HNF-1 Alpha Research Articles

Expression of Constitutive Androstane Receptor, Hepatic Nuclear Factor 4α, and P450 Oxidoreductase Genes Determines Interindividual Variability in Basal Expression and Activity of a Broad Scope of Xenobiotic Metabolism Genes in the Human Liver

Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4 alpha (HNF4 alpha) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4 alpha expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4 alpha, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

Macrosomia and Hyperinsulinaemic Hypoglycaemia in Patients with Heterozygous Mutations in the HNF4A Gene

BackgroundMacrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY) genes HNF4A (encoding HNF-4α) and HNF1A/TCF1 (encoding HNF-1α), and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice.Methods and FindingsWe examined birthweight and hypoglycaemia in 108 patients from families with diabetes due to HNF4A mutations, and 134 patients from families with HNF1A mutations. Birthweight was increased by a median of 790 g in HNF4A-mutation carriers compared to non-mutation family members (p < 0.001); 56% (30/54) of HNF4A-mutation carriers were macrosomic compared with 13% (7/54) of non-mutation family members (p < 0.001). Transient hypoglycaemia was reported in 8/54 infants with heterozygous HNF4A mutations, but was reported in none of 54 non-mutation carriers (p = 0.003). There was documented hyperinsulinaemia in three cases. Birthweight and prevalence of neonatal hypoglycaemia were not increased in HNF1A-mutation carriers. Mice with pancreatic β-cell deletion of Hnf4a had hyperinsulinaemia in utero and hyperinsulinaemic hypoglycaemia at birth.Conclusions HNF4A mutations are associated with a considerable increase in birthweight and macrosomia, and are a novel cause of neonatal hypoglycaemia. This study establishes a key role for HNF4A in determining foetal birthweight, and uncovers an unanticipated feature of the natural history of HNF4A-deficient diabetes, with hyperinsulinaemia at birth evolving to decreased insulin secretion and diabetes later in life.

Genotype phenotype classification of hepatocellular adenoma

Studies that compare tumor genotype with phenotype have provided the basis of a new histological/molecular classification of hepatocellular adenomas. Based on two molecular criteria (presence of a TCF1/HNF1 alpha or beta-catenin mutation), and an additional histological criterion (presence or absence of an inflammatory infiltrate), subgroups of hepatocellular adenoma can be defined and distinguished from focal nodular hyperplasia. Analysis of 96 hepatocellular adenomas performed by a French collaborative network showed that they can be divided into four broad subgroups: the first one is defined by the presence of mutations in TCF1 gene inactivating the hepatocyte nuclear factor 1 (HNF1 alpha); the second by the presence of beta-catenin activating mutations; the category without mutations of HNF1 alpha or beta-catenin is further divided into 2 subgroups depending on the presence or absence of inflammation. Therefore, the approach to the diagnosis of problematic benign hepatocytic nodules may be entering a new era directed by new molecular information. It is hoped that immunohistological tools will improve significantly diagnosis of liver biopsy in our ability to distinguish hepatocellular adenoma from focal nodular hyperplasia (FNH), and to delineate clinically meaningful entities within each group to define the best clinical management. The optimal care of patients with a liver nodule will benefit from the recent knowledge coming from molecular biology and the combined expertise of hepatologists, pathologists, radiologists, and surgeons.

Aetiological heterogeneity of asymptomatic hyperglycaemia in children and adolescents

Randomly estimated fasting hyperglycaemia in an asymptomatic individual may represent the first sign of pancreatic beta-cell dysfunction. We aimed at specifying the genetic aetiology of asymptomatic hyperglycaemia in a cohort of children and adolescents. We analysed the aetiological diagnosis in 82 non-obese paediatric subjects (38 males) aged 0.2-18.5 years (median: 13.1) who were referred for elucidation of a randomly found blood glucose level above 5.5 mmol/l. In addition to fasting glycaemia and circulating levels of insulin and C-peptide, the subjects were tested by an oral glucose tolerance test and an intravenous glucose tolerance test and screened for mutations in the genes encoding glucokinase (GCK), HNF-1alpha (TCF1), Kir6.2 (KCNJ11) (if aged <2 years) and HNF-4alpha (HNF4A) (those with a positive family history of diabetes). We identified 35 carriers of GCK mutations causing MODY2, two carriers of TCF1 mutations causing MODY3, one carrier of a HNF4A mutation causing MODY1 and one carrier of a KCNJ11 mutation causing permanent neonatal diabetes mellitus. Of the remaining patients, 11 progressed to type 1 diabetes mellitus (T1DM) and 9 had impaired glucose tolerance or diabetes mellitus of unknown origin. In 23 subjects, an impairment of blood glucose levels was not confirmed. We conclude that 39 of 82 paediatric patients (48%) with randomly found fasting hyperglycaemia suffered from single gene defect conditions, MODY2 being the most prevalent. An additional 11 patients (13%) progressed to overt T1DM. The aetiological diagnosis in asymptomatic hyperglycaemic children and adolescents is a clue to introducing an early and effective therapy or, in MODY2, to preventing any future extensive re-investigations.

PII-27Genetic polymorphism of HNF-4 alpha in a Korean population: Identification of a novel functional HNF-4 alpha variant with altered transactivation activity toward CYP2D6.

PII-27Genetic polymorphism of HNF-4 alpha in a Korean population: Identification of a novel functional HNF-4 alpha variant with altered transactivation activity toward CYP2D6.

Bile acids and cytokines inhibit the human cholesterol 7α‐hydroxylase gene via the JNK/c‐jun pathway in human liver cells†

Cholesterol 7 alpha-hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen-activated protein kinase/c-Jun N-terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c-Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c-Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin-1 beta (IL-1 beta) regulation of human CYP7A1 gene expression via real-time polymerase chain reaction, reporter assays, co-immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL-1 beta and CDCA reduced CYP7A1 but induced c-Jun messenger RNA expression in human primary hepatocytes. IL-1beta inhibited human CYP7A1 reporter activity via the HNF4 alpha binding site. A JNK-specific inhibitor blocked the inhibitory effect of IL-1 beta on HNF4 alpha expression and CYP7A1 reporter activity. c-Jun inhibited HNF4 alpha and PPARgamma coactivator-1 alpha (PGC-1 alpha) coactivation of CYP7A1 reporter activity, whereas a dominant negative c-Jun did not. Co-immunoprecipitation and ChIP assays revealed that IL-1 beta and CDCA reduced HNF4 alpha bound to the CYP7A1 chromatin, and that c-Jun interacted with HNF4 alpha and blocked HNF4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin. In conclusion, IL-1 beta and CDCA inhibit HNF4 alpha but induce c-Jun, which in turn blocks HNF 4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents.

The Human Organic Anion Transporter 2 Gene Is Transactivated by Hepatocyte Nuclear Factor-4α and Suppressed by Bile Acids

The human organic anion transporter 2 (hOAT2, SLC22A7) mediates the sodium-independent uptake of numerous drugs, including cephalosporins, salicylates, dicarboxylates, and prostaglandins, and is mainly expressed in hepatocytes. Because the regulation of hOAT2 expression is poorly understood, we characterized cis-acting elements in the 5'-flanking region that regulate hOAT2 transcription. A consensus binding motif for the hepatocyte nuclear factor-4 alpha (HNF-4 alpha), arranged as a direct repeat (DR)-1, is located at nucleotides -329/-317 relative to the transcription initiation site. This element specifically binds HNF-4 alpha in electrophoretic mobility shift assays. A luciferase-linked hOAT2 promoter fragment containing the HNF-4 alpha binding site was transactivated upon cotransfection of an HNF-4 alpha expression vector in Huh7 cells, whereas site-directed mutagenesis of the DR-1 element abolished activation by HNF-4 alpha. Short interfering RNAs inhibiting endogenous HNF-4 alpha expression markedly reduced endogenous expression of hOAT2 in Huh7 cells. Because HNF-4 alpha is a known target for bile acid-mediated repression of gene transcription, we studied whether chenodeoxycholic acid (CDCA) suppresses hOAT2 gene expression by inhibiting HNF-4 alpha-mediated transactivation. Treatment of Huh7 cells with CDCA or the synthetic farnesoid X receptor (FXR) agonist GW4064 decreased mRNA and protein levels and also nuclear binding activity of HNF-4 alpha. The FXR-inducible transcriptional repressor small heterodimer partner inhibited transactivation of hOAT2 promoter constructs and of endogenous hOAT2 expression by HNF-4 alpha. We conclude that the hOAT2 gene is critically dependent on HNF-4 alpha and that bile acids repress the hOAT2 gene by inhibiting HNF-4 alpha. Hepatic uptake of hOAT2 substrates may thus be decreased in disease conditions associated with elevated intracellular levels of bile acids.

Oligonucleotide chip for the diagnosis of HNF-1α mutations

Mutations in HNF-1 alpha cause maturity-onset diabetes of the young (MODY) type 3, which is the most prevalent MODY subtype in most countries. In the present study, we investigated an oligonucleotide microchip for the detection of the known HNF-1 alpha mutations. We first optimized the coupling chemistries for covalent immobilization of allele-specific oligonucleotides on aldehyde (CHO)- and thiocyanate (NCS)-activated glass slides and compared their hybridization efficiencies. CHO-glass was found to provide a more favorable environment for hybridization than NCS-glass, whereas the binding capacity of NCS-glass for amine-activated oligonucleotide was much greater than with CHO-glass. We also investigated the effects of the length of the capture probes on the hybridized signals. To determine the presence of HNF-1 alpha mutations in a human sample, we prepared an oligonucleotide chip from selected mutation sites of exon2 from HNF-1 alpha. Cy3-labeled RNA target probes were obtained by in vitro transcription of promoter-tagged PCR products from a wild-type blood sample and subsequent fragmentation. Hybridization of the chip with the RNA target probes successfully identified all of the genotypes for the tested sites. This work demonstrates that oligonucleotide chip-based analysis is a good candidate for routine clinical testing for HNF-1 alpha mutations.

A New Mutation in the Hepatocyte Nuclear Factor-1-Alpha Gene (P224S) in a Newly Discovered German Family with Maturity-Onset Diabetes of the Young 3 (MODY 3). Family Members Carry Additionally the Homozygous I27L Amino Acid Polymorphism in the HNF1 Alpha Gene

Mutations in the hepatocyte nuclear factor-1-alpha gene cause maturity onset diabetes of the young 3 (MODY 3). Here we describe a new family affected by this disorder carrying the so far unknown mutation Pro224Ser in exon 3. First we identified a 17-year-old patient. OGTT demonstrated that insulin secretion was severely impaired: basal insulin was 3.7 uU/ml and 60 min after an oral glucose load plasma insulin peaked only threefold to 10.7 uU/ml. In addition, this patient carries the homozygous polymorphism Ile27Leu (exon1) in the hepatocyte nuclear factor-1-alpha gene that was shown to be associated with insulin resistance. So far, we have no evidence for insulin resistance in this individual patient. Additionally, two other family members carry the hepatocyte nuclear factor-1-alpha mutation Pro224Ser and the homozygous polymorphism Ile27Leu. A similar case with these two mutations in the HNF-1-alpha gene has not been described before. This data will allow to discover more patients with MODY 3.

Hepatocyte Nuclear Factor-1α Gene Inactivation: Cosegregation between Liver Adenomatosis and Diabetes Phenotypes in Two Maturity-Onset Diabetes of the Young (MODY)3 Families

Heterozygous germline mutations of the hepatocyte nuclear factor (HNF)-1 alpha are associated with maturity-onset diabetes of the young (MODY)3. Recently, the biallelic inactivation of the HNF-1 alpha gene was reported in liver adenomas. We show the occurrence of liver adenomatosis in six MODY3-affected patients from two unrelated and large families. Liver adenomatosis was characterized by the presence of numerous adenomas within a normal hepatic parenchyma. The HNF-1 alpha hot-spot germline mutation P291fs was identified in the two probands and in 16 relatives from the two families. The six patients affected by liver adenomatosis and diabetes exhibited the mutation. The analysis of liver-cell tumors from two affected patients evidenced the biallelic inactivation of HNF-1 alpha. The familial screening confirmed the clinical heterogeneity of the liver phenotype, from silent liver adenomatosis to fatal hemorrhage. These observations warrant the systematic screening for liver adenomatosis in MODY3 families to prevent its potentially deadly complications. Moreover, such screening may help to determine if a particular mutational spectrum of HNF-1 alpha is associated with liver adenomatosis and to establish its prevalence in this frequent form of diabetes in the young adult.

Physical and Functional Interaction of Acyl-CoA-binding Protein with Hepatocyte Nuclear Factor-4α

Although acyl-CoA-binding protein (ACBP) has been detected in the nucleus, the physiological significance of this observation is unknown. As shown herein for the first time, ACBP in the nucleus physically and functionally interacted with hepatocyte nuclear factor-4 alpha (HNF-4 alpha), a nuclear binding protein that regulates transcription of genes involved in both lipid and glucose metabolism. Five lines of evidence showed that ACBP bound HNF-4 alpha in vitro and in the nucleus of intact cells. (i) ACBP interaction with HNF-4 alpha elicited significant changes in secondary structure. (ii) ACBP and HNF-4 alpha were coimmunoprecipitated by antibodies to each protein. (iii) Double immunolabeling and laser scanning confocal microscopy (LSCM) of rat hepatoma cells and transfected COS-7 cells significantly colocalized ACBP and HNF-4 alpha within the nucleus and in the perinuclear region close to the nuclear membrane. (iv) LSCM fluorescence resonance energy transfer determined an intermolecular distance of 53 A between ACBP and HNF-4 alpha in rat hepatoma cell nuclei. (v) Immunogold electron microscopy detected ACBP within 43 A of HNF-4 alpha. These interactions were specific since ACBP did not interact with Sp1 or glucocorticoid receptor in these assays. The functional significance of ACBP interaction with HNF-4 alpha was evidenced by mammalian two-hybrid and transactivation assays. ACBP overexpression in COS-7 or rat hepatoma cells enhanced transactivation of an HNF-4 alpha-dependent luciferase reporter plasmid by 3.2- and 1.6-fold, respectively. In contrast, cotransfection with antisense ACBP expression vector inhibited transactivation. LSCM of the individual triple fluorescent-labeled (HNF-4 alpha, ACBP, and luciferase) rat hepatoma cells showed a high correlation (r2, 0.936) between the level of luciferase and the level of ACBP expression. In summary, ACBP physically interacted with HNF-4 alpha in vitro and in intact cells, although ACBP expression level directly correlated with HNF-4 alpha-mediated transactivation in individual cells.

Expression of HNFs and C/EBP alpha is correlated with immunocytochemical differentiation of cell lines derived from human hepatocellular carcinomas, hepatoblastomas and immortalized hepatocytes.

Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver-enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBP alpha using real-time reverse-transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B-17/Icrj-scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF-3beta, HNF-4 alpha, HNF-1 alpha, and C/EBP alpha were specific to HCCs with well-differentiated function and morphology. Furthermore, among these four transcription factors, HNF-4 alpha and HNF-1 alpha expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF-4 alpha and HNF-1 alpha are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBP alpha in HCCs.

Association of I27L polymorphism of hepatocyte nuclear factor-1 alpha gene with high-density lipoprotein cholesterol level.

The serum level of high-density lipoprotein cholesterol (HDL-c), which protects against the development of atherosclerosis, is under genetic control. However, the genetic components responsible for the serum HDL-c level are yet to be determined. A recent knockout mouse study demonstrated that hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is an essential transcriptional regulator of HDL-c metabolism. In this study, the association of an HNF-1 alpha gene polymorphism, isoleucine (Ile) 27 leucine (Leu), with lipid parameters, in particular with serum HDL-c level, was studied in 356 unrelated Japanese men. Though no significant difference was observed in total cholesterol and triglyceride levels among the three genotypes, the serum HDL-c level was significantly associated with the genotype (P < 0.01, trend test). Subjects with the Ile/Ile genotype had low serum HDL-c levels, and those with the Leu/Leu genotype had high serum HDL-c levels. These results demonstrate that the HNF-1 alpha gene locus is associated with serum HDL-c level and suggest that the Ile27 allele is a risk marker for atherosclerosis.

Hepatocyte nuclear factor 4 alpha isoforms originated from the P1 promoter are expressed in human pancreatic beta-cells and exhibit stronger transcriptional potentials than P2 promoter-driven isoforms.

The nuclear receptor hepatocyte nuclear factor (HNF) 4 alpha is involved in a transcriptional network and plays an important role in pancreatic beta-cells. Mutations in the HNF4 alpha gene are correlated with maturity-onset diabetes of the young 1. HNF4 alpha isoforms result from both alternative splicing and alternate usage of promoters P1 and P2. It has recently been reported that HNF4 alpha transcription is driven almost exclusively by the P2 promoter in pancreatic islets. We observed that transcripts from both P1 and P2 promoters were expressed in human pancreatic beta-cells and in the pancreatic beta-cell lines RIN m5F and HIT-T15. Expression of HNF4 alpha proteins originating from the P1 promoter was confirmed by immunodetection. Due to the presence of the activation function module AF-1, HNF4 alpha isoforms originating from the P1 promoter exhibit stronger transcriptional activities and recruit coactivators more efficiently than isoforms driven by the P2 promoter. Conversely, activities of isoforms produced by both promoters were similarly repressed by the corepressor small heterodimer partner. These behaviors were observed on the promoter of HNF1 alpha that is required for beta-cell function. Our results highlight that expression of P1 promoter-driven isoforms is important in the control of pancreatic beta-cell function.

Hepatocyte nuclear factor-1 alpha gene mutations and diabetes in Norway.

Mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene cause maturity-onset diabetes of the young (MODY), type 3. To estimate the prevalence of MODY3 in Norwegian diabetic pedigrees, we screened a total of 130 families for HNF-1 alpha mutations; 42 families with clinical MODY, 75 with suspected MODY, and 13 pedigrees with multiplex type 1 diabetes. Twenty-two families with clinical MODY, 15 families with suspected MODY, and one family with type 1 diabetes multiplex harbored HNF-1 alpha mutations. Thus, in about half of Norwegian families with clinical MODY, mutations in the HNF-1 alpha gene could be detected. Eight of the 18 different mutations identified were novel (G47E, T196fsdelCCAA, IVS3-1G>A, S256T, A276D, S445fsdelAG, M522V, and S531T). Haplotypes were determined for recurrent mutations, indicating a founder effect in Norway for the hot-spot mutation P291fsinsC and possibly also for P112L and R131W. To examine the molecular mechanisms underlying MODY3, we investigated the functional properties of 13 HNF-1 alpha mutations. Two mutant HNF-1 alpha proteins (R171X, R263C) were unable to bind DNA and at least five mutants (R131W, R171X, P379fsdelCT, S445fsdelAG, and Q466X) showed defective nuclear translocation. Transcriptional activation was reduced for most of the MODY3-associated mutants. Accordingly, the functional studies of HNF-1 alpha mutants indicate that beta-cell dysfunction in MODY3 is caused by loss-of-function mechanisms like reduced DNA binding, impaired transcriptional activation, and defects in subcellular localization.

Hepatocyte nuclear factor-4 alpha/gamma and hepatocyte nuclear factor-1 alpha as causal factors of interindividual difference in the expression of human dihydrodiol dehydrogenase 4 mRNA in human livers.

Human dihydrodiol dehydrogenase (DD) catalyses the oxidation of trans-dihydrodiols of polycyclic aromatic hydrocarbons and the reduction of several ketone-containing drugs. About 40-fold interindividual difference in DD activities has been noted. Recently, we found that transcriptional factors, hepatocyte nuclear factor (HNF)-1 alpha, HNF-4 alpha and HNF-4 gamma were essential for the expression of DD4 mRNA, which is a major form of DDs. Thus, to clarify a possible mechanism(s) underlying the interindividual difference in DD activities, we investigated the sequences of genes and the expression levels of mRNA for DD4 and HNFs in human livers. We found no clear relationship between the genotypes of DD4 and HNF genes and the expression levels of DD4 mRNA in the subjects. The expression level of DD4 mRNA significantly correlated with that of HNF-1 alpha, HNF-4 alpha or HNF-4 gamma. These results suggest that the expression level of DD4 mRNA is cooperatively regulated by the amounts of HNF-1 alpha, HNF-4 alpha and HNF-4 gamma.

Characterization of a naturally occurring mutation (L107I) in the HNF1 alpha (MODY3) gene.

Maturity onset diabetes of the young type 3 (MODY3) is a monogenic form of diabetes mellitus caused by mutations in the gene encoding for hepatocyte nuclear factor 1 alpha, HNF1 alpha. In this study we have examined the in vivo and in vitro effects of a mutation (L107I) outside the DNA binding and dimerization domains in the N terminal part of the HNF1 alpha gene. Beta-cell function of the affected family members was assessed by an oral glucose tolerance test. Functional tests were carried out to explain the role of the mutation in vitro by transcriptional activity assay, Western blotting, DNA-binding assays and subcellular localization experiments. Affected family members showed an 86% decreased insulin response to glucose when compared to age-matched healthy control subjects. In vitro the mutation showed a 79% decrease in transcriptional activity as compared to wild type HNF1 alpha in HeLa cells lacking HNF1 alpha. The transcriptional activity was not suppressed when the mutant was co-expressed with wild type HNF1 alpha suggesting that the decreased activity was not mediated by a dominant negative mechanism. The L107I/HNF1alpha protein showed normal nuclear targeting but impaired binding to an HNF1 alpha consensus sequence. Our results suggest that the L107I substitution represents a MODY3 mutation which impairs beta-cell function by a loss-of-function mechanism.

The Coactivator PGC-1 Is Involved in the Regulation of the Liver Carnitine Palmitoyltransferase I Gene Expression by cAMP in Combination with HNF4α and cAMP-response Element-binding Protein (CREB)

Liver carnitine palmitoyltransferase I catalyzes the transfer of long-chain fatty acids into mitochondria. L-CPT I is considered the rate-controlling enzyme in fatty acid oxidation. Expression of the L-CPT I gene is induced by starvation in response to glucagon secretion from the pancreas, an effect mediated by cAMP. Here, the molecular mechanisms underlying the induction of L-CPT I gene expression by cAMP were characterized. We demonstrate that the cAMP response unit of the L-CPT I gene is composed of a cAMP-response element motif and a DR1 sequence located 3 kb upstream of the transcription start site. Our data strongly suggest that the coactivator PGC-1 is involved in the regulation of this gene expression by cAMP in combination with HNF4 alpha and cAMP-response element-binding protein (CREB). Indeed, (i) cotransfection of CREB or HNF4 alpha dominant negative mutants completely abolishes the effect of cAMP on the L-CPT I promoter, and (ii) the cAMP-responsive unit binds HNF4 alpha and CREB through the DR1 and the cAMP-response element sequences, respectively. Moreover, cotransfection of PGC-1 strongly activates the L-CPT I promoter through HNF4 alpha bound at the DR1 element. Finally, we show that the transcriptional induction of the PGC-1 gene by glucagon through cAMP in hepatocytes precedes that of L-CPT-1. In addition to the key role that PGC-1 plays in glucose homeostasis, it may also be critical for lipid homeostasis. Taken together these observations suggest that PGC-1 acts to coordinate the process of metabolic adaptation in the liver.

Crystal Structure of the HNF4α Ligand Binding Domain in Complex with Endogenous Fatty Acid Ligand

HNF4 alpha is an orphan member of the nuclear receptor family with prominent functions in liver, gut, kidney and pancreatic beta cells. We have solved the x-ray crystal structure of the HNF4 alpha ligand binding domain, which adopts a canonical fold. Two conformational states are present within each homodimer: an open form with alpha helix 12 (alpha 12) extended and collinear with alpha 10 and a closed form with alpha 12 folded against the body of the domain. Although the protein was crystallized without added ligands, the ligand binding pockets of both closed and open forms contain fatty acids. The carboxylic acid headgroup of the fatty acid ion pairs with the guanidinium group of Arg(226) at one end of the ligand binding pocket, while the aliphatic chain fills a long, narrow channel that is lined with hydrophobic residues. These findings suggest that fatty acids are endogenous ligands for HNF4 alpha and establish a framework for understanding how HNF4 alpha activity is enhanced by ligand binding and diminished by MODY1 mutations.

Abnormal splicing of hepatocyte nuclear factor 1 alpha in maturity-onset diabetes of the young.

Mutations in the HNF-1 alpha gene result in maturity-onset diabetes of the young (MODY); an early-onset, dominantly inherited form of diabetes caused by pancreatic beta-cell dysfunction. Splice site mutations represent approximately 10% of reported HNF-1 alpha mutations. No studies to date have investigated the effect of splice site mutations on mRNA processing because the tissues with abundant HNF-1alpha expression (liver, pancreas, kidney and gut) are not easily accessible for analysis. This study aimed to define the pathogenic mechanism in three novel splice site mutations by analysing illegitimate transcripts. To assess the consequence of potential HNF-1 alpha splice site mutations we developed a nested reverse transcriptase PCR (RT-PCR) assay for the amplification of illegitimate HNF-1 alpha transcripts in Epstein Barr virus transformed lymphoblastoid cell lines. Sequencing the illegitimate HNF-1 alpha transcripts showed that the splice donor site mutation IVS8nt+1G>A leads to complete skipping of exon 8, the splice acceptor site mutation IVS4nt-2A>G causes skipping of exon 5 with the recruitment of a cryptic splice acceptor site within intron 5 and the cryptic splice acceptor site mutation (IVS7nt-6G>A) resulted in the skipping of exon 7. All three changes are predicted to result in premature termination of the HNF-1alpha protein, providing further evidence for their role as pathogenic mutations. We conclude that the sequencing of illegitimate transcripts from lymphoblastoid cell lines is helpful in the assessment of intronic variation in HNF-1 alpha that could alter splicing. This analysis of the mRNA is required to define mutational mechanisms and confirm pathogenic status.