Howard C. Towle

ChREBP•Mlx Is the Principal Mediator of Glucose-induced Gene Expression in the Liver

In mammals, glucose-regulated gene expression has been best characterized in the liver, where increased glucose metabolism induces transcription of genes encoding enzymes involved in de novo lipogenesis. ChREBP and Mlx dimerize and function together as a glucose-responsive transcription factor to regulate target genes, such as liver-type pyruvate kinase, acetyl-CoA carboxylase 1, and fatty acid synthase. To identify additional glucose-responsive genes in the liver, we used microarray analysis to compare gene expression patterns in low and high glucose conditions in hepatocytes. Target genes of ChREBP·Mlx were simultaneously identified by gene profiling in the presence or absence of a dominant negative Mlx. Of 224 genes that are induced by glucose, 139 genes (62%) were also inhibited by the dominant negative Mlx. Lipogenic enzyme genes involved in the entire pathway of de novo lipogenesis were found to be glucose-responsive target genes of ChREBP·Mlx. Genes encoding enzymes in other metabolic pathways and numerous regulators of metabolism were also identified. To determine if any of these genes are direct targets of ChREBP·Mlx, we searched for ChoRE-like sequences in the 5′-flanking regions of several genes that responded rapidly to glucose. ChoRE sequences that bound to ChREBP·Mlx and supported a glucose response were identified in two additional genes. Combining all of the known ChoRE sequences, we generated a modified ChoRE consensus sequence, CAYGNGN5CNCRTG. In summary, ChREBP·Mlx is the principal transcription factor regulating glucose-responsive genes in the liver and coordinately regulates a family of genes required for glucose utilization and energy storage.

Two CACGTG motifs with proper spacing dictate the carbohydrate regulation of hepatic gene transcription.

Regulatory sequences involved in the transcriptional induction of the rat S gene in response to increased glucose metabolism in the hepatocyte were investigated and compared with those of the liver-type pyruvate kinase (L-PK) gene. The carbohydrate response element (ChoRE) of the S gene was found to consist of two motifs related to the consensus binding site for the c-myc family of transcription factors, CACGTG. These two motifs are separated by five base pairs, a similar arrangement to that found in the L-PK ChoRE. In its natural context, the S ChoRE requires a novel accessory factor to support the full response to glucose. This factor, as well as the factor hepatic nuclear factor-4, are both capable of binding to the L-PK gene to enhance its carbohydrate regulation. The need for an accessory factor for supporting the glucose response can be overcome in two ways. First, multimers of the ChoREs of either the L-PK or S genes can function independently to support the glucose response. Second, mutations in the S ChoRE that create a perfect match to the consensus CACGTG motif at each locus no longer require an accessory factor site. The spacing of the two CACGTG motifs, but not the nature of the bases within the spacer, are critical for control. These observations suggest that a carbohydrate responsive factor binds to both motifs in a highly specific spatial orientation to confer the response to increased carbohydrate metabolism.

Metabolic Regulation of Gene Transcription in Mammals

All cells regulate gene expression in response to changes in the external environment. For unicellular organisms, specific mechanisms have evolved to allow these cells to metabolize various fuels based on their availability in the external milieu. In part, these mechanisms involve conditional transcription of genes encoding enzymes unique to specific metabolic pathways in the presence of appropriate nutrients. The study of such control mechanisms has led to several of the classic paradigms for transcriptional regulation present in today’s textbooks. Thus, the lac operon of Escherichia coli and the gal regulon of Saccharomyces cerevisiae are among the best understood regulatory pathways of gene expression. In multicellular organisms, the needs of not only the individual cell but also the whole organism must be managed. Consequently, much of the task of interpreting environmental cues in mammals is handled by hormonal and neuronal pathways. For example, the counterbalancing hormones insulin and glucagon play a major role in maintaining blood glucose levels within fairly narrow limits by controlling glucose utilization in several different tissues. Although not as widely appreciated, nutritional and metabolic signals also play an important role in controlling gene expression in multicellular organisms. This review will summarize recent work on two metabolic signals, cholesterol and glucose metabolism, which can lead to altered gene expression in mammals.

Glucose and Insulin Function through Two Distinct Transcription Factors to Stimulate Expression of Lipogenic Enzyme Genes in Liver

Transcription of a number of genes involved in lipogenesis is stimulated by dietary carbohydrate in the mammalian liver. Both insulin and increased glucose metabolism have been proposed to be initiating signals for this process, but the pathways by which these effectors act to alter transcription have not been resolved. We have previously defined by electrophoretic mobility shift assay a factor in nuclear extracts from rat liver, designated the carbohydrate-responsive factor (Cho- RF), that binds to liver-type pyruvate kinase and S14 promoters at sites critical for regulation by carbohydrate. The sterol regulatory element binding protein-1c (SREBP-1c) has also emerged as a major transcription factor involved in this nutritional response. In this study, we examined the relationship between SREBP-1c and ChoRF in lipogenic gene induction. The two factors were found to possess distinct DNA binding specificities both in vitro and in hepatocytes. Reporter constructs containing binding sites for ChoRF were responsive to glucose but not directly to insulin. On the other hand, reporter constructs with an SREBP-1c site responded directly to insulin. The S14 gene possesses binding sites for both ChoRF and SREBP, and both sites were found to be functionally important for the response of this promoter to glucose and insulin in hepatocytes. Consequently, we propose that SREBP-1c and ChoRF are independent transcription factors that mediate signals generated by insulin and glucose, respectively. For many lipogenic enzyme genes, these two factors may provide an integrated signaling system to support the overall nutritional response to dietary carbohydrate.

Glucose as a regulator of eukaryotic gene transcription

Glucose has essential metabolic roles as both a fuel for energy and a substrate for the biosynthesis of cell components. Because of its central importance, many cells have evolved mechanisms to sense glucose levels in their environment and to adapt the expression of their genetic information to glucose availability. This glucose signaling is vital in mammalian cells where derangements in glucose utilization might contribute to conditions such as obesity and type 2 diabetes. Two crucial issues stand out in understanding pathways of glucose-regulated gene transcription. First, how do cells sense changing glucose levels? Second, how is this signal transduced to the transcriptional apparatus of the cell? In mammalian cells, glucose sensing involves the detection of changes in glucose metabolism rather than glucose itself. A transcription factor that is involved in mediating responses to glucose, ChREBP, has been identified recently and studies have begun to elucidate the molecular basis of coupling between glucose metabolism and transcription factor activity.

Synergism of thyroid hormone and high carbohydrate diet in the induction of lipogenic enzymes in the rat. Mechanisms and implications.

We have investigated the relationship between the administration of triiodothyronine (T3) and a high carbohydrate (CHO) fat-free diet in the induction of lipogenic enzymes in two rat tissues, liver, and fat. Male thyroidectomized rats were treated with graded daily doses of T3 and either supplemented with a high CHO diet or left on a regular diet. Enzymes studied included malic enzyme (ME), fatty acid synthetase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. In the liver, all four lipogenic enzymes showed a synergistic response between T3 administration and high CHO feeding. In fat, ME also responded synergistically. The interaction was reflected in an increased sensitivity to T3. The dose of T3 required to achieve 50% maximal response was reduced three- to seven-fold by the high CHO diet. This phenomenon could not be attributed to a dietary-induced alteration either in T3 metabolism or in number or affinity of the T3-nuclear receptors. Moreover, studies of the relative rate of synthesis of ME suggested a simultaneous time of onset in the induction of ME, within 2 h after the application of either T3 or CHO. Thus, it is unlikely that either stimulus is secondary to the other. Since parallel experiments from this laboratory (Towle, Mariash, and Oppenheimer,1980. Changes in hepatic levels of messenger ribonucleic acid for malic enzyme during induction by thyroid hormone or diet. Biochemistry. 19: 579-585.) show that ME induction both by CHO and T3 is mediated by an increase in specific messenger RNA for ME, the interaction of T3 and the dietary factor occurs at a pretanslational level.

Carbohydrate Regulation of Hepatic Gene Expression EVIDENCE AGAINST A ROLE FOR THE UPSTREAM STIMULATORY FACTOR

Hepatic expression of the genes encoding L-type pyruvate kinase (L-PK) and S14 is induced in rats upon feeding them a high carbohydrate, low fat diet. A carbohydrate response element (ChoRE) containing two CACGTG-type E boxes has been mapped in the 5′-flanking region of both of these genes. The nature of the ChoRE suggests that a member of the basic/helix-loop-helix/leucine zipper family of proteins may be responsible for mediating the response to carbohydrate. Indeed, the upstream stimulatory factor (USF), a ubiquitous basic/helix-loop-helix/leucine zipper protein, is present in hepatic nuclear extracts and binds to the ChoREs of L-PK and S14 in vitro We have conducted experiments to determine whether USF is involved in the carbohydrate-mediated regulation of L-PK and S14. For this purpose, dominant negative forms of USF that are capable of heterodimerizing with endogenous USF but not of binding to DNA were expressed in primary hepatocytes. Expression of these forms did not block either S14 or L-PK induction by glucose. In addition, we have constructed mutant ChoREs that retain their carbohydrate responsiveness but have lost the ability to bind USF. Together, these data suggest that USF is not the carbohydrate-responsive factor that stimulates S14 and L-PK expression and that a distinct hepatic factor is likely to be responsible for the transcriptional response.

Glucose Regulation of Mouse S14 Gene Expression in Hepatocytes INVOLVEMENT OF A NOVEL TRANSCRIPTION FACTOR COMPLEX

Transcription of genes encoding enzymes required for lipogenesis is induced in hepatocytes in response to elevated glucose metabolism. We have previously mapped the carbohydrate-response elements (ChoREs) of the rat liver-type pyruvate kinase (L-PK) and S14 genes and found them to share significant sequence similarity. However, progress in unraveling this signaling pathway has been hampered due to the difficulty in identifying the key factor(s) that bind to these ChoREs. To gain further insight into the nature of the carbohydrate-responsive transcription factor, the glucose regulatory sequences from the mouse S14 gene were examined in primary hepatocytes. Three elements were found to be essential for supporting the glucose response: a thyroid hormone-response element between −1522 and −1494, an accessory factor site between −1421 and −1392, and the ChoRE between −1450 and −1425. Of these, only the accessory factor site was conserved between the rat and mouse S14 genes. Investigation of the ChoRE sequence indicated that two half E box motifs are critical for the response to glucose. Electrophoretic mobility shift assays revealed a complex formed between the mouse S14 ChoRE and liver nuclear proteins. This complex was also formed by ChoREs from the rat S14 and L-PK genes but not by mutants of these sites that are inactive in supporting the glucose response. These results suggest the presence of a novel transcription factor complex that mediates the glucose-regulated transcription of S14 and L-PK genes.

Glucose Activates ChREBP by Increasing Its Rate of Nuclear Entry and Relieving Repression of Its Transcriptional Activity

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that activates genes involved in de novo lipogenesis in mammals. The current model for glucose activation of ChREBP proposes that increased glucose metabolism triggers a cytoplasmic to nuclear translocation of ChREBP that is critical for activation. However, we find that ChREBP actively shuttles between the cytoplasm and nucleus in both low and high glucose in the glucose-sensitive β cell-derived line, 832/13. Glucose stimulates a 3-fold increase in the rate of ChREBP nuclear entry, but trapping ChREBP in the nucleus by mutagenesis or with a nuclear export inhibitor does not lead to constitutive activation. In fact, mutational studies targeting the nuclear export signal of ChREBP also identified a distinct function essential for glucose-dependent transcriptional activation. From this, we conclude that an additional event independent of nuclear translocation is required for activation. The N-terminal segment of ChREBP (amino acids 1-298) has previously been shown to repress activity under basal conditions. This segment has five highly conserved regions, Mondo conserved regions 1-5 (MCR1 to -5). Based on activating mutations in MCR2 and MCR5, we propose that these two regions act coordinately to repress ChREBP in low glucose. In addition, other mutations in MCR2 and mutations in MCR3 were found to prevent glucose activation. Hence, we conclude that both relief of repression and adoption of an activating form are required for ChREBP activation.

Glucose and Triiodothyronine Both Induce Malic Enzyme in the Rat Hepatocyte Culture EVIDENCE THAT TRIIODOTHYRONINE MULTIPLIES A PRIMARY GLUCOSE-GENERATED SIGNAL

We have stimulated in a cultured hepatocyte system the synergistic interaction between triiodothyronine (T3) and dietary carbohydrate in the induction of malic enzyme (ME). Kinetic studies revealed that isolated hepatocytes equilibrate with media T3 within 5 min; nuclei equilibrate with media T3 by 45 min; and the half-time of T3 metabolism was 10 h in 10% serum. We demonstrated nuclear T3 receptors in isolated hepatocytes and the induction of ME by T3 in physiological concentrations. However, in the complete absence of T3 glucose could still induce ME. At all concentrations of glucose (100-1,000 mg/dl), T3 (0.3 nM free T3) resulted in a relatively constant (1.4- to 1.7-fold) increase in ME response. The augmentation in ME activity was paralleled by an enhanced rate of enzyme synthesis as determined by [3H]leucine incorporation into immunoprecipitable ME. Cells cultured in serum free media also demonstrated a glucose-dependent increase in ME. Insulin greatly stimulated the glucose induction of ME, whereas dexamethasone had very little influence on ME induction. These studies demonstrate the usefulness of an adult hepatocyte tissue culture model for the study of the effects of T3 on gene expression in cells that are not derived from tumor. They clearly demonstrate that well established effects of T3 can be simulated in such a system at levels of free hormone that approximate those in extracellular body fluids. Our results indicate that an increased concentration of glucose per se can induce the formation of ME in the absence of alterations in extrahepatic hormones or factors. Moreover, our findings confirm inferences from in vivo studies that T3 acts as a multiplier of a glucose-induced signal.