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. Author manuscript; available in PMC: 2012 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2010 Nov 11;31(2):328–336. doi: 10.1161/ATVBAHA.110.217828

Hepatic HNF4α Is Essential for Maintaining Triglyceride and Cholesterol Homeostasis

Liya Yin 1, Huiyan Ma 1, Xuemei Ge 1, Peter A Edwards 2, Yanqiao Zhang 1
PMCID: PMC3079249  NIHMSID: NIHMS259655  PMID: 21071704

Abstract

Objective

Loss-of-function mutations in human hepatocyte nuclear factor 4α (HNF4α) are associated with maturity-onset diabetes of the young and lipid disorders. However, the mechanisms underlying the lipid disorders are poorly understood. In this report, we determined the effect of acute loss or augmentation of hepatic HNF4α function on lipid homeostasis.

Methods and Results

We generated adenovirus expressing LacZ (Ad-shLacZ) or small hairpin RNA of Hnf4α (Ad-shHnf4α). Tail vain injection of C57BL/6J mice with Ad-shHnf4α reduced hepatic Hnf4α expression and resulted in striking phenotypes including the development of fatty liver and a >80% decrease in plasma levels of triglycerides, total cholesterol and HDL-C. These latter changes were associated with reduced hepatic lipogenesis and impaired VLDL secretion. Deficiency in hepatic Hnf4α did not affect intestinal cholesterol absorption despite decreased expression of genes involved in bile acid synthesis. Consistent with the loss-of-function data, over-expression of Hnf4α induced numerous genes involved in lipid metabolism in isolated primary hepatocytes. Interestingly, many of these HNF4α-regulated genes were not induced in wild-type mice that over-expressed hepatic Hnf4α. Due to selective gene regulation, mice over-expressing hepatic Hnf4α had unchanged plasma triglyceride levels and decreased plasma cholesterol levels.

Conclusions

Loss of hepatic HNF4α results in severe lipid disorder as a result of dysregulation of multiple genes involved in lipid metabolism. In contrast, augmentation of hepatic HNF4α activity lowers plasma cholesterol levels but has no effect on plasma triglyceride levels due to selective gene regulation. Our data indicate that hepatic HNF4α is essential for controlling the basal expression of numerous genes involved in lipid metabolism and is indispensable for maintaining normal lipid homeostasis.

Introduction

Nuclear receptors are ligand-activated transcription factors that regulate diverse physiological processes such as reproduction, development and metabolism. Hepatocyte nuclear factor 4α (HNF4α, NR2A1) is a member of the nuclear receptor superfamily. It is highly expressed in the liver, with lower levels in the kidney, intestine and pancreatic β cells 1, 2. Like other members of the nuclear receptor superfamily, HNF4α has a highly conserved DNA-binding domain and a variable ligand-binding domain (LBD). However, HNF4α is known to be constitutively active. Structural analysis of the LBD of HNF4α indicates the C14–C18 long-chain fatty acids are tightly bound to the hydrophobic pocket 3, 4 and can not be dissociated from the receptor under non-denaturing condition 4. Recent data also show that linoleic acid selectively occupies the binding pocket of LBD, but does not affect the transcriptional activity of HNF4α 5. Together, these data indicate that under normal conditions, HNF4α activity is not affected by fatty acids, the endogenous ligands for HNF4α.

Loss-of-function mutations in human HNF4α are associated with maturity-onset diabetes of the young (MODY1) 6, characterized by autosomal dominant inheritance, early-onset diabetes and pancreatic β-cell dysfunction 7. The diabetes phenotype appears to result from reduced insulin secretion in response to glucose stimulation 8. In addition, patients with MODY1 also have reduced levels of plasma triglycerides and cholesterol 9–11. However, the mechanism underlying hypolipidemia remains poorly understood.

Homozygous mutations in whole body Hnf4α result in early embryonic lethality in mice 12, consistent with the central role of HNF4α in development. Liver-specific Hnf4α−/− (L-Hnf4α−/−) mice have increased plasma bile acid levels and reduced plasma levels of triglycerides and cholesterol 13. The disorder in bile acid homeostasis is a result of reduced hepatic expression of genes involved in bile acid biosynthesis and bile acid uptake from the blood 13, 14, whereas hypotriglyceridemia may result from reduced hepatic expression of microsomal transport protein (MTP) and apolipoprotein B (ApoB) 13, two proteins that play a critical role in very-low-density lipoprotein (VLDL) secretion. Hayhurst et al. suggested that the increased expression of scavenger receptor class B type I (SR-BI), an HDL receptor that selectively uptakes cholesteryl esters from plasma HDL 15, is responsible for hypocholesterolemia observed in L-Hnf4α −/− mice 13. By far, it is unknown whether other genes/pathways are also involved in HNF4α-regulated lipid homeostasis.

Both the liver and intestine play an important role in maintaining cholesterol homeostasis. Plasma cholesterol levels may be affected by intestinal cholesterol absorption, hepatic de novo cholesterol biosynthesis, VLDL secretion, plasma cholesterol uptake by the liver and subsequent hepatobiliary cholesterol secretion. HMG-CoA reductase (HMCR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway. Once synthesized, cholesterol and long-chain fatty acids are esterified to form cholesteryl esters by acyl-CoA:cholesterol acyltransferase 2 (ACAT2; SOAT2) for storage or secretion in the form of VLDL from the liver or chylomicrons from the intestine. Plasma HDL levels may be affected by many factors. The data from human Tangier disease and genetically engineered mice have clearly demonstrated that ATP-binding cassette (ABC) transporter A1 (ABCA1) is the major determinant of plasma HDL levels 16–20. Hepatic ABCA1 has been shown to be responsible for ~ 80% plasma total HDL 16. In addition, ApoA-I, the major component of HDL, is also required for maintaining plasma HDL-C levels 21.

Liver takes up plasma HDL-C or non-HDL-C via SR-BI, low-density lipoprotein receptor (LDLR) or LDLR-related protein (LRP). In the liver, cholesterol may be converted to bile acids by cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1), two key enzymes that control bile acid biosynthesis. Bile acids are subsequently secreted to the bile via transporters, such as bile acid export protein (BSEP). Hepatic cholesterol may also be secreted directly to the bile via ABC transporters G5 (ABCG5) and G8 (ABCG8) 22, 23, or SR-BI 24.

In this report, we demonstrate that acute loss of hepatic Hnf4α causes hypotriglyceridemia and the development of fatty liver via reducing VLDL secretion. We also demonstrate that hepatic Hnf4α deficiency results in hypocholesterolemia likely by reducing de novo cholesterol biosynthesis, VLDL secretion and HDL biogenesis. In contrast, hepatic over-expression of HNF4α reduces the levels of hepatic triglycerides and plasma cholesterol, but has no effect on plasma triglyceride levels. Together, these data demonstrate that hepatic HNF4α is essential for maintaining triglyceride and cholesterol homeostasis.

Materials and Methods

Mice

C57BL/6J mice and db/db mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and fed a standard chow diet. All experiments were approved by the Institutional Animal Care and Use Committee at the Northeastern Ohio Universities College of Medicine. All the mice were euthanized during the light cycle.

Adenovirus

Ad-Hnf4α was generated by cloning mouse Hnf4α cDNA to adenoviral vector pShuttle-IRES-hrGFP (Stratagene, CA) as described before 25. To generate adenovirus expressing small hairpin RNA corresponding to β-galactosidase (Ad-shLacZ, control) or Hnf4α (Ad-shHnf4α), oligonucleotides were designed using BLOCK-iT™ RNAi Designer (Invitrogen, CA), annealed and ligated to pEnter/U6 vector (Invitrogen, CA). Adenovirus was then generated following the instructions provided by Invitrogen. Three different shRNA oligonucleotides against murine Hnf4α were designed. The sequences that produced the most inhibitory effect on endogenous Hnf4α expression are: 5′-CACCGGTGCCAACCTCAATTCATCCCGAAGGATGAATT GAGGTTGGCACC-3′ (top strand) and 5′-AAAAGGTGCCAACCTCAATTCATCCTTCGG GATGAATTGAGGTTGGCACC-3′ (bottom strand). Adenovirus was grown in 293A cells and purified using a BD Adeno-X Virus Purification Kit (BD Biosciences, CA) or by cesium chloride density gradient centrifugation. To infect primary hepatocytes, adneovirus was added at a multiplicity of infection (m.o.i.) of 10 and cells were harvested after 48 h. To over-express genes in mice, 108–109 plaque formation units (pfu) of adenovirus was transfused into each mouse via tail vain injection. To knock down hepatic genes in mice, 1010 pfu of Ad-shLacZ or Ad-shHnf4α was transfused to each mouse via tail vain injection. In general, 6–7 days post infection, mice were fasted for 6 h and then euthanized.

Primary Hepatocytes

Mouse primary hepatocytes were isolated and cultured as described 26. Three days after isolation, hepatocytes were infected with adenovirus for 48 h prior to RNA extraction.

Real-Time PCR

RNA was isolated using TRIzol Reagent (Invitrogen) and mRNA levels determined by quantitative reverse-transcriptionpolymerase chain reaction (qRT-PCR) using SYBR Green Supermixand a 7500 real-time PCR machine from Applied Biosystems (Foster City, CA). The primer sequences for qRT-PCR are providedin Supplementary Table 1. Results were normalized to 36B4 mRNA.

Western Blot Assay

Whole liver lysates were prepared and Western blot assays were performed as described previously 25. β-actin antibody was from Novus Biologicals (CO). HNF4α and MTP antibodies were from Santa Cruz Biotechnology (CA). ApoB48/100 and ApoA-I antibodies were from Biodesign (Maine). Cyp7a1 antibody was a kind gift from David Russell at University of Texas Southwestern Medical Center.

Chromatin Immunoprecipitation (ChIP) Assay

Ad-GFP or Ad-HNF4α was used to infect primary hepatocytes for 48 h, or C57BL/6 mice via intravenous injection. The protein lysates from the primary hepatocytes or the liver (6 days post infection) were incubated with IgG or HNF4α antibody, and ChIP assays were performed as described previously (n=3 per treatment) 27.

Glucose and Insulin Tolerance Test

Nine-weeks old db/db mice were injected intravenously with Ad-shLacZ or Ad-shHnf4α. On day 7, glucose tolerance test was performed. Briefly, mice were fasted for 6 h, followed by intraperitoneal injection of glucose (2 g/kg). Blood glucose levels were determined at indicated time points using an AlphaTrak glucometer (Abbott Laboratories, IL). On day 9, insulin tolerance test was performed. Mice were injected intraperitoneally with insulin (HumulinR, 0.85 u/kg) after a 6-h fast and blood glucose levels determined at indicated time points.

Lipid Analysis

Approximately 100 mg liver was homogenized in methanol and lipids were extracted in chloroform/methanol (2:1 v/v) as described 28. Hepatic triglyceride and cholesterol levels were then quantified using kits from Wako Chemicals (Richmond, VA). Plasma lipid levels were determined as described previously 26.

VLDL Secretion

C57BL/6J mice were injected with either Ad-shLacZ or Ad-shHnf4α via tail vain injection. On day 6, these mice were fasted overnight, followed by intravenous injection of Tyloxapol (500 mg/kg). Blood was taken at indicated time points and plasma triglyceride levels determined.

De Novo Lipogenesis

C57BL/6 mice were injected with either Ad-shLacZ or Ad-shHnf4α. After 8 days and a 4-h fast, mice were injected intraperitoneally with 2H2O to get 3% enrichment. After 4 h, livers were collected. Labeled and unlabeled palmitate and cholesterol were analyzed by mass spectrometry as previously described 29. The fractional synthesis rate is expressed as: [newly synthesized palmitate or cholesterol per gram liver/total palmitate or cholesterol per gram liver] X 100%.

Intestinal Cholesterol Absorption

Intestinal cholesterol absorption was performed as described previously 27, 30. Briefly, C57BL/6J mice were injected with Ad-shLacZ or Ad-shHnf4α via tail vain. After 6 days, mice were injected with 2.5 μCi 3H-cholesterol in Intralipid (Sigma) via tail vein injection, followed by immediate gavage with 1 μCi 14C-cholesterol in median-chain triglycerides (MCT oil, Mead Johnson, Evansville, IN). After 72 h, blood and tissue were collected. The percent of the intestinal cholesterol absorption was calculated using the formula [percent of intragastric dose (14C-cholesterol) per ml plasma]÷[percent of intravenous dose (3H-cholesterol) per ml plasma].

Statistical Analysis

Statistical significance was analyzed using unpaired Student t test, one-way ANOVA or Mann-Whitney test (GraphPad InStat3 software). All valuesare expressed as mean±SEM. Differences were consideredstatistically significant at P<0.05.

Results

Acute Loss of Hepatic Hnf4α Results in Severe Lipid Disorder

To determine the effect of acute loss of hepatic HNF4α on lipid homeostasis, we generated three adenovirus expressing small hairpin RNA (shRNA) corresponding to Hnf4α (Ad-shHnf4α) and one to galactosidase Z (Ad-LacZ, control). One out of three Hnf4α shRNAs was chosen for further studies as it was particularly efficient in knocking-down endogenous Hnf4α mRNA (Supplemental Figure 1A) and protein (Supplemental Figure 1B).

To test the physiological consequences of endogenous Hnf4α deficiency, we transfused Ad-shLacZ or Ad-shHnf4α to C57BL/6 mice via tail vain injection. Tail vain injection essentially limits adenovirus to the liver. As shown in Figure 1A, expression of Hnf4α shRNA significantly reduced hepatic Hnf4α mRNA levels by 67% but had no effect on the mRNA levels of Bsep and many other genes (data not shown), indicating that Hnf4α shRNA specifically and efficiently targets Hnf4α in vivo.

Figure 1.

Figure 1

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Hepatic HNF4α deficiency causes severe lipid disorder. C57BL/6 mice were transfused with Ad-shLacZ or Ad-shHnf4α via tail vain injection (n=7 mice per group). After 6 days, mice were euthanized after a 5-h fast. (A) Hepatic mRNA levels were determined by quantitative real-time PCR (qRT-PCR). (B) Representative livers from both genotypes are shown. (C–F) Hepatic triglycerides (C), hepatic total cholesterol (D), plasma triglycerides (E), and plasma total cholesterol (TC) and HDL-C (F) were determined. * P<0.05, ** P<0.01 vs Ad-shLacZ treatment.

The livers of mice receiving Ad-shHnf4α were enlarged and white in color (Figure 1B). Analysis of hepatic lipid levels indicated that Hnf4α-deficient mice had ~ 4 fold increase in triglyceride levels (Figure 1C) and unchanged cholesterol levels in the liver (Figure 1D). These data demonstrate that hepatic Hnf4α deficiency leads to the development of fatty liver as a result of increased triglyceride accumulation.

In addition, Hnf4α-deficient mice had strikingly low levels of plasma lipids; plasma triglyceride levels decreased by 84% (Figure 1E) and plasma total cholesterol and HDL-C levels decreased by ~ 87% (Figure 1F). Together, the data of Figure 1 demonstrate that hepatic Hnf4α deficiency results in severe lipid disorder in both the liver and plasma.

Hepatic Hnf4α Deficiency Results in Dysregulation of Multiple Genes Involved in Lipid Metabolism

To understand the mechanism by which hepatic Hnfα deficiency results in lipid disorder, we analyzed hepatic gene expression by quantitative real-time PCR (qRT-PCR). The data show that the mRNA levels of genes involved in VLDL secretion (Mtp, Apob), de novo cholesterol biosynthesis (Hmgcr, Hmgcs, Srebp-2), cholesterol catabolism (Cyp7a1, Cyp8b1), cholesterol esterification (Acat2, Lcat), and cholesterol uptake (Ldlr, SR-BI) were all significantly reduced in Hnf4α-deficient mice (Figure 2A). In addition, hepatic mRNA levels of genes encoding transporters (Abca1, Abcg5, Abcg8, Mdr2) (Figure 2B), apolipoproteins (Apoa1, Apoa2, Apoc2, Apoc3 and Apoe) (Figure 2C) or nuclear receptors (Pparα, Pparγ) (Figure 2D) were also significantly reduced in Hnf4α-deficient mice. The finding that VLDL receptor (Vldr) (Figure 2A) and Abcg1 (Figure 2B) were induced in Hnf4α-deficient mice suggests that Hnf4α deficiency does not cause a global repression of genes involved in lipid metabolism. In addition, hepatic protein levels of Hnf4α, Mtp (Figure 2E) and ApoB48/100 (Figure 2F) were also significantly reduced in Hnf4α-deficient mice. Interestingly, hepatic Hnf4α deficiency did not significantly alter Cyp7a1 protein levels (Figure 2E), consistent with a previous report that Cyp7a1 protein levels are unchanged in L-Hnf4α−/− mice during the light cycle 14. Together, these data indicate that HNF4α is important for normal expression of multiple genes that are involved in lipid metabolism.

Figure 2.

Figure 2

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Hepatic HNF4α deficiency affects the expression of multiple genes involved in lipid metabolism. C57BL/6 mice were infected with Ad-shLacZ or Ad-shHnf4α for 6 days (n=7 mice per group). Hepatic mRNA levels were determined by qRT-PCR (A–D) and protein levels determined by Western blot assays (E, F). * P<0.05, # P<0.01 vs Ad-shLacZ treatment.

Hnf4α-deficient Mice Have Impaired VLDL Secretion

The significant reduction in hepatic Mtp and Apob in Hnf4α-deficient mice (Figure 2) suggested that these mice likely had reduced VLDL secretion. We tested this hypothesis by injecting Tyloxapol, an inhibitor for lipoprotein lipase (Lpl), to mice that had been infected with Ad-shLacZ or Ad-shHnf4α for 6 days. Hnf4α-deficient mice had markedly reduced levels of plasma triglycerides (Figure 3A) and ApoB48/100 secretion (Figure 3B) at 30, 60, or 90 min after injection of Tyloxapol, which corresponded to a 3.5-fold reduction in VLDL TG production rate (Figure 3C). Thus, the data of Figure 3A–C demonstrate that hepatic HNF4α deficiency causes severely impaired VLDL secretion.

Figure 3.

Figure 3

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Effect of hepatic HNF4α deficiency on VLDL secretion, lipogenesis, intestinal cholesterol absorption and insulin sensitivity. (A–D) C57BL/6 mice were injected with Ad-shLacZ or Ad-shHnf4α via tail vain injection (n=6 mice per group). After 6 days, mice were fasted overnight, followed by intravenous injection of Tyloxapol (500 mg/kg). Blood was taken at indicated time points and plasma triglyceride levels determined (A). Plasma ApoB48/100 levels were determined by Western blot assays (B). The relative VLDL triglyceride (TG) production rate was determined by the slopes of linear increases of plasma TG (C). Hepatic mRNA levels were determined by qRT-PCR (D). (E, F) C57BL/6 mice were infected with Ad- shLacZ or Ad-shHnf4α (n=5–6). After 8 days, mice were injected intraperitoneally with 2H2O and hepatic palmitate (E) and cholesterol synthesis (F) were determined. (G) Intestinal cholesterol absorption was determined using plasma dual-isotope ration method (n=5). (H–J) Nine-week old db/db mice were injected intravenously with Ad-shLacZ or Ad-shHnf4α (n=6 mice per group). Body weight (H) was determined, and glucose tolerance test (I) and insulin tolerance test (J) performed. * P<0.05, ** P<0.01 vs Ad-shLacZ treatment.

Hepatic HNF4α Deficiency Inhibits Lipogenesis and De Novo Cholesterol Biosynthesis

To determine whether Hepatic HNF4α deficiency affects lipogenesis, we determined the mRNA levels of genes involved in lipogenesis. The data of Figure 3D show that sterol regulatory element-binding protein 1c (Srebp1c), acetyl-CoA carboxylase (Acc) and steroyl-CoA desaturase 1 (Scd-1) were unaffected whereas the mRNA levels of fatty acid synthase (Fas), diacylglycerol acyltransferase (DGAT) 1 and DGAT2 were reduced in Hnf4α-deficient mice. To determine the physiological consequences of such changes, we analyzed hepatic lipogenesis by injecting mice with 2H2O; the data show newly synthesized paltimate/fatty acid (Figure 3E) and cholesterol (Figure 3F) were significantly reduced in Hnf4α-deficient mice. Together, these data demonstrate that hepatic HNF4α deficiency results in a significant reduction in lipogenesis and de novo cholesterol synthesis.

Hepatic Hnf4α Deficiency Does Not Affect Intestinal Cholesterol Absorption

Bile acids are involved in emulsification and absorption of dietary lipids. Loss of Cyp7a1 31, 32 or Cyp8b1 33 has been shown to reduce intestinal cholesterol absorption. Interestingly, hepatic Hnf4α deficiency reduced Cyp7a1 mRNA but not protein levels (Fig. 2). Previous data have shown that the ratio of cholic acid-derived metabolites to muricholic acid is similar between L-Hnf4α −/− mice and the control mice 14, indicating that hepatic Hnf4α deficiency does not alter the hydrophobicity index of the bile acid pool and thus may not affect cholesterol absorption. Consistent with this latter finding, intestinal cholesterol absorption, determined by plasma dual-isotope ratio method 30, was unchanged between Hnf4α-deficient mice and the control mice (p=0.23) (Figure. 3G). Thus, intestinal cholesterol absorption does not contribute to the reduced plasma cholesterol levels in hepatic Hnf4α-deficient mice.

Effect of Hepatic HNF4α Deficiency on Body Weight and Insulin Sensitivity

The striking phenotypes of fatty liver and hypolipidemia (Figure 1) led us to investigate the role of hepatic Hnf4α deficiency in glucose homeostasis. In wild-type mice, hepatic Hnf4α deficiency had no effect on food intake, body weight, glucose tolerance or insulin sensitivity (Supplementary Figure 2 and data not shown). Interestingly, in 9-week old db/db mice, infection with Ad-shHnf4α for 9 days caused a significant reduction in body weight (Figure 3H) but had no effect on food intake (data not shown). In addition, db/db mice receiving Ad-shHnf4α had increased glucose intolerance (Figure 3I) and increased insulin insensitivity (Figure 3J). These data indicate that hepatic HNF4α deficiency may be sufficient to cause insulin insensitivity.

Ex Vivo Expression of HNF4α Induces Many Genes Involved in Lipid Metabolism

Our loss-of-function studies have demonstrated that hepatic HNF4α is required for maintaining the expression of numerous genes involved in lipid metabolism (Figures 2 and 3). To determine whether over-expression of HNF4α also alters these genes, we generated adenovirus expressing Hnf4α (Ad-Hnf4α). Ad-Hnf4α or Ad-GFP (control) was subsequently used to infect primary hepatocytes isolated from wild-type mice. Consistent with the loss-of-function data, over-expression of Hnf4α significantly induced the mRNA levels of Mtp, Apob (VLDL secretion), Cyp8b1 (bile acid synthesis), Lrp, Ldlr, SR-BI (cholesterol uptake), Acat2, Lcat (cholesterol esterification), Abca1, Abcg5, Abcg8 (transporters), Apoa1, Apoa2, and Apoc2 (apolipoproteins) (Table 1). However, over-expression of Hnf4α had no significant effect on Srebp-2, Hmgcr, Srebp-1c, or Fas (Table 1), suggesting that augmentation of hepatic HNF4α activity may not affect lipogenesis.

Table 1.

HNF4α regulates the expression of numerous genes involved in lipid metabolism in primary hepatocytes

Pathway Gene Fold change P value
VLDL secretion Mtp 11.3 <0.01
Apob 2.0 <0.01
Cholesterol biosynthesis Hmgcr 1.2 >0.05
Hmgcs 2.6 <0.05
Srebp-2 0.9 >0.05
Cholesterol catabolism Cyp7a1 ND
Cyp8b1 5.6 <0.05
Cholesterol uptake Lrp 1.5 <0.05
Ldlr 2.1 <0.01
SR-BI 3.1 <0.01
cholesterol esterification Acat2 2.0 <0.01
Lcat 11.3 <0.01
Transporters Abca1 2.7 <0.01
Abcg5 9.2 <0.01
Abcg8 13.3 <0.01
Apolipoproteins Apoa1 47.9 <0.01
Apoa2 5.6 <0.05
Apoc2 16.9 <0.01
Fatty acid synthesis Srebp-1c 1.3 >0.05
Fas 1.0 >0.05
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Primary hepatocytes were infected with Ad-GFP or Ad-HNF4α for 48 h. mRNA levels were quantified by qRT-PCR. Fold changes indicate the relative mRNA levels induced by HNF4α vs GFP (control).

Hepatic Over-expression of HNF4α Lowers Plasma Cholesterol Levels But Not Triglyceride Levels

To determine the physiological consequences of over-expression of hepatic HNF4α in vivo, we transfused Ad-GFP or Ad-Hnf4α to C57BL/6 mice via tail vain injection. Over-expression of hepatic Hnf4α significantly increased hepatic Hnf4α protein levels (Figure 4A), modestly reduced plasma total cholesterol levels but had no effect on plasma triglyceride levels (Figure 4B). Interestingly, over-expression of hepatic Hnf4α also reduced hepatic triglyceride levels but had no effect on hepatic total cholesterol levels (Figure 4C). In addition, over-expression of hepatic Hnf4α had no effect on intestinal cholesterol absorption (data not shown). Analysis of plasma by fast protein liquid chromatography (FPCL) indicated that over-expression of Hnf4α in the liver primarily lowered plasma HDL-C, and had no much effect on VLDL or LDL-C (Figure 4D). Over-expression of hepatic Hnf4α also tended to reduce VLDL triglycerides (Figure 4E). In addition, over-expression of hepatic Hnf4α had no effect on intestinal cholesterol absorption (data not shown). Together, the data of Figure 4 indicate that over-expression of hepatic HNF4α lowers plasma cholesterol levels primarily by lowering HDL-C, and reduces hepatic triglyceride levels.

Figure 4.

Figure 4

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Over-expression of HNF4α in the liver lowers plasma cholesterol levels but has no effect on plasma triglyceride levels. C57BL/6 mice were injected intravenously with adenovirus expressing GFP (Ad-GFP) or HNF4α (Ad-HNF4α) (n=5 mice per group). After 6 days, mice were fasted for 5 h prior to euthanization. Hepatic protein levels were determined by Western blot assays (A). Triglyceride (TG) and total cholesterol (TC) levels in the plasma (B) and liver (C) were determined. Plasma cholesterol lipoprotein profile (D) and triglyceride lipoprotein profile (E) were determined by FPLC analysis. * P<0.05 vs Ad-GFP treatment.

Over-expression of Hepatic HNF4α Selectively Regulates Gene Expression

The data of Figure 5A show that over-expression of hepatic Hnf4α increases the mRNA levels of Apob, Cyp8b1, SR-BI and Apoc2 (Figure 5A), but did not affect the mRNA levels of Hmgcr, Hmgcs, Cyp7a1, Abca1, Abcg5, Abcg8, Apoa1, Acat2, Lcat, or Srebp-1c (Figure 5A). The levels of hepatic Mtp were highly variable; hepatic Mtp mRNA (Figure 5A; p=0.06) and protein levels (Figure 5B) tended to increase following over-expression of HNF4α in the liver. Over-expression of Hnf4α in the liver had no effect on plasma ApoB48/100 levels (Figure 5C), consistent with unchanged plasma triglyceride levels (Figure 4). In addition, the induction of hepatic SR-BI following over-expression of Hnf4α in the liver (Figure 5A) is consistent with reduced plasma HDL-C levels (Figure 4).

Figure 5.

Figure 5

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Over-expression of HNF4α in the liver selectively regulates gene expression. (A–C) C57BL/6 mice were injected intravenously with Ad-GFP or Ad-HNF4α (n=5 mice per group). After 6 days, mice were fasted for 5 h prior to euthanization. Hepatic mRNA levels were determined by qRT-PCR (A). Hepatic (B) and plasma (C) protein levels were determined by Western blot assays. (D) Protein levels in isolated primary hepatocytes and the liver were determined. (E) ChIP assays were used to determine the enrichment of HNF4α protein in the promoter of the Abcg5 gene (n=3 per treatment). * P<0.05 vs Ad-GFP treatment. NS, not significant.

The finding that HNF4α over-expression induces many genes in isolated primary hepatocytes (Table 1) but not in the liver (Figure 5A) is intriguing. Compared to the liver, primary hepatocytes had much lower levels of Hnf4α mRNA (< 6%; data not shown) and protein (Figure 5D). We hypothesized that many genes were inducible in primary hepatocytes by exogenous HNF4α because endogenous HNF4α expression was low in these cells; we also hypothesized that many of these genes were not inducible in the liver by exogenous HNF4α because endogenous HNF4α expression was already high in vivo. We utilized Abcg5, which is a known HNF4α target gene 34 and is highly inducible by HNF4α in isolated primary hepatocytes (Table 1) but not in the liver (Figure 5A), as an example to test our hypothesis.

HNF4α regulates Abcg5 expression through binding to a specific DR-1 element in the Abcg5 promoter 34. We infected primary hepatocytes with Ad-GFP (control) or Ad-HNF4α, followed by chromatin immunoprecipitation (ChIP) assays; the data indicated that HNF4α protein bound to the known DR-1 element only when HNF4α was over-expressed in isolated primary hepatocytes (Figure 5E, left panel). In contrast, HNF4α protein bound to the known DR-1 element both in the absence or presence of exogenous HNF4α protein and the expression of exogenous HNF4α protein did not further increase the recruitment of HNF4α protein to the DR-1 element in Abcg5 promoter (Figure 5E, right panel). Thus, the data of Figure 5D, E suggest that the difference in the abundance of endogenous HNF4α between the liver and primary hepatocytes is responsible for the differential regulation of certain genes by exogenous HNF4α.

Discussion

In this report, we have utilized loss-of-function and over-expression approaches to determine the role of hepatic HNF4α in lipid homeostasis. We show that acute loss of hepatic HNF4α results in striking phenotypes, including low blood triglyceride and cholesterol levels, fatty liver and hepatomegaly (Figure 1). These changes are associated with reduced lipogenesis, de novo cholesterol synthesis and VLDL secretion. Consistent with changes, the expression of numerous genes that are involved in lipid metabolism is significantly altered. In addition, acute loss of hepatic HNF4α is also associated with increased glucose intolerance (Fig. 3). Interestingly, over-expression of HNF4α in the liver also moderately lowers plasma cholesterol levels but has no effect on plasma triglyceride levels (Figure 4). Together, these data demonstrate that hepatic HNF4α is indispensable for maintaining normal triglyceride and cholesterol homeostasis.

The hypolipidemic effect of acute loss of hepatic Hnf4α reported in the current study is consistent with a previous study that utilized L-Hnf4α −/− mice 13. In the latter report, however, the investigators attributed the hypocholesterolemia to increased hepatic SR-BI expression 13. In the current study, we demonstrate that acute loss of hepatic Hnf4α reduces SR-BI expression (Figure 2A). We noted the methodological difference between our studies and the studies by Hayhurst et al. 13, which includes knock-down methods (shRNA vs. Cre-loxP), time (6–7 days vs. 45 days) and the degree of Hnf4α deletion (67% vs. >90%). Importantly, our loss-of-function data are supported by the finding that over-expression of HNF4α increases SR-BI expression in both isolated primary hepatocytes (Table 1) and the liver (Figure 5A). We also demonstrate that hepatic Hnf4α deficiency results in reduced expression of genes involved in de novo cholesterol biosynthesis (Hmgcs, Hmgcr), VLDL secretion (Mtp, Apob) and HDL biogenesis (Abca1, Apoa1) (Figure 2). We further demonstrate that such changes in gene expression have functional consequences; hepatic HNF4α deficiency reduces de novo cholesterol synthesis and VLDL secretion (Figure 3). In addition, we demonstrate that hepatic HNF4α deficiency has no effect on intestinal cholesterol absorption (Figure 3G). Thus, our data have demonstrated that the reduced plasma cholesterol levels in Hnf4α-deficient mice may result from reduced de novo cholesterol synthesis and VLDL secretion.

Both the current study and a previous study 13 have demonstrated that loss of hepatic Hnf4α decreases hepatic Mtp and Apob expression. However, the functional significance of such changes has not been documented before. In this report, we demonstrate that hepatic VLDL secretion is severely impaired in Hnf4α-deficient mice (Figure 3A–C). Consistent with reduced levels of Fas, Dgat1 and Dgat2 (Figure 3D), hepatic HNF4α deficiency also significantly reduces lipogenesis (Figure 3E). Consequently, the current data demonstrate that the development of fatty liver and hypotriglyceridemia following acute loss of hepatic HNF4α, result from severely impaired VLDL secretion.

Loss of HNF4α in pancreatic β-cells is known to impair glucose-stimulated insulin secretion 8. The role of loss of hepatic HNF4α in insulin sensitivity has not been investigated before. In this report, we show that acute loss of hepatic Hnf4α in diabetic db/db mice aggravates glucose intolerance and insulin insensitivity (Figure 3I, J). Such a phenotype may partially result from massive lipid accumulation in the liver and changes in the expression of genes involved in glucose metabolism. Detailed investigation on the role of hepatic HNF4α in glucose metabolism is one of our future directions. Nonetheless, our data suggest that hepatic HNF4α deficiency may also cause insulin insensitivity.

Compared to the emerging data from loss-of-function studies, little is known about the role of increased hepatic HNF4α activity in lipid metabolism. Here we show that over-expression of HNF4α in isolated primary hepatocytes induces many genes that are involved in lipid metabolism (Table 1). Interestingly, only some of these genes are also induced in mice over-expressing hepatic Hnf4α (Figure 5A). The data of Figure 5E suggest that the difference in endogenous HNF4α protein levels between primary hepatocytes and the liver may account, at least in part, for the differential gene regulation by exogenous HNF4α. Certain genes remain inducible in the liver by exogenous HNF4α (Figure 5A), which may be accounted for by the existence of multiple DR-1 elements in these genes. For instance, both SR-BI 27 and Abcg5 34 are known HNF4α target genes and have three DR-1 elements and one DR-1 element, respectively; SR-BI, but not Abcg5, can be induced by exogenous HNF4α in the liver (Figure 5A). Consistent with the selective induction of certain genes in vivo (Figure 5), hepatic over-expression of Hnf4α does not affect plasma triglyceride levels but modestly reduces plasma cholesterol levels (Figure 4). Since hepatic over-expression of SR-BI is known to reduce plasma cholesterol levels 24, our data suggest that the reduced plasma cholesterol levels in Hnf4α-over-expressing mice may result from increased hepatic SR-BI expression (Figure 5). Interestingly, over-expression of HNF4α in the liver also reduces hepatic triglyceride levels, which may be accounted for in part by increased hepatic ApoB expression (Figure 5A).

In summary, we have utilized loss-of-function and over-expression approaches to demonstrate that hepatic HNF4α is required for maintaining basal expression of many genes involved in lipid metabolism and is indispensable for maintaining normal lipid homeostasis. Our data indicate that augmentation of hepatic HNF4α activity only have modest effects on lipid homeostasis.

Supplementary Material

Supp1

Acknowledgments

We appreciate the mouse metabolic phenotyping center (MMPC) at Vanderbilt University (supported by DK59637) for providing assistance with the FPLC analysis, the MMPC at Case Western Reserve University (supported by DK76769) for providing assistance with the de novo lipogenesis assays.

Sources of Funding

This work was supported by a Scientist Development Grant 0830255N from the American Heart Association (AHA) (to Y.Z.) and R15DK088733 from NIH (to Y.Z), and HL30568 and HL68445 from NIH (to P. A. E.).

Footnotes

Disclosures

None.

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