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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Diabetologia. 2008 Jun 19;51(8):1505–1514. doi: 10.1007/s00125-008-1050-8

Sequence Variation Between the Mouse and Human Glucose-6-Phosphatase Catalytic Subunit Gene Promoters Results in Differential Activation by Peroxisome Proliferator Activator Receptor Gamma Coactivator-1α

Marcia M Schilling 1, James K Oeser 1, Joshua K Chandy 1, Brian P Flemming 1, Shelley R Allen 1, Richard M O’Brien 1,§
PMCID: PMC2590337  NIHMSID: NIHMS78561  PMID: 18563384

Abstract

Aims/Hypothesis

The glucose-6-phosphatase catalytic subunit (G6PC) plays a key role in hepatic glucose production by catalyzing the final step in gluconeogenesis and glycogenolysis. Peroxisome proliferator activator receptor gamma coactivator-1α (PGC-1α) markedly stimulates mouse G6PC fusion gene expression through hepatocyte nuclear factor-4α (HNF-4α), which binds an element located between −76 and −64 in the promoter. The aim of this study was to compare the regulation of mouse and human G6PC gene expression by PGC-1α.

Methods & Results

Surprisingly we show here that, in H4IIE cells, PGC-1α alone fails to stimulate human G6PC fusion gene expression even though the sequence of the −76/−64 HNF-4α binding site is perfectly conserved in the human promoter. Mutational analyses demonstrated that the observed difference could be explained, in part, by a 3 bp sequence variation between the mouse and human promoters. Introducing the human sequence into the mouse G6PC promoter reduced PGC-1α stimulated fusion gene expression whereas the inverse experiment, in which the mouse sequence was introduced into the human G6PC promoter, resulted in the generation of a G6PC fusion gene that was now induced by PGC-1α. This critical 3 bp region is located immediately adjacent to a consensus nuclear hormone receptor half site that is perfectly conserved between the mouse and human G6PC promoters. Gel retardation experiments revealed that this 3 bp region influences the affinity of HNF-4α binding to the half site.

Conclusions

These observations suggest that PGC-1α may be more important in the control of mouse than human G6PC gene expression.

Keywords: glucose-6-phosphatase, PGC-1alpha, diabetes, HNF-4alpha, transcription, promoter

Introduction

Glucose-6-phosphatase catalyzes the terminal step of gluconeogenesis and glycogenolysis, the hydrolysis of inorganic phosphate from glucose-6-phosphate [1]. Glucose-6-phosphatase is a multi-component enzyme system consisting of transporters for glucose-6-phosphate, glucose, and inorganic phosphate, and a catalytic subunit whose activity site is orientated towards the lumen of the endoplasmic reticulum [1]. In mammals, glucose-6-phosphatase activity is highest in the liver and kidney, but activity is also detected at lower levels in the brain, small intestine, and pancreatic islets [1]. Hepatic glucose-6-phosphatase activity is critical for maintaining plasma glucose levels between meals. Mutations in the gene encoding the glucose-6-phosphatase catalytic subunit (G6PC) result in glycogen storage disease type-1a (von Gierke’s disease), which is characterized by life-threatening hypoglycemia, growth retardation, renal dysfunction, and hepatomegaly [2]. In contrast, increased G6PC mRNA and activity are thought to contribute to the elevated hepatic glucose production (HGP) characteristic of both type 1 and type 2 diabetes [3, 4].

Peroxisome proliferator-activated receptor gamma (PPAR)-coactivator 1α (PGC-1α) is an important regulator of metabolism, contributing to transcriptional activation of genes involved in adaptive thermogenesis, mitochondrial biogenesis, respiration, and gluconeogenesis [5, 6]. In the liver PGC-1α is upregulated during fasting and in diabetes and it stimulates both G6PC and phosphoenolpyruvate carboxykinase (PEPCK) gene expression [7]. Two groups have generated mice with a global deletion of the PGC-1α gene. Both reported that the mice were viable but had multi-system abnormalities that included diminished mitochondrial respiratory capacity, vacuolar lesions in the central nervous system and cold intolerance [8, 9]. However, these groups reported variable effects of PGC-1α deletion on hepatic gluconeogenic gene expression and fasting blood glucose levels. Lin et al. [8] observed hypoglycemia in the fasted state and reduced hepatic glucose production, however, instead of the expected impairment, G6PC and PEPCK gene expression were surprisingly elevated in the fed state to a level equivalent to that in the fasted state. This elevation in G6PC and PEPCK gene expression was potentially caused by elevated hepatic C/EBPβ gene expression [8]. In contrast, in their mice, Leone et al. [9] observed euglycemia in the fasted state with no elevation of G6PC, PEPCK or C/EBPβ gene expression in the fed state and the same induction following fasting as seen in wild type mice. The reasons for these differences are unclear. Interestingly, although Leone et al. [9] observed no change in G6PC and PEPCK gene expression and euglycemia in the fasted state, hepatic glucose production and gluconeogenic flux are altered in these mice as a consequence of altered tricarboxylic acid cycle flux [10]. In mice with a liver-specific deletion of the PGC-1α gene G6PC and PEPCK gene expression were normal in the fed state but their induction by fasting was impaired [11]. The observation that the effect of fasting was not abolished is likely not explained by the presence of PGC-1β because, though induced by fasting [12], it only weakly activates G6PC and PEPCK gene expression [13]. However, the observation is consistent with the fact that on-going protein synthesis is not required for the hormonal regulation of these genes [14]. As such, PGC-1α is not essential for G6PC and PEPCK gene expression but plays an important role as a transcriptional amplifier [15].

At the molecular level, PGC-1α interacts with multiple transcription factors including PPARα, PPARγ, nuclear respiratory factor-1, the glucocorticoid receptor, estrogen-related receptors, liver X receptor, pregnane X receptor, myocyte enhancer factor-2, and hepatic nuclear factor-4α (HNF-4α) [5, 6]. HNF-4α is a member of the steroid hormone receptor superfamily and is expressed in the liver, intestine, kidney, pancreatic islets, stomach, and skin [16, 17]. HNF-4α knockout mice have an embryonic lethal phenotype [18] whereas liver-specific HNF-4α deficient mice exhibit defective lipid homeostasis and weight loss [19]. Mutation of HNF-4α results in maturity onset diabetes of the young type 1 which is also characterized by altered lipid homeostasis as well as defective glucose-stimulated insulin secretion [17]. HNF-4α is essential for the induction of G6PC and PEPCK gene expression by PGC-1α [20].

We have previously shown that in rat H4IIE hepatoma cells the activation of mouse G6PC gene expression by PGC-1α was mediated through an HNF-4α binding site located between −76 and −64 [21] whereas Rhee et al. [20] showed in immortalized mouse hepatocytes that the activation of human G6PC gene expression by PGC-1α was mainly mediated through two HNF-4α binding sites located between −296 and −284 and −256 and −244. Because these latter sites are poorly conserved in the mouse G6PC promoter it appeared to explain the differences between the reported results. However, in the current study we demonstrate that the PGC-1α responsiveness of the mouse and human G6PC promoters are fundamentally different. Thus, when the mouse and human G6PC promoters were compared in the same H4IIE cell line only mouse G6PC fusion gene expression was induced by PGC-1α. The molecular basis for this difference was mapped to a sequence variation between the mouse and human promoters that affects the affinity of HNF-4α binding. These results suggest that PGC-1α may be more important for the regulation of mouse than human G6PC gene expression, a difference that could contribute to the observed higher rate of fasting HGP in mice than humans.

Materials and Methods

Plasmid construction

The generation of a mouse G6PC-luciferase fusion gene, containing promoter sequence located between −484 and +66, relative to the transcription start site, in the pGL3 MOD vector has been previously described [22]. PCR reactions with this plasmid as the template were used to generate mouse G6PC-luciferase fusion genes containing wild type or mutated promoter sequence between −85 and +1. A human G6PC-luciferase fusion gene, containing promoter sequence located between −826 and +63, was generated by PCR using human genomic DNA as the template. PCR reactions with this plasmid as the template were used to generate human G6PC-luciferase fusion genes containing wild type or mutated promoter sequence between −83 and +7. All promoter fragments generated by PCR were completely sequenced to verify the absence of polymerase errors.

Expression vectors encoding the rat glucocorticoid receptor (pRSV-GR; Ref. [23]), rat HNF-4α (pSG5-HNF4α; Ref. [24]) and human FOXO1 (pcDNA3-FKHR; Ref. [25]) were generously provided by Drs. Keith Yamamoto, Daryl Granner and David Powell, respectively. A pBJ5 expression vector encoding a putative mouse PGC-1α splice variant representing the entire 797 amino acid PGC-1α open reading frame [26] minus amino acids 476 to 487 has been previously described [21]. A full-length pBJ5-PGC-1α expression vector was constructed by inserting the missing PGC-1α sequence, which was isolated from I.M.A.G.E. clone # 4193174.

Cell culture and transient transfections

Rat H4IIE hepatoma cells were cultured and transiently transfected in suspension using the calcium phosphate-DNA co-precipitation method as previously described [27].

Luciferase assays

Luciferase assays were performed using the Promega Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions. In these experiments we saw no effect of PGC-1α or HNF-4α overexpression on Renilla luciferase activity. Therefore, for comparisons of PGC-1α- and HNF-4α-regulated gene expression, firefly luciferase activity directed by the various fusion gene constructs was expressed relative to Renilla luciferase activity in the same sample. Each construct was analyzed in duplicate in multiple transfections, as specified in the Figure Legends, using multiple independent plasmid preparations.

Nuclear extract preparation and gel retardation assays

H4IIE nuclear extract was prepared as previously described [28]. Oligonucleotides representing sense and antisense wild type and mutated G6PC promoter sequences as described in the text were synthesized with BamH I compatible ends by Operon Technologies, Inc. (Alameda, CA) and subsequently annealed and labeled with [α32P]dATP using the Klenow fragment of Escherichia coli DNA Polymerase I to a specific activity of approximately 9 × 104 Bq/pmol [29]. Gel retardation assays were then performed exactly as previously described [21].

Statistical analyses

The transfection data were analyzed for differences from the control values, as specified in the Figure Legends. Statistical comparisons were calculated using an unpaired Student’s t-test. The level of significance was as indicated (two-sided test).

Results and Discussion

PGC-1α can selectively stimulate mouse G6PC-luciferase fusion gene expression

The rate of fasting hepatic glucose production (HGP) is 10 fold higher in mice than humans [30, 31]. We have been interested as to whether differences in the regulation of G6PC gene transcription between mice and humans could partly explain this observation. A carbohydrate response element identified in the mouse and rat G6PC promoters is not conserved in the human promoter [32]. However, the mechanism by which insulin suppresses both mouse and human G6PC fusion gene expression in human HepG2 hepatoma cells appears identical [33]. Likewise, the synthetic glucocorticoid dexamethasone equally stimulates both mouse and human G6PC fusion gene expression in rat H4IIE hepatoma cells (Fig. 1). Since PGC-1α markedly induces G6PC gene expression and HGP [7], we next compared the ability of PGC-1α to stimulate mouse and human G6PC fusion gene expression in rat H4IIE hepatoma cells. As previously reported [21], co-transfection of H4IIE cells with an expression vector encoding PGC-1α robustly induces the expression of a mouse G6PC-luciferase fusion gene containing promoter sequence between −484 and +66 (Fig. 2A). In contrast, expression of a human G6PC-luciferase fusion gene containing promoter sequence between −826 and +63 was unaffected (Fig. 2A). Although mouse PGC-1α was used in this experiment, mouse and human PGC-1α share 95% amino acid sequence homology (758/797) suggesting the existence of limited differences in structural characteristics and binding properties between species. Indeed, the observation that the human promoter was unresponsive to PGC-1α at all concentrations tested (Fig. 2B), suggests that there is a fundamental difference between the mouse and human G6PC promoters rather than just a difference in sensitivity towards the action of PGC-1α.

Figure 1. Dexamethasone stimulates both mouse and human G6PC fusion gene expression.

Figure 1

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H4IIE cells were transiently co-transfected, as described in Research Design and Methods, with the indicated mouse or human G6PC-luciferase fusion genes (15 μg), and expression vectors encoding Renilla luciferase (0.15 μg) and the glucocorticoid receptor (5 μg). Following transfection, cells were incubated for 18–20 hr in serum-free medium in the presence or absence of 500 nM dexamethasone (Dex). Cells were then harvested and luciferase activity assayed as described in Research Design and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase in the cell lysate, in dexamethasone versus control cells and are expressed as fold induction. Results represent the mean ± S.E.M. of 3–8 experiments using three independent preparations of both fusion gene constructs in which each experimental condition was assayed in duplicate.

Figure 2. PGC-1α selectively stimulates mouse but not human G6PC fusion gene expression.

Figure 2

Figure 2

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H4IIE cells were transiently co-transfected, as described in Research Design and Methods, with the indicated mouse or human G6PC-luciferase fusion genes (12 μg), an expression vector encoding Renilla luciferase (0.15 μg), and either a pBJ5 expression vector encoding PGC-1α or the empty pBJ5 vector control (Panel A: 3 μg; Panel B: as indicated). Following transfection, cells were incubated for 18–20 hr in serum-free medium. Cells were then harvested and luciferase activity assayed as described in Research Design and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase in the cell lysate, in PGC-1α transfected versus empty vector transfected cells and are expressed as fold induction. Results represent the mean ± S.E.M. of 12–35 experiments (Panel A) or 3 experiments (Panel B) using three independent preparations of both fusion gene constructs in which each experimental condition was assayed in duplicate. Panel A: *, p < 0.05 versus −484/+66 mG6P WT; Panel B: *, p < 0.05 versus matching pBJ5. White columns: −484/+66 mG6P + pBJ5; black columns: −484/+66 mG6P + pBJ5-PGC-1α; light gray columns: −826/+63 hG6P + pBJ5; dark gray columns: −826/+63 hG6P + pBJ5-PGC-1α.

Identification of sequence variations that contribute to the selective effect of PGC-1α on mouse G6PC-luciferase fusion gene expression

The results suggested that either the human promoter contained a species-specific element that repressed the PGC-1α response or that the mouse promoter contained a species-specific PGC-1α responsive element that is absent in the human promoter. Before addressing these possibilities, we first sought to define shorter regions of the mouse and human G6PC promoters that demonstrated a selective effect of PGC-1α.

The effect of PGC-1α on mouse G6PC-luciferase fusion gene expression was previously mapped to the promoter region between −85 and +66 and was shown to require an HNF-4α binding site located between −76 and −64 [21]. The 5′ untranslated regions (5′ UTRs) of the mouse and human genes contain numerous sequence variations whereas the mouse promoter sequence between −85 and +1 and the equivalent human sequence between −83 and +1 are highly conserved (Fig. 3). Deletion of the mouse 5′ UTR between +2 and +66 reduced the magnitude of PGC-1α-induced mouse G6PC-luciferase fusion gene expression indicating that element(s) in the 5′ UTR contribute to the PGC-1α response (data not shown). However, the −85 to +1 promoter region still mediated a PGC-1α response (Fig. 4A). In contrast, a similar region of the human G6PC promoter, located between −83 and +7 was unresponsive to PGC-1α (Fig. 4B). The 5′ UTR sequence between + 1 and +7 is identical between mouse and human so this cannot explain the observed difference. This result implies that at least part of the differential regulation of mouse and human G6PC fusion genes by PGC-1α must be explained by sequence differences within the proximal promoter regions shown in Fig. 3.

Figure 3. Sequence alignment of the mouse and human G6PC gene promoters.

Figure 3

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The human and mouse proximal G6PC promoter sequences are labeled relative to the experimentally determined transcription start sites, designated as +1 [46, 47]. The conserved HNF-4α binding site [21] is boxed. The G6PC promoter region shown contains 7 base pairs that differ between the human and mouse species. Site directed mutations were generated in the mouse and human G6PC promoters, designated MUT 1, 2, & 4, in which the mouse sequence was substituted for that of the human and vice versa. In addition, a site directed mutation of a conserved consensus nuclear receptor half-site, designated MUT 3, was generated in the mouse G6PC promoter, in which the sequence of this half-site was changed to GTCAAG.

Figure 4. Identification of sequences that contribute to the selective stimulation of mouse but not human G6PC fusion gene expression by PGC-1α.

Figure 4

Figure 4

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H4IIE cells were transiently co-transfected, as described in Research Design and Methods, with the indicated mouse (Panel A) or human (Panel B) G6PC-luciferase fusion genes (12 μg), an expression vector encoding Renilla luciferase (0.15 μg), and either a pBJ5 expression vector encoding PGC-1α or the empty pBJ5 vector control (3 μg). The fusion genes contained either the indicated wild type mouse and human promoter regions or site directed mutations of these promoters as described in the text. These mutations represented substitutions of mouse sequence for that of the human (hMUT) or vice versa (mMUT) (Fig. 3). Following transfection, cells were incubated for 18–20 hr in serum-free medium. Cells were then harvested and luciferase activity assayed as described in Research Design and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase in the cell lysate, in PGC-1α transfected versus empty vector transfected cells and are expressed as fold induction. Results represent the mean ± S.E.M. of 6–9 experiments (Panel A) or 3 experiments (Panel B) using three independent preparations of all fusion gene constructs in which each experimental condition was assayed in duplicate. Panel A: *, p < 0.05 versus −85/+1 mG6P WT; Panel B: *, p < 0.05 versus −83/+7 hG6P WT.

Inspection of the mouse promoter sequence between −85 and +1 with the equivalent human sequence between −83 and +1 reveals only 7 nucleotide differences, specifically one deletion and six substitutions (Fig. 3). The previously identified mouse HNF-4α binding site [21] is perfectly conserved in the human promoter (Fig. 3). To identify the sequence required for the selective effect of PGC-1α on mouse G6PC fusion gene expression site directed mutations were generated in the context of the mouse −85/+1 G6PC promoter, designated hMUT 1, 2 & 4, in which the mouse sequence was substituted for that of the human (Fig. 3). G6PC-luciferase fusion genes containing these mutations were then transiently transfected into H4IIE cells and the effect of PGC-1α co-transfection was assessed. Figure 4A shows that switching the sequence of the MUT 2 and MUT 4 regions to that of the human had no effect on the PGC-1α response whereas switching the sequence of the MUT 1 region to that of the human reduced the PGC-1α response. Significantly, Figure 4B shows that when the inverse experiment was performed, in which the sequence of the MUT 1 region in the human promoter was switched to that of the mouse, expression of the resulting G6PC-luciferase fusion gene, designated −83/+7 hG6P mMUT 1, was induced by PGC-1α. This result suggests that the selective effect of PGC-1α on mouse G6PC-luciferase fusion gene expression can be explained, at least in part, by the differential binding of a factor to the MUT 1 region.

A conserved nuclear receptor half-site is required for the effect of PGC-1α on mouse G6PC fusion gene expression

Analysis of the G6PC promoter sequence encompassing the MUT 1 region using MatInspector software [34] revealed the existence of a consensus nuclear receptor half-site adjacent to the MUT 1 region (Fig. 3). This element is perfectly conserved in the mouse and human G6PC promoters (Fig. 3). A mouse G6PC-luciferase fusion gene containing a site directed mutation of this element, designated MUT 3, was transiently transfected into H4IIE cells and the effect of PGC-1α co-transfection was assessed. Figure 5 shows that mutating the MUT 3 region markedly reduced the PGC-1α response. These data demonstrated that, in addition to the HNF-4α binding site located between −76 and −64 [21], this nuclear receptor half-site was critical for the PGC-1α response.

Figure 5. A conserved nuclear receptor half-site is required for the stimulation of G6PC fusion gene expression by PGC-1α.

Figure 5

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H4IIE cells were transiently co-transfected, as described in Research Design and Methods, with the indicated mouse G6PC-luciferase fusion genes (12 μg), an expression vector encoding Renilla luciferase (0.15 μg), and either a pBJ5 expression vector encoding PGC-1α or the empty pBJ5 vector control (3 μg). The fusion genes contained either the wild type mouse promoter sequence between −85 and +1 or a site directed mutation of the conserved nuclear receptor half-site (MUT 3), as described in the legend to Fig. 3. Following transfection, cells were incubated for 18–20 hr in serum-free medium. Cells were then harvested and luciferase activity assayed as described in Research Design and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase in the cell lysate, in PGC-1α transfected versus empty vector transfected cells and are expressed as fold induction. Results represent the mean ± S.E.M. of 7 experiments using three independent preparations of both fusion gene constructs in which each experimental condition was assayed in duplicate. *, p < 0.05 versus −85/+1 mG6P WT.

Altered HNF-4α binding to a conserved nuclear receptor half-site contributes to the selective effect of PGC-1α on mouse G6PC-luciferase fusion gene expression

We reasoned that the most likely scenario that explained the data was one in which PGC-1α interacted with a member of the nuclear receptor superfamily bound to the MUT 3 region with the flanking sequence, as represented by the MUT 1 region, affecting binding affinity. Studies on PPARα binding to nuclear receptor half-sites have established a precedent for such a model [35]. Gel retardation assays using the mouse G6PC promoter sequence between −62 and −29, which includes both the MUT 3 and MUT 1 regions (Fig. 3), as the labeled probe failed to detect specific protein-DNA binding (data not shown). However, this presumably reflects the low affinity of interaction of nuclear receptors with half-sites.

Since chromatin immunoprecipitation experiments have shown that HNF-4α binds this region of the G6PC promoter in intact H4IIE cells [21] the simplest model to explain the data is one in which HNF-4α binds the −76/−64 element as well as the MUT 3 region, with the MUT 1 flanking region affecting the affinity of HNF-4α binding (Fig. 3). To explore this possibility an oligonucleotide representing the mouse G6PC promoter sequence between −82 and −37, which contains the high affinity −76/−64 HNF-4α binding site [21] and the putative HNF-4α binding half-site (Fig. 6A), was used as the labeled probe in gel retardation assays and the ability of oligonucleotides representing the mouse G6PC promoter sequence between −82 and −55, which includes just the high affinity −76/−64 HNF-4α binding site, or between −62 and −29, which includes just the MUT 3 and MUT 1 regions (Fig. 6A), to compete for HNF-4α binding was assessed. When the labeled double-stranded wild type (WT) G6P −82 and −37 oligonucleotide was incubated with nuclear extract prepared from H4IIE cells, a single major protein-DNA complex was detected (Fig. 6B). Supershift experiments using specific antisera have demonstrated that this complex contains HNF-4α [21]. Competition experiments, in which a 100-fold molar excess of unlabeled DNA was included with the labeled probe, were used to correlate protein binding with the PGC-1α response. The WT G6P −82/−37 oligonucleotide competed effectively for formation of the protein-DNA complex as did the −82/−55 oligonucleotide that includes just the high affinity −76/−64 HNF-4α binding site (Fig. 6B). Significantly, the WT G6P −62/−29 oligonucleotide also competed for formation of the protein-DNA complex confirming that this promoter region can bind HNF-4α (Fig. 6B). Oligonucleotides, designated mG6P hMUT 1 and MUT 3 (Fig. 6A), that contains mutations identical to those described in the hMUT 1 (Fig. 4A) and MUT 3 (Fig. 5) fusion genes competed poorly with the labeled probe for protein binding and less effectively than the WT G6P −62/−29 oligonucleotide (Fig. 6B). Figure 6C shows the results from multiple experiments using a variable molar excess of the various competitors. The observation that the MUT 3 oligonucleotide still competes slightly at high molar excess may be explained by the presence of a partial nuclear receptor half-site, AGGgCA, located between −35 and 30 (Fig. 6A). These results show that the binding of HNF-4α to the MUT 1 and MUT 3 regions correlates with the PGC-1α response.

Figure 6. The conserved nuclear receptor half-site required for the stimulation of G6PC fusion gene expression by PGC-1α binds HNF-4α.

Figure 6

Figure 6

Figure 6

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Panel A: Sequences of the sense strands of oligonucleotides used in gel retardation studies. The G6Pase nucleotide positions are numbered relative to the transcription start site of the mouse G6Pase gene. The HNF-4, MUT 3 and MUT 1 motifs are boxed and mutations introduced into these motifs are shown in color. The same mutations as shown here were introduced into the G6Pase-luciferase fusion genes described in Figs. 4, 5 & 7.

Panel B: A labeled, double-stranded oligonucleotide representing the wild type (WT) mouse G6PC promoter region between −82 and −37 was incubated in the absence (−) or presence of a 100-fold molar excess of the unlabeled oligonucleotides shown. H4IIE nuclear extract was then added and protein binding analyzed using the gel retardation assay as described in Research Design and Methods. In the representative autoradiograph shown only the retarded complex is visible and not the free probe, which was present in excess. Previous experiments have shown that the specific complex detected represents HNF-4α binding [21].

Panel C: Gel retardation experiments were performed as described in Panel A except that a variable molar excess unlabeled competitor DNAs was used as shown and data was quantitated using scintillation counting. Results represent the mean ± S.E.M. of at least three experiments. Black squares: −82/−37 WT; white squares: −82/−55 WT; black triangles: −62/−29 mG6P WT; white triangles: −62/−29 mG6P hMUT1; black circles: −62/−29 mG6P MUT3.

In an attempt to further support this conclusion, protein binding to labeled probes representing the mouse −82/−37 region and equivalent human −81/−36 region was compared. No difference was detected (data not shown) suggesting that the use of labeled probes only detects HNF-4α binding to the conserved high affinity HNF-4α binding site and not the HNF-4α half-site. This is consistent with the observation that protein binding to the labeled −62/−29 probe was not detectable (see above). This result indicates that, while HNF-4α binding to the half-site can be implied from competition experiments (Fig. 6), it cannot be directly demonstrated using labeled probes in the gel retardation assay. Presumably this reflects the low affinity of HNF-4α binding to the half-site such that HNF-4α dissociates from this site during the electrophoretic separation of the bound and free probe. Similar situations have been reported in studies on the glucocorticoid receptor [36] and FOXO1 [37] where binding to low affinity sites could only be implied through competition experiments and not directly demonstrated using labeled probes.

Overexpression of HNF-4α or FOXO1 enhances the action of PGC-1α on both mouse and human G6PC fusion gene expression

Since the data support a model in which the MUT 1 region affects the affinity of HNF-4α binding to the MUT 3 region we reasoned that overexpression of HNF-4α might, through a mass action effect, result in binding of HNF-4α to the MUT 3 region in the human G6PC promoter despite the suboptimal MUT 1 flanking sequence. Figure 7 shows that this is indeed the case. Although PGC-1α and HNF-4α had little effect alone, co-transfection of both PGC-1α and HNF-4α together resulted in a synergistic induction of human G6PC fusion gene expression (Fig. 7). The effects of both HNF-4α and PGC-1α alone or in combination were enhanced when the sequence of the MUT 1 region in the human promoter was switched to that of the mouse (Fig. 7). Similarly, co-transfection of both PGC-1α and HNF-4α also resulted in a synergistic induction of mouse G6PC fusion gene expression but in this case the effects of both HNF-4α and PGC-1α alone or in combination were reduced when the sequence of the MUT 1 region in the mouse promoter was switched to that of the human (Fig. 7). This again suggests that the sequence of the MUT 1 region affects both the affinity of HNF-4α binding and the PGC-1α response. The observation that overexpression of HNF-4α affects the PGC-1α response implies that the results of G6PC fusion gene analyses will be dependent on variations in endogenous HNF-4α expression in H4IIE cells. This may explain why in some experiments the presence of the human MUT 1 sequence is associated with a complete lack of a PGC-1α response (Fig. 4B) whereas in other experiments a partial response remains (Fig. 4A).

Figure 7. Combined expression of HNF-4α and PGC-1α synergistically stimulates both mouse and human G6PC fusion gene expression.

Figure 7

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H4IIE cells were transiently co-transfected, as described in Research Design and Methods, with the indicated mouse or human G6PC-luciferase fusion genes (12 μg), and expression vectors encoding Renilla luciferase (0.15 μg) and either pBJ5 (3 μg) or pBJ5-PGC-1α (3 μg), or pSG5 (3 μg) or pSG5-HNF-4α (3 μg) as indicated. Following transfection the cells were incubated for 18–20 hours in serum-free medium. The cells were then harvested and luciferase assays were performed as described in Research Design and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase activity in the cell lysate, in HNF-4α- and/or PGC-1α-stimulated versus empty vector transfected cells, expressed as fold induction. Results represent the mean ± S.E.M. of 3 experiments using three independent preparations the fusion gene plasmids with each sample assayed in duplicate. *, p < 0.05 versus −83/+7 hG6P WT; #, p < 0.05 versus −85/+1 mG6P WT. Black columns: pSG5-HNF-4α; white columns: pBJ5-PGC-1α; gray columns: pSG5-HNF-4α + pBJ5-PGC-1α.

Figure 8 shows similar experiments in which the action of PGC-1α on G6PC fusion gene expression was assessed in the presence or absence of FOXO1. As previously reported, both mouse [38] and human [39] G6PC fusion gene expression was stimulated by overexpression of FOXO1 (Fig. 8). In addition, co-transfection of both PGC-1α and FOXO1 together resulted in a synergistic induction of both mouse and human G6PC fusion gene expression (Fig. 8).

Figure 8. Combined expression of FOXO1 and PGC-1α synergistically stimulates both mouse and human G6PC fusion gene expression.

Figure 8

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H4IIE cells were transiently co-transfected, as described in Research Design and Methods, with the indicated mouse or human G6PC-luciferase fusion genes (12 μg), and expression vectors encoding Renilla luciferase (0.15 μg) and either pBJ5 (1.5 μg) or pBJ5-PGC-1α (1.5 μg), or pcDNA3 (1.5 μg) or pcDNA3-FOXO1 (1.5 μg) as indicated. Following transfection the cells were incubated for 18–20 hours in serum-free medium. The cells were then harvested and luciferase assays were performed as described in Research Design and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase activity in the cell lysate, in FOXO1- and/or PGC-1α-stimulated versus empty vector transfected cells, expressed as fold induction. Results represent the mean ± S.E.M. of 9 experiments using three independent preparations the fusion gene plasmids with each sample assayed in duplicate. *, p < 0.05 versus −826/+63 hG6P WT + pcDNA3-FOXO1; #, p < 0.05 versus −231/+66 mG6P WT + pcDNA3-FOXO1. Black columns: pcDNA-FOXO1; white columns: pBJ5-PGC-1α; gray columns: pcDNA-FOXO1 + pBJ5-PGC-1α.

Our results demonstrate that, in H4IIE cells, PGC-1α can selectively stimulate mouse but not human G6PC fusion gene expression (Fig. 2). This differential regulation can be explained by a sequence variation between the mouse and human promoters that results in reduced binding of HNF-4α to the human promoter (Figs. 3–6). When HNF-4α (Fig. 7) or FOXO1 (Fig. 8) are overexpressed, PGC-1α interacts synergistically with these factors to induce human G6PC fusion gene expression, though the induction achieved is still less than seen with the mouse promoter. These results imply that PGC-1α may be more important for the regulation of G6PC gene expression in mice than in humans and that the difference in PGC-1α regulation observed has the potential to contribute to the 10 fold higher rate of hepatic glucose production in mice [30, 31]. In addition, the results suggest that the regulation of G6PC gene expression by PGC-1α is likely to be more sensitive to changes in the expression of HNF-4α and FOXO1 in humans than mice.

Spiegelman and colleagues [20, 40] previously noted that PGC-1α alone had little effect on human G6PC fusion gene expression in immortalized mouse hepatocytes and the data presented here provide an explanation for that observation. They also reported that co-transfection of PGC-1α with either FOXO1 [40] or HNF-4α [20] resulted in a synergistic induction of fusion gene expression. The same synergistic interaction between PGC-1α and HNF-4α (Fig. 7) and FOXO1 (Fig. 8) is seen in H4IIE cells. With respect to the action of FOXO1, Puigserver et al. [40] presented a model in which PGC-1α interacted directly with FOXO1. However, mutation [20] or deletion [21] of the three insulin response sequences in the G6PC promoter had no effect on the PGC-1α response. Moreover, we recently showed that PGC-1α stimulates G6PC fusion gene expression through HNF-4α and that the binding of FOXO1 to the G6PC promoter is neither required nor sufficient for this induction [41]. We hypothesize that the synergism seen between FOXO1 and PGC-1α occurs at the level of the preinitiation transcription complex. We envisage that FOXO1, bound to multiple sites in the G6PC promoter, and PGC-1α tethered to the promoter through HNF-4α, synergize to increase pre-initiation complex formation. This concept is consistent with the observation that both proteins can interact with other non-DNA bound factors [5, 6, 42]. For example, FOXO1 can bind the CBP KIX domain, located within the N-terminal activation domain [43] whereas PGC-1α can bind the C-terminal activation domain on the related protein p300 [44]. The synergistic action of FOXO1 and PGC-1α on G6PC fusion gene transcription could then feasibly be mediated through combined binding to CBP/p300. More recently, Accili and colleagues [45] demonstrated in primary hepatocytes that lacked FOXO1 that the induction of G6PC expression by PGC-1α was markedly reduced. This is consistent with the synergistic interaction between FOXO1 and PGC-1α seen in both H4IIE cells [41] and immortalized mouse hepatocytes [40]. However, while the effect of PGC-1α on G6PC gene expression was markedly reduced in primary hepatocytes lacking FOXO1, a ~20-fold induction remained [45]. In contrast, the ability of PGC-1α to stimulate G6PC expression in hepatocytes derived from mice lacking HNF-4α is completely lost [20]. The results of Accili and colleagues [45] are therefore consistent with our model in which PGC-1α stimulates G6PC fusion gene expression through HNF-4α with the binding of FOXO1 to the G6PC promoter being neither required nor sufficient for this induction, though it does act synergistically to enhance the response [41].

Published reports suggest that this model is also consistent with the mechanism of action of PGC-1α on PEPCK gene expression. Thus, the action of PGC-1α on PEPCK fusion gene expression is mediated through HNF-4α and glucocorticoid receptor binding sites whereas the FOXO1 binding site is not required for the PGC-1α response [7, 15]. In addition, the ability of PGC-1α to stimulate PEPCK gene expression in hepatocytes derived from mice lacking HNF-4α is also completely lost [20].

Acknowledgments

We thank Jared N. Boustead for constructing the full length PGC-1α expression vector and several human G6PC-luciferase fusion genes. We also thank Drs. Keith Yamamoto, Daryl Granner and David Powell for providing the glucocorticoid receptor, HNF-4α and FOXO1 expression vectors, respectively. Research in the laboratory of R.O’B. was supported by NIH grant DK56374 and by NIH grant P60 DK20593, which supports the Vanderbilt Diabetes Center Core Laboratory. Marcia M. Schilling was supported by the Vanderbilt Molecular Endocrinology Training Program (5 T 32 DK07563).

Abbreviations

G6PC

glucose-6-phosphatase catalytic subunit

HGP

hepatic glucose production

PGC-1α

peroxisome-proliferator activated receptor gamma coativator-1α

HNF-4α

hepatocyte nuclear factor-4α

PEPCK

phosphoenolpyruvate carboxykinase, 5′ UTR, 5′ untranslated region

Footnotes

The authors have no financial interests that would result in a conflict of interest with respect to this work.

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