HNF-1alpha Gene Research Articles

Defective Pancreatic β-Cell Glycolytic Signaling in Hepatocyte Nuclear Factor-1α-deficient Mice

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1alpha gene, insulin secretion and intracellular calcium ([Ca2+]i) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+ currents (KATP) in beta-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+]i, and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1alpha diabetes results from defective beta-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.

Mutation screening in 18 Caucasian families suggest the existence of other MODY genes.

Maturity-onset diabetes of the young (MODY) is a heterogeneous subtype of non-insulin-dependent diabetes mellitus characterised by early onset, autosomal dominant inheritance and a primary defect in insulin secretion. To date five MODY genes have been identified: hepatocyte nuclear factor-4 alpha (HNF-4alpha/MODY1/TCF14) on chromosome 20q, glucokinase (GCK/MODY2) on chromosome 7p, hepatocyte nuclear factor-1 alpha (HNF-1alpha/MODY3/TCF1) on chromosome 12q, insulin promoter factor-1 (IPF1/MODY4) on chromosome 13q and hepatocyte nuclear factor-1 beta (HNF-1beta/MODY5/TCF2) on chromosome 17cen-q. We have screened the HNF-4alpha, HNF-1alpha and HNF-1beta genes in members of 18 MODY kindreds who tested negative for glucokinase mutations. Five missense (G31D, R159W, A161T, R200W, R271W), one substitution at the splice donor site of intron 5 (IVS5nt + 2T-->A) and one deletion mutation (P379fsdelT) were found in the HNF-1alpha gene, but no MODY-associated mutations were found in the HNF-4alpha and HNF-1beta genes. Of 67 French MODY families that we have now studied, 42 (63%) have mutations in the glucokinase gene, 14 (21%) have mutations in the HNF-1alpha gene, and 11 (16%) have no mutations in the HNF-4alpha, IPF1 and HNF-1beta genes. Eleven families do not have mutations in the five known MODY genes suggesting that there is at least one additional locus that can cause MODY.

Phenotypic effects of the forced expression of HNF4 and HNF1alpha are conditioned by properties of the recipient cell.

Tagged versions of HNF4 or HNF1alpha cDNAs in expression vectors have been introduced by transient and stable transfection into three cell lines of hepatic origin that all fail to express these two liver-enriched transcription factors and hepatic functions. C2 and H5 cells are dedifferentiated rat hepatoma variants and WIF12-E cells are human fibroblast-rat hepatoma hybrids with a reduced complement of human chromosomes. Transfectants were analyzed for the expression state of the endogenous genes coding for these transcription factors and for hepatic functions. Each cell line showed a different response to the forced expression of the transcription factors. In C2 cells, no measurable effect was observed, either upon transitory or stable expression. H5 cells reexpressed the endogenous HNF4 gene only upon transient HNF1alpha transfection, and the endogenous HNF1alpha gene only in stable HNF4 transfectants. WIF12-E cells responded to the forced transient or stable expression of either HNF1alpha or HNF4 by cross-activation of the corresponding endogenous gene. In addition, the stable transfectants reexpress HNF3alpha and C/EBPalpha, as well as all of the hepatic functions examined. Hybrid cells similar to WIF12-E had previously been observed to show pleiotropic reexpression of the hepatic phenotype in parallel with loss of human chromosome 2. For the stable WIF12-E transfectants, it was verified that reexpression of the hepatic phenotype was not due to loss of human chromosome 2. The demonstration of reciprocal cross-regulation between HNF4 and HNF1alpha in transient as well as stable transfectants implies that direct effects are involved.

Linkage and molecular scanning analyses of MODY3/hepatocyte nuclear factor-1 alpha gene in typical familial type 2 diabetes: evidence for novel mutations in exons 8 and 10.

Mutations of the hepatocyte nuclear factor-1 alpha (HNF1 alpha) gene are an important cause of autosomal dominant diabetes with onset before age 25 yr [maturity-onset diabetes of the young (MODY)], and some regions of the HNF1 alpha gene appear to be hot spots for mutations. To evaluate the role of HNF1 alpha in the more common familial type 2 diabetes, we studied 62 families of Northern European origin by linkage analysis and molecular screening. Linkage was rejected under dominant models consistent with either late-onset type 2 diabetes or early-onset dominant diabetes. We used single strand conformation polymorphism analysis to screen 53 diabetic members of 36 families who reported diabetes diagnosed before age 40 yr, 9 members of 2 Utah families with typical MODY, and 24 additional members of families with possible linkage. One MODY family showed the previously reported frameshift mutation (P291fsinsC) in exon 4. Among the individuals with more typical type 2 diabetes, we identified the previously reported common polymorphisms, a new intronic polymorphism, and 3 common amino acid variants. We also identified 2 novel missense mutations that segregated with type 2 diabetes in 1 family each: lysine for glutamic acid substitution at codon 619 in exon 10 (E619K), and an arginine for threonine substitution at codon 537 in exon 8 (R537T) in a second family. The exon 8 mutation showed relatively low penetrance, and the role in this family remains uncertain. No coding mutations were identified in the family members screened on the basis of linkage but without early-onset diabetes. Although HNF1 alpha mutations are not a common cause of familial type 2 diabetes, they may account for 5% of families in which at least 1 member has onset of type 2 diabetes before age 40 yr. Incomplete penetrance and a high sporadic frequency make linkage an inefficient screening tool.

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.

A missense mutation in hepatocyte nuclear factor-4 alpha, resulting in a reduced transactivation activity, in human late-onset non-insulin-dependent diabetes mellitus.

Non-insulin-dependent diabetes mellitus (NIDDM) is a heterogeneous disorder characterized by hyperglycemia resulting from defects in insulin secretion and action. Recent studies have found mutations in the hepatocyte nuclear factor-4 alpha gene (HNF-4alpha) in families with maturity-onset diabetes of the young (MODY), an autosomal dominant form of diabetes characterized by early age at onset and a defect in glucose-stimulated insulin secretion. During the course of our search for susceptibility genes contributing to the more common late-onset NIDDM forms, we observed nominal evidence for linkage between NIDDM and markers in the region of the HNF-4alpha/MODY1 locus in a subset of French families with NIDDM diagnosed before 45 yr of age. Thus, we screened these families for mutations in the HNF-4alpha gene. We found a missense mutation, resulting in a valine-to-isoleucine substitution at codon 393 in a single family. This mutation cosegregated with diabetes and impaired insulin secretion, and was not present in 119 control subjects. Expression studies showed that this conservative substitution is associated with a marked reduction of transactivation activity, a result consistent with this mutation contributing to the insulin secretory defect observed in this family.

Hepatic function in a family with a nonsense mutation (R154X) in the hepatocyte nuclear factor-4alpha/MODY1 gene.

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic disorder characterized by autosomal dominant inheritance, onset usually before 25 yr of age, and abnormal pancreatic beta-cell function. Mutations in the hepatocyte nuclear factor(HNF)-4alpha/MODY1, glucokinase/MODY2, and HNF-1alpha/MODY3 genes can cause this form of diabetes. In contrast to the glucokinase and HNF-1alpha genes, mutations in the HNF-4alpha gene are a relatively uncommon cause of MODY, and our understanding of the MODY1 form of diabetes is based on studies of only a single family, the R-W pedigree. Here we report the identification of a second family with MODY1 and the first in which there has been a detailed characterization of hepatic function. The affected members of this family, Dresden-11, have inherited a nonsense mutation, R154X, in the HNF-4alpha gene, and are predicted to have reduced levels of this transcription factor in the tissues in which it is expressed, including pancreatic islets, liver, kidney, and intestine. Subjects with the R154X mutation exhibited a diminished insulin secretory response to oral glucose. HNF-4alpha plays a central role in tissue-specific regulation of gene expression in the liver, including the control of synthesis of proteins involved in cholesterol and lipoprotein metabolism and the coagulation cascade. Subjects with the R154X mutation, however, showed no abnormalities in lipid metabolism or coagulation except for a paradoxical 3.3-fold increase in serum lipoprotein(a) levels, nor was there any evidence of renal dysfunction in these subjects. The results suggest that MODY1 is primarily a disorder of beta-cell function.

Mutations in the hepatocyte nuclear factor-1alpha/MODY3 gene in Japanese subjects with early- and late-onset NIDDM.

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-1alpha gene are the cause of maturity-onset diabetes of the young type 3 (MODY3). We have screened 193 unrelated Japanese subjects with NIDDM for mutations in this gene: 83 with early-onset NIDDM (diagnosis at <30 years of age) and 110 with late-onset NIDDM (diagnosis > or = 30 years of age). All of the members of the latter group also had at least one sibling with NIDDM. The 10 exons, flanking introns, and promoter region were amplified using polymerase chain reaction and were sequenced directly. Mutations were found in 7 of the 83 (8%) unrelated subjects with early-onset NIDDM. The mutations were each different and included four missense mutations (L12H, R131Q, K205Q, and R263C) and three frameshift mutations (P379fsdelCT, T392fsdelA, and L584S585fsinsTC). One of the 110 subjects with late-onset NIDDM was heterozygous for the missense mutation G191D. This subject, who was diagnosed with NIDDM at 64 years of age, also had a brother with NIDDM (age at diagnosis, 54 years) who carried the same mutation, suggesting that this mutation contributed to the development of NIDDM in these two siblings. None of these mutations were present in 50 unrelated subjects with normal glucose tolerance (100 normal chromosomes). Mutations in the HNF-1alpha gene occur in Japanese subjects with NIDDM and appear to be an important cause of early-onset NIDDM in this population. In addition, they are present in about 1% of subjects with late-onset NIDDM.

Studies of the genetic variability of the coding region of the hepatocyte nuclear factor-4alpha in Caucasians with maturity onset NIDDM.

Mutations in the hepatocyte nuclear factor-4alpha (HNF-4alpha) gene cause the type 1 form of maturity onset diabetes of the young (MODY1). To address the question of whether genetic variability of HNF-4alpha is associated with late onset non-insulin-dependent diabetes mellitus (NIDDM) we have sequenced the coding region and intron/exon boundaries of the gene in 36 randomly recruited Danish NIDDM patients. Two nucleotide substitutions that changed the sequence of HNF-4alpha were identified: Thr/Ile130, which has been reported previously and a novel Val/Met255. The Val/Met 255 mutation was found in 4 of 477 Danish NIDDM patients and in none of 217 glucose tolerant control subjects; thus it cannot be excluded that this mutation may have an impact on NIDDM susceptibility. Among 509 NIDDM patients the allelic frequency of the Thr/Ile130 variant was 4.7% (95% confidence interval: 3.4-6.0%) compared to 1.9% (0.7-3.1%) among 239 control subjects (p = 0.008). However, in a population sample of 942 Swedish men with an average age of 70 years the allelic frequency of the variant was similar in 246 men with either impaired glucose tolerance (5.6% [2.6-8.6%]) or NIDDM (5.4% [2.7-8.1%]) as compared to 666 glucose tolerant men (5.1% [3.9-6.3%]). Also in a population sample of 369 young healthy Danes the prevalence of the codon 130 variant (4.7% [3.2-6.2%]) was similar to what was found in Swedish Caucasians. Thus, the allelic frequency of the Thr/Ile130 variant among the control subjects in the Danish case-control study deviates from the prevalence in the two other studies which is why we consider the significant association between the codon 130 variant and NIDDM an incidental finding. In glucose tolerant subjects the codon 130 variant in its heterozygous form had no major effect on glucose-induced insulin and C-peptide release although a tendency to a lower insulin secretion during an oral glucose tolerance test was seen in middle-aged subjects. In conclusion, variability in the coding region of the HNF-4alpha gene is not a common cause of NIDDM among whites of Danish ancestry. However, a Val/Met255 mutation was found exclusively in NIDDM patients (0.8% of cases) and functional as well as family segregation studies are needed to determine whether this HNF-4alpha variant is a NIDDM causing mutation.

Hepatocyte nuclear factor-4 prevents silencing of hepatocyte nuclear factor-1 expression in hepatoma x fibroblast cell hybrids.

Hepatocyte nuclear factors-1alpha (HNF1alpha) and -4 (HNF4) are components of a liver-enriched transcription activation pathway which is thought to play a critical role in hepatocyte-specific gene expression, including activation of alpha1-antitrypsin gene expression. HNF1alpha, HNF4 and alpha1-antitrypsin (alpha1AT) genes are extinguished in hepatoma/fibroblast somatic cell hybrids, suggesting that fibroblasts contain a repressor-like activity. To determine the molecular basis for silencing of these genes in cell hybrids, ectopic expression of HNF1alpha and HNF4 was used. Results show that constitutive expression of HNF4 prevents extinction of HNF1alpha gene expression in hepatoma/fibroblast hybrids. In contrast, forced HNF1alpha expression failed to prevent extinction of the HNF4 locus in cell hybrids. Likewise, the alpha1AT gene remained silent in the presence of both HNF1alpha and HNF4. These results suggest that extinction of HNF1alpha is a simple lack-of-activation phenotype, whereas extinction of HNF4 andalpha1AT loci is more complex, perhaps involving negative regulation.

Mutations in the hepatocyte nuclear factor-1alpha gene are a common cause of maturity-onset diabetes of the young in the U.K.

Mutations in the hepatocyte nuclear factor-1alpha (HNF1alpha) gene have recently been shown to cause maturity-onset diabetes of the young (MODY). We have examined 15 U.K. MODY families for mutations in the coding region of the HNF-1alpha gene. Eight different mutations, three frameshift (P291fsinsC, P379fsdelCT, and A443fsdelCA) and five missense mutations (P129T, R131W, R159W, P519L, and T620I), were identified in eleven families (73%). The previously reported mutation P291fsinsC was found in four pedigrees. A screen of a further 32 probands with early onset (<40 years of age) NIDDM showed the mutation in two additional families. This common mutation was present on at least three different haplotypes, suggesting that its high frequency is due to recurrent mutation rather than a founder effect. We have demonstrated that mutations in the HNF-1alpha gene are a common cause of MODY in U.K. families and result in early onset NIDDM with a progressive clinical course. Mutation-based genetic counseling can now be considered for the majority of patients with MODY.

Identification of nine novel mutations in the hepatocyte nuclear factor 1 alpha gene associated with maturity-onset diabetes of the young (MODY3).

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous subtype of non-insulin-dependent diabetes mellitus (NIDDM) characterised by early onset, autosomal dominant inheritance and a primary defect in insulin secretion. Recent studies have shown that mutations in the two functionally related transcription factors, hepatocyte nuclear factor 4 alpha (HNF-4alpha) and hepatocyte nuclear factor 1 alpha (HNF-1alpha) are associated with the MODY1 and MODY3 forms of diabetes respectively, whereas mutations in the enzyme glucokinase are the cause of the MODY2 form. We have examined 10 unrelated Caucasian families in which MODY/NIDDM co-segregated with markers for MODY3 for mutations in the HNF-1alpha gene (TCF1). Ten different mutations were observed in these families, all of which co-segregated with diabetes. There were no obvious relationships between the nature of the mutations observed (i.e. frameshift, nonsense, or missense) or their location in the gene with clinical features of diabetes (age at onset, severity) in these families. The mechanisms by which mutations in the HNF-1alpha gene cause diabetes mellitus are unclear but might include abnormal pancreatic islet development during foetal life thereby limiting their later function, as well as impaired transcriptional regulation of genes that play a key role in normal pancreatic beta cell function.

Genetic variation in the hepatocyte nuclear factor-1 alpha gene in Danish Caucasians with late-onset NIDDM.

Non-insulin-dependent diabetes mellitus (NIDDM) is a phenotypically and genetically heterogeneous disorder. A recent random genome mapping study has localized a locus termed NIDDM2 that maps to the region of chromosome 12 that includes MODY3, one of the three genes responsible for maturity-onset diabetes of the young, a monogenic form of NIDDM characterized by early age of onset and autosomal dominant inheritance. These findings suggest that NIDDM2 and MODY3 may represent different alleles of the same gene. MODY3 has recently been shown to be the gene encoding the transcription factor hepatocyte nuclear factor-1 alpha (HNF-1 alpha) thereby allowing us to determine whether mutations in the HNF-1 alpha gene are present in subjects with late-onset NIDDM. We screened 84 white NIDDM patients of Danish ancestry and found four nucleotide substitutions that changed the sequence of HNF-1 alpha, Ile27-->Leu, Ala98-->Val, Ser487-->Asn and Arg583-->Gln, five nucleotide substitutions that were silent and did not change the amino acid, Leu17, Gly288, Leu459 and Thr515, and five substitutions in the intron regions. The frequencies of the codon 27, 98 and 487 amino acid variants were similar in 245 unrelated NIDDM patients and 242 age-matched control subjects. The Arg583-->Gln mutation was found in 2 of 245 NIDDM patients and in none of the control subjects. Thus, genetic variation in the HNF-1 alpha gene is not a common factor contributing to NIDDM susceptibility in white subjects of Danish ancestry.

Regulation and function of the tissue-specific transcription factor HNF1 alpha (LFB1) during Xenopus development.

We review the data available on the structure, developmental appearance and embryonic regulation of the tissue-specific transcription factor HNF1 alpha (LFB1) in Xenopus. The expression of the HNF1 alpha gene starts early in embryogenesis shortly after mid-blastula transition and the protein accumulates in the region of the embryo where liver, pronephros and gut--tissues that contain HNF1 alpha in the adult--are developing. The cofactor DCoH, known to stabilize dimer formation of HNF1 alpha, is present as a maternal factor in the egg and has a partially distinct tissue distribution compared to HNF1 alpha. This implies that DCoH does not only modulate HNF1 alpha dimerization but may also cooperate with other transcription factors. By injecting HNF1 alpha promoter CAT constructs into fertilized Xenopus eggs we obtained activation of the injected gene restricted to the region of the developing larvae expressing endogenous HNF1 alpha. Deletion analysis allowed to define the OZ-element that is essential for embryonic activation. This element also occurs in other promoters activated at mid-blastula transition in the embryo and interacts with the maternal factor OZ-1. As the HNF1 alpha promoter also contains functional binding sites for HNF4 and HNF1, we postulate that all of these transcription factors contribute to the cascade leading to proper embryonic activation of the HNF1 alpha gene.