Liver X Receptor α Is Involved in the Transcriptional Regulation of the 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase Gene

Insulin, dexamethasone, and forskolin were obtained from Sigma (St. Louis, MO), and the specific liver X receptor (LXR) α agonist TO-901317 was from Calbiochem (San Diego, CA). The 5′-promoter regions of the human PFK2/FBP2 gene (−1197/+33 bp; +1 designates the transcription start site) and the human LXRα gene (−2191/+77 bp) were cloned by PCR and incorporated into a pA3Luc luciferase reporter plasmid (PFK2/FBP2-Luc and LXRα-Luc, respectively). A variety of deletion mutants of the PFK2/FBP2 promoter-reporter plasmids (−698, −448, and −282/+33 bp), a heterologous promoter construct containing only the LXR response element (LXRE) 2 of the PFK2/FBP2 gene (−191/−165 bp) and the thymidine kinase (TK) minimal promoter, and expression plasmids for the human glucocorticoid receptor, insulin receptor, LXRα, LXRβ, retinoid X receptor α (RXRα, sterol response element–binding protein 1c (SREBP1c) (12), carbohydrate response element–binding protein (ChREBP), and max-like protein X (Mlx) (13) also were constructed by standard molecular cloning techniques.

The human hepatocyte–derived HuH7 cells were grown in DMEM (Sigma) supplemented with 10% fetal bovine serum. Cultured cells were maintained at 37°C in a humid atmosphere with 5% CO₂.

In each experiment, cells were plated and cultured in 24-well plates with 50% confluency. Twenty-four hours after plating, the cells were transfected with the test plasmids (reporter plasmid with/without expression plasmid[s] when necessary) using the FuGene 6 transfection reagent (Roche, Indianapolis, IN). When different amounts of an expression plasmid(s) were used in an experiment, a complementary amount of the same plasmid without complementary DNA was used so that the net amount of the plasmids transfected was equal among the groups. On the next day, the medium was changed to serum-free medium with 0.1% BSA.

To see the effects of the hormones involved in glucose metabolism on PFK2/FBP2 gene 5′-promoter activity, each factor/reagent, in 1,000× concentration, was added directly into the culture medium, and the cells were incubated for a defined time interval. Dexamethasone was used to see the effect of pure glucocorticoid. Forskolin, an activator of adenylate cyclase, was used as a surrogate of glucagon/cAMP. At the end of incubation, the culture medium was removed, and the cells were harvested for the determination of promoter activity.

To see the effects of the coexpression of various transcription factors on PFK2/FBP2 gene 5′-promoter activity, both the PFK2/FBP2-Luc reporter plasmid and the test expression plasmid(s) were cotransfected as above. The cells were harvested 24–48 h after the end of transfection, followed by the determination of promoter activity.

The luciferase assay was performed as previously described (14), and light output was measured for 20 s at room temperature using a luminometer (Berthold Lumat LB9507; Berthold, Bad Wildbad, Germany).

Endogenous expression of PFK1 (PFKL; liver type) and each isoform of PFK2/FBP2 mRNAs in HuH7 cells was examined by RT-PCR analysis. The cells were plated in 3.5-cm-diameter dishes, and total RNA was extracted using the RNeasy RNA extraction kit (Qiagen, Hilden, Germany). One microgram each was then applied for the reverse transcription reaction using Superscript III reverse transcriptase (Invitrogen, San Diego, CA), followed by PCR reaction (25–30 cycles; 94°C, 30 s; 60°C, 30 s; and 72°C 2 min) using Blend Taq DNA polymerase (Toyobo, Tokyo, Japan). The following were the primer sets used: for PFK1 sense, 5′-GGAGAACTTCATGTGGTGTGAGAG-3′, antisense, 5′-GAGAAGTTAGACTTCTCCTTGG-3′; PFKFB1 sense, 5′-CCAACACTACCAGAGAACGAC-3′, antisense, 5′-CTCTGATGTTGAGTTCACTCTC-3′; PFKFB2 sense, 5′-CTAACACGCTACCTCAACTGG-3′, antisense, 5′-GACTATCTTGCTCTGGATGTAG-3′; PFKFB3 sense, 5′-GTGAAGCAGTACAGCTCCTAC-3′, antisense, 5′-GGATGTTCATCAGGTAGTACAC-3′; and PFKFB4 sense, 5′-CTGACTCGATACCTGAACTGG-3′, antisense, 5′-CCTATCCAGGTCCTCATCTAG-3′. The sequences for both sense and antisense primers in each mRNA were obtained from a different exon to distinguish the mRNA-derived PCR product from the genomic DNA–derived product.

Endogenous expression of PFK2/FBP2 mRNA in the liver in vivo was examined by quantitative real-time PCR analysis. C57/BL6 mice were divided into two groups (fasting [F] and fasting/refeeding [FR]; n = 6 in each group). Following the 24-h fast, mice in the FR group were fed for 3.5 h with standard laboratory chow and then killed. Mice from the F group were killed without refeeding. Trunk blood was obtained for the measurements of blood glucose and plasma insulin, and liver tissue was isolated, followed by the extraction of total RNA for the determination of endogenous PFK2/FBP2 mRNA. Total RNA was extracted using the RNEasy RNA extraction kit (Qiagen).

For real-time RT-PCR, reagents, software, and equipment were from Applied Biosystems (Foster City, CA). TaqMan reactions were performed using the TaqMan Universal PCR Master Mix and the StepOnePlus Real-Time PCR System, and data analysis was performed using sequence detection system software. TaqMan probes for the mouse PFK2/FBP2 mRNA (Applied Biosystems), and those for the mouse glyceraldehyde-3-phosphate dehydrogenase mRNA (Applied biosystems) as an internal control, were used. The quantity of the PFK2/FBP2 mRNA in an unknown sample was determined from the C_t value using the standard curve. A control without a template was included in each experiment. Nontemplate controls, standard dilutions, and samples were assayed in duplicate.

Electromobility shift assay (EMSA) analysis was carried out to see the binding of LXRα/RXRα on the putative LXRE identified. Except for the supershift experiment (see below), we used a commercially available non–RI EMSA kit (LightShift Chemiluminescent EMSA kit; Pierce, Rockford, IL), according to the manufacturer’s instructions. In brief, the nuclear extract of the HuH7 cells transfected with LXRα/RXRα expression plasmids was prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL) and then incubated for 20 min with the double-stranded 3′-end-biotinylated oligonucleotide probes (50 fmol) encompassing the LXR binding sites. The probes used are shown in Fig. 6A. The mixture was subjected to 6% nondenaturing polyacrylamide gel (160 V for 4 h). Finally, the biotinylated DNA was transferred to a nylon membrane and cross-linked, and then the biotin-labeled DNA was detected with a digital-imaging apparatus (LightCapture; Atto, Tokyo, Japan).

To confirm the specificity of the band identified, a supershift experiment was performed, as described previously (15). Mouse LXRα and human RXRα recombinant proteins were synthesized from constructs in the pSG5 expression plasmid (16), using the TNT T7 Quick Coupled Transcription/Translation System (Promega). Binding reactions contained 20 mmol/L HEPES (pH 7.6), 50 mmol/L KCl, 12% glycerol, 1 mmol/L dithiothreitol, 1 μg poly(dI-dC)(dI-dC), and 4 μL of each of the synthesized nuclear receptors or unprogrammed reticulocyte lysates. Double-stranded oligonucleotides (PFK2 LXRE2 region: 5′-AGCTCTCCTGACCTCTGCCCAACCTCTC-3′) were labeled with [α-³²P] deoxy-cytidine triphosphate by a fill-in reaction using a Klenow fragment of DNA polymerase. Binding reactions were performed at room temperature for 30 min. Then, 1 μL of anti-human LXRα ligand–binding domain mouse monoclonal antibody (Perseus Proteomics, Tokyo, Japan) was added as indicated in the figure, and the mixture was incubated for an additional 30 min at room temperature. The protein-DNA complexes were resolved on a 5% polyacrylamide gel in 0.5× Tris-base EDTA (TBE, 45 mmol/L).

Chromatin immunoprecipitation (ChIP) assay was carried out using the ChIP-IT Express chromatin immunoprecipitation kit (Active Motif, Carlsbad, CA), according to the manufacturer’s instructions, to further confirm the binding of LXRα/RXRα on the putative LXRE in vivo. In brief, HuH7 cells transfected with LXRα/RXRα expression plasmids were homogenized by a Dounce homogenizer and centrifuged, and the shared chromatin in the supernatant was incubated with protein G–coated magnetic beads, protease inhibitor cocktail, and anti-LXRα antibody (Perseus Proteomics) for 4 h at 4°C. The complex then was washed twice, treated with the reverse cross-liking buffer, and then incubated with protease K for 1 h at 37°C. Finally, the reaction solution was applied for PCR. The primer sets used are shown in Fig. 7A. Primer sets for negative control also were used following the manufacturer’s instructions.

Blood glucose was measured by the glucose oxidase method. Plasma insulin was measured by the mouse insulin enzyme-linked immunosorbent assay kit (Morinaga Institute of Biological Science, Tokyo, Japan).

Samples in each group of the experiments were in triplicate or quadruplicate. All data were expressed as means ± SEM. When the statistical analyses were performed, data were compared by one-way ANOVA with the Fisher's protected least significant difference test, and P values <0.05 were considered significant.

There are four PFK2/FBP2 isoenzymes in humans (PFKFB1, PFKFB2, PFKFB3, and PFKFB4) (4) and at least three PFK1 isoenzymes (PFKL, PFKM, and PFKP/PFKC) (17–19). Among them, PFKL and PFKFB1 are known to be expressed in the liver in vivo. Indeed, we found endogenous expression of PFKL and PFKFB1 mRNA in HuH7 cells analyzed by RT-PCR (Fig. 1B), indicating that this cell line is appropriate for the transcriptional regulation of the PFKFB1 isoform of the PFK2/FBP2 gene in vitro.

We first examined the effects of hormones related to glucose metabolism, such as insulin, glucagon/cAMP, and glucocorticoid, on the transcriptional activity of the PFK2/FBP2 gene. We found that insulin significantly stimulated the transcription of the PFK2/FBP2 gene in time- and dose-dependent manners (Fig. 2A). In contrast, forskolin/cAMP, a surrogate of glucagon, and dexamethasone showed significant inhibitory effects (Fig. 2B). Considering the fact that insulin is an anabolic/glycolytic hormone, whereas glucagon/cAMP is a gluconeogenic hormone, our results suggest that the regulation of the PFK2/FBP2 gene is similar to that of other glycolytic rather than gluconeogenic genes, at the transcriptional level.

Because the 5′-promoter activity of the PFK2/FBP2 gene is found to be regulated like that of other anabolic/glycolytic enzyme genes, transcription factors under the control of insulin/glucose, such as SREBP1c, LXRα/β, and ChREBP/Mlx, seem to be involved in its transcription. Therefore, we next tried to determine which transcriptional factor(s) is regulating the expression of the PFK2/FBP2 gene by coexpression experiments. The results showed that, among the factors examined, LXRα/RXRα potently (>900% increase) stimulated the 5′-promoter activity of the PFK2/FBP2 gene (Fig. 3A). Other factors also had significant positive effects but much less (~1.5- to 2-fold increase) so than that of LXRα/RXRα. The effect of LXRα/RXRα coexpression was time dependent (Fig. 3B, left panel) and also dose dependent, which was further augmented by a specific LXRα ligand, TO-901317 (Fig. 3B, right panel). These results suggest that LXRα/RXRα is one of the key regulatory factors involved in PFK 2/FBP2 gene transcription. Of interest, however, when LXRα/RXRα and other factors were simultaneously expressed, LXRβ/RXRα augmented, whereas SERBP1c or ChREBP/Mlx rather inhibited, the positive effect of LXRα/RXRα (Fig. 3C). The precise molecular mechanisms and the physiological significance of the interactions among the factors observed here remain unresolved in this study.

To confirm the involvement of LXRα/RXRα in the regulation of the PFK2/FBP2 gene, we next examined the effects of a synthetic LXRα ligand, TO-901317, and a putative endogenous LXRα activator, glucose (or its metabolite[s]) (20), on PFK2/FBP2 gene transcription. In naïve HuH7, treatment of the cells with high glucose (330 mg/dL) or TO-901317 had no significant effect on PFK2/FBP2 gene 5′-promoter activity. On the other hand, when LXRα/RXRα was coexpressed, mild but significant transcriptional induction was observed by either treatment (Fig. 4A). We then examined the role of insulin in LXRα/RXRα-mediated PFK2/FBP2 gene transcription. Under the condition where LXRα/RXRα was coexpressed, insulin as well as TO-901317 caused significant positive effects, and a further increase was observed when cells were treated with both insulin and TO-901317 (Fig. 4B). These results suggest that a factor(s) other than LXRα/RXRα also is involved in the positive effect of insulin. In addition, the transcriptional activity of the LXRα gene was significantly stimulated by insulin (Fig. 4C), as reported previously (21).

We next tried to identify the LXRE(s) in the PFK2/FBP2 gene 5′-promoter. We first performed deletion analysis. Our initial experiment showed that deletion up to −282 bp did not eliminate LXRα/RXRα responsiveness (Fig. 5A), suggesting that the LXRE seems to be located in the proximal portion of the PFK2/FBP2 gene promoter. We then analyzed this region more precisely using a site-directed mutagenesis technique. Within −282 bp of the promoter, we found two putative LXRE consensus sequences, LXRE1 and LXRE2 (Fig. 5B, upper panel). We introduced mutation in either or both of the elements (Fig. 5B, middle panel) and then examined the effect of LXRα/RXRα coexpression. We found that the mutation of LXRE1 did not affect the responsiveness, whereas the mutation of LXRE2 markedly diminished it, although a significant increase still remained (Fig. 5B, lower panel). In addition, we recognized a significant positive effect of LXRα/RXRα coexpression in a heterologous construct containing only LXRE2 (Fig. 5C). No effect was found in empty plasmid (TK-Luc alone) (data not shown). These results suggest that LXRE2 plays a significant role in the LXRα/RXRα-mediated transcriptional activation of the PFK2/FBP2 gene.

We then tried to confirm the LXR binding of the LXRE2 element by EMSA. We found a clear single band, which was completely competed by an excess amount of unlabeled probe containing consensus LXRE element but not by that with the mutated element (Fig. 6A and B, left panel). We also observed a supershift using specific anti-LXR antibody (Fig. 6B, right panel). These data strongly suggest that LXRα/RXRα binds to the LXRE2 located in the proximal promoter of the PFK2/FBP2 gene.

Because binding of LXR was recognized by EMSA, we also tried to confirm the binding of LXRα/RXRα on the LXRE2 by ChIP assay using anti-LXRα antibody. We found a PCR product using a primer set spanning LXRE2 but none with negative control primers (Fig. 7). The results confirm the in vivo binding of LXRα on the LXRE2 of the PFK2/FBP2 gene.

Finally, we performed an in vivo experiment to determine whether nutritional status indeed influences the expression level of PFK2/FBP2 mRNA in the mouse liver by quantitative RT-PCR. We found that fasting/refeeding caused a significant increase in plasma insulin (Fig. 8A), whereas no difference was found in blood glucose levels (Fig. 8B). In this condition, RT-PCR analysis showed that PFK2/FBP2 mRNA was markedly increased in fasting/refeeding compared with fasting alone (Fig. 8C). These in vivo results are in accordance with our in vitro data, suggesting that PFK2/FBP2 gene transcription is regulated in a similar way as other anabolic/glycolytic enzyme genes rather than gluconeogenic enzyme genes.

PFK2/FBP2, a bifunctional enzyme involved in both glycolysis and gluconeogenesis, plays an important role in the regulation of glucose metabolism in the liver. In this study, we comprehensively examined the transcriptional regulation of the PFK2/FBP2 gene, using the HuH7 human hepatocyte–derived cell line in vitro and the mice liver tissue in vivo. We found that the promoter activity of the PFK2/FBP2 gene was stimulated by insulin and inhibited by forskolin/cAMP at the transcriptional level, suggesting that the PFK2/FBP2 gene is regulated in a similar way as other glycolytic enzyme genes rather than gluconeogenic enzyme genes (22–24). Furthermore, among the insulin/glucose-related transcription factors, we found that LXRα/RXRα is one of the key regulators of PFK2/FBP2 gene transcription. In addition, our in vivo experiment also showed that endogenous PFK2/FBP2 mRNA in the liver is induced by fasting/refeeding compared with fasting alone, in agreement with the previous report obtained in the rat liver estimated by Northern blot analysis (8).

Glycolysis/gluconeogenesis is known to be controlled not only by the regulation of enzyme activities but also by their expression levels. Regarding the PFK2/FBP2 gene, previous studies have shown that insulin, a representative anabolic/glycolytic hormone, increased the mRNA of the liver-type PFK2/FBP2 gene in the adult rat hepatocyte in vitro (9), in the rat-derived FAO-1 hepatoma cells in vitro (10), and also in the rat liver in vivo (8). Because insulin and/or glucose and its metabolite(s) is known to play an important role in the regulation of glycolytic enzyme genes, we surveyed the role of the insulin/glucose-related representative transcription factors, such as SREBP1c, LXRα/β, and ChREBP/Mlx, and identified LXRα as one of the key regulatory factors in PFK2/FBP2 gene expression.

LXRα (NR1H3) and LXRβ (NR1H2) belong to the nuclear receptor superfamily and are known to be involved in glucose and fatty acid synthesis as well as intracellular cholesterol metabolism (25–27). Insulin stimulates LXRα expression in hepatocytes (21), and LXRα is known to induce a variety of metabolism-related target genes directly or indirectly (27–30). To confirm that the involvement of LXRα on PFK2/FBP2 gene transcription is functional, we examined the effects of a specific LXR ligand, TO-901317, and found that in the presence of LXRα/RXRα, the ligand significantly increased the transcriptional activity of PFK2/FBP2 gene by 50–100%. A similar but weaker effect was observed when cells were treated with high glucose, a putative activator of LXRα (20). To further confirm that the PFK2/FBP2 gene is a direct target of LXRα, deletion/mutation analyses and an experiment using heterologous promoter (TK) construct were carried out, with the result that LXRE2 was identified as a cis-acting element for LXRα/RXRα. EMSAs and ChIP assays also provided convincing data supporting the direct binding of LXRα/RXRα on the element. Our data do not directly support the idea that LXRα is the predominant factor mediating the insulin stimulation of PFK2/FBP2 gene expression. However, considering the facts that insulin is known to be a potent inducer of LXRα (21), and that the current data show a marked stimulatory effect of LXRα/RXRα on PFK2/FBP2 gene promoter activity, we assume that LXRα is at least partly mediating the positive effect of insulin on PFK2/FBP2 gene transcription.

The present results suggest the possible role of other transcription factors as well. Indeed, coexpression of insulin-related transcription factors, such as SREBP1c and LXRβ, and the glucose metabolite(s)-stimulated factor ChREBP/Mlx also caused a significant increase in the transcriptional activity of the PFK2/FBP2 gene, although the effects were much weaker than that of LXRα/RXRα. Furthermore, simultaneous treatment of insulin and TO-901317 under the coexpression of LXRα/RXRα had a minimal but significant additive effect, suggesting that a factor(s) other than LXRα/RXRα is mediating the additional effect of insulin. The presence of different pathway(s) and/or element(s) also is suggested by the fact that a weaker but significant stimulatory effect of LXRα/RXRα still exists in the mutant promoter construct in which the confirmed LXR binding site (LXRE2) is deleted or mutated. Because no possible LXR binding site was found in the nucleotide sequence of the remaining promoter region, we assume that some unknown factor(s) induced by LXRα, possibly SREBP1c, is responsible for the remaining positive effect observed in the shortest construct (−282/+33 bp), because the expression of SREBP1c is partly dependent on LXRα/RXRα (29,30).

LXRα is expressed dominantly in the liver and regulates genes involved not only in sterol metabolism but also in fatty acid synthesis, such as acetyl CoA carboxylase and fatty acid synthase via LXREs located in the promoter of each gene (24,28,31). It generally is recognized that the glycolytic pathway is a part of the lipogenic pathway, and, indeed, under the control of PFK2/FBP2, PFK1 produces dihydroxyacetone phosphate, which is a precursor of glycrol-3-phospate, a basic component of triacylglycerol. In this sense, it is not surprising that LXRα regulates gene expression related to not only fatty acid synthesis but also to glycolysis. To our knowledge, however, this is the first work showing that a gene (PFK2/FBP2) belonging to the Embden-Meyerhof pathway also is a direct target of LXRα.

In contrast to insulin, the glucagon/cAMP pathway is known to have positive and negative effects on gluconeogenic and glycolytic enzyme gene expression, respectively (32), and, indeed, in this study, we found an inhibitory effect of forskolin on PFK2/FBP2 gene transcription. On the other hand, the effect of glucocorticoid is complex because the hormone facilitates gluconeogenesis during starvation, whereas it exhibits lipogenic effects in the anabolic condition. Regarding the effect of glucocorticoid on the PFK2/FBP2 gene, previous studies show conflicting results: dexamethasone decreased PFK2/FBP2 mRNA in adult rat hepatocytes (9), whereas opposite effects were observed in rat hepatoma-derived FAO and FTO-2B cells (10,11). The human PFK2/FBP2 gene has no clear glucocorticoid response element(s) in the promoter region examined in this study, and we found inhibitory rather than stimulatory effects. Additional studies using human primary hepatocyte cell culture are necessary to clarify the issue.

Finally, from the clinical standpoint, activation of LXRα is assumed to have a beneficial effect on glucose and cholesterol metabolism but at the same time can potentially facilitate fatty acid and triglyceride synthesis by inducing lipogenic genes, such as acetyl CoA carboxylase, fatty acid synthase, and PFK2/FBP2. Indeed, previous animal experiments showed that administration of the LXR ligand, TO-901317, improved diabetes but caused severe fatty liver and obesity (33,34). In contrast, selective inhibition of PFK2 causes decreased PFK1 activity, which decreases adipogenesis, as shown in PFK1-M knockout mice (19), but causes glycogenosis and diabetes, as observed in PFK1-M–deficient humans (18). However, moderate inhibition of both PFK2 and FBP2 activities using LXR inhibitor may possibly be beneficial, because it will suppress both adipogenesis and gluconeogenesis without complete suppression of the mainstream glycolytic pathway. In this context, pharmacological inhibitors of LXRα or PFK2/FBP2 might be a promising therapeutic tool for the prevention of obesity and diabetes in the future.

This work was supported in part by the Kochi University President’s Discretionary Grant, and Grants-in-Aid for Scientific Research (Research on Hypothalamo-Hypophyseal Disorders) from the Ministry of Health, Labor, and Welfare of Japan.

No potential conflicts of interest relevant to this article were reported.

L.-F.Z. performed research, analyzed the data, wrote the manuscript, and contributed to the discussion. Y.I. designed and performed research, analyzed the data, contributed to the discussion, and reviewed and edited the manuscript. M.N., T.T., M.T., M.O., S.N., K.H., and K.M. helped perform research and contributed to the discussion. M.K. performed research. S.F. and Y.T. reviewed the manuscript. Y.I. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors thank Ms. Reiko Matsumoto (Department of Endocrinology, Metabolism, and Nephrology, Kochi Medical School, Kochi University) for her excellent technical support.