Mutations in hepatocyte nuclear factor 1α (HNF-1α) lead to maturity-onset diabetes of the young type 3 as a result of impaired insulin secretory response in pancreatic β-cells. The expression of 50 genes essential for normal β-cell function was studied to better define the molecular mechanism underlying the insulin secretion defect in Hnf-1α−/− mice. We found decreased steady-state mRNA levels of genes encoding glucose transporter 2 (Glut2), neutral and basic amino acid transporter, liver pyruvate kinase (L-Pk), and insulin in Hnf-1α−/− mice. In addition, we determined that the expression of several islet-enriched transcription factors, including Pdx-1, Hnf-4α, and Neuro-D1/Beta-2, was reduced in Hnf-1α−/− mice. These changes in pancreatic islet mRNA levels were already apparent in newborn animals, suggesting that loss of Hnf-1α function rather than chronic hyperglycemia is the primary cause of the altered gene expression. This expression profile was pancreatic islet–specific and distinct from hepatocytes, where we found normal expression of Glut2, L-Pk, and Hnf-4α in the liver of Hnf-1α−/− mice. The expression of small heterodimer partner (Shp-1), an orphan receptor that can heterodimerize with Hnf-4α and inhibit its transcriptional activity, was also reduced in Hnf-1α−/− islets. We characterized a 0.58-kb Shp-1 promoter and determined that the decreased expression of Shp-1 may be indirectly mediated by a downregulation of Hnf-4α. We further showed that Shp-1 can repress its own transcriptional activation by inhibiting Hnf-4α function, thereby establishing a feedback autoregulatory loop. Our results indicate that loss of Hnf-1α function leads to altered expression of genes involved in glucose-stimulated insulin secretion, insulin synthesis, and β-cell differentiation.

Maturity-onset diabetes of the young (MODY) is characterized by autosomal dominant inheritance, onset of diabetes usually before 25 years of age, and deficient insulin secretory response (1). Genetic studies have identified mutations in at least six genes associated with different forms of MODY. The MODY2 gene encodes the glycolytic enzyme glucokinase (GCK), and MODY subtypes 1, 3, 4, 5, and 6 are caused by mutations in transcription factors hepatocyte nuclear factor (HNF)-4α, -, PDX-1, HNF-1β, and NEURO-D/BETA-2, respectively (2,3,4,5,6,7). In addition, patients with mutations in the insulin gene itself are predisposed to impaired glucose tolerance (8).

There is increasing evidence suggesting that a transcriptional network is crucial for normal pancreatic β-cell function. In vitro studies have shown that the forkhead transcription factor HNF-3β is a transcriptional activator of PDX-1, HNF-4α, and HNF-1α, suggesting that HNF-3β is a master regulator of this transcriptional hierarchy (9,10,11,12). The HNF-1α promoter contains evolutionarily conserved binding sites for transcription factors HNF-4 and HNF-3. The presence of these binding sites is crucial for HNF-1α activity both in vivo and in vitro (13,14,15,16). Furthermore, Hnf-1β regulates the expression of Hnf-4α and Hnf-1α in extraembryonic visceral endoderm cells, indicating that it may also be an upstream regulator of the MODY1 and MODY3 genes in pancreatic islets (17,18). Less information is available on the transcriptional regulation of HNF-1α on other islet-enriched transcription factors.

HNF-1α is a homeodomain transcription factor composed of an NH2-terminal dimerization domain, a POU-homeobox DNA binding domain, and a COOH-terminal transactivation domain (19). HNF-1α is expressed in liver, kidney, intestine, and pancreatic islets (20). Mice with heterozygous mutations in Hnf-1α gene are phenotypically normal. However, homozygous inactivating mutations have been shown to have type 2 diabetes, dwarfism, renal Fanconi syndrome, and hypercholesterolemia (21,22,23). Clinical studies in humans have shown that heterozygous mutations in HNF-1α are associated with impaired pancreatic β-cell function that is characterized by impaired insulin secretion (24,25). The majority of mutations in the HNF-1α gene that have been identified in individuals with MODY3 can be predicted to result in loss of function, although some mutations may exhibit a weak dominant negative activity (26). The spectrum of mutations (missense, insertions, deletions, and splicing) also suggests that a gene dosage mechanism is responsible for the development of this autosomal dominant disorder. Decreased levels of HNF-1α are likely to result in reduced transactivation of HNF-1α target genes.

Previous physiological studies using pancreatic islets from Hnf-1α–deficient mice showed that β-cell dysfunction in these animals is likely to result from its defective glycolytic signaling proximal to mitochondrial oxidation (27,28). This conclusion is based on the following findings: 1) insulin secretion and intracellular calcium responses to glucose and glyceraldehyde are reduced; 2) glucose flux through glycolysis and the generation of ATP and NADH are decreased; 3) ATP-sensitive K+-currents (KATP) in β-cells from Hnf-1α−/− mice are not suppressed by glucose but normally sensitive to ATP; and 4) mitochondrial substrates can suppress KATP and correct the insulin secretion defect of Hnf-1α−/− islets. The results are also supported by recent studies showing reduced expression of liver pyruvate kinase (L-Pk) and glucose transporter 2 (Glut2) in islets of Hnf-1α−/− mice (29) and downregulation of Glut2, L-Pk, and insulin in cell lines that express a dominant negative form of HNF-1α (30). In contrast to the above reports, a recent in vitro study suggested that a mitochondrial defect may contribute to the β-cell defect in HNF-1α–deficient diabetes (31). In this report, a dominant negative Hnf-1α, HNF-1α-P291fsinsC, was expressed in insulinoma INS-1 cells and resulted in reduced expression levels of mitochondrial 2-oxoglutarate dehydrogenase (Ogdh) E1 subunit and increased expression of uncoupling protein 2 (Ucp2), thereby leading to impaired mitochondrial oxidation and consequently defective insulin secretory response (31). These inconsistent findings have yet to be resolved.

In this study, we explored the molecular basis by which Hnf-1α deficiency causes pancreatic β-cell dysfunction by comparing pancreatic islet gene expression profiles from wild-type and Hnf-1α−/− mice in vivo. Our results indicate that Hnf-1α deficiency results in a pleiotropic defect that includes dysregulation of glycolytic genes as well as other transcription factors of the pancreatic islet transcription factor network. Our results localize the transcriptional defect in islets of Hnf-1α−/− mice to the glycolytic pathway and provide evidence for a wider role of Hnf-1α in the regulation of islet-enriched transcription factors that are critical for β-cell differentiation than has previously been anticipated.

Vectors.

The construction of expression vector pCMV-pHNF-4α was described previously (15). The vector pcDNA3-pHNF-1α was generated by cloning a 2.8-kb NotI/BamHI fragment from pBJ5-pHNF-1α (a gift from G. Crabtree, Stanford University, Stanford, CA) containing the entire rat HNF-1α coding sequence into pcDNA3. The pcDNA3-pSHP-1 expression vector was constructed by polymerase chain reaction (PCR) amplification of an 850-bp mouse Shp fragment that contained the entire open reading frame using mouse liver cDNA as a template. This fragment was cloned into the EcoRI site of pcDNA3.

The reporter plasmid pGL2-pSHP-1 was constructed by PCR amplification of the human SHP-1 promoter (Genbank accession no. AF044316), using oligonucleotides SHP-Pm-1 (5′-TCCTAGACTGGACAGTGGGCA-3′) and SHP-Pm-2 (5′-CTTCCAGCTCTCTG GCTCTGT-3′) and subsequent cloning of the 582-bp fragment into plasmid pPCR2.1. A KpnI/XhoI fragment from this plasmid was then subcloned into the pGL2, inserting the SHP promoter upstream of the luciferase reporter gene. Vector pGL2-pSHPmut was generated by PCR using oligonucleotides SHPmut-F (5′-TCCTAGACTGGACAGTAA AAAAAGCCT-3′) and SHP-Pm-2, as well as pGL2-pSHP as a template. This fragment, containing a mutated HNF-4 binding site, was cloned into pPCR2.1 before insertion into pGL2. The sequences of all PCR-generated clones were confirmed by dideoxynucleotide sequencing.

Pancreatic islet and RNA isolation.

Pancreatic islets were isolated from 4- to 6-week-old Hnf-1α−/− mice and their heterozygous (Hnf-1α+/−) and wild-type (Hnf-1α+/+) littermates. We used collagenase digestion and differential centrifugation through Ficoll gradients, with a modification of procedures previously described (32). Islets were cultured overnight in RPMI 1640 medium supplemented with 10% fetal calf serum and 11.1 mmol/l glucose. Whole pancreata were removed from newborn mice. Total RNA was then extracted using TRIzol reagent (Gibco-BRL) and following the manufacturer’s instructions. Contaminating genomic DNA was removed using 1 μl RNase free DNase-I (Boehringer) per 5 μg RNA.

Reverse transcriptase–PCR.

Conditions used for reverse transcriptase (RT)-PCR followed the method of Wilson and Melton (33), with minimal modifications. cDNAs were synthesized using MMLV-RT (Stratagene) with dNTPs and random hexamer primers (Stratagene). These cDNAs provided templates for PCRs using specific primers at annealing temperatures of 60–65°C in the presence of dNTPs and Taq polymerase. Typically, between 20 and 28 cycles were used for amplification in the linear range. The sequences of the primers that were used for the amplification of cDNAs are available upon request from M.S.

Immunohistochemistry.

Glut2 immunofluorescence was performed on 5-μm sections from 6-week-old Hnf-1α+/+, Hnf-1α+/−, and Hnf-1α−/− mice that were prepared as previously described (27). Sections were blocked for 30 min with normal rabbit serum at a concentration of 1:100 in phosphate-buffered saline (PBS). Sections were stained with rabbit anti-mouse Glut2 antibody (Chemicon International, Temecula, CA) in PBS at a dilution of 1:200. The secondary antibody, a TRITC-labeled guinea pig anti-rabbit IgG, was applied at a concentration of 1:100 in PBS. Sections were washed twice in PBS between each step and after the application of the secondary antibody and were mounted in PBS:glycerol, 1:9.

Measurements of pyruvate kinase activity.

Pyruvate kinase enzyme activity was measured in extracts of islets isolated from four mice. After overnight culture in RPMI 1640 containing 11.6 mmol/l glucose, islets were hand picked, washed twice in PBS, and transferred to a homogenization buffer consisting of 200 mmol/l mannitol, 70 mmol/l sucrose, 5 mmol/l potassium HEPES buffer (pH 7.5), and 1 mmol/l dithiothreitol. The islets were washed twice in buffer containing 1 mmol/l EGTA and then twice in buffer without EGTA. The islets were then sonicated (Ultrasonics Sonicator Cell Disrupter, Model W 185 F; Heat Systems, Plainview, NY) and centrifuged at 105,000 × g for 60 min at 4°C. The protein content of the islet extracts was measured (Biorad, #500-0006), and the activity was measured in equivalent amounts of protein from the different mice. Pyruvate kinase enzyme activity was measured in a reaction buffer consisting of 80 mmol/l triethanolamine chloride buffer (pH 7.5), 5 mmol/l MgSO4, 100 mmol/l KCl, 2.5 mmol/l ADP, 0.14 mmol/l NADH, 1 mmol/l dithiothreitol, and 7 mmol/l phosphoenolpyruvate and 2.5 units of lactic dehydrogenase (all reagents were purchased from Sigma, St. Louis, MO). Each reaction was carried out in a final volume of 1 ml and was started by the addition of the islet extract. NADH levels were measured as fluorescence emitted at 448 nm in response to excitation at 340 nm for 10 min (31). The rates of consumption of NADH were similar in Hnf-1α+/+ (n = 4), Hnf-1α+/− (n = 5), and Hnf-1α−/− (n = 4) mice.

Electrophoretic mobility shift analysis.

In vitro translated proteins or nuclear cell extracts were incubated with 32P-labeled ds-stranded oligonucleotide probes containing the wild-type or mutant HNF-4 binding sites in the SHP promoter (sequences: 5′-TGGACAGTGGGCAAAGTCC-TCCC-3′ and 5′-TGGACAGTAAAAAAAGTCCT CCC-3′, respectively). The 15-μl reaction mixture contained 20 mmol/l Hepes buffer (pH 7.9), 40 mmol/l KCl, 1 mmol/l MgCl2, 0.1 mmol/l EGTA, 0.5 mmol/l DTT, 4% Ficoll, and 2 μg of poly(dIdC) at 25°C for 20 min. Supershift analysis was performed by incubating the antibody with the protein or nuclear extracts for 5 min on ice before adding the probe. The reaction mixture was loaded on a 6% nondenaturing polyacrylamide gel containing 0.25× TBE buffer (0.023 mol/l Tris-borate and 0.5 mmol/l EDTA) and run at 4°C. Nuclear extracts were prepared as described (15).

Tissue culture, transient transfections, and luciferase assay.

Cos7 and HepG2 cells were grown on minimum essential medium supplemented with 10% fetal calf serum and Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal calf serum, respectively. HIT-T15 cells were grown in DMEM supplemented with 5% fetal calf serum and 10% horse serum. A modified calcium phosphate precipitation procedure was used for transient transfections (15). In short, 0.5 ml of the precipitate, containing 1 μg CMV-LacZ, 0.5 μg luciferase reporter construct, and the indicated amount of expression vectors and carrier DNA (up to 10 μg), were added per 60-mm dish. Luciferase was normalized for transfection efficiency by the corresponding β-galactosidase activity (34).

Statistical analysis.

Results are given as mean ± SE. Statistical analyses were performed by using a Student’s t test, and the null hypothesis was rejected at the 0.05 level.

Impaired expression of genes involved in glucose-stimulated insulin secretion.

Insulin secretory responses to glucose are markedly reduced in Hnf-1α−/− mice compared with littermate controls. Methylpyruvate, an insulin secretagogue that freely permeates the mitochondrial membrane, can suppress ATP-dependent K+-channel activity in Hnf-1α−/− pancreatic islets, suggesting that the defect in glucose metabolism is upstream of mitochondrial glucose metabolism. To test which genes involved in glucose metabolism were affected by the disruption of Hnf-1α, we measured the mRNA levels of glucose transporters, amino acid transporters, and all glycolytic enzymes. We also measured the proximal rate-limiting enzymes of oxidative phosphorylation, including components of the pyruvate dehydrogenase complex (pyruvate decarboxylase and dihydrolipoyl dehydrogenase), aconitase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase complex (2-oxoglutarate dehydrogenase, dihydrolipoyl succinyl-transferase, and dihydrolipoyl dehydrogenase) as well as Ucp2. Furthermore, we determined the mRNA levels of the sulfonylurea receptor-1 (Sur-1), ATP-dependent K+-channel (Kir6.2), prohormone convertase-2 and -3 (Pc2, Pc3), pterin-4α-carbinolamine dehydratase (Pcd, DCoh), insulin, glucagon, somatostatin, pancreatic polypeptide (YY), and pancreatic peptide (PP).

Hypoxanthine phosphoribosyltransferase expression levels were determined to show that each sample contained equivalent amounts of mRNA. No products were amplified in the absence of reverse transcriptase, indicating the absence of genomic DNA in the mRNA samples. The cDNA of each sample was derived from one animal, and five to six animals of each genotype (Hnf-1α+/+, Hnf-1α+/−, and Hnf-1α−/−) were studied. RNAs for genes encoding L-Pk, Gck, Glut2, neutral and neutral and basic amino acid transporter (Nbat), insulin (Ins-1), glucagon, and Dcoh were significantly reduced (Fig. 1A). The absence of Glut2 protein in mice that lack Hnf-1α was also confirmed by immunohistochemistry of pancreatic sections (Fig. 2). The expression of other glucose (Glut1) and amino acid (neutral amino acid transporter) transporters remained unchanged (data not shown). The expression of the remaining glycolytic enzymes, including phosphoglucose isomerase, phosphofructokinase, aldolase B, triosephosphate isomerase, glucosephosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, 2,3-bisphosphoglycerate mutase, and α-, β-, and γ-enolases were also similar in islets of Hnf-1α+/+ and Hnf-1α−/− animals (Fig. 1B, data not shown). Expression of mitochondrial genes involved in the TCA cycle, such as Ogdh, as well as Kir6.2, Sur-1, Pc2, and Pc3, were also unchanged (Fig. 1B, data not shown). The expression of different islet cell type markers, such as Iapp, somatostatin, and YY genes, did not differ, suggesting that the various islet cell types contributed equally in wild-type and Hnf-1α−/− animals (data not shown). As expected, glucagon expression was slightly reduced in hyperglycemic Hnf-1α−/− mice (Fig. 1A).

Because hyperglycemia and hypoinsulinemia in Hnf-1α−/− animals can lead to secondary changes in gene expression (35), the expression of the above-mentioned genes were also studied in pancreata of newborn animals that had not been exposed to chronic hyperglycemia. We found that the expression of Glut2, L-Pk, Dcoh, and Ins-1 was already decreased shortly after birth (Fig. 1A), suggesting that Hnf-1α deficiency leads to the reduced expression of these genes. In contrast, glucagon and Gck mRNA levels were not altered in newborn pancreata (Fig. 1A), demonstrating that the decreased expression in Hnf-1α−/− adult islets is likely due to hyperglycemia or hypoinsulinemia in these mice. Finally, we found that the expression of L-Pk was markedly reduced in islets of adult and newborn Hnf-1α−/− animals. This transcriptional defect was specific for L-Pk, and expression levels of the muscle isoform (M2Pk) were unchanged in Hnf-1α−/− mice. To estimate the effect of the reduced L-Pk expression on overall pyruvate kinase activity, we measured total pyruvate kinase activity in islets of Hnf-1α+/+ and Hnf-1α−/− mice (data not shown). No differences were detected in total pyruvate kinase activity, supporting previous findings that M2Pk is the major pyruvate kinase isoform in pancreatic islets (36,37).

Abnormal gene expression of islet-enriched transcription factors in Hnf-1α−/− mice.

The effect of Hnf-1α deficiency on the expression of islet-enriched transcription factors was determined in islets of 4-week-old Hnf-1α−/−, Hnf-1α+/−, and Hnf-1α+/+ littermates by RT-PCR. Steady-state mRNA levels of Hnf-4α, an upstream regulator of Hnf-1α, were reduced in islets of Hnf-1α−/− mice, suggesting that Hnf-1α is critical for the normal expression of Hnf-4α in pancreatic islets (Fig. 1A). We also found that expression levels of several other islet-enriched transcription factors, including Pdx-1, Neuro-D1, and Shp-1, were reduced ∼2- to 11-fold in islets of Hnf-1α−/− mice (Fig. 1A). In contrast, the expression levels of Hnf-1β, -3α, -3β, -3γ, -4γ, -6, Nkx-2.2, Pax-4, Pax-6, Isl-1, Cdx2/3, and Lim-1 did not differ between Hnf-1α−/− and Hnf-1α+/+ mice (Fig. 1A, data not shown). Steady-state mRNA levels were also measured in pancreata of newborn mice. The expression levels of Pdx-1 and Neuro-D1 were already reduced in newborn mice (Fig. 1A). This indicates that Hnf-1α deficiency rather than nonspecific effects as a result of hyperglycemia is responsible for the abnormal expression of Pdx-1 and Neuro-D1.

Liver gene expression profiles in Hnf-1α+/+ and Hnf-1α−/− mice.

To test whether the changes in gene expression profiles of pancreatic islets are also present in the liver of Hnf-1α−/− animals, we measured mRNA concentrations of all the above-mentioned genes in Hnf-1α null, heterozygous, and wild-type mice. In contrast to pancreatic islets, the expression of Glut1, Glut2, and L-Pk was increased in Hnf-1α−/− mice (Fig. 3). The expression of the remaining glycolytic and mitochondrial genes was similar in mice of different Hnf-1α genotypes (data not shown). Consistent with previous observations (21,22), transcript levels of Hnf-1β were increased, whereas steady-state mRNA of DCoH and Igf-1 were reduced in Hnf-1α null liver (Fig. 3). The expression of Hnf-3α, -3β, -3γ, and -4α were unchanged, whereas Hnf-6 was reduced eightfold in Hnf-1α−/− mice compared with wild-type littermates (Fig. 3). We also tested 100 additional genes, including genes of apolipoprotein, amino acid, and lipid metabolism. We found that in the liver of Hnf-1α−/− mice, mRNA levels of Nbat were reduced 2.2-fold compared with wild-type littermates (Fig. 3). In summary, these data demonstrate that differences exist in the gene expression profiles of islets versus liver in Hnf-1α−/− mice. Therefore, in the liver, other mechanisms that can activate Hnf-1α target genes, such as Glut2 and L-Pk, and compensate for the loss of Hnf-1α function must be operative.

The transcriptional activation and inhibition of small heterodimer partner (Shp-1) is mediated by Hnf-4α in vitro.

Shp-1 is an orphan nuclear receptor that heterodimerizes with various nuclear hormone receptors, including Hnf-4α (38,39). Our in vivo expression data indicate that Shp-1 expression was reduced in adult islets of Hnf-1α null mice but not in newborn islets (Fig. 1). We then analyzed the promoter regions of human and murine Shp-1 but found no Hnf-1 binding sites (40). In contrast, a putative HNF-4α consensus binding sequence was identified at position −551 to −570 bp in the human SHP-1 promoter. This binding site was also conserved at a similar location in the murine Shp-1 promoter (Genbank accession nos. for murine and human sequences are AF044315 and AF044316, respectively). We therefore hypothesized that the downregulation of Shp-1 may be due to the reduced Hnf-4α message in Hnf-1α−/− mice.

To further study the transcriptional regulation of Shp-1, we cloned a 582 bp of the 5′ human SHP-1 promoter containing the putative HNF-4 binding site and linked it to a luciferase reporter gene (pGL2-pSHP-1). Cotransfection of this reporter plasmid with an HNF-4α expression vector (pcDNA3-pHNF-4α) showed an approximate two- to fivefold dose-dependent activation of the SHP-1 promoter in three different cell lines, Cos7, HepG2, and HIT-T15 (Figs. 4A–C). In contrast, HNF-1α expression did not result in the transcriptional activation of this reporter construct (Figs. 4A–C). To confirm that HNF-4α can directly activate the SHP-1 promoter, we mutated one hexamer half of the HNF-4 binding site in the reporter plasmid pGL2-pSHP-1. Transcriptional activation of HNF-4α on the resulting SHP-1 reporter plasmid (pGL2-pSHPmut) was abolished (Fig. 4D–F), indicating that the HNF-4 binding site in the SHP-1 promoter is functionally important for its expression.

Electrophoretic mobility shift assays were performed to investigate whether HNF-4α can bind to the putative HNF-4 binding site in the SHP-1 promoter. A major DNA/protein complex was detected with a 32P-labeled oligonucleotide that contained the HNF-4 binding site (Fig. 5, lane 2). This binding activity could be competed by an unlabeled excess of cold HNF-4 binding oligonucleotide (Fig. 5, lane 3). Furthermore, a supershift of the complex was observed after preincubation of extract with a monospecific anti–HNF-4α antiserum (α-445) but not with anti–STAT-1 antibodies (Fig. 5, lanes 4 and 5), demonstrating that HNF-4α can bind to the HNF-4 site in the SHP-1 promoter. As expected, HNF-4α was not able to bind to the mutated HNF-4 site (Fig. 5, lane 7).

To test whether SHP-1 can inhibit its own transcriptional activation, we cotransfected SHP-1 expression vector pcDNA3-pSHP-1 with constant quantities of pCMV-pHNF-4α and pGL2-pSHP-1 into Cos7, HIT-T15, and HepG2 cells. Luciferase activity decreased in a dose-dependent manner when increasing amounts of pcDNA3-pSHP-1 vector were coexpressed with HNF-4α (Figs. 4G–I). These data suggest that SHP-1 can inhibit its own transcription through a negative transcriptional feedback loop that is mediated by HNF-4α (Fig. 6).

Previous studies demonstrated that the insulin secretory responses to glucose are impaired in Hnf-1α−/− mice (27). Physiological studies have shown that KATP in β-cells of mutant mice are insensitive to suppression by glucose or glyceraldehyde but are normally sensitive to ATP. Flux of glucose through glycolysis in islets of mutant mice is reduced, but mitochondrial substrates, such as methylpyruvate, can inhibit KATP, elevate [Ca2+], and restore the insulin secretion defect (28). These results indicate that insulin secretion is impaired by a defect proximal to mitochondrial metabolism in the β-cell glycolytic signaling pathway. However, a recent in vitro study showed that expressing a dominant negative form of Hnf-1α, HNF1α-P291fsinsC, in insulinoma INS-1 cells affects mitochondrial function by reducing the expression of Ogdh and increasing the expression of Ucp2 (31). The net result of these expressing alterations is to impair mitochondrial function such as oxidation and ATP generation and to dissipate the mitochondrial membrane potential, leading to decreased glucose-stimulated insulin secretion (GSIS). These inconsistent findings have not been resolved. In addition, the molecular basis by which Hnf-1α deficiency causes impaired GSIS are poorly understood.

In this study, we investigated the transcriptional defect resulting from Hnf-1α deficiency. We studied steady-state mRNA levels in adult and newborn islets of genes that are critical for β-cell function. Gene transcript levels were also measured in the pancreas of newborn animals to control for secondary effects that hyperglycemia may have on gene expression profiles in HNF-1α−/− mice. Newborn Hnf-1α−/− mice are normoglycemic (87 vs. 90 mg/dl in HNF-1α+/+ and HNF-1α−/− newborn mice, respectively), and blood glucose levels of HNF-1α+/− females are indistinguishable from HNF-1α+/+ females during the last week of pregnancy (118 vs. 121 mg/dl in HNF-1α+/+ and HNF-1α+/− pregnant mice, respectively). This indicates that newborn islets of HNF-1α−/− mice were not exposed to chronic hyperglycemia. We found reduced expression of three genes involved in the glycolytic signaling pathway, namely Glut2, Gck, and L-Pk in Hnf-1α−/− mice (Fig. 1A). In addition, we found diminished expression of Nbat, an amino acid transporter that transports both neutral and basic amino acids into the cell (Fig. 1A). Reduced expression of Glut2, Gck, and Nbat are likely to contribute to the defect in GSIS because mutant mice that lack Glut2 or Gck are hyperglycemic and hypoinsulinemic as a result of impaired GSIS (41,42). The downregulation of Nbat may reduce the transport of basic and neutral amino acids into the β-cell. Furthermore, the reduced expression of Nbat offers a molecular explanation of the decreased cellular ATP production in response to leucine in cells that express a dominant negative form of the HNF-1α protein (31). In contrast, it is unlikely that decreased L-Pk expression levels contribute significantly to the β-cell defect in HNF-1α−/− mice because we did not observe a significant reduction in the overall enzyme activity of islet pyruvate kinase in Hnf-1α−/− mice. Therefore, our findings agree with previous studies that indicated that M2Pk is the major pyruvate kinase isoform in pancreatic islets (36,37). Our data suggest, then, that the lack of Glut2, Gck, and Nbat, which was not compensated for by increased expression levels of other glucose and amino acid transporters, may contribute to the diminished glucose or amino acid–stimulated insulin secretion rates in HNF-1α−/− animals.

To study whether Hnf-1α deficiency also impairs the expression of genes that are critical for mitochondrial function, we measured mRNA levels of numerous mitochondrial genes, including Ogdh and Ucp2. Ogdh constitutes the rate-limiting enzyme in the mitochondrial TCA cycle, and Ucp2 uncouples respiration from oxidative phosphorylation and inhibits the efficiency of ATP synthesis. In contrast to a previous report (31), the expression of Ogdh and Ucp2 were not changed in the absence of Hnf-1α (Fig. 1B). We also did not find the expression of other mitochondrial enzymes involved in the TCA cycle to be affected by Hnf-1α deficiency (Fig. 1B). Several reasons may account for these discrepancies. First, the previous study investigated the consequences of Hnf-1α deficiency by using a deoxycycline-inducible dominant negative Hnf-1α. Deoxycycline has been shown to be cytotoxic and thus may alter gene expression in treated cells (43,44). Second, because Hnf-1α can either homodimerize or heterodimerize with Hnf-1β, the dominant negative Hnf-1α used in the previous study may inhibit the function of both Hnf-1α and Hnf-1β (45). As a result, reduced Ogdh and increased Ucp2 expression may be due to impaired Hnf-1β function and not solely to Hnf-1α. Finally, the previous study was done in a transformed insulinoma cell line (INS-1), and there are differences between INS-1 cell line (46) and islets (used in the present study) that may explain the differences in Hnf-1α–regulated genes in the mitochondria.

In addition to the transcriptional defect in genes regulating β-cell glycolytic signaling, we found the expression levels of several other transcription factors to be significantly reduced in mice with Hnf-1α deficiency. The steady-state mRNAs of Hnf-4α, Pdx-1, and Neuro-D1/Beta-2, as well as the dimerization cofactor Dcoh, were reduced shortly after birth in islets of Hnf-1α−/− mice, suggesting that these changes are caused by HNF-1α deficiency and are likely to contribute to the molecular defects in Hnf-1α−/− islets. Decreased expression of several of these genes may be mediated directly by Hnf-1α. For instance, a functional HNF-1 binding site was identified recently in the HNF-4α promoter P2 that is located ∼46 kb upstream of the hepatic (P1) promoter. Transcription from the P2 promoter is responsible for the expression of the most abundant HNF-4α isoform in pancreatic β-cells, and HNF-1α is a potent activator of HNF-4α transcription from the P2 promoter in vitro (47). Our finding suggests that in pancreatic islets, Hnf-1α and Hnf-4α form an autoregulatory loop and that a haploinsufficiency in either of these transcription factors causes an expression disequilibrium (Fig. 6). This observation may also explain a possible molecular mechanism for the similar pancreatic β-cell defects in MODY1 and MODY3 patients (25,48,49). It is interesting that the expression of the insulin promoter factor Ipf-1/Pdx-1 was reduced in a dose-dependent manner in Hnf-1α+/+, Hnf-1α+/−, and Hnf-1α−/− mice. Pdx-1 is an essential transcription factor for pancreas development and β-cell function, including insulin gene transcription (50,51). Expression levels of Pdx-1 in Hnf-1α−/− islets were reduced 30–50% (Fig. 1A). This reduction was sufficient for a significant downregulation of Pdx-1 target genes, such as Gck and insulin (51,52,53,54). Functional Hnf-1α as well as Hnf-4α binding sites have been reported in evolutionarily conserved enhancer elements of the Pdx-1 promoter (11,12). Therefore, decreased Pdx-1 expression in Hnf-1α−/− islets may also be the result of reduced HNF-1α and HNF-4α gene expression. We also found that the mRNA levels of Neuro-D1, a helix-loop-helix transcription factor that can activate insulin gene transcription in vitro (55), were reduced 80% in Hnf-1α−/− mice. Despite the reduced expression of Pdx-1 and Neuro-D1, mRNA levels of Ins-2 were indistinguishable in islets of HNF-1α+/+ and HNF-1α−/− mice (Fig. 1A). This finding is in agreement with other reports (27,29) that did not find significantly reduced insulin mRNA levels in HNF-1α−/− mice and may be explained by compensation through other transcriptional activators of the insulin gene, such as Nkx2.2, Cdx2/3, Isl-1, and Pax4. In contrast, we found that Ins-1 expression was reduced ∼50% in islets of in HNF-1α−/− mice (Fig. 1A). This reduction is most likely a direct effect of Hnf-1α deficiency, mediated by an Hnf-1 binding element in the Ins-1 promoter that is lacking in the murine Ins-2 promoter (56).

It is worth noting that expression levels of other islet transcription factors that are important for β-cell metabolism, differentiation, and insulin gene transcription, including Hnf-1β, -3α, -3β, -3γ, -4γ, -6, Cdx-2, Nkx2.2, Isl-1, Pax4, and Pax6, were unchanged in Hnf-1α−/− mice. These findings support our hypothesis that loss of Hnf-1α function causes a pleiotropic but distinct defect in the β-cell transcriptional network essential for its normal function. It is interesting that the transcriptional control of this network and the regulation of downstream target genes are distinct in pancreatic islets and in the liver. For instance, the expression of Glut2, Gck, and L-Pk was normal or increased in the liver but significantly decreased in islets of Hnf-1α−/− mice compared with wild-type littermates. These differences may be explained by compensatory upregulation of transcriptional regulators (e.g., Hnf-1β) in the liver, reduced expression of liver-enriched transcription factors absent in adult pancreatic islets (e.g., Hnf-6), or differential regulation of transcription factors and downstream targets through tissue-specific promoters (e.g., Hnf-4α and Gck).

Finally, in vitro studies have suggested that SHP-1 may heterodimerize with HNF-4α and inhibit its transcriptional activity (39). Mutations in SHP-1 have also been identified in humans and may be associated with mild obesity in Japanese study subjects (57). Shp-1 expression was markedly reduced in islets of adult Hnf-1α−/− mice, suggesting that Shp-1 may be regulated by Hnf-1α, Hnf-4α, Pdx-1, or Neuro-D. To better understand the molecular basis of Shp-1 gene regulation, we studied the Shp-1 promoter. The 5′ regulatory sequence of Shp-1 lacked binding sites for Hnf-1α, Pdx-1, and Neuro-D but contained a conserved and functional Hnf-4 site. The biochemical characterization of this site and in vitro transactivation studies indicate that Hnf-4α is a transcriptional activator of the Shp-1 and that the reduced Shp-1 mRNA levels in pancreatic islets of HNF-1α−/− mice likely are due to the reduced expression of HNF-4α in Hnf-1α–deficient islets. In addition, our data indicate that Shp-1 may regulate its own expression by inhibiting Hnf-4α function, thus forming a negative feedback loop, and indirectly (through Hnf-4α) regulate the activity of Hnf-1α, Pdx-1, and Neuro-D (Fig. 6). Additional studies, including targeted mutagenesis of the Shp-1 gene in mice, are needed to shed light on the biological role of Shp-1 in pancreatic islet function.

FIG. 1.

Steady-state mRNA levels of target genes in Hnf-1α+/+, Hnf-1α+/−, and Hnf-1α−/− pancreatic islets of 4- to 6–week-old mice. A: RT-PCR analysis of islet-enriched transcription factors and candidate HNF-1α target genes in pancreata of newborn mice and adult islets. B: RT-PCR analysis of glycolytic and mitochondrial gene mRNA levels in the adult islets. At least five different animals for each genotype were measured by RT-PCR using [α-32P]dCTP (expression levels of only two animals per genotype are shown). PCR products were separated by PAGE, and bands were visualized by autoradiography. Quantitative measurements were obtained by densitometry. WT/Mut indicates the ratio of expression levels of the means of wild-type (Hnf-1α+/+) and null (Hnf-1α−/−) mice (n = 5–6). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

FIG. 1.

Steady-state mRNA levels of target genes in Hnf-1α+/+, Hnf-1α+/−, and Hnf-1α−/− pancreatic islets of 4- to 6–week-old mice. A: RT-PCR analysis of islet-enriched transcription factors and candidate HNF-1α target genes in pancreata of newborn mice and adult islets. B: RT-PCR analysis of glycolytic and mitochondrial gene mRNA levels in the adult islets. At least five different animals for each genotype were measured by RT-PCR using [α-32P]dCTP (expression levels of only two animals per genotype are shown). PCR products were separated by PAGE, and bands were visualized by autoradiography. Quantitative measurements were obtained by densitometry. WT/Mut indicates the ratio of expression levels of the means of wild-type (Hnf-1α+/+) and null (Hnf-1α−/−) mice (n = 5–6). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

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FIG. 2.

Immunofluorescence staining of pancreatic islets for Glut2 protein. Pancreatic tissue sections showing representative islets from 4-week-old Hnf-1α+/+ and Hnf-1α−/− mice are shown. Sections were stained with antibodies against Glut2, as described in research design and methods. Pancreas sections from HNF-1α wild-type littermates demonstrated red fluorescence staining on the membranes of the cells at the center of the pancreatic islets, indicating the presence of Glut2 in the membranes of pancreatic β-cells. In contrast, there was no staining evident in the islets of Hnf-1α−/− mice. Staining in HNF-1α heterozygous mice was similar to that in the wild-type mice (data not shown).

FIG. 2.

Immunofluorescence staining of pancreatic islets for Glut2 protein. Pancreatic tissue sections showing representative islets from 4-week-old Hnf-1α+/+ and Hnf-1α−/− mice are shown. Sections were stained with antibodies against Glut2, as described in research design and methods. Pancreas sections from HNF-1α wild-type littermates demonstrated red fluorescence staining on the membranes of the cells at the center of the pancreatic islets, indicating the presence of Glut2 in the membranes of pancreatic β-cells. In contrast, there was no staining evident in the islets of Hnf-1α−/− mice. Staining in HNF-1α heterozygous mice was similar to that in the wild-type mice (data not shown).

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FIG. 3.

Steady-state mRNA levels of HNF-1α target genes in livers of 4- to 6-week-old Hnf-1α+/+, Hnf-1α+/−, and Hnf-1α−/− mice. RT-PCR was performed from three separate RNA samples of each genotype as described in research design and methods. WT/Mut indicates the ratio of expression levels of the means of wild-type (Hnf-1α+/+) and null (Hnf-1α−/−) mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005. Differences in L-Pk and Glut2 expression in wild-type versus mutant livers reached marginal significance (P = 0.06).

FIG. 3.

Steady-state mRNA levels of HNF-1α target genes in livers of 4- to 6-week-old Hnf-1α+/+, Hnf-1α+/−, and Hnf-1α−/− mice. RT-PCR was performed from three separate RNA samples of each genotype as described in research design and methods. WT/Mut indicates the ratio of expression levels of the means of wild-type (Hnf-1α+/+) and null (Hnf-1α−/−) mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005. Differences in L-Pk and Glut2 expression in wild-type versus mutant livers reached marginal significance (P = 0.06).

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FIG. 4.

Transient expression and transcriptional activity of HNF-4α using a reporter plasmid that contains the SHP promoter upstream of the luciferase gene. AC: The reporter construct pGL2-pSHP was cotransfected with pCMV-pHNF-4α or pcDNA3-pHNF-1α into Cos7, HepG2, and HIT-T15 cells. D–F: The HNF-4 binding site in the SHP promoter is mutated in pGL2-pSHPmut. GI: Cotransfection in various cell types of pCMV-pHNF-4α and increasing amounts of pCMV-pSHP in the presence of reporter pGL2-pSHP. Cells were harvested 48 h after transfections and assayed for luciferase and β-galactosidase activities. The average fold inductions from two independent transfections done in duplicate and normalized to β-galactosidase activity are shown. Error bars indicate SD.

FIG. 4.

Transient expression and transcriptional activity of HNF-4α using a reporter plasmid that contains the SHP promoter upstream of the luciferase gene. AC: The reporter construct pGL2-pSHP was cotransfected with pCMV-pHNF-4α or pcDNA3-pHNF-1α into Cos7, HepG2, and HIT-T15 cells. D–F: The HNF-4 binding site in the SHP promoter is mutated in pGL2-pSHPmut. GI: Cotransfection in various cell types of pCMV-pHNF-4α and increasing amounts of pCMV-pSHP in the presence of reporter pGL2-pSHP. Cells were harvested 48 h after transfections and assayed for luciferase and β-galactosidase activities. The average fold inductions from two independent transfections done in duplicate and normalized to β-galactosidase activity are shown. Error bars indicate SD.

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FIG. 5.

Electrophoretic mobility shift assay analysis of the HNF-4 binding site in the SHP promoter. The DNA binding activity was measured using a 32P-labeled double-stranded oligonucleotide containing the HNF-4 binding site as a probe and nuclear extracts of HIT-T15 cells that were transfected with pCMV-pHNF-4α. Competition was carried out with a 20-fold excess of cold probe (lane 3), and supershifts were performed with a monospecific anti–HNF-4α antiserum or an anti–Stat-1 control antiserum (lanes 4 and 5, respectively). No binding activity was detected with a probe containing a mutated HNF-4 binding site (lane 7). Free probe on the bottom of the gel is not shown.

FIG. 5.

Electrophoretic mobility shift assay analysis of the HNF-4 binding site in the SHP promoter. The DNA binding activity was measured using a 32P-labeled double-stranded oligonucleotide containing the HNF-4 binding site as a probe and nuclear extracts of HIT-T15 cells that were transfected with pCMV-pHNF-4α. Competition was carried out with a 20-fold excess of cold probe (lane 3), and supershifts were performed with a monospecific anti–HNF-4α antiserum or an anti–Stat-1 control antiserum (lanes 4 and 5, respectively). No binding activity was detected with a probe containing a mutated HNF-4 binding site (lane 7). Free probe on the bottom of the gel is not shown.

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FIG. 6.

Proposed model for a hierarchical transcriptional network in pancreatic islets. This diagram shows the transcriptional regulation of genes essential for pancreatic islet function by hepatocyte nuclear factors. Arrows show positive regulation. Bold arrows indicate transcriptional regulation described in this study; numbers adjacent to arrows indicate references of previous in vitro and in vivo studies.

FIG. 6.

Proposed model for a hierarchical transcriptional network in pancreatic islets. This diagram shows the transcriptional regulation of genes essential for pancreatic islet function by hepatocyte nuclear factors. Arrows show positive regulation. Bold arrows indicate transcriptional regulation described in this study; numbers adjacent to arrows indicate references of previous in vitro and in vivo studies.

Close modal

This work was supported in part by the American Diabetes Association, National Institutes of Health Grant R01-DK-55033-01 (M.S.), Human Frontier Science Program Organization research grant (M.P. and M.S.), and National Institutes of Health Medical Scientists Training Program Grant GM07739 (D.Q.S.). M.S. is an Irma Hirschl Scholar, Pew Scholar, and Robert and Harriet Heilbrunn Professor. K.N.M. was supported by the GATEWAYS to the Laboratory Program.

1.
Fajans SS: Maturity-onset diabetes of the young (MODY).
Diabetes Metab Rev
5
:
579
–606,
1989
2.
Vionnet N, Stoffel M, Takeda J, Yasuda K, Bell GI, Zouali H, Lesage S, Velho G, Iris F, Passa P, Froguel P, Cohen D: Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus.
Nature
356
:
721
–722,
1992
3.
Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, Fajans SS, Signorini S, Stoffel M, Bell GI: Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young.
Nature
384
:
458
–460,
1996
4.
Yamagata K, Oda N, Kaisaki PJ, Furuta H, Vaxillaire M, Southam L, Cox RD, Lathrop GM, Boriraj VV, Chen X, Cox NJ, Oda Y, Yano H, Le Beau MM, Yamada S, Nishigori H, Takeda J, Fajans SS, Hattersley AT, Iwasaki N, Hansen T, Pedersen O, Polonsky KS, Bell GI: Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young.
Nature
384
:
455
–458,
1996
5.
Stoffers A, Ferrer J, Clarke WL, Habener JF: Early-onset type-II diabetes mellitus (MODY4) linked to IPF1.
Nat Genet
17
:
138
–139,
1997
6.
Horikawa Y, Iwasaki N, Hara M, Furuta H, Hinokio Y, Cockburn BN, Lindner T, Yamagata K, Ogata M, Tomonaga O, Kuroki H, Kasahara T, Iwamoto Y, Bell GI: Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY.
Nat Genet
17
:
384
–385,
1997
7.
Malecki MT, Jhala US, Antonellis A, Fields L, Doria A, Orban T, Saad M, Warram JH, Montminy M, Krolewski AS: Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus.
Nat Genet
23
:
323
–328,
1999
8.
Steiner DF, Tager HS, Chan SJ, Nanjo K, Sanke T, Rubenstein AH: Lessons learned from molecular biology of insulin-gene mutations.
Diabetes Care
13
:
600
–609,
1990
9.
Duncan SA, Navas MA, Dufort D, Rossant J, Stoffel M: Regulation of a transcription factor network required for differentiation and metabolism.
Science
281
:
692
–695,
1998
10.
Wu KL, Gannon M, Peshavaria M, Offield MF, Henderson E, Ray M, Marks A, Gamer LW, Wright CV, Stein R: Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.
Mol Cell Biol
17
:
6002
–6013,
1997
11.
Gerrish K, Gannon M, Shih D, Henderson E, Stoffel M, Wright CV, Stein R: Pancreatic beta cell-specific transcription of the pdx-1 gene: the role of conserved upstream control regions and their hepatic nuclear factor 3beta sites.
J Biol Chem
275
:
3485
–3494,
2000
12.
Ben-Shushan E, Marshak S, Shoshkes M, Cerasi E, Melloul D: A pancreatic β-cell-specific enhancer in the human PDX-1 gene is regulated by hepatocyte nuclear factor 3β (HNF-3β), HNF-1α, and SPs transcription factors.
J Biol Chem
276
:
17533
–17540,
2001
13.
Kuo CJ, Conley PB, Chen L, Sladek FM, Darnell JE Jr, Crabtree GR: A transcriptional hierarchy involved in mammalian cell-type specification.
Nature
355
:
457
–461,
1992
14.
Duncan SA, Nagy A, Chan W: Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4α(−/−) embryos.
Development
124
:
279
–287,
1997
15.
Stoffel M, Duncan SA: The maturity-onset diabetes of the young (MODY1) transcription factor HNF4alpha regulates expression of genes required for glucose transport and metabolism.
Proc Natl Acad Sci U S A
94
:
13209
–13214,
1997
16.
Gragnoli C, Lindner T, Cockburn BN, Kaisaki PJ, Gragnoli F, Marozzi G, Bell GI: Maturity-onset diabetes of the young due to a mutation in the hepatocyte nuclear factor-4 alpha binding site in the promoter of the hepatocyte nuclear factor-1 alpha gene.
Diabetes
46
:
1648
–1651,
1997
17.
Barbacci E, Reber M, Ott M, Breillat C, Huetz F, Cereghini S: Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification.
Development
126
:
4795
–4805,
1999
18.
Coffinier C, Thepot D, Babinet C, Yaniv M, Barra J: Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation.
Development
126
:
4785
–4794,
1999
19.
Mendel DB, Crabtree GR: A member of a novel class of dimerizing homeodomain proteins.
J Biol Chem
266
:
677
–680,
1991
20.
Blumenfeld M, Maury M, Chouard T, Yaniv M, Condamine H: Hepatic nuclear factor 1 (HNF1) shows a wider distribution than products of its known target genes in the developing mouse.
Development
113
:
589
–599,
1991
21.
Pontoglio M, Barra J, Hadchouel M, Doyen A, Kress C, Bach JP, Babinet C, Yaniv M: Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome.
Cell
84
:
575
–585,
1996
22.
Lee YH, Sauer B, Gonzalez FJ: Laron dwarfism and non-insulin-dependent diabetes mellitus in the Hnf-1alpha knockout mouse.
Mol Cell Biol
18
:
3059
–3068,
1998
23.
Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ, Shefer S, Bollileni JS, Gonzalez FJ, Breslow JL, Stoffel M: Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism.
Nat Genet
27
:
375
–382,
2001
24.
Lehto M, Tuomi T, Mahtani MM, Widen E, Forsblom C, Sarelin L, Gullstrom M, Isomaa B, Lehtovirta M, Hyrkko A, Kanninen T, Orho M, Manley S, Turner RC, Brettin T, Kirby A, Thomas J, Duyk G, Lander E, Taskinen MR, Groop L: Characterization of the MODY3 phenotype: early-onset diabetes caused by an insulin secretion defect.
J Clin Invest
99
:
582
–591,
1997
25.
Byrne MM, Sturis J, Menzel S, Yamagata Y, Fajans SS, Dronsfield MJ, Bain SC, Hattersley AT, Velho G, Froguel P, Bell GI, Polonsky KS: Altered insulin secretory response to glucose in diabetic and nondiabetic subjects with mutations in the diabetes susceptibility gene MODY3 on chromosome 12.
Diabetes
46
:
1503
–1511,
1996
26.
Yamagata K, Yang Q, Yamamoto K, Iwahashi H, Miyagawa J, Okita K, Yoshiuchi I, Miyazaki J, Noguchi T, Nakajima H, Namba M, Hanafusa T, Matsuzawa Y: Mutation P291fsinsC in the transcription factor hepatocyte nuclear factor-1alpha is dominant negative.
Diabetes
47
:
1231
–1235,
1998
27.
Pontoglio M, Sreenan S, Roe M, Pugh W, Ostrega D, Doyen A, Pick AJ, Baldwin A, Velho G, Froguel P, Levisetti M, Bonner-Weir S, Bell GI, Yaniv M, Polonsky KS: Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice. J Clin Invest
101
:
2215
–2222,
1998
28.
Dukes ID, Sreenan S, Roe MW, Levisetti M, Zhou YP, Ostrega D, Bell GI, Pontoglio M, Yaniv M, Philipson L, Polonsky KS: Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice. J Biol Chem
273
:
24457
–24464,
1998
29.
Parrizas M, Maestro MA, Boj SF, Paniagua A, Casamitjana R, Gomis R, Rivera F, Ferrer J: Hepatic nuclear factor 1-α directs nucleosomal hyperacetylation to its tissue-specific transcriptional targets.
Mol Cell Biol
21
:
3234
–3243,
2001
30.
Wang H, Maechler P, Hagenfeldt KA, Wollheim CB: Dominant-negative suppression of HNF-1α function results in defective insulin gene transcription and impaired metabolism-secretion coupling in a pancreatic β-cell line.
EMBO J
17
:
6701
–6713,
1998
31.
Wang H, Antinozzi PA, Hagenfeldt KA, Maechler P, Wollheim CB: Molecular targets of a human HNF1α mutation responsible for pancreatic β-cell dysfunction.
EMBO J
19
:
4257
–4264,
2000
32.
Sturis J, Pugh WL, Tang J, Polonsky KS: Prevention of diabetes does not completely prevent insulin secretory defects in the ZDF rat.
Am J Physiol
269
:
E786
–E792,
1995
33.
Wilson PA, Melton DA: Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication.
Curr Biol
4
:
676
–686,
1994
34.
Alam J, Cook JL: Reporter genes: application to the study of mammalian gene transcription.
Anal Biochem
188
:
245
–254,
1990
35.
Webb GC, Akbar MS, Zhao C, Steiner DF: Expression profiling of pancreatic beta cells: glucose regulation of secretory and metabolic pathway genes. Proc Natl Acad Sci U S A
97
:
5773
–5778,
2000
36.
MacDonald MJ, Kowluru A: Evidence for calcium enhanced phosphorylation of pyruvate kinase by pancreatic islets. Mol Cell Biochem
68
:
107
–114,
1985
37.
MacDonald MJ, Kowluru A, Chang CM: Evidence for phosphorylation of pancreatic islet pyruvate kinase.
Metabolism
34
:
600
–603,
1985
38.
Seol W, Choi HS, Moore DD: An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors.
Science
272
:
1336
–1369,
1996
39.
Lee YK, Dell H, Dowhan DH, Hadzopoulou-Cladaras M, Moore DD: The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression.
Mol Cell Biol
20
:
187
–195,
2000
40.
Lee HK, Lee YK, Park SH, Kim YS, Lee JW, Kwon HB, Soh J, Moore DD, Cho HS: Structure and expression of the orphan nuclear receptor SHP gene.
J Biol Chem
273
:
14398
–14402,
1998
41.
Grupe A, Hultgren B, Ryan A, Ma YH, Bauer M, Stewart TA: Transgenic knockouts reveal a critical requirement for pancreatic beta cell glucokinase in maintaining glucose homeostasis.
Cell
83
:
69
–78,
1995
42.
Guillam MT, Hummler E, Schaerer E, Yeh JI, Birnbaum MJ, Beermann F, Schmidt A, Deriaz N, Thorens B, Wu JY: Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2.
Nat Genet
17
:
327
–330,
1997
43.
Tsukuda N, Gabler WL: The influence of doxycycline of the attachment of fibroblasts to gelatin-coated surfaces and its cytotoxicity.
J Periodontol
64
:
1219
–1224,
1993
44.
Saez E, No D, West A, Evans RM: Inducible gene expression in mammalian cells and transgenic mice.
Curr Opin Biotechnol
8
:
608
–616,
1997
45.
Chouard T, Blumenfeld M, Bach I, Vandekerckhove J, Cereghini S, Yaniv M: A distal dimerization domain is essential for DNA-binding by the atypical HNF1 homeodomain.
Nucleic Acids Res
18
:
5853
–5863,
1990
46.
Asfari M, Janijic D, Meda P, Li G, Halban PA, Wollheim CB: Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines.
Endocrinology
130
:
167
–178,
1992
47.
Mitchell SMS, Thomas H, Jaschkowitz K, Bulman MP, Frayling TM, Ellard S, Tack C, Ryffel GU, Hattersley AT: A mutation of a novel splice variant of HNF-4α co-segregates with MODY.
Diabetologia
In press
48.
Byrne MM, Sturis J, Fajans SS, Stoltz A, Oritz FJ, Stoffel M, Smith MJ, Bell GI, Halter JB, Polonsky KS: Altered insulin secretory responses to glucose in subjects with mutation in the MODY1 gene on chromosome 20.
Diabetes
44
:
699
–705,
1994
49.
Shih DQ, Dansky HM, Fleisher M, Assmann G, Fajans SS, Stoffel M: Genotype phenotype relationships in HNF-4a/MODY1. Haploinsufficiency is associated with reduced apolipoprotein (AII), apolipoprotein (CIII), lipoprotein (a), and triglyceride levels.
Diabetes
49
:
832
–837,
2000
50.
Jonsson J, Carlson L, Edlund T, Edlund H: Insulin-promoter-factor 1 is required for pancreas development in mice.
Nature
371
:
606
–609,
1994
51.
Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H: Beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes.
Genes Dev
12
:
1763
–1768,
1998
52.
Waeber G, Thompson N, Nicod P, Bony C: Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor.
Mol Endocrinol
10
:
1327
–1334,
1998
53.
Carty MD, Lillquist JS, Peshavaria M, Stein R, Soeller WC: Identification of cis- and trans-active factors regulating human islet amyloid polypeptide gene expression in pancreatic beta-cells.
J Biol Chem
272
:
11986
–11993,
1997
54.
Melloul D, Ben-Neriah Y, Cerasi E: Glucose modulates the binding of an islet-specific factor to a conserved sequence within the rat 1 and the human insulin promoters.
Proc Natl Acad Sci U S A
90
:
3865
–3969,
1993
55.
Dumonteil E, Laser B, Constant I, Philippe J: Differential regulation of the glucagon and insulin I gene promoters by the basic helix-loop-helix transcription factors E47 and BETA2.
J Biol Chem
273
:
19945
–19954,
1998
56.
Emens LA, Landers DW, Moss LG: Hepatocyte nuclear factor 1 alpha is expressed in a hamster insulinoma line and transactivates the rat insulin I gene. Proc Natl Acad Sci U S A
89
:
7300
–7304,
1992
57.
Nishigori H, Tomura H, Tonooka N, Kanamori M, Yamada S, Sho K, Inoue I, Kikuchi N, Onigata K, Kojima I, Kohama T, Yamagata K, Yang Q, Matsuzawa Y, Miki T, Seino S, Kim MY, Choi HS, Lee YK, Moore DD, Takeda J: Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects.
Proc Natl Acad Sci U S A
98
:
575
–580,
2001
58.
Vaulont S, Puzenat N, Levrat B, Cognet M, Kahn A, Raymondjean M: Proteins binding to the liver specific pyruvate kinase gene promoter: a unique combination of known factors.
J Mol Biol
209–215,
1989
59.
Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR, Kaufman S: Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. Proc Natl Acad Sci U S A
89
:
11891
–11894,
1992

Address correspondence and reprint requests to Markus Stoffel MD, Laboratory of Metabolic Diseases, The Rockefeller University, 1230 York Ave., Box 292, New York, NY 10021. E-mail: stoffel@rockvax.rockefeller.edu.

Received for publication 26 March 2001 and accepted in revised form 26 July 2001.

D.Q.S. and S.S. contributed equally to the work.

DMEM, Dulbecco’s modified Eagle’s medium; GLUT2, glucose transporter 2; GSIS, glucose-stimulated insulin secretion; HNF-1α, hepatocyte nuclear factor 1α; IPF-1/PDX-1, insulin promoter factor 1; L-Pk, liver pyruvate kinase; MODY, maturity-onset diabetes of the young; Ogdh, 2-oxoglutarate dehydrogenase; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase–polymerase chain reaction; SHP, small heterodimer partner; Ucp2, uncoupling protein 2.