The maturity-onset diabetes of the young (MODY1) transcription factor HNF4alpha regulates expression of genes required for glucose transport and metabolism

Abstract

Hepatocyte nuclear factor 4alpha (HNF4alpha) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4alpha gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4alpha protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4alpha function. The effect of loss of function on HNF4alpha target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic beta-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4alpha. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4alpha. These data provide direct evidence that HNF4alpha is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.

Figure 1

Transient expression and transcriptional activity of HNF4α and the C-terminal deletion mutants (Q268X) and (E360X) in HepG2 cells. (A) The reporter plasmid pZLHIV-A1-4 was cotransfected into HepG2 cells with CMV-LacZ plasmid and effector plasmids expressing HNF4α and the indicated deletion mutants. Cells were harvested 48 hr later 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. (B and C) Cotransfections as described for A but with a constant amount of vectors CMV-HNF4 and an increasing amount of CMV-HNF4(Q268X) (B) or CMV-HNF4(E360X) (C) as indicated. Error bars indicate range.

Figure 2

Expression, DNA binding, and dimerization properties of HNF4α and deletion mutant (Q268X). (A) SDS/PAGE analysis of in vitro-translated wild-type HNF4α and mutant HNF4(Q268X), followed by autoradiography. Numbers indicate molecular mass protein markers in kDa. (B) EMSA analysis of DNA binding activity of in vitro-translated HNF4α and deletion mutant (Q268X) (Top) by using the ³²P-labeled double-stranded oligonucleotide LF-A1 as a probe. Supershift analysis was carried out with a polyclonal anti-HNF4 antiserum, an anti-Flag mAb, or an anti-STAT-2 control antiserum. (C) EMSA analysis of DNA binding activity of in vitro-translated wild-type HNF4α with increasing amounts of HNF4(Q268X) protein. (D) Both in vitro-translated wild-type HNF4 and HNF4(E360X), but not HNF4(Q268X), dimerize with HNF4. FLAG-tagged HNF4(Q268X), HNF4(E360X), or wild-type HNF4α bound to a anti-FLAG mAb attached to agarose was incubated with [³⁵S]-methionine-labeled HNF4α protein. Immunoprecipitates were run on a 10% SDS/PAGE and bound HNF4α was detected by autoradiography. Each reaction was performed in buffers containing 100 and 200 mM NaCl, respectively (32). (C) ³⁵S-methionine-labeled HNF4α.

Figure 3

VE-specific expression of HNF4α and aldoB. Morphological section of HNF4+/+ EBs and in situ hybridization with antisense RNA of HNF4α (B) and aldoB (D) probe. Phase contrast micrograph of 5-μm sections through 14 days ES cell EBs, stained with hematoxylin and eosin (A and C), and the corresponding dark field on the right (B and D). The cuboidal epithelium of the VE is identified by an arrow.

Figure 4

HNF4 regulates gene expression of genes involved in glucose transport and glucose metabolism in vitro and in vivo. (A) HNF4+/+ (J1), HNF4 +/− (2–69 and B16), and HNF4−/− (A8, B9, B13) ES-cell EBs were assayed for the presence of mRNAs derived from genes encoding glucose transporters, enzymes of glycolysis, and intracellular storage proteins. (B) Steady-state mRNA levels of HNF4α target genes were measured in E8.5 HNF4+ and HNF4−/− embryos by reverse transcriptase-PCR. Because HNF4−/− embryos have a block in gastrulation, a greater proportion of the starting material in HNF4−/− embryos is VE tissue, as confirmed by the greater levels of Gata-4 mRNA.

Figure 5

Gene regulation in the VE is glucose responsive. Steady-state mRNA levels of HPRT, Gata-4, aldoB, and L-PK of wild-type (R1) and HNF4−/− (A8) EBs, cultured for 6 hr in medium containing 1, 5, 10, 15 or 20 mmol/liter of glucose.