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. 2006 Oct 15;399(2):285-95.
doi: 10.1042/BJ20060659.

Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase

Aaron W Adamson  1 , Gabriela Suchankova, Caterina Rufo, Manabu T Nakamura, Margarita Teran-Garcia, Steven D Clarke, Thomas W Gettys
Affiliations

Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase

Aaron W Adamson et al. Biochem J. .

Abstract

Refeeding a carbohydrate-rich meal after a fast produces a co-ordinated induction of key glycolytic and lipogenic genes in the liver. The transcriptional response is mediated by insulin and increased glucose oxidation, and both signals are necessary for optimal induction of FAS (fatty acid synthase). The glucose-regulated component of FAS promoter activation is mediated in part by ChREBP [ChoRE (carbohydrate response element)-binding protein], which binds to a ChoRE between -7300 and -7000 base-pairs in a carbohydrate-dependent manner. Using in vivo footprinting with nuclei from fasted and refed rats, we identify an imperfect DR-1 (direct repeat-1) element between -7110 and -7090 bp that is protected upon carbohydrate refeeding. Electrophoretic mobility-shift assays establish that this DR-1 element binds HNF-4alpha (hepatocyte nuclear factor 4alpha), and chromatin immunoprecipitation establishes that HNF-4alpha binding to this site is increased approx. 3-fold by glucose refeeding. HNF-4alpha transactivates reporter constructs containing the distal FAS promoter in a DR-1-dependent manner, and this DR-1 is required for full glucose induction of the FAS promoter in primary hepatocytes. In addition, a 3-fold knockdown of hepatocyte HNF-4alpha by small interfering RNA produces a corresponding decrease in FAS gene induction by glucose. Co-immunoprecipitation experiments demonstrate a physical interaction between HNF-4alpha and ChREBP in primary hepatocytes, further supporting an important complementary role for HNF-4alpha in glucose-induced activation of FAS transcription. Taken together, these observations establish for the first time that HNF-4alpha functions in vivo through a DR-1 element in the distal FAS promoter to enhance gene transcription following refeeding of glucose to fasted rats. The findings support the broader view that HNF-4alpha is an integral component of the hepatic nutrient sensing system that co-ordinates transcriptional responses to transitions between nutritional states.

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Figures

Figure 1
Figure 1. In vivo DNase I footprint analysis of the rat liver FAS gene region of −7150/−7050
(A) Nuclei isolated from liver of fasted and fasted-refed rats were digested with DNase I as described in the Experimental section. The Figure represents the DNase I footprint of the sense strand of the FAS promoter from −7150 to −7050. The end points of the protected regions were identified by chemical sequencing (C/T and G/A lanes). Areas affected by refeeding are labelled as 1–5. Sequences 1, 2, 4 and 5 bind unknown factors, while sequence 3 represents the DR1. (B) The sequence of the FAS DR-1 element was compared with that of the consensus DR-1 and L-PK gene.
Figure 2
Figure 2. HNF-4α binds the FAS DR-1 element
(A) Sequences of the oligonucleotides used as probes and competitors in the gel-shift reactions. The sequence of the DR-1 elements are highlighted in boldface and the mutated bases in the −7110/−7074 mut DR-1 are displayed in lower-case. (B) 1.0 ng of −7110/−7074 radiolabelled oligonucleotide was incubated with either 5 μg of fasted or refed liver nuclear extract and subjected to electrophoresis on a 5% non-denaturing polyacrylamide gel. (C) All gel-shift reactions were incubated with 5 μg of refed liver nuclear extract and employed the following radiolabelled oligonucleotides: −7110/−7074 (lanes 1, 2 and 3) and −7110/−7074 mut DR1 (lane 4). The competing oligonucleotides AOX DR-1 (lane 2) and −7110/−7074 mut DR1 (lane 3) were added at 100× molar excess to the binding reaction. (D) Immunological characterization of HNF-4α binding to the DR-1 element. Gel-shift reactions included 5 μg of refed liver nuclear extract and employed the following radiolabelled oligonucleotides: −7110/−7074 (lanes 1 and 2) and −7110/−7089 (lanes 3 and 4). Antibody (Ab) directed against HNF-4α was added and arrows indicate positions of specific complexes.
Figure 3
Figure 3. In vivo binding of HNF-4α to the FAS gene promoter is increased following the refeeding of carbohydrate to fasted rats
(A) ChIP assay of FAS promoter using nuclei isolated from liver of fasted or carbohydrate-refed rats. Chromatin fragments immunoprecipitated with HNF-4α antibodies were amplified by PCR with primers spanning the distal carbohydrate region of the rat FAS gene promoter (−7314 to −6980). Immunoprecipitation with normal goat IgG (mock) was used as a negative control and 1% purified input DNA (input) was used a positive control for the PCR reaction. The data shown are representative of three to six individual experiments. (B) Chromatin immunoprecipitated by the HNF-4α antibody was amplified using primers spanning the DR-1 sites of the L-PK and PEPCK promoters. (C) The results of three to six individual experiments were quantified by measuring the density of the PCR products separated on an agarose gel. The numbers are derived from the average density of the PCR products from the refed liver samples compared with the average intensity of those from the fasted liver samples, which are set at 1.0. (D) Whole cell protein extracts from fasted and refed rat liver were analysed by immunoblotting for HNF-4α and actin as described in the Experimental section.
Figure 4
Figure 4. The DR-1 element of FAS is required for full glucose responsiveness
(A) Schematic representation of the reporter constructs used in the subsequent luciferase assays. (B) COS-1 cells were transfected with the listed FAS luciferase reporter constructs and empty vector (open bars) or vector expressing HNF-4α (solid bars). Data are normalized to the luciferase activity of the luciferase constructs in the presence of vector alone, which is equal to 1. The results shown represent the means±S.E.M. for four independent experiments with three replicate transfections per experiment. (C) COS-1 cells were transfected with the listed FAS/L-PK luciferase reporter constructs as outlined in (B). (D) Rat primary hepatocytes were transfected with the listed luciferase reporter constructs. Cells were cultured in 5.5 mM (open bars) or 27.5 mM glucose (solid bars) for 48 h in the presence of 100 nM insulin and 100 nM dexamethasone. The data are expressed as fold increase in luciferase activity over 5.5 mM glucose. The results shown represent the means±S.E.M. for four independent experiments with three replicate transfections per experiment.
Figure 5
Figure 5. Effects of HNF-4α gene silencing on FAS gene expression in primary hepatocytes
After plating, hepatocytes were then transfected with 100 pmol of either HNF-4α siRNA or scrambled siRNA and incubated for 48 h in medium supplemented with 100 nM dexamethasone, in the presence of 5 mM glucose. (A) Following the 48 h incubation, a sample of both HNF-4α siRNA and scrambled siRNA transfected hepatocytes were harvested for immunoblot analysis of HNF-4α protein levels. Actin was used as a loading control. After knockdown of HNF-4α protein, the hepatocytes were cultured in M199 medium containing 5 or 25 mM glucose, with or without 100 nM insulin, and supplemented with 100 nM dexamethasone. After 24 h, total RNA was extracted and analysed for FAS (B) and LPK (C) gene expression by real-time quantitative PCR. Results are the means±S.E.M for four independent cultures.
Figure 6
Figure 6. HNF-4α physically interacts with ChREBP in vivo
(A) HEK-293 cells were co-transfected with expression vectors for HNF-4α and FLAG-tagged ChREBP. Nuclear protein extracts were prepared, 42 h following transfection, and subjected to co-immunoprecipitation with anti-HNF-4α antibody. After incubation at 4 °C overnight and washing three times with PBS containing 0.1% Nonidet P40, the samples were analysed on a 10% gel by SDS/PAGE and Western blotted with anti-FLAG antibody. The top and middle panels are Western blots of 5% of the nuclear extract before immunoprecipitation, and the bottom panel is a Western blot of the immunoprecipitated proteins. (B) Nuclear protein extracts from the liver of refed rats were prepared, subjected to immunoprecipitation with anti-HNF-4α antibody or preimmune IgG (PI), and Western blotted with anti-ChREBP antibody (top panel). The blot was then stripped and immunoblotted with a separate anti-HNF-4α antibody (bottom panel). (C) The inverse of the experiment displayed in (B) was performed, with nuclear protein extracts subjected to immunoprecipitation with anti-ChREBP antibody, and immunoblotted with anti-HNF-4α antibody (top panel). The blot was stripped and immunoblotted with anti-ChREBP antibody (bottom panel).
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