A Molecular Link between the Common Phenotypes of Type 1 Glycogen Storage Disease and HNF1α-null Mice*

The clinical manifestations of type 1 glycogen storage disease (GSD-1) in patients deficient in the glucose-6-phosphatase (G6Pase) system (e.g. growth retardation, hepatomegaly, hyperlipidemia, and renal dysfunction) are shared by Hnf1α−/− mice deficient of a transcriptional activator, hepatocyte nuclear factor 1α (HNF1α). However, the molecular mechanism is unknown. The G6Pase system, essential for the maintenance of glucose homeostasis, is comprised of glucose 6-phosphate transporter (G6PT) and G6Pase. G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum where it is metabolized by G6Pase to glucose and phosphate. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively.Hnf1α−/− mice also develop noninsulin-dependent diabetes mellitus caused by defective insulin secretion. In this study, we sought to determine whether there is a molecular link between HNF1α deficiency and function of the G6Pase system. Transactivation studies revealed that HNF1α is required for transcription of the G6PT gene. Hepatic G6PT mRNA levels and microsomal G6P transport activity are also markedly reduced in Hnf1α−/− mice as compared withHnf1α+/+ andHnf1α+/− littermates. On the other hand, hepatic G6Pase mRNA expression and activity are up-regulated inHnf1α−/− mice, consistent with observations that G6Pase expression is increased in diabetic animals. Taken together, the results strongly suggest that metabolic abnormalities in HNF1α-null mice are caused in part by G6PT deficiency and by perturbations of the G6Pase system.

genesis and glycogenolysis, from the cytoplasm to the lumen of the endoplasmic reticulum (ER). Inside the ER, G6Pase with its active site facing the lumen (4) catalyzes the hydrolysis of G6P to glucose and phosphate. Therefore, G6PT and G6Pase work in concert to maintain glucose homeostasis. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively (1,2). Both groups of patients manifest growth retardation, hepatomegaly, hyperlipidemia, and renal dysfunction, clinical features associated with Hnf1␣ Ϫ/Ϫ mice (5-7) that are completely deficient in hepatocyte nuclear factor 1␣ (HNF1␣), a dimeric homeodomain-containing transcriptional activator (8 -10). HNF1␣ is expressed in the liver, kidney, pancreas, and digestive tract (5,8,10) and is required for the expression of many liver genes (11)(12)(13)(14). In this study, we establish a molecular link between HNF1␣ deficiency and function of the G6Pase system. We show that HNF1␣ binds to the G6PT promoter and is required for activation of G6PT gene transcription. Further, we show that hepatic G6PT mRNA expression in Hnf1␣ Ϫ/Ϫ mice is markedly reduced, resulting in a near abolishment of microsomal G6P transport activity. These data indicate that Hnf1␣ Ϫ/Ϫ mice, similar to GSD-1b patients, are deficient in the G6PT.
Hnf1␣ Ϫ/Ϫ mice also develop noninsulin-dependent diabetes mellitus (NIDDM) (6) caused by defective insulin secretion and ␤-cell glycolytic signaling (15,16). This finding is consistent with observations showing that mutations in the Hnf1␣ Ϫ/Ϫ gene in humans cause type 3 maturity-onset diabetes of the young, an autosomal dominant form of NIDDM characterized by impaired insulin secretion (17)(18)(19)(20). It has been speculated that overexpression of G6Pase might contribute to the pathophysiology of NIDDM. In animal models of diabetes, G6Pase mRNA expression and enzymatic activity are increased, resulting in an elevation in hepatic glucose production (21,22). Moreover, rats overexpressing the G6Pase gene exhibit several metabolic abnormalities associated with NIDDM, including glucose intolerance and hyperinsulinemia (23). Insulin has been shown to inhibit G6Pase gene transcription, and this effect is mediated through a cluster of insulin-response elements in the G6Pase promoter (24). Further, HNF1␣ is required for maximal repression of G6Pase gene transcription by insulin (25). In this study, we show that G6Pase activity and mRNA levels are elevated in Hnf1␣ Ϫ/Ϫ mice. Taken together, our data indicate that metabolic abnormalities in Hnf1␣ Ϫ/Ϫ mice are caused in part by perturbation of the G6Pase system.

Construction of Promoter-CAT Fusion Genes, Transfection, and CAT
Assays-The G6PT promoter-chloramphenicol acetyltransferase (CAT) fusion gene constructs were synthesized by polymerase chain reaction using the G6PT gene (26) as the template. The 3Ј primer for the G6PT 5Ј deletion mutants consisted of nucleotides Ϫ21 to Ϫ1, and the 5Ј primers consisted of nucleotides Ϫ609 to Ϫ589, Ϫ200 to Ϫ180, and Ϫ152 to Ϫ132. Each primer contained an additional HindIII or XbaI site at the 5Ј-end. After digestion with HindIII and XbaI, the amplified fragments were inserted upstream of the bacterial CAT gene of a modified promoter-and enhancer-less pCAT-Basic-N plasmid (27). The G6PT(Ϫ200/Ϫ1M)CAT construct was generated by site-directed mutagenesis using a pair of primers (nucleotides Ϫ172 to Ϫ149) that contain TAA3 GGG mutations at nucleotides Ϫ163/Ϫ161. All constructs were verified by DNA sequencing. The pSVCAT, which contains both the SV40 enhancer and promoter and pCAT-Basic-N plasmids were used as positive and negative controls, respectively. Additionally, we constructed G6PT promoter-CAT constructs in the reverse orientation, G6PT(Ϫ1/Ϫ609)CAT, and showed that it directed no CAT expression (data not shown).
HepG2 human hepatoma cells were grown at 37°C in ␣-modified minimal essential medium supplemented with 4% fetal bovine serum. Cells in 25-cm 2 flasks were transfected with the G6PT promoter-CAT constructs by the calcium phosphate-DNA coprecipitate method as previously described (27). The CAT activity was assayed by incubating total cellular protein in a buffer containing 250 mM Tris-HCl, pH 7.8, 4 mM acetyl coenzyme A, and 0.1 Ci [ 14 C]chloramphenicol (56 Ci/mmol, Amersham Pharmacia Biotech). The acetylated compounds were separated from chloramphenicol by thin-layer chromatography (95% chloroform, 5% methanol; v/v) on silica gel IB2 (Gilman Sciences). Spots were quantitated on an AMBIS Radioanalytic Imaging System (San Diego, CA).
Electromobility Shift Assays-HepG2 nuclear extracts were prepared essentially as described (28). End-labeled oligonucleotide probes (2 ng; 0.2-0.5 ϫ 10 6 cpm) were incubated for 20 min at room temperature in binding reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.05% Nonidet P-40, 1 mM EDTA, 0.5 mM dithiothreitol, and 10% glycerol) containing 0.4 -1 g of poly(dI-dC) and 3 g of nuclear extracts. Following binding, the mixture was electrophoresed through a 5% nondenaturing polyacrylamide gel, dried, and autoradiographed. For competition experiments, competitor DNA was incubated in the mixture prior to the addition of probe. For gel supershift assays, specific antisera were preincubated with HepG2 extracts at 4°C for 20 min before the addition of probe.
Animals-Hnf1␣ Ϫ/Ϫ mice were generated by Cre-loxP-mediated deletion to remove exon 1 of the Hnf1␣ gene (6). All animal studies were conducted under an animal protocol approved by the NIH Animal Care and Use Committee.
Northern Blot, Phosphohydrolase, and G6P Uptake Analyses-Total RNA was isolated by the guanidinium thiocyanate/CsCl method, fractionated by electrophoresis through 1.2% agarose gels containing 2.2 M formaldehyde and transferred to a Nytran membrane by electroblotting. The membranes were hybridized to cDNA probes for G6Pase, G6PT, or ␤-actin.
Phosphohydrolase assays were performed as previously described (4). Disrupted microsomal membranes were prepared by incubating intact microsomes in 0.2% deoxycholate for 20 min at 0°C. Nonspecific phosphatase activity was estimated by preincubating microsomal preparations at pH 5 for 10 min at 37°C, a condition that inactivates the thermally labile G6Pase. G6P uptake measurements were performed as previously described (26). Microsomes permeabilized with 0.2% deoxycholate, which abolished G6P uptake, were used as negative controls. Statistical analysis using the unpaired t test was performed with the GraphPad Prism Program (GraphPad Software, San Diego, CA).

HNF1␣ Binds to the G6PT Promoter and Transactivates
G6PT Gene Transcription-To determine whether HNF1␣ regulates G6PT gene expression, we analyzed the 5Ј-flanking region of the gene and identified a HNF1 motif at nucleotides Ϫ165 to Ϫ153 followed by a TATA-box at nucleotides Ϫ141 to Ϫ136 upstream of the translation start site at ϩ1 (Fig. 1A). To determine whether HNF1␣ activates transcription of the G6PT gene, we examined expression of the CAT gene directed by the G6PT promoter. Whereas both G6PT(Ϫ609/Ϫ1)CAT and G6PT(Ϫ200/Ϫ1)CAT constructs directed significant levels of CAT expression, CAT activity was found to be barely detectable with the G6PT(Ϫ152/Ϫ1)CAT construct (Fig. 1B). These data indicate that nucleotides Ϫ200 to Ϫ1 constitute a minimal G6PT promoter, which contains an activating element at nucleotides Ϫ200 to Ϫ153 encompassing the HNF1 motif (Ϫ165/Ϫ153).
To demonstrate whether HNF1␣ is the protein factor that binds to this activating element, electromobility shift assays were performed using HepG2 nuclear extracts. A protein-DNA complex, C1, was formed between the G6PT(Ϫ173/Ϫ145) oligo and HepG2 extracts ( Fig. 2A, lane 2). The formation of complex C1 was efficiently blocked by the addition of an excess of unlabeled target DNA (lanes 3-5) and by an oligonucleotide containing the HNF1 motif (lanes 6 -8), but not by an unrelated HNF4 (lane 21) and C/EBP oligonucleotide (lane 22). An HNF1-M1 oligonucleotide containing a mutated HNF1 site (TAA3 GGG) had markedly reduced ability to block complex C1 formation (lanes 9 -13) and an HNF1-M2 oligonucleotide that disrupts the entire DNA binding motif (TAA3 GGG and TAA3 GGG conversions) was completely incapable of blocking complex C1 formation (lanes 14 -18). Further, an antiserum to HNF1␣ (lane 19), but not HNF1␤ (lane 20), caused a shift in the mobility of complex C1, demonstrating that a protein factor in this complex is HNF1␣.
To determine whether binding of HNF1␣ to the G6PT promoter activates gene transcription, we examined CAT expression after cotransfecting G6PT promoter-CAT fusion genes with pBJ5 or pBJ5-HNF1␣. HNF1␣ elicited a 9.2-fold increase in CAT expression directed by the G6PT(Ϫ200/Ϫ1)CAT construct (Fig. 2B). In contrast, HNF1␣ elicited only a 3.8-fold increase in CAT activity directed by G6PT(Ϫ200/Ϫ1M)CAT, which contains a mutated HNF1 site (TAA3 GGG conversion at nucleotides Ϫ163 to Ϫ161). Moreover, CAT expression directed by G6PT(Ϫ200/Ϫ1M)CAT was also markedly reduced when compared with G6PT(Ϫ200/Ϫ1)CAT in the absence of a cotransfected HNF1␣ (Fig. 2B). Thus, our data indicate that HNF1␣ binds to its cognate site within the promoter and activates G6PT gene transcription.
Sequence analysis also predicts the presence of a binding site for HNF3 at nucleotides Ϫ62 to Ϫ56 and a C/EBP motif at nucleotides Ϫ50 to Ϫ42 of the G6PT promoter (Fig. 1A). HNF3 belongs to the forkhead or winged helix family of transcription factors (29,30), and it has been shown that HNF3␥ is required for transcription of the G6Pase gene (27). The C/EBP family belongs to the bZIP class of transcription factors that contain a FIG. 1. The G6PT promoter. A, nucleotides Ϫ200 to ϩ3 of the 5Ј-flanking region of the human G6PT gene. The numbers indicate the distance in nucleotides from the translation start site (ϩ1). The TATAbox and motifs for HNF1, HNF3, and C/EBP are boxed. The transcription start site is denoted by an arrow. B, promoter activity of the G6PT 5Ј-flanking region. The G6PT promoter-CAT fusion genes (10 g/25-cm 2 flask) were transfected into HepG2 cells, and CAT activity was expressed as percentage of activity expressed by the G6PT(Ϫ609/Ϫ1)CAT construct. Specific CAT activities directed by G6PT(Ϫ609/Ϫ1)CAT, pS-VCAT, and pCAT-Basic-N plasmids were 7.6, 4.8, and 0.02 nmol/ min/mg protein, respectively. Five independent experiments were conducted with two preparations of each construct.
basic DNA-binding region adjacent to a leucine zipper dimerization domain (31,32). The roles of HNF3 and C/EBP in G6PT gene transcription are currently under investigation.
Expression of G6PT and G6Pase Genes in Hnf1␣ Ϫ/Ϫ Mice-The vital role of HNF1␣ in transactivation of the G6PT gene and the clinical features common to both GSD-1 patients and Hnf1␣ Ϫ/Ϫ mice suggest that the expression of the G6PT gene is likely to be perturbed in these mice. We therefore examined G6PT mRNA expression in the liver of Hnf1␣ Ϫ/Ϫ mice by Northern blot analysis. The results show that the levels of hepatic G6PT mRNA were markedly reduced in Hnf1␣ Ϫ/Ϫ mice as compared with their wild-type and heterozygous littermates (Fig. 3). Additionally, whereas hepatic microsomes isolated from Hnf1␣ ϩ/ϩ or Hnf1␣ ϩ/Ϫ mice actively transported G6P, G6P uptake activities in intact hepatic microsomes from Hnf1␣ Ϫ/Ϫ animals were markedly reduced (Fig. 4), confirming that G6P transport function of G6PT is impaired in Hnf1␣ Ϫ/Ϫ mice.
It has been demonstrated that HNF1␣ is required for trans-activation of the G6Pase gene (27), and it acts as an accessory factor for maximal suppression of G6Pase transcription by insulin (25). In this study, we show that levels of hepatic G6Pase mRNA were increased by 2-to 6-fold in Hnf1␣ Ϫ/Ϫ mice (Fig. 3), resulting in an increase in G6Pase enzymatic activity in deoxycholate-disrupted microsomes where the G6PT function is not required ( Table I). The results suggest that HNF1␣ is not required for transcription of the G6Pase gene in vivo. On the other hand, the results are consistent with observations that G6Pase expression is increased in diabetic animals (21,22) and that HNF1␣ is required for suppression of G6Pase transcription by insulin (25). The active site of G6Pase faces the lumen of the ER (4) and for G6Pase catalysis in vivo, G6P must be translocated from the cytoplasm into the lumen by the G6PT (3,26). Biochemically, G6Pase activity in intact hepatic microsomes of GSD-1b patients, deficient in G6PT, is low or nondetectable, consistent with a functional G6Pase deficiency manifested by these patients. On the other hand, high levels of G6Pase activity were detected in disrupted hepatic microsomes of GSD-1b patients where G6PT function was abolished. The difference in G6Pase enzymatic activity in intact versus disrupted microsomes is best evaluated by measuring the G6Pase latency value, defined as the portion of enzymatic activity that is not expressed unless the microsomes are disrupted (33). G6Pase latency values in hepatic microsomes of GSD-1b patients are significantly higher than that in normal individuals (34). The apparent G6PT deficiency manifested by Hnf1␣ Ϫ/Ϫ mice prompted us to charac- FIG. 2. Activation of G6PT transcription by HNF1␣. A, binding of HepG2 nuclear proteins to nucleotides Ϫ173/Ϫ145 in the G6PT promoter. The G6PT(Ϫ173/Ϫ145) fragment encompassing a HNF1 site (Ϫ165/Ϫ153) was labeled and used in electromobility shift assays with HepG2 nuclear extracts. Reaction mixtures were preincubated with a competitor oligonucleotide, an antiserum to HNF1␣ (supershift), or antiserum to HNF1␤ (supershift), and analyzed on a 5% nondenaturing polyacrylamide gel. Consensus sequences that bind to transcription factors are underlined. B, stimulation of CAT expression directed by G6PT promoter constructs by HNF1␣. G6PT promoter-CAT constructs (5 g/25-cm 2 flask)) were transfected into HepG2 cells in the presence of 1 g each of pBJ5 (solid bar) or pBJ5-HNF1␣ (open bar). The G6PT(Ϫ200/Ϫ1M)CAT construct contains a mutated HNF1 site (TAA3 GGG conversion at nucleotides Ϫ163/Ϫ161). Specific CAT activities directed by G6PT(Ϫ609/Ϫ1)CAT, pSVCAT, and pCAT-Basic-N plasmids were 0.55, 0.33, and 0.001 nmol/min/mg protein, respectively. Four independent experiments were conducted with two preparations of each construct.  terize the G6Pase system in these mice. As expected, G6Pase activity in intact hepatic microsomes of Hnf1␣ Ϫ/Ϫ mice was only 53% of that found in Hnf1␣ ϩ/ϩ or Hnf1␣ ϩ/Ϫ mice (Table I). Moreover, hepatic G6Pase latency value was 87.9% in Hnf1␣ Ϫ/Ϫ mice, which was markedly higher than the value of 41.7% found in Hnf1␣ ϩ/ϩ /Hnf1␣ ϩ/Ϫ mice (Table I). Taken together, these results indicate that Hnf1␣ Ϫ/Ϫ mice, like GSD-1b patients, are deficient in the G6PT.

DISCUSSION
In this study, we have investigated the molecular mechanisms of phenotypic similarities between GSD-1 patients deficient in the G6Pase system (1, 2) and Hnf1␣ Ϫ/Ϫ mice lacking the transactivator, HNF1␣ (5-7). We demonstrate that Hnf1␣ Ϫ/Ϫ mice, like GSD-1b patients, are deficient in G6PT, an ER-associated membrane protein that translocates G6P from cytoplasm to the lumen of ER and a member of the G6Pse system required for the maintenance of glucose homeostasis (1)(2)(3). The results establish for the first time a molecular link between the common phenotypes of GSD-1 and Hnf1␣ Ϫ/Ϫ mice. Further, we show that the expression of the G6Pase gene is also perturbed in Hnf1␣ Ϫ/Ϫ mice.
G6PT, encoded by a single copy gene located on human chromosome 11q23 (35), is expressed in nearly all tissues examined, including liver, kidney, pancreas, and digestive tract (36). In this study, we demonstrate that nucleotides Ϫ200 to Ϫ1 upstream of the translation start site constitute a minimal G6PT promoter and HNF1␣ is required for transcription of the G6PT gene. The minimal G6PT promoter contains an activating element at nucleotides Ϫ200 to Ϫ153 encompassing the HNF1 motif at nucleotides Ϫ165 to Ϫ153. We show that HNF1␣ activates transcription of the G6PT gene following binding to its cognate site. Consistent with this, hepatic G6PT mRNA expression was inhibited and microsomal G6P transport function in the liver was impaired in Hnf1␣ Ϫ/Ϫ mice. In GSD-1b patients, deficiency in G6PT results in an increase in hepatic G6Pase latency values (34). Likewise, G6Pase latency values in Hnf1␣ Ϫ/Ϫ mice are also markedly increased. Taken together, these data demonstrate that the G6PT function in Hnf1␣ Ϫ/Ϫ mice is impaired, resulting in a phenotype that closely resembles that of GSD-1b.
In addition to functional G6Pase deficiency, GSD-1b patients suffer additional infectious complications because of heritable neutropenia and functional deficiencies of neutrophils and monocytes (37), clinical features not associated with Hnf1␣ Ϫ/Ϫ mice. The results of a recent study showed that GSD-1b patients carrying either a homozygous splicing (794G3 A) mutation or heterozygous G339D and R415X mutations suffer no impairment in their polymorphonuclear leukocyte functions (38). The 794G3 A mutation was shown to be leaky because the mutated G6PT gene of the patient directed the expression of both mature and truncated G6PT transcripts (38). Likewise, the R415X mutation was shown to only partially inactive the transporter (39). These studies strongly suggest that neutropenia as well as neutrophil and monocyte dysfunctions occur only in patients that harbor null G6PT mutations. Therefore, Hnf1␣ Ϫ/Ϫ mice, which express a low level of the G6PT gene, do not manifest neutropenia or polymorphonuclear leukocyte dysfunction.
It is noteworthy that overexpression of G6Pase in primary hepatocytes creates the metabolic profile of liver cells derived from NIDDM patients (40). Moreover, rats overexpressing the G6Pase gene manifest glucose intolerance and hyperinsulinemia (23). Transient expression studies have shown that HNF1␣ is required for transcription of the G6Pase gene (27). However, HNF1␣ is also required for the maximal repression of G6Pase gene transcription by insulin (24,25). The increase in G6Pase expression in Hnf1␣ Ϫ/Ϫ mice strongly suggests that the in vivo role of HNF1␣ is to act as an accessory factor to enhance the inhibitory action of insulin on G6Pase gene transcription (25). It has been shown that in diabetic rats, prolonged hyperglycemia increases G6Pase gene expression independent of insulin (22) and that the glucose-stimulated increase in G6Pase mRNA depending upon the presence of glucokinase (41). It appears that HNF1␣ deficiency compounded with impaired insulin secretion and hyperglycemia contributes to G6Pase overexpression in Hnf1␣ Ϫ/Ϫ mice. Whether perturbations in G6Pase expression contribute to the pathogenesis of NIDDM in these mice remains to be elucidated.
Glucose 6-phosphate, the substrate of the G6Pase system, plays a pivotal role in metabolism. It is at the branch point of lipid biosynthesis and glycogen biosynthesis as well as facilitating energy homeostasis through glucose. Kinetic studies have suggested that G6P uptake is the rate-limiting step in G6Pase catalysis (3). This notion is further supported by functional G6Pase deficiency manifested by GSD-1b patients carrying inactivating mutations in the G6PT gene (26). Similarly, in Hnf1␣ Ϫ/Ϫ mice, G6P generated by glycogenolysis and gluconeogenesis could not be efficiently translocated to the lumen of the ER, resulting in an increase in hepatic glycogen deposition and stimulation of cholesterol and fatty acid synthesis. Taken together, our study demonstrates, for the first time that metabolic abnormalities in Hnf1␣ Ϫ/Ϫ mice are caused in part by G6PT deficiency and disruption in the balance of the G6Pase system.