GSD-1 (glycogen storage disease type 1) is caused by an inherited defect in glucose-6-phosphatase activity, resulting in a massive accumulation of hepatic glycogen content and an induction of de novo lipogenesis. The chlorogenic acid derivative S4048 is a pharmacological inhibitor of the glucose 6-phosphate transporter, which is part of glucose-6-phosphatase, and allows for mechanistic studies concerning metabolic defects in GSD-1. Treatment of mice with S4048 resulted in an ~60% reduction in blood glucose, increased hepatic glycogen and triacylglycerol (triglyceride) content, and a markedly enhanced hepatic lipogenic gene expression. In mammals, hepatic expression of lipogenic genes is regulated by the co-ordinated action of the transcription factors SREBP (sterol-regulatory-element-binding protein)-1c, LXRα (liver X receptor α) and ChREBP (carbohydrate-response-element-binding protein). Treatment of Lxra−/− mice and Chrebp−/− mice with S4048 demonstrated that ChREBP, but not LXRα, mediates the induction of hepatic lipogenic gene expression in this murine model of GSD-1. Thus ChREBP is an attractive target to alleviate derangements in lipid metabolism observed in patients with GSD-1.

INTRODUCTION

Patients with GSD-1 (glycogen storage disease type 1) have a defect in the G6Pase (glucose-6-phosphatase) enzyme complex that consists of the enzymes G6PT (glucose-6-phosphate transporter), known as SLC37A4 [solute carrier family 37 (glucose-6-phosphate transporter), member 4], and the catalytic subunit G6PC (G6Pase catalytic). As a result of this defect, the patients suffer from postprandial hypoglycaemia and have high levels of liver glycogen stores. Besides these effects on glucose metabolism, hepatic lipid metabolism is also severely disturbed; GSD-1 patients have increased de novo lipogenesis and suffer from hyperlipidaemia [1]. The chlorogenic acid derivative S4048 is an inhibitor of G6PT and has been shown to provide a model of GSD-1 when given to laboratory animals. For instance, treatment of rats with S4048 resulted in elevated hepatic glycogen and G6P (glucose 6-phosphate) contents [2], as well as induction of hepatic lipid synthesis [3].

In fed conditions, liver and muscle store excess glucose as glycogen [4], but a certain amount of glucose enters the glycolytic pathway to yield pyruvate, which is converted into acetyl-CoA, the precursor of fatty acids and triacylglycerol (triglycerides) in de novo lipogenesis. Acc1 (acetyl-CoA carboxylase 1), Acc2 and Fasn (fatty acid synthase) are crucial enzymes in de novo lipogenesis and the hepatic expression of the genes encoding these three enzymes and other lipogenic enzymes is tightly regulated by a number of transcription factors.

Important ‘lipogenic’ transcription factors are SREBP (sterol-regulatory-element-binding protein)-1c, LXRα (liver X receptor α) and ChREBP (carbohydrate-response-element-binding protein). SREBP-1c is a member of the bHLH-LZ (basic-helix-loop-helix–leucine zipper) transcription factor family and induces transcription of almost all lipogenic genes [5]. LXRα has been identified as an oxysterol-activated nuclear receptor [68] that, after ligand binding, heterodimerizes with RXR (retinoid X receptor). The LXRα–RXR heterodimer binds to LXREs (LXR-response elements) present in the promoters of target genes and subsequently regulates the expression of multiple lipogenic genes, in part by increasing Srebp-1c transcription [912], but also by direct effects on the transcription of Fasn [13] and Acc [14]. Recently, it was reported that LXRα also regulates mRNA levels and activity of the third lipogenic transcription factor, ChREBP [15], which is regulated by hepatic G6P and/or by the carbohydrate flux through the PPP (pentose 5-phosphate pathway).

The non-oxidative part of the PPP is in near equilibrium with glycolysis and comprises a cascade of biochemical reactions in which G6P and fructose 6-phosphate are converted into xylulose 5-phosphate, an activator of PP2A (protein phosphatase 2A), that dephosphorylates and thereby activates ChREBP [16,17]. Translocation of ChREBP into the nucleus is enhanced upon dephosphorylation of Ser196, whereas dephosporylation of Thr666 enhances the binding activity of ChREBP to DNA [18,19]. In the nucleus, ChREBP binds as a heterodimer with Mlx (Max-like factor X) [20] to the ChoRE (carbohydrate-response element) in promoters of target genes. A recent in vitro study showed that hepatic G6P itself activates ChREBP transcriptional activity via a PP2A-independent route that does not require a PPP flux [21].

Remarkably, most genes regulated by ChREBP are also regulated by SREBP-1c [22], e.g. Fasn and Acc. Yet, expression of the gene encoding PK (pyruvate kinase) is regulated by ChREBP, but not by SREBP-1c [23]. In contrast, expression of the gene encoding GK (glucokinase) is regulated by SREBP-1c, but not by ChREBP [24].

Altogether, ChREBP might be considered as a switch that controls the conversion of carbohydrates into lipids. Evidence is emerging that oxysterols are not the only endogenous LXR ligands, since both glucose and G6P have shown to bind and activate LXR [25]. Since both ChREBP and LXR might be activated by increased intracellular G6P concentrations, these transcription factors are likely to be key players in the metabolic perturbations associated with GSD-1. We therefore determined whether ChREBP and/or LXRα are involved in the induction of lipogenic gene expression upon S4048 treatment.

MATERIALS AND METHODS

In vivo experiments with C57BL/6J and Lxra−/− mice

Male C57BL/OlaHsd mice (Harlan) and Lxra−/− mice and their wild-type littermates on a mixed C57BL/6J Sv129/OlaHsd background (our own breeding colony) [26] were housed in a light- and temperature-controlled facility. The mice were fed on a standard laboratory chow diet (RMH-B; Hope Farms) and had free access to water. Experimental procedures were approved by the Ethics Committees for Animal Experiments of the University of Groningen.

Mice were equipped with a permanent catheter in the right atrium of the heart [27] to infuse S4048 and the entrance of the catheter was attached to the skull. The mice were allowed a recovery period of at least 5 days after surgery. Mice were kept in metabolic cages during the experiment and the preliminary fasting period, allowing frequent collection of small tail blood samples under unrestrained conditions [28]. After 9 h of fasting, the mice were infused for 6 h with S4048 (a gift from Sanofi-Aventis Deutschland) (5.5 mg of S4048/ml of PBS with 6% DMSO at 0.135 ml/h) or vehicle. Blood glucose concentrations were measured in a small blood sample obtained by tail bleeding every hour during the experiment. After 6 h of infusion, the mice were killed by cardiac puncture and the liver was immediately removed and freeze-clamped within 30 s of excision.

In vivo experiments with Chrebp−/− mice

Male Chrebp−/− mice and their wild-type littermates [24] on a C57BL/6J background were housed in a light- and temperature-controlled facility. The mice were fed a standard laboratory chow diet (diet 7002; Harlan Teklad Premier Laboratory Diets) and had free access to drinking water. The experiments were approved by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.

After 9 h of fasting, the mice were anaesthetized with sodium pentobarbital and received a 6-h infusion with S4048 (5.5 mg of S4048/ml of PBS with 6% DMSO at 0.135 ml/h) or vehicle via the jugular vein as described previously [29]. Blood glucose concentrations were measured and the liver removal was performed as described above.

Hepatic analyses

Hepatic concentrations of triglyceride, free cholesterol and total cholesterol were measured using commercial kits (Roche Diagnostics and Wako Chemicals) after lipid extraction according to Bligh and Dyer [30]. Hepatic glycogen and G6P contents were determined as described by Bergmeyer [31].

Gene expression

Total liver RNA was isolated using the TRI Reagent method (Sigma–Aldrich) according to manufacturer's protocol. The integrity and concentration of RNA were determined with the NanoDrop™ 1000 spectrophotometer (Thermo Scientific). cDNA was obtained using the reverse transcription procedure with Moloney-murine-leukaemia virus reverse transcriptase (Sigma–Aldrich) and random primers according to the protocol of the manufacturer. cDNA levels were measured by qRT-PCR (quantitative real-time PCR) amplification using an ABI PRISM 7700 sequence detector (Applied Biosystems) against a calibration curve of pooled cDNA solutions. Expression levels were normalized to the β-actin levels. The sequences of the primers and probes can be found on http://www.labpediatricsrug.nl and are deposited at the RTPrimerDB (http://www.rtprimerdb.org).

Statistics

All values are means±S.D. for the number of animals indicated. Statistical analysis was assessed using the Mann–Whitney U test and the level of significance was set at P< 0.05. Analyses were performed using SPSS for Windows software.

RESULTS

Pharmacological inhibition of the G6PT as a murine model of GSD-1

Previous experiments from our laboratory [2,3] showed that inhibition of G6PT with S4048 results in severe hepatic steatosis and massive hepatic glycogen stores in rats. In the present study, we treated mice with S4048. During S4048 administration, blood glucose concentrations were significantly reduced within 1 h (Figure 1). At the end of the 6-h infusion period, blood glucose concentrations were 64% lower in the treated mice compared with vehicle-treated mice. As expected, hepatic glycogen and G6P contents were massively elevated upon S4048 treatment, as were hepatic and plasma triglyceride concentrations (Table 1).

Infusion of the GP6T inhibitor S4048 results in hypoglycaemia

Figure 1
Infusion of the GP6T inhibitor S4048 results in hypoglycaemia

Relative blood glucose concentrations in C57BL/OlaHsd mice during infusion of 30 mg/kg of body weight per h of the G6PT inhibitor S4048 or vehicle. Results are means± S.D. (n=7).

Figure 1
Infusion of the GP6T inhibitor S4048 results in hypoglycaemia

Relative blood glucose concentrations in C57BL/OlaHsd mice during infusion of 30 mg/kg of body weight per h of the G6PT inhibitor S4048 or vehicle. Results are means± S.D. (n=7).

Table 1
Hepatic triglyceride, cholesterol, glycogen and G6P contents in mice after treatment with S4048

C57BL/OlaHsd mice after a 6-h infusion with a 30 mg/kg or body weight per h of the G6PT inhibitor S4048 or vehicle. Results are means±S.D. (n=5). *P< 0.05 for S4048 compared with vehicle.

Liver metabolite Vehicle S4048 
Triglycerides (nmol/mg of liver) 25.7±6.9 60.3±22.6* 
Free cholesterol (nmol/mg of liver) 8.1±1.5 8.2±1.6 
Cholesterylester (nmol/mg of liver) 1.9±0.7 2.5±1.7 
Glycogen (nmol/mg of liver) 16.1±3.4 254.8±85.4* 
G6P (nmol/g of liver) 21.4±15.0 574.9±133.7* 
Liver metabolite Vehicle S4048 
Triglycerides (nmol/mg of liver) 25.7±6.9 60.3±22.6* 
Free cholesterol (nmol/mg of liver) 8.1±1.5 8.2±1.6 
Cholesterylester (nmol/mg of liver) 1.9±0.7 2.5±1.7 
Glycogen (nmol/mg of liver) 16.1±3.4 254.8±85.4* 
G6P (nmol/g of liver) 21.4±15.0 574.9±133.7* 

In the livers of S4048-treated mice, expression of lipogenic genes, such as Fasn, Acc1 and Acc2 was markedly increased compared with control mice (Figure 2). Compared with control mice, hepatic mRNA levels of Pk and Gk in S4048-treated mice were increased by 400% and reduced by 70% respectively. Taken together, this strongly suggests that S4048-induced lipogenic gene expression is related to enhanced transcriptional ChREBP activity and not to SREBP-1c activity since Pk gene expression is regulated by ChREBP, but not by SREBP-1c [23], whereas Gk is regulated by SREBP-1c, but not by ChREBP [24]. Because it has been reported that LXRα might activate ChREBP transcriptional activity [15] and that cellular G6P might be an endogenous LXR ligand [25], we explored the effects of S4048-infusion on lipogenic gene expression in Lxra−/− and Chrebp−/− mice in comparison with their wild-type littermates.

Infusion of the G6PT inhibitor S4048 induces hepatic lipogenic gene expression

Figure 2
Infusion of the G6PT inhibitor S4048 induces hepatic lipogenic gene expression

Changes in hepatic gene expression patterns in C57BL/OlaHsd mice upon treatment with 30 mg/kg of body weight per h of the G6PT inhibitor S4048. Results were normalized to the β-actin mRNA concentrations, with results from untreated mice set as 1. Results are means±S.D. (n=5); *P< 0.05 for S4048 treatment compared with control. Fas, Fasn.

Figure 2
Infusion of the G6PT inhibitor S4048 induces hepatic lipogenic gene expression

Changes in hepatic gene expression patterns in C57BL/OlaHsd mice upon treatment with 30 mg/kg of body weight per h of the G6PT inhibitor S4048. Results were normalized to the β-actin mRNA concentrations, with results from untreated mice set as 1. Results are means±S.D. (n=5); *P< 0.05 for S4048 treatment compared with control. Fas, Fasn.

Induction of hepatic lipogenic genes upon pharmacological inhibition of G6PT is not mediated by LXRα

LXRα is the major isoform that controls hepatic lipogenic gene expression and we therefore treated Lxra−/− mice and their wild-type littermates with S4048. In both genotypes, S4048 infusion resulted in mildly increased hepatic glycogen and G6P concentrations (Table 2) and in inductions of hepatic Pk, Acc1, Acc2 and Fasn expression (Figure 3). Thus S4048-induced expression of hepatic lipogenic genes is independent of LXRα. In Lxra−/− and Lxra+/+ mice, S4048-infusion induced transcription of G6pt, G6pc and Chrebp. Hepatic Gk expression was not significantly affected upon S4048-infusion in either type of mice, although it tended to decrease upon S4048 treatment.

Induction of hepatic lipogenic gene expression by the G6PT inhibitor S4048 is not mediated by LXRα

Figure 3
Induction of hepatic lipogenic gene expression by the G6PT inhibitor S4048 is not mediated by LXRα

Changes in hepatic gene expression patterns in Lxra+/+ and Lxra−/− mice upon treatment with 30 mg/kg of body weight per h of the G6PT inhibitor S4048. Results were normalized to the β-actin mRNA concentrations, with results from untreated Lxra+/+ mice set as 1. Results are means±S.D. (n=3, control mice; n=5 S4048-treated mice). *P< 0.05 for S4048 treatment compared with control. Fas, Fasn.

Figure 3
Induction of hepatic lipogenic gene expression by the G6PT inhibitor S4048 is not mediated by LXRα

Changes in hepatic gene expression patterns in Lxra+/+ and Lxra−/− mice upon treatment with 30 mg/kg of body weight per h of the G6PT inhibitor S4048. Results were normalized to the β-actin mRNA concentrations, with results from untreated Lxra+/+ mice set as 1. Results are means±S.D. (n=3, control mice; n=5 S4048-treated mice). *P< 0.05 for S4048 treatment compared with control. Fas, Fasn.

Table 2
Hepatic triglyceride, cholesterol, glycogen and G6P contents in Lxra+/+ and Lxra−/− mice after treatment with S4048

Liver metabolite levels in Lxra+/+ and Lxra−/− mice after a 6-h infusion with 30 mg/kg or body weight per h of the G6PT inhibitor S4048 or vehicle. Results are means±S.D. (n=3, control mice; n=5, S4048-treated mice). *P< 0.05 for S4048 compared with vehicle.

 Lxra+/+ mice Lxra−/− mice 
Liver metabolite Vehicle S4048 Vehicle S4048 
Triglycerides (nmol/mg of liver) 18.1±11.0 38.1±2.5* 24.7±5.6 71.2±15.3* 
Free cholesterol (nmol/mg of liver) 7.4±0.6 8.6±1.4 7.2±0.4 7.6±0.3 
Cholesterylester (nmol/mg of liver) 1.1±0.1 1.9±0.4 0.9±0.4 3.1±0.3* 
Glycogen (nmol/mg of liver) 78.8±26.2 183.0±6.1* 101.6±53.0 336.0±40.1* 
G6P (nmol/g of liver) 265.5±66.0 998.8±288.9 340.7±136.8 624.5±50.6* 
 Lxra+/+ mice Lxra−/− mice 
Liver metabolite Vehicle S4048 Vehicle S4048 
Triglycerides (nmol/mg of liver) 18.1±11.0 38.1±2.5* 24.7±5.6 71.2±15.3* 
Free cholesterol (nmol/mg of liver) 7.4±0.6 8.6±1.4 7.2±0.4 7.6±0.3 
Cholesterylester (nmol/mg of liver) 1.1±0.1 1.9±0.4 0.9±0.4 3.1±0.3* 
Glycogen (nmol/mg of liver) 78.8±26.2 183.0±6.1* 101.6±53.0 336.0±40.1* 
G6P (nmol/g of liver) 265.5±66.0 998.8±288.9 340.7±136.8 624.5±50.6* 

Induction of hepatic lipogenic genes upon pharmacological inhibition of G6PT is mediated by ChREBP

Next, we repeated the S4048 infusion experiments in Chrebp−/− mice and their wild-type littermates. In both Chrebp−/− and the wild-type mice, the infusion of S4048 resulted in enhanced liver glycogen and extremely increased intracellular G6P concentrations (Table 3). Although the S4048-infusion did not significantly affect the liver triglyceride content in the Chrebp−/− mice and their wild-type littermates, there was a tendency towards increased liver triglyceride content upon S4048-infusion (Table 3). Expression of the lipogenic genes was increased upon S4048 treatment in the wild-type mice but failed to reach significance for Pk and Fasn, due to the lack of statistical power (Figure 4). Interestingly, the S4048-mediated induction of hepatic Pk, Acc1 and Fasn expression was completely abolished in the Chrebp−/− mice. In addition, the S4048-mediated induction of Acc2 in the Chrebp−/− mice was only minor compared with the effects of S4048 on Acc2 in the wild-type mice (29% induction in Chrebp−/− mice compared with 227% induction in the Chrebp+/+ mice). This, however, might be due to an already higher Acc2 expression in the vehicle-infused Chrebp−/− mice compared with the vehicle-infused Chrebp+/+ mice. Altogether, these results show that the S4048-induced hepatic lipogenic gene expression is mediated by ChREBP. Hepatic Chrebp and Srebp-1c expression were reduced in Chrebp−/− mice as compared with wild-type littermates. Finally we did not find significant differences in hepatic Chrebp, Srebp-1c and Gk expression in S4048-treated mice of either genotype.

Induction of hepatic lipogenic gene expression by the G6PT inhibitor S4048 is mediated by ChREBP

Figure 4
Induction of hepatic lipogenic gene expression by the G6PT inhibitor S4048 is mediated by ChREBP

Changes in hepatic gene expression patterns in Chrebp−/− and Chrebp+/+ mice upon treatment with 30 mg/kg of body weight per h of the G6PT inhibitor S4048. Results were normalized to the β-actin mRNA concentrations, with results from untreated Chrebp+/+ mice set as 1. Results are means±S.D. (n=4, S4048-treated Chrebp+/+ mice and control Chrebp−/− mice; n=5, control Chrebp+/+ mice and S4048-treated Chrebp−/− mice). *P< 0.05 for S4048 treatment compared with control. Fas, Fasn.

Figure 4
Induction of hepatic lipogenic gene expression by the G6PT inhibitor S4048 is mediated by ChREBP

Changes in hepatic gene expression patterns in Chrebp−/− and Chrebp+/+ mice upon treatment with 30 mg/kg of body weight per h of the G6PT inhibitor S4048. Results were normalized to the β-actin mRNA concentrations, with results from untreated Chrebp+/+ mice set as 1. Results are means±S.D. (n=4, S4048-treated Chrebp+/+ mice and control Chrebp−/− mice; n=5, control Chrebp+/+ mice and S4048-treated Chrebp−/− mice). *P< 0.05 for S4048 treatment compared with control. Fas, Fasn.

Table 3
Hepatic triglyceride, cholesterol, glycogen and G6P contents in Chrebp+/+ and Chrebp−/− mice

Liver metabolite levels in Chrebp+/+ and Chrebp−/− mice after a 6-h infusion with 30 mg/kg or body weight per h of the G6PT inhibitor S4048 or vehicle. Results are means±S.D. (n=4, S4048-treated Chrebp+/+ mice and vehicle-treated Chrebp−/− mice; n=5, vehicle-treated Chrebp+/+ mice and S4048-treated Chrebp−/− mice). *P< 0.05 for S4048 compared with vehicle.

 Chrebp+/+ mice Chrebp−/− mice 
Liver metabolite Vehicle S4048 Vehicle S4048 
Triglycerides (nmol/mg of liver) 82±55 121±51 96±54 135±43 
Free cholesterol (nmol/mg of liver) 6.1±0.5 6.7±2.0 6.3±0.9 6.5±1.0 
Cholesterylester (nmol/mg of liver) 7.2±1.3 6.0±1.1 6.5±0.2 5.7±0.7 
Glycogen (nmol/mg of liver) 30.8±12.9 91.8±43.8* 41.3±8.7 108.7±43.1* 
G6P (nmol/g of liver) 33±28 2442±1397* 56±20 3345±522* 
 Chrebp+/+ mice Chrebp−/− mice 
Liver metabolite Vehicle S4048 Vehicle S4048 
Triglycerides (nmol/mg of liver) 82±55 121±51 96±54 135±43 
Free cholesterol (nmol/mg of liver) 6.1±0.5 6.7±2.0 6.3±0.9 6.5±1.0 
Cholesterylester (nmol/mg of liver) 7.2±1.3 6.0±1.1 6.5±0.2 5.7±0.7 
Glycogen (nmol/mg of liver) 30.8±12.9 91.8±43.8* 41.3±8.7 108.7±43.1* 
G6P (nmol/g of liver) 33±28 2442±1397* 56±20 3345±522* 

DISCUSSION

Hepatic lipid metabolism, and especially de novo lipogenesis, is regulated by several transcription factors, including SREBP-1c, ChREBP and LXRα. Numerous studies have addressed the respective roles of SREBP-1c and LXR in control of lipogenic gene expression [5,3234]. Other studies have focused on the interplay between these two factors [10,14]. The role of ChREBP in control of hepatic de novo lipogenesis is mostly related to the adaptive induction of lipogenic genes in response to increased glucose availability [18,21,22,35] and thus ChREBP has become an attractive target in the treatment of hepatic steatosis and insulin resistance [36]. Only one study has addressed the role of LXR in ChREBP activation and transcription [15]. In the present work, we characterize a murine model of GSD-1. In this model, ChREBP but not LXRα mediated the induction of hepatic lipogenic gene expression. Furthermore, the phenotype depended in part on the mouse strain used. Collectively, our results point toward ChREBP as a possible target to treat a number of metabolic derangements in patients with GSD-1.

Since GSD-1 is caused by a lack of G6Pase activity, glucose production by glycogenolysis or gluconeogenesis is severely impaired in GSD-1 patients who, as a consequence, suffer from hypoglycaemia. GSD-1 is characterized further by increased liver glycogen storage and a fatty liver. The murine model of GSD-1 we developed, using short-term pharmacological inhibition of the G6PT by S4048, captured several of these characteristics. A 6-h S4048-infusion resulted in hypoglycaemia and dramatically increased hepatic glycogen and G6P content, accompanied by increased hepatic triglyceride concentrations. Previously two mouse models of GSD-1, by genetic modification of the G6PC, have been published by the group of Chou [37,38]. Mice from both these models die postnatally from very severe hypoglycaemia and suffer from major disturbances in lipid metabolism, such as fatty liver. Both hypoglycaemia and fatty liver are also seen in our acute GSD-1 model and are apparently direct consequences of G6P accumulation in the liver.

Hepatic carbohydrates regulate ChREBP transcriptional activity either by the direct effects of hepatic G6P [21] or xylulose 5-phosphate via the induction of the PPP due to a high G6P content [16,17]. Because our murine model of GSD-1 is characterized by massively elevated G6P contents, the first experiments already suggested ChREBP to be the most likely transcriptional regulator of the enhanced hepatic lipogenic gene expression under these conditions. The pivotal role of ChREBP was evident from studies performed in Chrebp−/− mice treated with S4048. From the results depicted in Figure 4 it is clear that ChREBP indeed mediates the effects of S4048 on hepatic lipogenic gene expression. The molecular mechanism of ChREBP activation, leading to the induction of lipogenic gene expression, in our model is not known. Further detailed studies are required to reveal whether PP2A-dependent and/or PP2A-independent ChREBP activation is involved in the S4048-induced lipogenic gene expression. The S4048-mediated increase of hepatic G6pc expression upon S4048 treatment was also dependent on ChREBP. Although the role of ChREBP in the regulation of the G6Pase enzyme complex has not been a major topic of research, Dentin et al. [35] showed that shRNA (short-hairpin RNA) against ChREBP normalized the elevated hepatic G6Pase gene expression in ob/ob mice. The authors speculate that this is due to a normalization of hepatic insulin signalling in the shRNA-treated ob/ob mice. Intriguingly, SREBP-1c and ChREBP control the transcription of similar sets of genes and it is therefore hard to distinguish the effects of both transcription factors on lipogenic gene expression in vivo. However, Pk expression is regulated by ChREBP, but not by SREBP-1c [23], and Gk expression is regulated by SREBP-1c, but not by ChREBP [24]. When focusing on changes in expression of these two genes in the first experiments (Figure 2), it appeared that our murine GSD-1 model is associated with enhanced hepatic transcriptional activity of ChREBP, but not of SREBP-1c.

The activity of LXRα is regulated by cholesterol metabolites [68], and the unaffected hepatic cholesterol concentrations (Table 1) suggested that the induced hepatic lipogenic gene expression in our murine GSD-1 model was independent of LXRα. We also did not observe an induction of the LXR target gene Srebp-1c upon S4048-infusion (Figure 2), further supporting the conclusion that the induction of lipogenic genes was not mediated by LXR. Accordingly, the studies with the Lxra−/− mice clearly showed that LXRα does not mediate S4048-induced hepatic lipogenic gene expression since the effects of S4048 on hepatic gene expression levels did not differ between Lxra−/− mice and their wild-type littermates (Figure 3). Although previous in vitro experiments suggested that G6P is an LXR agonist [25], the current studies clearly show that G6P does not activate LXRα in vivo. In addition, using Lxra/Lxrb double-knockout mice and ChREBP shRNA, Denechaud et al. [18] have shown that the effects of carbohydrates on hepatic gene expression, e.g. Acc1 and Pk, required ChREBP, but not LXR. These results are consistent with the present study showing that induced hepatic expression of Acc1 and Pk upon elevated hepatic carbohydrates was absent in Chrebp−/− mice, but not in Lxra−/− mice (Figures 3 and 4).

The differential effects of S4048 on hepatic glycogen, G6P and triglyceride concentrations between the different mouse models are striking. Already upon vehicle infusion, the Lxra−/− mouse strain (Table 2) already presented with elevated hepatic glycogen and G6P contents compared with the C57BL/OlaHsd mice (Table 1) and the Chrebp−/− mouse strain (Table 3). In a previous study we had found considerably high liver glycogen and G6P concentrations in the wild-type littermates of the Lxra−/− mouse strain [39]. Although this might lead to the conclusion that the high liver glycogen and G6P content might be due to the mixed C57BL/6J Sv129/OlaHsd background, further experiments need to be performed to investigate this in more detail. In the Chrebp−/− mice and their wild-type littermates, S4048-infusion resulted in a modest 3-fold induction of liver glycogen concentrations and a massive ~60-fold induction in liver G6P concentrations, whereas the induction of liver triglyceride content was severely blunted (Table 3). We speculate that differences in infusion protocols might have resulted in these differences. The C57BL/OlaHsd mice, the Lxra−/− mice and their wild-type littermates were infused without sedation, whereas the Chrebp−/− mice and their wild-type littermates received the infusion under pentobarbital sedation. Despite these differences in the effect of S4048 on liver glycogen and G6P between the strains, however, the effects of S4048 on the hepatic expression of Pk, Acc1, Acc2 and Fasn was comparable between the first set of experiments and the wild-type littermates of the Lxra−/− mice and Chrebp−/− mice.

In conclusion, the present study shows that ChREBP mediates the induction of lipogenic gene expression levels upon pharmacological inhibition of G6PT. Hence, increased de novo lipogenesis in GSD-1 patients [1] might also be due to effects on ChREBP activity and/or transcription, suggesting that ChREBP might be an interesting target for future pharmacological interventions to prevent or to treat severe hypertriglyceridaemia and hepatic steatosis in these patients.

The experiments involving the and Chrebp−/− mice and their wild-type littermates were performed in the laboratory of Dr Jay D. Horton at the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas. We thank Dr Kosuka Uyeda for providing the Chrebp−/− mice.

Abbreviations

     
  • Acc

    acetyl-CoA carboxylase

  •  
  • ChREBP

    carbohydrate-response-element-binding protein

  •  
  • Fasn

    fatty acid synthase

  •  
  • G6P

    glucose 6-phosphate

  •  
  • G6Pase

    glucose-6-phosphatase

  •  
  • G6PC

    G6Pase catalytic

  •  
  • G6PT

    glucose 6-phosphate transporter

  •  
  • Gk

    glucokinase

  •  
  • GSD-1

    glycogen storage disease type 1

  •  
  • LXRα

    liver X receptor α

  •  
  • LXRE

    LXR-response element

  •  
  • Pk

    pyruvate kinase

  •  
  • PP2A

    protein phosphatase 2A

  •  
  • PPP

    pentose 5-phosphate pathway

  •  
  • RXR

    retinoid X receptor

  •  
  • shRNA

    short-hairpin RNA

  •  
  • SREBP

    sterol-regulatory-element-binding protein

AUTHOR CONTRIBUTION

Aldo Grefhorst and Marijke Schreurs performed the experiments and analysis, and wrote the manuscript. Maaike Oosterveer performed analysis and was involved in writing and editing the manuscript. Victor Cortés and Rick Havinga performed some of the experiments. Andreas Herling provided S4048. Dirk-Jan Reijngoud, Albert Groen, and Folkert Kuipers were involved in the design of the studies and writing of the manuscript.

FUNDING

This work was supported by the Netherlands Organization for Scientific Research [grant number 903-39-291]; the Ter Meulen Fund of the Royal Netherlands Academy of Arts and Science (to A.G.); and by the Dutch Diabetes Research Foundation [grant number 2003-00-009 (to M.S.)].

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Author notes

1

These authors contributed equally to this work.