Changes in glucose levels are known to directly alter gene expression. A number of previous studies have found that these effects are in part mediated by modulating the levels and the activity of transcription factors. We have investigated an alternative mechanism by which glucose might regulate gene expression by modulating levels of a transcriptional repressor. We have focused on Id2, which is a protein that indirectly regulates gene expression by sequestering certain transcription factors and preventing them from forming functional dimers. Id2 targets include the class A basic helix-loop-helix transcription factors and the sterol regulatory element-binding protein (SREBP)-1. We demonstrate that increases in glucose levels cause a rapid increase in levels of Id2 in J774.2 macrophages, and a number of lines of evidence indicate that this is via the hexosamine pathway because 1) the effect of glucose requires glutamine; 2) the effect of glucose is mimicked by low levels of glucosamine; 3) the effect of glucose is inhibited by azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT); and 4) adenoviral mediated overexpression of GFAT increases levels of Id2. We go on to show that increases in Id2 can have functional effects on metabolic genes, because Id2 blocked the SREBP-1-induced induction of hormone-sensitive lipase (HSL) promoter activity, whereas Id2 alone does not modulate activity of the HSL promoter. In summary, these studies define a new mechanism by which glucose uses the hexosamine pathway to regulate gene expression by increasing levels of a transcriptional repressor.
- E proteins
it is well recognized that changes in glucose levels have direct effects of gene expression in a range of tissues (49). Gene expression events induced by normal physiological fluctuations in glucose levels play a critical role in the maintenance of proper glucose and lipid homeostasis. These effects have been thoroughly investigated mostly in liver, where changes in glucose levels play an important role in switching off gluconeogenesis and switching on glycolytic pathways (15, 17). Glucose also plays a key role in regulating the expression and release of insulin in the β-cells of the pancreas (45), and the insulin released subsequently plays an important role in the regulation of metabolic genes (17). However, persistent hyperglycemia associated with diabetes is known to contribute to a wide range of gene expression events associated with the development of diabetic complications (5). More recently, we and others (19, 21, 41, 44) have shown that glucose has direct effects on genes controlling cholesterol ester metabolism in macrophages, and these findings have suggested a possible link between hyperglycemia and foam cell formation. The mechanisms by which glucose regulates gene expression in macrophages have not been characterized.
A number of mechanisms have been implicated in glucose-mediated regulation of gene expression (5). The hexosamine pathway has drawn particular attention because increased activation of this pathway has been implicated in the glucose-mediated effects on leptin gene expression (52). However, prolonged activation of the hexosamine pathway can lead directly to the development of insulin resistance (2, 24, 25, 28, 31, 32, 34, 50). The mechanism by which the hexosamine pathway regulates gene expression has been studied and is known to involve regulation of the activity of some transcription factors. For example, the hexosamine pathway mediates the glycosylation of both myc and Sp1, resulting in increases in transactivating potential (10, 13). Furthermore, cellular levels of myc are upregulated by glucose in some cell types (9, 27). However, there are currently no studies investigating the effects of the hexosamine pathway on other modifiers of transcription, such as transcriptional repressor proteins.
We have investigated the effects of glucose on the expression of the transcriptional repressor Id2 because this is one of the genes that is upregulated in hyperglycemic ob/ob mice (51), and our preliminary gene expression studies (22) found that Id2 mRNA levels increase with glucose in cell models. Id2 is able to act as a transcriptional repressor because it has high homology to the helix-loop-helix region of the basic helix-loop-helix (bHLH) transcription factors (38, 56). This homology allows Id2 to dimerize with the class A bHLH transcription factors (E proteins) and sterol regulatory element-binding protein (SREBP)-1c (36), but because Id2 lacks the basic DNA binding domain, the heterodimers containing Id2 are nonfunctional. Thus Id2 acts as a dominant-negative regulator of these transcription factors and is likely to have the capacity to regulate the expression of a range of genes involved in glucose metabolism and insulin action. In the present study, we find that glucose increases levels of Id2 protein in J774 macrophages and that this occurs via the hexosamine pathway. We go on to show that changes in Id2 levels can have functional consequences for the expression of metabolic genes. These findings identify upregulation of transcriptional repressors as a new mechanism by which the hexosamine pathway contributes to changes in gene expression.
MATERIALS AND METHODS
RPMI 1640 medium and PBS without calcium or magnesium were obtained from GIBCO-BRL (Gaithersburg, MD). Anti-Id2 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine and TRIzol were from Invitrogen. Luciferase assay kits were from Promega. Anti-rabbit horseradish peroxidase (HRP) secondary antibody was obtained from DAKO. All other chemicals were obtained from Sigma unless stated otherwise.
The murine J774.2 cell line was obtained from the European Culture Collection. These cells were grown in RPMI 1640 medium in the presence of 10 mM glucose supplemented with 10% fetal calf serum (heat inactivated at 50°C for 30 min) and 1% antibiotic-antimycotic. The medium was changed every 48 h. Before the experiments, cells were serum starved and then incubated in RPMI containing either zero glucose, low glucose (5 mM), or high (20 mM) glucose. Chinese hamster ovary cells that overexpress the insulin receptor (CHO-IR) were kindly provided by Prof. K. Siddle, University of Cambridge.
Immunoprecipitation and Western blotting analysis.
J774.2 cells were washed once with sterile calcium and magnesium-free PBS. Cell culture medium was replaced with serum-free RPMI 1640 medium containing 0.2% fatty acid free BSA and the appropriate level of glucose or other chemicals. This was replaced with fresh medium after overnight incubation. Experiments were performed on confluent J774.2 macrophages. After stimulation, the cells were washed once with ice-cold PBS and lysed in buffer containing 300 mM sucrose, 3 mM MgCl2, 0.5% NP-40, 10 mM Tris·HCl, pH 8.8, 10 mM NaCl supplemented with 2 μg/ml of aprotinin, 1 μM pepstatin, 10 μM leupeptin, 30 μM N-acetyl-leucinyl-leucinyl-norleucinal, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride. The insoluble fraction was removed from lystate by centrifugation at 14,000 rpm for 10 min at 4°C. Cell lysate (500 μl) was incubated at 4°C with 5 μl of rabbit anti-Id2 antibody for 2 h before incubation with 30 μl of protein G-agarose for an additional hour. The immunoprecipitate was washed extensively before it was resuspended in Western loading buffer (20% glycerol, 4% SDS, 200 mM dithiothreitol, 0.2 M Tris-buffered saline, pH 6.8) and subjected to SDS polyacrylamide gel electrophoresis. Samples were then transferred onto polyvinylidene difluoride membrane and immunoblotted overnight with Id2 antibody and then anti-rabbit HRP antibody. Immunoreactive bands were visualized by using enhanced chemiluminescence, with the signal being detected by the Fuji LAS1000 imaging system. Bands were quantified with Image gauge software.
The expression plasmid encoding mouse SREBP-1a was a generous gift from Dr. T. F. Osborne (University of California, Irvine, CA) (43). A fragment of the human hormone-sensitive lipase (HSL) promoter (−2,682 to 301) was inserted into the BglII sites of the pGL3basic luciferase expression vector. An expression plasmid encoding human Id2 was a generous gift from Dr. E. Hara (Department of Applied Biological Science, Science University of Tokyo, Noda, Japan).
Luciferase reporter studies.
One day before transfection, CHO-IR cells were passaged and maintained at 37°C, 5% CO2 in F-12 medium containing 10% FBS. Transient transfections were performed using the lipofectamine reagent according to the manufacturer's instructions. All transfections to analyze HSL promoter function contained 0.5 μg of HSL promoter-luciferase vector and 20 ng of thymidine kinase promoter-linked Renilla luciferase vector (pRL-TK). Cotransfections contained 100 ng of SREBP-1a expression plasmid and 500 ng of CMV-Id2 expression plasmid. Total amounts of DNA were adjusted to 2 μg with empty pcDNA3 vectors. A control plasmid (pGL3basic) was used in place of the HSL-Luc construct in the experiments to study nonspecific effects of SREBP-1 and Id2 as indicated in the legend for Fig. 9. Cells were incubated with DNA-lipofectamine complexes for 6 h before transfection medium was replaced by F-12 medium containing 10% FBS. Cells were harvested after 24 h for dual luciferase assay. Firefly luciferase data were normalized for Renilla luciferase activity, driven by the internal pRL-TK control expressed as fold induction.
Adenoviral expression of glutamine:fructose-6-phosphate amidotransferase.
Adenovirus expressing glutamine:fructose-6-phosphate amidotransferase (GFAT) was created and propagated as described previously (6). J774.2 cells were grown to confluence in high-glucose medium with serum and then infected with β-galactosidase or GFAT virus at ∼100 MOI (multiplicity of infection) for 4 h . Virus was removed and cells were then incubated overnight in low-glucose medium (5.5 mM) with serum overnight. In the morning, cell medium was again changed to 5.5 mM medium with serum, and cells were then lysed after 8 h.
Glucose increases levels of Id2 protein in a concentration-dependent manner.
To test the effects of glucose on Id2 protein levels in J774.2 cells, the cells were transferred directly from standard RPMI (10 mM glucose) to RPMI containing 5 or 20 mM glucose, and cell extracts were prepared at various time points. Western blotting of these cell lysates revealed a rapid divergence in Id2 levels between these two conditions, with cells in 20 mM glucose having 60% more Id2 within 2 h (Fig. 1A). This relative difference was maintained for 24 h. The differential effects were even more dramatic when cells were grown in medium lacking glucose overnight and then transferred to medium containing either 5 or 20 mM glucose (Fig. 1B). This identified Id2 as a protein whose levels change rapidly in response to changes in glucose levels.
Mechanisms of glucose-mediated increase in Id2 protein levels.
We next sought to identify the mechanism by which glucose was causing the upregulation of Id2. One possibility is that the slight change in osmolarity caused by increasing extracellular glucose concentrations could regulate the expression of Id2, but this was ruled out by the finding that 20 mM mannitol was unable to mimic the effects of 20 mM glucose (Fig. 2A). The finding that the effect of glucose could not be mimicked by the transportable but nonmetabolizable glucose analog 3-O-methyl-d-glucose indicates that the effects on Id2 expression required further metabolism of glucose (Fig. 2, A and B). It has been reported (18) that glucose 6-phosphate can promote some of the effects of glucose on gene expression. Evidence for this finding comes from the observation that the effects of glucose can be mimicked by 2-deoxyglucose, a glucose analog that can be phosphorylated but not further metabolized. However, in J774 macrophages, 2-deoxyglucose did not mimic the effects of glucose on Id2 expression (Fig. 2, B and C). These effects were the same when cells were cultured in both low-glucose (0.5 mM) medium or medium containing a physiologically relevant level of glucose (5 mM). These findings indicate that the effect of glucose on Id2 protein levels was most likely being mediated by a metabolite of glucose.
De novo diacylglycerol production in cells is dependent on elevations in glucose concentrations, and indeed, diacylglycerol levels rise in proportion to glucose levels in a range of cell types, including monocytes (8). This, in turn, results in an increase in the activation of protein kinase C (PKC) isoforms that can result in changes in gene expression (29). However, the PKC pathway does not appear to be involved in the effects of glucose on Id2 expression in macrophages because overnight treatment with the phorbol ester PMA was able to completely downregulate cPKC isoforms in J774 cells, but this did not block the glucose-induced increase in Id2 levels (Fig. 3).
The hexosamine biosynthetic pathway is a key pathway involved in mediating the effects of glucose on cellular function (31, 32). In this pathway the enzyme GFAT diverts a small amount of fructose 6-phosphate from the glycolytic pathways at the level of fructose 6-phosphate and converts it to glucosamine 6-phosphate in a reaction that uses glutamine as the donor for the amine group. Uridyl diphosphate (UDP) and acetyl groups are then added to create UDP-N-acetylglucosamine (UDP-GlcNAc) (23, 42, 53). To determine whether this pathway could be involved in regulating Id2 levels, we first determined whether the effects of glucose were blocked by the GFAT inhibitor azaserine. As is seen in Fig. 4, the effects of glucose were greatly attenuated by azaserine, which implies a role for the hexosamine pathway. Furthermore, we find that adenovirus-mediated expression of GFAT causes a significant increase in levels of Id2 (Fig. 5). The metabolism of glucose via the hexosamine pathway requires the availability of glutamine, and the effects of glucose on Id2 expression were not seen in cells incubated in glutamine-free medium (Fig. 6). Glucosamine 6-phosphate is the product of the reaction catalyzed by GFAT, but this molecule can also be produced by the direct phosphorylation of glucosamine. Glucosamine can be transported into cells via the GLUT family of facilitative glucose transporters, which means that the addition of glucosamine to the cell culture medium can mimic the effects of glucose acting via the hexosamine pathway. Here, we find that low concentrations of glucosamine cause increases in levels of Id2 protein that are similar to those induced by glucose (Fig. 7). These data together provide strong evidence that the glucose-induced increase in Id2 levels is mediated via the hexosamine pathway.
Id2 is a protein that is known to have a rapid turnover in the cell, with its degradation mediated by ubiquitination and proteasomal pathways (4, 14). The available literature suggests that the hexosamine pathway might increase the levels of cellular proteins by causing them to be glycated and thus less susceptible to proteasomal degradation (30, 38). We have found that both high glucose and high glucosamine increase the level of GlcNAc incorporated into Id2 (data not shown), which suggests that such protein stabilization might occur. To test this, we incubated J774.2 cells for a short time in medium containing either 5 or 20 mM glucose and then added cycloheximide to block new protein synthesis and observed the rate of decrease in cellular levels of Id2 (Fig. 8). These studies showed that Id2 indeed had a short half-life but that the half-life was greater in cells that were grown in high-glucose solution. Over three independent experiments, we found that after 30 min of cycloheximide treatment, 45% of initial Id2 remained in cells incubated in 5 mM glucose, whereas in cells incubated in 20 mM glucose, 75% of Id2 remained.
Effects of Id2 on activity of HSL promoter.
A key question was whether the glucose-induced increase in Id2 levels could explain any of the glucose-mediated effects on gene expression. We (41) had previously shown that glucose-attenuated insulin induced increases in HSL levels in J774 cells, so we investigated the effect of Id2 on the activity of the HSL gene promoter. We chose to investigate the effects of SREBP-1 and Id2 on the HSL promoter because this promoter has sites for SREBP-1 binding (48), and Id2 is known to bind to SREBP-1 and attenuate its effects (36). Furthermore, SREBP-1 expression is known to be increased by insulin (16, 47). We transfected CHO-IR cells with an HSL pomoter-luciferase reporter construct and an SREBP-1 expression construct. SREBP-1 increased luciferase activity driven by the HSL promoter by approximately twofold, and this was significantly attenuated by coexpression of Id2, although Id2 alone had no effect on promoter activity (Fig. 9). This was specific, because neither Id2 nor SREBP-1 caused any change in the expression of the PGL3basic control luciferase construct. These data argue for a role for Id2 in the glucose-mediated downregulation of HSL gene expression that we have observed previously (41).
It is well established that increased activity of the hexosamine pathway induced by elevated levels of glucose can regulate expression of a range of genes in many cell types (23, 31, 53). The mechanism by which this is achieved is partly understood and in some cases involves the direct glycosylation of certain transcription factors that subsequently regulate both the stability of these proteins and their transactivating potential (10, 20, 55). However, it is presently not clear how transcription factors that control glucose and lipid metabolism might be regulated. New light is shed on this question by the finding of the present study that, in macrophages, the level of expression of the transcriptional repressor Id2 is highly regulated by changes in glucose, and that this effect is mediated via the hexosamine pathway.
Id2 is a member of the helix-loop-helix transcriptional repressor protein family (Id1–4) (40, 56). One functionally important role for these proteins is to block differentiation and promote proliferation. For example, overexpression of Id2 is known to promote myoblast proliferation while it blocks its differentiation into myotubes (35). Id2 levels fall during differentiation of adipocytes, and evidence has been presented to indicate that this is necessary for differentiation to proceed (37). The mechanisms by which Id2 works are reasonably well documented. In part, Id2's effects are mediated by interacting with retinoblastoma protein, which helps maintain a proliferative state (30). However, Id2's best-characterized effects relate to its ability to dimerize with E proteins, a subset of bHLH transcription factors (39). Because Id2 lacks a basic DNA binding domain, the resulting dimer is inactive, and thus Id2 acts as a dominant negative regulator of the function of these transcription factors (40). Id2 does not bind to most of the other bHLH transcription factors, but it is able to regulate their function indirectly by sequestering the E proteins, which are required to form functional bHLH transcription factor heterodimers (39). Thus Id2 influences the function of other bHLH proteins such as MyoD, even though it does not bind directly to them.
However, any effects of Id2 on metabolic pathways are likely to be mediated by binding to transcription factors that regulate expression of metabolic genes. One candidate is the bHLH-L/Z transcription factor SREBP, because it is reported (36) that Id2 binds to and inhibits the activity of SREBP-1c. This insulin-responsive transcription factor is essential for adipocyte differentiation and the expression of genes controlling lipid metabolism (36). In support of this, we have found that Id2 antagonizes the stimulatory effects of SREBP-1 on the HSL promoter. Therefore, it is reasonable to conclude that the increases observed in Id2 protein levels in macrophages after their exposure to glucose are likely to have important effects on gene expression and cell function. It is interesting to note that the recently identified glucose-regulated transcription factor named carbohydrate-responsive element-binding protein (ChREBP) is also a bHLH-L/Z class transcription factor (11, 12, 26). Given the structural similarities to SREBP, this suggests that Id2 and ChREBP could potentially interact. However, it is presently not known whether such interactions can occur, and it is not known whether ChREBP is expressed in macrophages (12).
This study provides the first evidence that Id2 levels can be increased by glucose, although previous studies have indicated that the closely related proteins Id1 and Id3 are regulated by glucose (54) and fatty acids (7) in β-cells. Previous studies have shown that both IGF-I (3) and cAMP (36, 46) also cause increases in Id2 levels in some cell types. Together, these findings imply that the Id proteins might play a wider role in allowing cells to respond to changes in nutrient status. In this regard, it is of note that Id2 is one of the genes upregulated in the muscle, fat, and liver of the obese ob/ob mice (51). Furthermore, a recent preliminary report (1) also indicates that Id2 knockout mice are resistant to the development of atherosclerosis, which suggests that Id2 contributes significantly to the atherogenic process under normal circumstances. It is also of interest that Id2 levels in aortic smooth muscle cells are reduced by treatment with the thiazolidinedione drugs, which suggests that this might play a role in the anti-diabetic effects these drugs have (57). These findings suggest that elevations in Id2 levels could contribute to the changes in cellular function that occur in the insulin-resistant and diabetic states.
In the present study, we have not identified the detailed mechanism by which the hexosamine pathway upregulates Id2 levels. However, our present data and previous findings together indicate that there are effects at the levels of both transcription and protein stabilization. The effect on transcription was not investigated here but could well involve glucose-mediated effects on the function of transcription factors. For example, both Sp1 and c-myc upregulate Id2 expression (30, 38), and the hexosamine pathway is known to regulate the activity of both myc and Sp1 via post translational modifications that increase their transactivating potential (10, 13). Furthermore, cellular levels of myc are increased by glucose in some cell types (9, 27).
In summary, it is well known that flux through the hexosamine pathway provides a mechanism by which changes in glucose concentration can result in changes in gene expression, although the details of how this is achieved are not fully understood. Our findings that the levels of Id2 in macrophages are proportional to glucose levels in the cell provide evidence for a previously undescribed mechanism by which the hexosamine pathway can contribute to gene-regulatory events and thus allow cells to respond appropriately to the nutrients in their environment.
This work was supported by a UK Medical Research Council Studentship awarded to R. Tingsabadh, a Diabetes UK Research Grant awarded to P. R. Shepherd, and a Norwegian Research Council Fellowship awarded to L. M. Grønning
The technical assistance of Mina Edwards is greatly appreciated.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 by American Physiological Society