) was arbitrarily set at 1.0. Results are expressed as the mean ± SEM of three independent experiments. ** and ***, Difference statistically significant at P < 0.01 and P < 0.001, respectively.

 
Insulin resistance induced by glucosamine requires the presence of insulin for glucosamine to enter the cells and be phosphorylated by hexokinase 2 (35, 36). Glucosamine, which bypasses the rate-limiting reaction in the hexosamine pathway, competes with glucose for transport into cells via glucose transporters located at the plasma membrane. To determine the effects of glucosamine on C/EBP{alpha} gene expression, 3T3-L1 adipocytes were incubated in low glucose (5.7 mM) supplemented with or without 2 mM glucosamine in the presence of 10 nM insulin for 12 h. The medium was replaced with fresh medium supplemented under the same experimental conditions, and the cells were incubated for a second 12-h period (Fig. 4CGo). Under these experimental conditions, glucosamine-treated cells were able to consume exogenous glucose with 0.48 vs. 1.39 mM glucose remaining in the culture medium of control and glucosamine-treated cells, respectively. In the presence of glucosamine, C/EBP{alpha} mRNA levels decreased significantly by 65% (P < 0.001), whereas the expression of C/EBPß mRNA remained unaltered (data not shown), thus ruling out a global effect by this glucose metabolite.

Effect of xylitol on C/EBP{alpha} mRNA levels
The possibility that the nonoxidative branch of the pentose phosphate pathway is involved in the regulation of glucose-mediated gene expression was examined using the sugar alcohol xylitol, a precursor of xylulose-5-phosphate (37). 3T3-L1 adipocytes were incubated in glucose-free DMEM medium with a range of concentrations of xylitol (0.5–10 mM) and insulin for 24 h, and the steady state levels of C/EBP{alpha} mRNA were compared with those in insulin-treated cells maintained in either low or high glucose. In cells incubated with 0.5 mM xylitol, the mRNA content for the C/EBP{alpha} gene was similar to that seen with 5.7 mM glucose (Fig. 5AGo). However, the response of cells treated with higher xylitol concentrations was significantly blunted. A 50–60% decrease in C/EBP{alpha} mRNA levels occurred at a xylitol concentration of 10 mM (P < 0.01), similar to that observed with 24 mM glucose. Thus, the down-regulating effect of high glucose on the expression of C/EBP{alpha} mRNA can be reproduced by xylitol.



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  		Figure 5. Effect of xylitol on C/EBP{alpha} mRNA expression. A, 3T3-L1 adipocytes in glucose-free medium were maintained in glucose-free medium supplemented with a range of concentrations of xylitol (0.5–10 mM) or transferred to either low (5.7 mM) or high (24 mM) glucose in the presence of 10 nM insulin for 8 h. The medium was replaced with fresh medium supplemented with the same mixture every 8 h for the next 16 h. B, One group of cells in glucose-free medium was incubated in the absence or presence of 20 µM azaserine for 30 min before the addition of 10 mM xylitol and insulin for 24 h. A second group of cells was maintained for 24 h in glutamine- and glucose-free medium supplemented with 10 mM xylitol and insulin. Total RNA was isolated, and Northern blot analysis was performed. The results are expressed as the mean ± SEM of three independent experiments, where the relative expression of C/EBP{alpha} mRNA in the presence of 10 mM xylitol was arbitrarily set at 1.0. **, P < 0.01.

 
It has been reported that high xylitol increases glucose-6-phosphate concentrations, as metabolites from the pentose phosphate pathway can be recycled back to this glycolytic intermediate (17). Because GFAT transfers the amide group from glutamine to fructose-6-phosphate to form glucosamine-6-phosphate, we hypothesized that transferring cells to glutamine-free medium or selective GFAT inhibition might prevent the attenuated response of high xylitol on C/EBP{alpha} mRNA levels. The expression of C/EBP{alpha} mRNA was augmented significantly (P < 0.01) when insulin-treated cells were incubated for 24 h with 10 mM xylitol either in the absence of glutamine or after inhibition of GFAT by azaserine (Fig. 5BGo).

Analysis of nucleotide-linked hexosamines and protein glycosylation
Previous studies have shown that treatment with glucosamine (19, 21) or overexpression of GFAT enzyme (20) results in an increase in the intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a substrate for protein glycosylation. Here, we found that UDP-GlcNAc concentrations were increased 2.9-fold in insulin-treated 3T3-L1 adipocytes incubated with low glucose and 2 mM glucosamine (28.7 ± 5.9 nmol/mg protein), but not high glucose (8.3 ± 0.5) compared with cells maintained in low glucose (10.4 ± 0.4; P < 0.001). In the presence of xylitol (0.5 and 10 mM), the levels of UDP-GlcNAc were 98 ± 4% and 96 ± 5% of those found in high glucose-treated cells. Hence, it appears that the concentrations of UDP-GlcNAc are not significantly changed when glucose flux via GFAT is increased. Of interest, inhibition of GFAT did not decrease UDP-GlcNAc concentrations in response to these treatments.

The level of O-glycosylation of intracellular proteins can be measured with the monoclonal antibody RL2 (38). The GlcNAc content of proteins was examined by RL2 immunoblotting of lysates of 3T3-L1 adipocytes cultured under various experimental conditions. There were multiple proteins detected with RL2 in cells maintained in low glucose and 10 nM insulin for 24 h (Fig. 6Go). The intensity of the RL2 signal was augmented in several of these protein bands after cell incubation in high glucose or low glucose supplemented with 2 mM glucosamine; the latter treatment gave rise to the strongest signal (Fig. 6Go, lane 3 vs. lane 1 or 2). Moreover, insulin-treated cells incubated in glucose-free medium that was supplemented with xylitol led to a dose-dependent increase in RL2 signal. To control for protein loading, the blot was probed with an antibody directed against the 89-kDa protein of the basal transcription factor, TFIIH. The fact that changes were noted in the RL2 signal under condition where the TFIIH p89 signal remained constant is consistent with a change in the content of GlcNAc in proteins rather than a perturbation in the relative protein abundance. These results indicate that ambient concentrations of glucose, glucosamine, or xylitol can alter intracellular protein glycosylation.



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  		Figure 6. RL2 Western blot of glycoproteins in 3T3-L1 adipocytes. 3T3-L1 adipocytes in glucose-free medium were transferred to high glucose or low glucose supplemented with or without 2 mM glucosamine (GlcN) or were maintained in glucose-free medium supplemented with 0.5 or 10 mM xylitol. After an 8-h incubation with 10 nM insulin, the medium was replaced with fresh medium supplemented with the same mixture for the next 16 h. After 24-h incubation, equal quantities of protein extracts were separated by SDS-PAGE under reducing conditions and then electrotransferred onto a PVDF membrane. The resulting blot was probed with the RL2 monoclonal antibody and detected by chemiluminescence. A second blot was probed with an anti-TFIIH p89 polyclonal antibody to confirm the protein content across all lanes. The left margin indicates the Mr x 10-3. *, Increased RL2 signal in response to high glucose or glucosamine treatment.

 
Effects of glucose, glucosamine, and xylitol on intracellular ATP levels
A recent report from Hresko et al. (39) has indicated that the glucosamine-induced insulin resistance can be ascribed to lowering of ATP levels. The effects of glucose, glucosamine, and xylitol on intracellular ATP levels in insulin-treated 3T3-L1 adipocytes are shown in Table 1Go. The ATP concentration of insulin-treated cells incubated in low glucose (121.6 ± 19.6 pmol/assay) was normalized to 1.0. The results clearly indicate that cells incubated in low glucose supplemented or not with 2 mM glucosamine had ATP levels similar to those observed with high glucose or xylitol at 0.5 and 10 mM.


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  		Table 1. Glucose consumption and ATP levels in 3T3-L1 adipocytes after an 8-h incubation with glucose, glucosamine, or xylitol in the presence of 10 nM insulin

 
Glucose metabolism regulates promoter activity of the C/EBP{alpha} gene
To assess the effect of glucose metabolites on the expression of a C/EBP{alpha} promoter-reporter construct, we used 3T3-L1 preadipose cells that were stably transfected with the 468-bp proximal region of the murine C/EBP{alpha} promoter linked to the reporter gene luciferase (31). In the absence of insulin, the induction of basal C/EBP{alpha} reporter activity by low glucose was similar to the basal activity seen under high glucose conditions (Fig. 7AGo). Of interest, down-regulation of C/EBP{alpha} reporter gene expression by insulin was apparent in cells maintained in high glucose for 5 h, in agreement with the findings of Tang et al. (31). However, only a modest 15% reduction in C/EBP{alpha} promoter activity was observed in cells treated with insulin and low glucose. These findings are consistent with our results that showed the permissive effect of insulin toward glucose-mediated reduction in C/EBP{alpha} gene expression. As illustrated in Fig. 7BGo, there was a 1.6 ± 0.1-fold increase in reporter gene expression in insulin-treated cells maintained in low glucose compared with those in high glucose (P < 0.001). Moreover, glucosamine in the presence of low glucose attenuated C/EBP{alpha} reporter activity by 60 ± 3% in insulin-treated cells (P < 0.001). Under these experimental conditions, cells incubated with xylitol in the absence of glucose led to a significant 40 ± 2% decrease in promoter activity when comparing 10 mM vs. 0.5 mM xylitol (P < 0.001). These results emphasize the presence of a regulatory element within this short region of the C/EBP{alpha} promoter that is influenced in part by metabolic signals.



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  		Figure 7. Analysis of C/EBP{alpha} promoter activity in 3T3-L1 cells. A, Cells harboring a 468-bp 5'-flanking/5'-untranslated region of the mouse C/EBP{alpha} promoter fused to the luciferase gene were transferred to either high (24 mM) or low (5.7 mM) glucose and then incubated in the absence (open bars) or presence (filled bars) of 10 nM insulin. Five hours later, cell lysates were prepared, and luciferase assays were conducted. The results are expressed as the mean ± SEM of three independent observations. B, Effects of glucosamine (GlcN) and xylitol on C/EBP{alpha} promoter. Insulin-treated cells were incubated for 5 h in medium containing glucose, GlcN, or xylitol as indicated. The results are expressed as the mean ± SEM of six to nine experiments, where the relative reporter activity in the presence of 24 mM glucose was arbitrarily set at 1.0. **, P < 0.01.

 
Hormonal/metabolic regulation of C/EBP{alpha} mRNA levels in white adipose tissue
Glucosamine and elevated concentrations of glucose have deleterious effect on insulin-stimulated expression of C/EBP{alpha} in 3T3-L1 adipocytes. We decided to examine this further using an in vivo setting. Infusion of glucosamine or glucose for 48 h resulted in a rise in plasma glucose correlated with higher levels of plasma insulin compared with those in saline-infused rats (Table 2Go). Rats infused with low dose of insulin (with a well tolerated hypoglycemia of 5.2 ± 0.2 mM) alone or together with glucosamine produced sustained hyperinsulinemia. At the end of the respective treatments, the relative abundance of mRNA coding for C/EBP{alpha} was assessed in both omental and epididymal white adipose tissues by Northern blot analysis. As illustrated in Fig. 8AGo, infusion of glucose or glucosamine resulted in a decrease in C/EBP{alpha} gene expression in epididymal fat, but had no effect in omental adipose tissue compared with saline-infused controls. The treatment with insulin alone had no effect per se on C/EBP{alpha} mRNA levels in the two fat depots (compare lane 1 vs. 5 and lane 6 vs. 10). In contrast, the combined addition of glucosamine and insulin attenuated the adverse effect of glucosamine in epididymal fat while causing a significant reduction in the levels of C/EBP{alpha} mRNA in omental adipose tissue. Total RNA was not modified by the treatments in the two tissues studied. The signal was quantified by densitometry and expressed as the ratio between the relative amount of mRNA from treated animals and that from control animals (Table 3Go).


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  		Table 2. Plasma concentrations of glucose and insulin during a 48-h infusion with saline, glucose, insulin, glucosamine, or glucosamine plus insulin

 


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  		Figure 8. Effect of 48-h infusion of glucose, insulin, glucosamine, or glucosamine plus insulin on the mRNA levels of C/EBP{alpha} and aP2 (A) or PPAR{gamma}2, leptin, and GLUT4 (B) in two rat adipose tissues. At the completion of the in vivo studies, RNA was isolated from omental and epididymal fat depots, as described in Materials and Methods, and subjected to Northern blot analysis. The labeled probes used were as indicated. An equivalent amount of RNA (15 µg) was applied to each lane, as evidenced by the 18S ribosomal RNA signal. A representative autoradiogram is shown.

 

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  		Table 3. Effect of a 48-h infusion with saline, glucose, insulin, glucosamine, or glucosamine plus insulin on the abundance of C/EBP{alpha} and aP2 mRNAs in two fat depots

 
The adipocyte fatty acid-binding protein, aP2, is a small cytosolic protein whose promoter is transcriptionally activated by C/EBP{alpha} (3). Therefore, analysis of aP2 mRNA levels from these five groups of rats was performed, and the results indicated that its pattern of expression was similar to that of C/EBP{alpha} (Fig. 8AGo). The same results were obtained after reprobing the membranes with cDNAs for leptin and the {gamma}2-subtype of the PPAR{gamma}, another important modulator of aP2 and other genes that are involved in glucose and lipid metabolism (Fig. 8BGo). In contrast, perfusion with glucosamine, insulin, or the combination of both agents increased the expression of GLUT4 mRNA in the two fat depots. These data indicate that factors other than suppression of C/EBP{alpha} and/or PPAR{gamma} mRNA are required for the regulation of GLUT4 mRNA in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of metabolic signals in the regulation of C/EBP{alpha} expression in cultured adipocytes and white adipose tissue has not been previously examined. In this study, it was found that exposure to insulin for 5–24 h down-regulated C/EBP{alpha} gene expression when fully differentiated 3T3-L1 adipocytes were incubated in 24 mM glucose, but not in low glucose. Glucose metabolism is the dominant signal, and insulin is required for efficient hexose transport and its biotransformation into cells. Investigation of the molecular mechanisms underlying the glucose-mediated reduction in C/EBP{alpha} mRNA in our system demonstrated that gene transcription, as assessed by nuclear run-on and promoter activity assays, was the primary pathway responsible for the mRNA down-regulation elicited by the combination of insulin and high glucose. In good agreement with previous observations (6, 10), the decrease in C/EBP{alpha} mRNA level was accompanied by transcriptional repression of GLUT4 gene. In contrast, GLUT1 and C/EBPß gene transcription and mRNA levels were identical in insulin-treated 3T3-L1 adipocytes incubated in low and high glucose.

The present study shows that the level of the 42-kDa C/EBP{alpha} protein was markedly reduced by high glucose in insulin-treated 3T3-L1 adipocytes. Moreover, we did not detect expression of the C/EBP{alpha} 30-kDa isoform, in agreement with the results of Lincoln et al. (40) but not with those of McDougald et al. (10). Interestingly, it was found that altering the cell lysis protocol led to a diminution and/or elimination of the p30 C/EBP{alpha} protein (40). Thus, our failure to detect the truncated C/EBP{alpha} protein may be due to the way whole cell lysates were prepared. Recent studies have shown the rapid dephosphorylation of C/EBP{alpha} in insulin-treated 3T3-L1 adipocytes, with near-complete dephosphorylation after a 2-h incubation with insulin (11, 41). However, these researchers relied largely on the phosphorylation status of the 30-kDa C/EBP{alpha} protein but not that of the 42-kDa isoform due to the difficulty of measuring changes in phosphorylation level of the latter (11). None of the experiments carried out in our study was performed with incubation periods of less than 5–24 h with glucose and insulin. Under these conditions, it is apparent that the addition of high glucose to insulin-treated 3T3-L1 adipocytes failed to alter the extent of 42-kDa C/EBP{alpha} protein phosphorylation, as evidenced by the absence of additional bands on Western blot.

Glucose-6-phosphate per se is known to transduce the glucose effect on transcriptional regulation of some genes (12, 42); however, other glucose-regulated genes require the progression of glucose-6-phosphate through metabolic pathways (17, 18, 43). In the study herein, we found that the removal of glutamine from the high glucose-containing medium or pretreatment of 3T3-L1 adipocytes with azaserine prevented the reduction in C/EBP{alpha} mRNA levels by the combination of high glucose and insulin. These findings are consistent with the idea that increased flux of glucose through the hexosamine pathway may have a major role in the regulation of C/EBP{alpha} gene expression. However, the same treatments increased the level of C/EBP{alpha} mRNA in insulin-treated cells maintained either in low glucose or in the presence of 10 mM xylitol in glucose-free medium. There are numerous synthetic pathways that could be affected by glutamine depletion or addition of azaserine. In fact, glutamine starvation may trigger a regulatory signal, and azaserine is a known inhibitor of all transamidases, which includes GFAT, CTP synthase, and glutamine phosphoribosylpyrophosphate amidotransferase (44). For these reasons and the fact that incubation of insulin-treated 3T3-L1 adipocytes in medium supplemented with high glucose or xylitol (10 mM) did not significantly increase UDP-GlcNAc levels to levels higher than those in cells exposed to low glucose and insulin, we believe that the regulation of C/EBP{alpha} gene expression is not likely to be mediated by products of the hexosamine pathway in the system described here.

Another finding of this study was that glucosamine (2 mM) in the presence of low glucose markedly reduced the C/EBP{alpha} mRNA level in insulin-treated 3T3-L1 adipocytes while inhibiting C/EBP{alpha} promoter activity. Moreover, we show here that the infusion of glucosamine in rats, a procedure known to induce peripheral insulin resistance (25, 45), resulted in significant down-regulation of C/EBP{alpha} gene expression in epididymal fat. In agreement with previous observations (38, 43), glucosamine markedly increased the levels of UDP-GlcNAc concomitant with enhanced O-glycosylation of proteins. Given the differences in the levels of hexosamine metabolites and protein glycosylation patterns among glucose, glucosamine, and xylitol, it is unlikely that protein glycosylation will modulate C/EBP{alpha} mRNA levels.

It has been recently reported that desensitization of the insulin-stimulated glucose transport effector system in 3T3-L1 adipocytes by glucosamine treatment was due solely to a reduction in intracellular ATP levels (39), possibly by trapping ATP as glucosamine-6-phosphate. In sharp contrast, we found that ATP levels were unaffected. The 3T3-L1 adipocytes metabolize glucose very rapidly in the presence of insulin, such that no glucose can be measured at the end of a 16-h incubation in low glucose medium (our unpublished data). We believe that under the experimental conditions used by Hresko et al. (see Fig. 12, Ref. 39), where insulin (1 µM)-treated cells were maintained in low glucose and glucosamine for 16 h without medium replenishment, the apparent approximately 5-fold increase in sensitivity to glucosamine was merely the result of glucose starvation. Our observation clearly indicates that glucose protected insulin-treated 3T3-L1 adipocytes from ATP depletion by glucosamine.

Previous observations made in 3T3-L1 adipocytes indicated that inhibition of phosphatase 2A and 1 led to transcriptional repression of the C/EBP{alpha} gene (6). Also, it has been recently shown that an inhibitor of mitogen-activated kinase kinase blocked the reduction in the level of both C/EBP{alpha} mRNA and protein in insulin-treated 3T3-L1 adipocytes (41). Activated MAP kinase has been shown to localize in the nucleus (46), where it may modulate the activity of transcription factors. Therefore, it is possible that the synergism between insulin and a high concentration of glucose on C/EBP{alpha} gene transcription can be explained in part through phosphorylation of transcription complexes by insulin receptor-mediated activation of the p21Ras/mitogen-activated protein kinase phosphorylation cascade. Moreover, glucose metabolites may also act as glucose sensors in regulating translocation of selective protein kinase C isozymes (47), a mechanism known to occur in hyperglycemia-induced insulin resistance.

The observed decrease in C/EBP{alpha} gene expression in epididymal fat, but not in omental adipose tissue, after infusion of glucosamine or high glucose in rat is interesting and suggests that expression of the C/EBP{alpha} gene can be regulated in divergent fashion by specific fat depots. Indeed, the expression of leptin and PPAR{gamma} genes was higher in sc than in visceral adipose tissue (48, 49). Furthermore, heterogeneity in the expression and function of adrenergic receptors has been observed among various fat depots (50). In the work herein, infusion of insulin (in combination with mild hypoglycemia) had no effect on C/EBP{alpha} gene expression in fat. This finding is consistent with our in vitro study, which showed that insulin when in combination with 5.7 mM glucose had negligible effects on gene expression in 3T3-L1 adipocytes. The reduction in C/EBP{alpha} mRNA and protein by metabolic signals is expected to be associated with transcriptional repression of several genes, including the lipid-binding protein aP2 (6). Consistent with this hypothesis, the pattern of aP2 mRNA expression after infusion with glucosamine or glucose was depot specific and correlated with that of C/EBP{alpha} mRNA. The observation that insulin infusion did not have a significant effect on aP2 gene expression in white adipose tissue supports the earlier findings of Lowell and Flier (51), who showed that aP2 mRNA levels were unaffected after treatment of cultured adipocytes with insulin. Because of the central role of aP2 in coupling obesity to insulin resistance (52), such depot-specific differences in its expression could have physiological implications for the control of regional adiposity and fat distribution, with altered and/or preferential substrate utilization and local production of tumor necrosis factor-{alpha} (52), a cytokine implicated in obesity-linked insulin resistance and diabetes. Although this in vivo study focused on C/EBP{alpha} and aP2 mRNA levels, we also analyzed the mRNA expression levels of other genes that are involved in glucose and lipid metabolism, including PPAR{gamma}2, leptin, and GLUT4. The PPAR{gamma}2 and leptin gene expression followed that of C/EBP{alpha}; however, it was found that reduction in C/EBP{alpha} mRNA may not be required to suppress GLUT4 mRNA level. This finding is in agreement with the conclusions of Hemati et al. (11, 41). The mechanism of hyperglycemia-mediated insulin resistance is still poorly understood. In any rate, integration of a complex array of transcriptional activities elicited by C/EBP{alpha}, PPAR{gamma}2, and other factors appears to be central to the overall control of energy homeostasis by nutrients and hormones.


   Acknowledgments
 
We are grateful to Drs. Morris J. Birnbaum, Steven L. McKnight, Bruce M. Spiegelman, and M. Daniel Lane for kindly providing the cDNA probes. We also thank Dr. M. Daniel Lane for the gift of 3T3-L1 transfected cells, and Sutapa Kole for her expert technical assistance.

Received October 16, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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