Hyperhomocysteinemia induces hepatic cholesterol biosynthesis and lipid accumulation via activation of transcription factors

Connie W. H. Woo,1,3 Yaw L. Siow,1,3,4 Grant N. Pierce,1,3 Patrick C. Choy,4 Gerald Y. Minuk,5 David Mymin,4,5 and Karmin O1,2,3,4

Departments of 1Physiology and 2Animal Science, 3National Centre for Agri-Food Research in Medicine, St. Boniface Hospital Research Centre, 4Centre for Research and Treatment of Atherosclerosis, Faculty of Medicine, and 5Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 29 October 2004 ; accepted in final form 29 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hyperhomocysteinemia is an independent risk factor for cardiovascular disorders. Elevated plasma homocysteine (Hcy) concentration is associated with other cardiovascular risk factors. We previously reported that Hcy stimulated cholesterol biosynthesis in HepG2 cells. In the present study, we investigated the underlying mechanisms of Hcy-induced hepatic cholesterol biosynthesis in an animal model. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 wk. The mRNA expression and the enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were significantly increased in livers of hyperhomocysteinemic rats. There were marked hepatic lipid accumulation and an elevation of plasma cholesterol concentration in hyperhomocysteinemic rats. Three transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element-binding protein (CREB), and nuclear factor Y (NF-Y) were activated in livers of hyperhomocysteinemic rats. Upon Hcy treatment of hepatocytes, there was a significant increase in HMG-CoA reductase mRNA expression in these cells. The activation of SREBP-2, CREB, and NF-Y preceded the increase in HMG-CoA reductase expression in Hcy-treated cells. Pretreatment of hepatocytes with inhibitors for transcription factors not only blocked the activation of SREBP-2, CREB, and NF-Y but also attenuated Hcy-induced HMG-CoA reductase mRNA expression. These results suggested that hyperhomocysteinemia-induced activation of SREBP-2, CREB, and NF-Y was responsible for increased cholesterol biosynthesis by transcriptionally regulating HMG-CoA reductase expression in the liver leading to hepatic lipid accumulation and subsequently hypercholesterolemia. In conclusion, the stimulatory effect of Hcy on hepatic cholesterol biosynthesis may represent an important mechanism for hepatic lipid accumulation and cardiovascular disorder associated with hyperhomocysteinemia.

homocysteine; 3-hydroxy-3-methylglutaryl coenzyme A reductase; cAMP response element-binding protein; sterol regulatory element-binding protein-2; nuclear factor Y


HYPERHOMOCYSTEINEMIA, an elevation of blood homocysteine (Hcy) concentration, is considered an independent risk factor for cardiovascular and cerebrovascular disorders (7, 37, 48). The mechanisms responsible for hyperhomocysteinemia-associated cardiovascular disorders are still under investigation. McCully (25) reported the autopsy findings of extensive arterial thrombosis and atherosclerosis in two pediatric patients with severe hyperhomocysteinemia and proposed a pathogenic link between elevated blood Hcy concentrations and atherogenesis. Hypercholesterolemia is another independent risk factor for cardiovascular diseases. A positive correlation between the plasma concentrations of Hcy and cholesterol was found in hyperhomocysteinemic patients (30, 33) as well as in experimental animals (47, 49). Patients with severe hyperhomocysteinemia exhibited a moderately fatty liver upon necropsy (25, 26). In addition, elevated Hcy concentrations due to an alteration of methionine metabolism were observed in patients with liver cirrhosis (2). In mice hyperhomocysteinemic caused by cystathionine {beta}-synthase deficiency, there was excessive accumulation of lipid droplets in hepatocytes due to increased endoplasmic reticulum stress (49). Liver is an important organ for the de novo synthesis of cholesterol (16). It is possible that changes in hepatic lipid metabolism may affect plasma cholesterol concentrations in patients with hyperhomocysteinemia. We previously reported that Hcy stimulated the production and secretion of cholesterol in HepG2 cells via activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (31). We postulated on the basis of this in vitro study that Hcy caused the activation of HMG-CoA reductase at the level of gene expression rather than by a direct interaction between Hcy and the enzyme (31). However, the molecular mechanism of Hcy-induced changes in HMG-CoA reductase activity is unclear.

HMG-CoA reductase is the rate-limiting enzyme, catalyzing the conversion of HMG-CoA to mevalonate, in cholesterol biosynthesis (36). The activity of this enzyme is regulated by several mechanisms involving transcriptional regulation, posttranslational modification, allosteric regulation, and levels of endogenous and exogenous cholesterol (3, 19, 27). Sterol regulatory element-binding proteins (SREBPs), a subfamily of basic helix-loop-helix zipper proteins, are important transcription factors regulating lipid homeostasis (14, 45). Two genes, SREBP-1 and -2 encode three SREBP proteins, namely SREBP-1a, -1c, and -2 (14). SREBP-2 preferentially activates enzymes involved in cholesterol production such as HMG-CoA reductase rather than those for fatty acid biosynthesis (14, 15). Upon stimulation, SREBP-2 (125 kDa) is cleaved by the action of site-1-protease (S1P) and site-2-protease (S2P) (14). The truncated form of SREBP-2 (68 kDa) can enter the nucleus to activate target genes. However, SREBP-2 is a weak transcription activator by itself, and the optimal activation of a gene transcription depends on the presence of additional transcription factors (4, 17, 29). Two transcription factors, cAMP response element-binding protein (CREB) and nuclear factor Y (NF-Y), have been identified as important SREBP-2 coregulators and act synergistically for upregulation of HMG-CoA reductase gene expression (4, 29). The involvement of these transcription factors in cholesterol biosynthesis during hyperhomocysteinemia remains to be examined.

Several diet-induced hyperhomocysteinemia animal models have been used to investigate mechanisms of Hcy-induced cardiovascular disorders (1, 46, 49). We (1, 46) recently reported that Hcy stimulated nuclear factor-{kappa}B (NF-{kappa}B) activation and chemokine expression in the vasculature, leading to endothelial dysfunction in hyperhomocysteinemic rats. Although several mechanisms for Hcy-induced endothelial dysfunction have been proposed, little information is available regarding the effect of hyperhomocysteinemia on other organs. The objective of the present study was to investigate the effect of hyperhomocysteinemia on cholesterol biosynthesis in the liver and the molecular mechanism of Hcy-induced HMG-CoA reductase expression via transcriptional regulation in cultured hepatocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) aged 8 wk were divided into three groups and maintained for 4 wk on the following diets before the experiments: 1) control diet (regular diet), consisting of Lab Diet Rat Diet 5012 (PMI Nutrition International, St. Louis, MO); 2) high-methionine diet, consisting of regular diet plus 1.7% (wt/wt) methionine; and 3) high-cysteine diet, consisting of regular diet plus 1.2% (wt/wt) cysteine. Results from our previous studies (1, 46) demonstrated that hyperhomocysteinemia could be induced in rats by feeding a high-methionine diet. The Hcy concentrations in the plasma, in livers, and in hepatocytes were measured with the IMx Hcy assay, which was based on fluorescence polarization immunoassay technology (Abbott Diagnostics, Abbott Park, IL) (1, 44, 46). The cholesterol and triacylglycerol levels in the plasma, liver, and hepatocytes were determined by using enzymatic kits (Wako Chemicals) after overnight fasting. All procedures were performed in accordance with the Guide to the Care and Use of Experimental Animals published by Canadian Council on Animal Care and approved by University of Manitoba Protocol Management and Review Committee.

Histological analysis. The liver was excised and a portion of it frozen immediately in liquid nitrogen. Cryosections of the liver tissue were prepared from 12 rats per treatment group and stained with Oil Red O to visualize lipid droplets. Another portion of the liver was immersion fixed in 10% neutral buffered formalin (10% formalin, 25 mM NaH2PO4, 45 mM Na2HPO4) overnight and then embedded in paraffin. Sequential 5-µm paraffin-embedded sections were prepared and stained with hematoxylin and eosin (H&E) to evaluate the morphological changes. The H&E- and Oil Red O-stained sections (5 per liver) were captured and analyzed by using an Axioskop2 MOT microscope (Carl Zeiss Microimaging, Thornwood, NY), an Axiocam camera, and Photoshop 6.0 (Adobe, San Jose, CA) (23). The experimental rats were coded so that the analysis was performed by individuals without any knowledge of which treatment each rat had received.

Determination of HMG-CoA reductase activity. HMG-CoA reductase activity was measured by a radiochemical method using [3-14C]HMG-CoA as a substrate (50). In brief, a portion of the liver was homogenized in a buffer containing 9.5 mM KH2PO4-40.5 mM K2HPO4 (pH 7.4), 5 mM dithiothreitol, and 1 mM EDTA with a hand-operated Fisher Tissuemiser Homogenizer (Fisher Scientific, Pittsburgh, PA) according to the manufacturer's instructions. The homogenate was centrifuged at 10,000 g for 10 min. The resulting supernatant was collected and the protein concentration determined. An aliquot of the supernatant (1 mg of protein) was used for the enzyme assay. The enzyme assay mixture contains 19 mM KH2PO4, 81 mM K2HPO4 (pH 7.4), 20 mM glucose 6-phosphate, 2.5 mM NADP, 1 unit of glucose-6-phosphate dehydrogenase, 8 mM dithiothreitol, 1.2 mM EDTA, and 0.004 µCi [3-14C]HMG-CoA (PerkinElmer Life Sciences, Boston, MA). The reaction was carried out at 37°C for 60 min. The assay tubes were placed on ice, and mevalonolactone (1 mg) and 5 M HCl were then added to the reaction mixture (50). Radiolabeled HMG-CoA and mevalonolactone were separated by thin-layer chromatography using a solvent system of chloroform-acetone (2:1, vol/vol). The thin-layer chromatographic plate was dried, and the location of mevalanolactone was visualized after staining with iodine vapor. The area containing mevalanolactone was scraped from the plate, and the amount of radioactivity associated with it was measured using a scintillation counter.

Electrophoretic mobility shift assay. Nuclear proteins were prepared according to a method described previously (8, 41). The CREB-DNA or NF-Y-DNA binding activity was determined by electrophoretic mobility shift assay (EMSA) (1, 8, 41, 42). In brief, a portion of the liver or cultured hepatocytes was homogenized in a buffer containing Tris-buffered saline (pH 7.4) followed by centrifugation at 3,000 g for 5 min. The pellet was resuspended in buffer A (1.5 ml), containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, and 10% Nonidet P-40. The mixture was centrifuged at 15,000 g for 15 min. The resulting nuclear pellet was resuspended in buffer B (0.1 ml) containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µM aprotinin, 1 µM pepstatin, and 1 µM leupeptin. The mixture was centrifuged again at 15,000 g for 15 min. The resulting supernatant contained nuclear proteins (1, 8, 41, 42). Nuclear proteins were then incubated with 32P-end-labeled oligonucleotides containing the consensus sequence specific for the CREB-DNA binding site (5'-GACGTCAGAGAGC-3'; Promega, Madison, WI) or for the NF-Y-DNA binding site (5'-ATTGGT-3'; Santa Cruz Biotechnology, Santa Cruz, CA). The reaction mixture was separated in nondenaturing polyacrylamide gel (6%) followed by autoradiography. Results were analyzed with Bio-Rad Quantity One image analysis software (Bio-Rad, Hercules, CA).

Western immunoblotting analysis. For determination of SREBP-2 protein, nuclear proteins were isolated from the homogenized liver or hepatocytes. Western blot analysis (1) was performed with rabbit anti-SREBP-2 polyclonal antibodies (Santa Cruz Biotechnology). In brief, nuclear proteins (100 µg) were separated by electrophoresis on a 7.5% sodium dodecyl sulfate polyacrylamide gel. Proteins on the gel were then transferred to a nitrocellulose membrane. Bands corresponding to SREBP-2 were visualized with enhanced chemiluminescence reagents and exposed to Kodak X-Omat Blue XB-1 film. Films were analyzed with Bio-Rad Quantity One image analysis software (version 4.2.1). The same membranes were reprobed with anti-{beta}-actin antibodies (Santa Cruz Biotechnology) to confirm the equal loading of nuclear proteins prepared from livers or hepatocytes after different treatments (18, 43).

Isolation of hepatocytes. Hepatocytes were isolated from rat liver as previously described (35). Briefly, the Sprague-Dawley rat was killed, and the liver was perfused with phosphate-buffered saline (PBS; Sigma-Aldrich, St Louis, MO) containing 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, and 0.9% NaCl, pH 7.4, for 30 min to flush out blood cells, followed by perfusion with PBS containing 0.5 mM EGTA for 15 min. The liver was further perfused with PBS containing type I collagenase (95 U/ml, Sigma) for another 15 min. The perfused liver was cut into small pieces and digested with type I collagenase (95 U/ml). The digested tissue was then minced and filtered. Cells in the filtrate were collected after centrifugation at 260 g for 5 min at 25°C. Hepatocytes were collected and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum in the Thermo Forma Direct Heat CO2 incubator with 5% CO2-95% O2 at 37°C. L-Hcy was prepared from L-Hcy thiolactone (Sigma) by a method described previously (6, 10, 13, 39). In brief, L-Hcy thiolactone was dissolved and hydrolyzed in potassium hydroxide (5 M) to remove thiolactone group (6, 10, 13, 39). The preparation was then neutralized with HCl. The concentration of L-Hcy in the preparation was quantified with the IMx Hcy assay. Freshly prepared L-Hcy was used in all of the experiments.

Ribonuclease protection assay. Total RNAs were isolated from livers or hepatocytes with TRIzol reagent (Invitrogen, Carlsbad, CA). Ribonuclease (RNase) protection assay was performed with the BD RiboQuant Multi-Probe RNase Protection Assay System (BD Biosciences Pharmingen, San Diego, CA). Briefly, total RNA (10 µg) was hybridized with a 32P end-labeled HMG-CoA reductase oligonucleotide probe (256 bp) overnight at 56°C, followed by ribonuclease digestion to remove nonhybridized probe. After digestion, the protected fragments (183 bp) were resolved on a denaturing 4.75% polyacrylamide gel containing 8 M urea, followed by autoradiography. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide probe (126 bp, protected fragment 97 bp) was used as an internal control. The ratio of HMG-CoA reductase to GAPDH mRNA was calculated, and values were expressed as a percentage of control.

Immunofluorescence staining. For detection of hepatic HMG-CoA reductase, livers were excised from rats fed different types of diet. A portion of the liver was immersion fixed in 10% neutral buffered formalin (10% formalin, 25 mM NaH2PO4, 45 mM Na2HPO4) overnight and then embedded in paraffin. Sequential 5-µm paraffin-embedded sections were prepared and incubated with rabbit anti-HMG-CoA reductase polyclonal antibodies (Upstate, Lake Placid, NY) at 4°C overnight followed by incubation with fluorescein (FITC)-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA) as secondary antibodies at 25°C for 45 min. For detection of activated transcription factors, isolated hepatocytes (2 x 105 cells) were cultured on the collagen-coated chamber slides in DMEM in the absence or presence of Hcy. After incubation for 15 min, cells were fixed on the slides and permeabilized with methanol. The fixed cells were incubated with primary antibodies against SREBP-2, phosphorylated-CREB (Ser133), and/or NF-Y (Santa Cruz Biotechnology) at 4°C overnight. After a washing with PBS, cells were incubated with FITC-conjugated goat anti-rabbit IgG, Cy3-conjugated rabbit anti-goat IgG (Zymed Laboratories) and Alexa Fluor 405 goat anti-mouse IgG (Molecular Probes) as secondary antibodies at 25°C for 45 min. Bound antibodies were viewed under Zeiss fluorescence microscopy (Axioskop2 MOT).

Protein assay. The protein content in livers, hepatocytes, and nuclear fractions was quantified by the method developed by Lowry et al. with BSA as a standard (9, 24).

Statistical analysis. The results were analyzed using a two-tailed independent Student's t-test. The level of statistical significance was set at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of hyperhomocysteinemia on plasma and hepatic lipid content. Hyperhomocysteinemia was induced in rats fed a high-methionine diet for 4 wk. A significant increase in plasma Hcy concentrations was detected in this group of rats (Table 1). The concentration of plasma total cholesterol was significantly elevated in hyperhomocysteinemic rats (Table 1). The plasma triacylglycerol concentration was similar among all groups of rats. The Hcy concentrations in livers isolated from hyperhomocysteinemic rats were threefold higher than those in the control group (Table 1). The lipid contents were also quantified in livers isolated from the three groups of rats. Compared with the controls, the high-methionine diet resulted in a significant increase in the total cholesterol content in the liver (Table 1). The high-cysteine diet did not cause a significant change in the total cholesterol content in the liver (Table 1). There was no significant difference in the concentration of triacylglycerol in livers isolated from all groups of rats. Hepatic cholesterol accumulation was further evaluated by the H&E and Oil Red O staining analyses. The H&E staining revealed microvesicular structure (lipid vacuoles) in the livers of hyperhomocysteinemic rats (Fig. 1). Such structural changes were not observed in the livers of rats fed a high-cysteine diet (Fig. 1). In contrast to the control group, the cryosections of livers isolated from hyperhomocysteinemic rats displayed a marked increase in the intensity of Oil Red O staining and fluorescence staining for HMG-CoA reductase protein, reflecting the accumulation of lipid droplets and increased reductase protein levels in these livers (Fig. 1). There was no change in hepatic and plasma lipid contents in rats fed a high-cysteine diet compared with the control group (Fig. 1 and Table 1). There were no significant differences in body weights among rats fed the three different diets. Taken together, these results suggested that hyperhomocysteinemia induced by a high-methionine diet led to hypercholesterolemia and cholesterol accumulation in the liver.


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Table 1. Plasma and hepatic levels of Hcy, cholesterol, and triacylglycerol in rats

 


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Fig. 1. Hematoxylin & eosin (H&E), Oil Red O, and immunofluorescence staining of livers. Formalin-fixed livers from rats fed with regular (Control), high-methionine (Met), or high-cysteine (Cys) diet were examined with H&E staining for morphological changes and with fluorescence staining for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein. Cryosections of livers from 3 groups of rats were stained with Oil Red O and counterstained with H&E to visualize the lipid droplets (in red). All staining analyses were performed in livers isolated from 12 rats per treatment group, and 5 cryosections were prepared for each liver. Representative photos are shown here. Bars, 50 µm. In H&E staining, arrowheads point to lipid vacuoles; in Oil Red O staining, arrowhead points to hepatocytes; in immunofluorescence staining, HMG-CoA reductase was visualized as green fluorescence.

 
Activation of HMG-CoA reductase and transcription factors in the liver. We investigated whether an elevation of cholesterol content in the liver and in the plasma of hyperhomocysteinemic rats was due to the activation of HMG-CoA reductase. There was a significant increase in the HMG-CoA reductase activity (Fig. 2A) and the mRNA level (Fig. 2B) in livers isolated from rats fed a high-methionine diet. These results suggested that increased HMG-CoA reductase expression contributed to increased hepatic cholesterol biosynthesis in hyperhomocysteinemic rats. On the other hand, a high-cysteine diet did not alter HMG-CoA reductase expression (Fig. 2), indicating that hyperhomocysteinemia-induced HMG-CoA reductase expression was not due to a general effect produced by sulfur-containing amino acids.



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Fig. 2. Enzymatic activity and mRNA expression of HMG-CoA reductase. A: HMG-CoA reductase activity was determined in livers isolated from rats fed with Control, Met, or Cys diet. B: HMG-CoA reductase mRNA (HMGCR) and GAPDH mRNA were determined by RNase protection assay. Results (n = 12) are expressed as means ± SE. *P < 0.05 compared with control values (expressed as 100%).

 
To investigate whether transcription factors were involved in hyperhomocysteinemia-induced HMG-CoA reductase mRNA expression, nuclear proteins were prepared from livers isolated from rats fed different diets. The SREBP-2 protein in the nucleus was quantified by Western immunoblotting analysis. As shown in Fig. 3, the protein level of SREBP-2 was significantly elevated in the nuclear fraction (referred to as nSREBP-2, 68 kDa) of the liver tissue isolated from rats fed a high-methionine diet. Once inside the nucleus, SREBP-2 could bind to the promoter region of the target gene such as HMG-CoA reductase and regulate its expression. Therefore, an increase in the nuclear level of SREBP-2 was an indication that this transcription factor was activated in the liver of hyperhomocysteinemic rat. The activation of CREB and NF-Y in the liver was determined by EMSA. As shown in Fig. 3, there was a significant increase in the CREB-DNA and NF-Y-DNA binding activities in the liver nuclear fraction prepared from rats fed a high-methionine diet, indicating that both CREB and NF-Y were also activated in livers of hyperhomocysteinemic rats.



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Fig. 3. Activation of transcription factors in the liver. Nuclear proteins were isolated from livers of rats fed Control, Met, or Cys diet. Nuclear sterol regulatory element-binding protein-2 (nSREBP-2) was determined by Western immunblotting analysis. {beta}-Actin was used to confirm equal loading amount of proteins from each sample. cAMP response elemt-binding protein (CREB)-DNA or nuclear factor Y (NF-Y)-DNA binding activity was determined by EMSA. The levels of nSREBP-2, {beta}-actin, and CREB-DNA or NF-Y-DNA binding activity were quantified on the basis of the densitometric unit and are expressed as means ± SE (n = 12). *P < 0.05 compared with control values (expressed as 100%).

 
Transcriptional regulation of HMG-CoA reductase expression in hepatocytes. To investigate the relationship between the activation of transcription factors and Hcy-induced HMG-CoA reductase expression, experiments were performed in hepatocytes isolated from rats fed a control diet. Cells were incubated with Hcy for various time periods, and nuclear proteins were prepared to determine the activation of transcription factors. After cells were incubated with Hcy for only 5 min, the CREB-DNA and NF-Y-DNA binding activities were markedly enhanced as assayed by EMSA (Fig. 4A). The activation of SREBP-2 became evident after cells were incubated with Hcy for 10 min, as determined by Western immunoblotting analysis (Fig. 4A). An increase in HMG-CoA reductase mRNA expression was observed in cells incubated with Hcy for 2–8 h (Fig. 4B). There was a significant increase in HMG-CoA reductase mRNA expression in cells incubated with Hcy at concentrations of 0.5 mM and 1.0 mM (Fig. 4C). There were 1.7- and 2-fold elevations of the intracellular Hcy concentrations after hepatocytes were incubated with Hcy (1 mM) for 15 min (2.17 ± 0.02 vs. 1.26 ± 0.35 nmol/mg cellular protein in control cells) and 60 min (2.59 ± 0.04 vs. 1.26 ± 0.35 nmol/mg cellular protein in control cells), respectively. Addition of Hcy (1 mM) to the culture medium did not affect the viability of hepatocytes. On the other hand, incubation of hepatocytes with methionine or cysteine did not result in a significant increase in HMG-CoA reductase mRNA expression (Fig. 4D). These results further indicated that the stimulatory effect observed was not a generalized effect of sulfur-containing amino acids but was related specifically to Hcy. In addition, there was a significant increase in the cholesterol concentration in hepatocytes after incubation with Hcy (Fig. 5). The amount of cholesterol secreted by hepatocytes into the culture medium was also significantly elevated upon Hcy treatment (Fig. 5). Triple staining with antibodies against SREBP-2, phosphorylated CREB (pCREB), and NF-Y was performed to further demonstrate the activation of these factors in hepatocytes (Fig. 6). The immunofluorescence imaging analysis revealed that a marked increase in the nuclear staining intensity for SREBP-2 (in green), pCREB (in red) or NF-Y (in blue) in hepatocytes treated with Hcy. Activation of CREB by phosphorylation (40) allows its efficient interaction with SREBP-2 along with NF-Y to stimulate the transcription of HMG-CoA reductase gene. Taken together, these results suggested that Hcy was able to activate these three transcription factors and subsequently to induce HMG-CoA reductase expression in hepatocytes.



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Fig. 4. Determination of transcription factor activation and HMG-CoA reductase mRNA in hepatocytes. Hepatocytes were isolated from rats fed a regular diet. A: cells were incubated in the absence or presence of homocysteine (Hcy; 1 mM) for various time periods. Nuclear proteins were isolated, and the level of SREBP-2 in the nucleus (nSREBP-2) was determined by Western immunoblotting analysis, whereas CREB-DNA or NF-Y-DNA binding activity was determined by EMSA. B: cells were incubated with Hcy (1 mM) for various time periods. C: cells were incubated with Hcy at various concentrations for 4 h. D: cells were incubated in the absence (Control) or presence of Hcy (1 mM), methionine (Met, 1 mM), or cysteine (Cys, 1 mM) for 4 h. HMG-CoA reductase mRNA and GAPDH mRNA were determined by RNase protection assay. Results are expressed as ratio of HMG-CoA reductase mRNA to GAPDH mRNA and depicted as means ± SE (n = 6, each done in duplicate). *P < 0.05 compared with control values (expressed as 100%).

 


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Fig. 5. Determination of cholesterol concentrations in hepatocytes and in culture medium. Hepatocytes were isolated from rats fed a regular diet. Cells were incubated in the absence (Control) or presence of Hcy (1 mM) for 4 h. Cholesterol concentrations in cells and in culture medium were determined. Results are expressed as means ± SE (n = 5, each done in duplicate). *P < 0.05 compared with control values.

 


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Fig. 6. Immunofluorescence staining of transcription factors in hepatocytes. Hepatocytes were isolated from rats fed a regular diet. Cells were incubated in the absence (Control) or presence of Hcy (1 mM) for 15 min. Immunofluorescence staining was performed. Specific antibodies were used to identify SREBP-2 (green fluorescence), phosphorylated-CREB (pCREB, red fluorescence) or NF-Y (blue fluorescence). Representative photos were obtained from 5 separate experiments. Bar, 20 µm.

 
To further investigate whether Hcy-induced HMG-CoA reductase expression was mediated via the activation of SREBP-2, CREB, and NF-Y, individual inhibitors for these transcription factors were included in the culture medium. First, a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) was added to cultured hepatocytes to test whether inhibition of SREBP-2 alone could prevent Hcy-induced HMG-CoA reductase expression. This inhibitor was shown to inhibit the production of a mature form of SREBP-2 in HeLa cells (32). In the present study, addition of AEBSF to the culture medium not only prevented Hcy-induced SREBP-2 activation (Fig. 7) but also abolished Hcy-stimulated HMG-CoA reductase mRNA expression in hepatocytes (Fig. 8). Next, the involvement of CREB in Hcy-induced HMG-CoA reductase expression was further examined by adding a cAMP-dependent protein kinase (PKA) inhibitor to cultured hepatocytes. It has been shown that phosphorylation of CREB by PKA can lead to an activation of this transcription factor (40). In the present study, addition of the PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) (38) not only blocked Hcy-induced CREB-DNA binding activity (Fig. 7) but also prevented Hcy-induced HMG-CoA reductase mRNA expression in hepatocytes (Fig. 8). Finally, addition of N-ethylmaleimide (NEM), a known inhibitor for NF-Y (28), to the culture medium prevented Hcy-induced NF-Y activation (Fig. 7) as well as HMG-CoA reductase expression (Fig. 8). These inhibitors did not significantly alter the basal level of HMG-CoA redutase mRNA expression in hepatocytes (Fig. 8). These results indicated that Hcy-induced HMG-CoA reductase expression in hepatocytes was likely to be mediated via transcriptional regulation by SREBP-2, CREB, and NF-Y. Taken together, these results suggest that activation of CREB and NF-Y along with SREBP-2 by Hcy plays an essential role in the transcriptional regulation of HMG-CoA reductase.



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Fig. 7. Effect of inhibitors on transcription factor activation in hepatocytes. Hepatocytes were isolated from rats fed a regular diet. Cells were incubated with Hcy (1 mM) in the absence or presence of SREBP-2 inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF, 300 µM), CREB inhibitor (H89, 5 µM), or NF-Y inhibitor N-ethylmaleimide (NEM, 1 mM). Nuclear proteins were isolated from cells after 15 min of incubation. The level of SREBP-2 in the nucleus (nSREBP-2) was determined by Western immunoblotting analysis, whereas CREB-DNA or NF-Y-DNA binding activity was determined by EMSA. The levels of nSREBP-2 and CREB-DNA or NF-Y-DNA binding activity were quantified on the basis of the densitometric unit and are expressed as means ± SE (n = 6, each done in duplicate). *P < 0.05 compared with control values (expressed as 100%). #P < 0.05 compared with values obtained from Hcy-treated cells.

 


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Fig. 8. Effect of inhibitors on HMG-CoA reductase mRNA expression in hepatocytes. Hepatocytes were isolated from rats fed a regular diet. Cells were incubated with or without Hcy (1 mM) in the absence or presence of SREBP-2 inhibitor AEBSF (300 µM), CREB inhibitor H89 (5 µM) or NF-Y inhibitor NEM (1 mM). Total RNA was prepared from cells after 4 h of incubation. Expression of HMG-CoA reductase mRNA (HMGCR), which was normalized to GAPDH mRNA, was determined by RNase protection assay. Results were expressed as the ratio of HMG-CoA reductase mRNA to GAPDH mRNA and are depicted as means ± SE (n = 6, each done in duplicate). *P < 0.05 compared with control values (expressed as 100%). #P < 0.05 compared with values obtained from Hcy-treated cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The novel findings of the present study are that 1) in the absence of other known risk factors, hyperhomocysteinemia can upregulate HMG-CoA reductase gene expression in the liver via the activation of SREBP-2, CREB, and NF-Y in hepatocytes; 2) hyperhomocysteinemia stimulates cholesterol biosynthesis in the liver and increases the cholesterol concentration in the plasma. Congruent with the finding of fatty liver in hyperhomocysteinemic patients and in experimental animals (25, 26, 47, 49), the present study revealed that small lipid droplets accumulated in the liver of hyperhomocysteinemic rat. Lipid accumulation in the liver appeared to arise from the upregulation of HMG-CoA reductase mRNA expression. This resulted in an enhanced hepatic cholesterol biosynthesis leading to an elevation of cholesterol concentration in the plasma of hyperhomocysteinemic rats. Although a 22% increase in the HMG-CoA reductase mRNA level was found in livers of hyperhomocysteinemic rats, a more than 50% increase in the HMG-CoA reductase activity was observed in the same tissue (Fig. 2). Besides transcriptional regulation, the activity of HMG-CoA reductase can also be affected by other mechanisms. The discrepancy between the level of HMG-CoA reductase mRNA and the enzyme activity might be due to posttranslational modification that warrants further investigation. Abnormal cholesterol metabolism can cause serious complications of many diseases, including hepatic fatty infiltration, which can progress to fibrosis and cirrhosis leading to liver failure (11, 12). The ability of Hcy to promote cholesterol biosynthesis in hepatocytes would provide one of the plausible mechanisms of hyperhomocysteinemia-associated liver pathology. However, it should be noted that pathological changes in the liver caused by short-term hyperhomocysteinemia induced by dietary manipulation might be different from that caused by long-standing hyperhomocysteinemia due to a congenital enzyme defect in patients.

Recently, several transcription factors that regulate HMG-CoA reductase gene expression have been identified (4, 29, 45). SREBP-2 is thought to play a critical role in cholesterol biosynthesis (14, 15, 45). Upon stimulation, the full-length SREBP-2 (125 kDa) in rough endoplasmic reticulum membrane is cleaved in Golgi apparatus to a truncated form (68 kDa), which enters the nucleus and activates transcription of target genes such as HMG-CoA reductase. In the present study, the level of SREBP-2 in the nucleus was found to be significantly elevated in the liver of hyperhomocysteinemic rat as well as in Hcy-treated hepatocytes. To efficiently activate HMG-CoA reductase gene expression, the activation of other transcriptional factors (coregulators) along with SREBP-2 is essential. The two known coregulators for HMG-CoA reductase are CREB and NF-Y (4, 29). Both CREB-DNA and NF-Y-DNA binding activities were significantly elevated in the liver of hyperhomocysteinemic rat as well as in Hcy-treated hepatocytes. These results suggest that activation of SREBP-2 and its coregulators (CREB and NF-Y) by Hcy plays an important role in hepatic expression of HMG-CoA reductase in hyperhomocysteinemic rats. It was reported that insulin, a known activator of HMG-CoA reductase, could enhance CREB transcriptional activity in HepG2 cells through the induction of CREB phosphorylation (20). Once CREB has been activated, it interacts efficiently with SREBP-2 to stimulate the transcription of the HMG-CoA reductase gene in the presence of NF-Y. In the present study, treatment of cultured hepatocytes with individual inhibitors for SREBP-2, CREB, or NF-Y not only blocked Hcy-mediated activation of corresponding transcription factors but also prevented Hcy-induced HMG-CoA reductase mRNA expression. These results suggest that the activation of these transcription factors is essential for Hcy-induced HMG-CoA reductase expression.

Under normal conditions, the intracellular homeostasis of Hcy is tightly regulated. Hcy can be metabolized by two major pathways, namely the transsulfuration pathway to form cysteine and the remethylation pathway to form methionine (26). Factors that perturb steps in Hcy metabolic pathways can cause an increase in its cellular concentration and lead to an elevation of Hcy concentration in the blood. It has been suggested that cellular dysfunction is caused by elevation of intracellular concentrations of Hcy itself and that elevated plasma total Hcy level is a marker of increased intracellular homocysteine (49). In many in vitro studies, exogenous Hcy in concentrations of 5–10 mM was applied to increase intracellular Hcy levels (21, 22, 34, 49). For example, Hcy at concentrations of 1–5 mM was required to add to the cultured HepG2 cells to attain a two- to sixfold transient increase in intracellular Hcy concentrations and to cause endoplasmic reticulum stress in these cells (49). In the present study, the Hcy concentration was increased up to twofold in hepatocytes after incubation with 1 mM Hcy for 60 min. The concentrations of Hcy in livers isolated from hyperhomocysteinemic rats were threefold higher than that in control livers. However, it remains to be further investigated whether the stimulatory effect of Hcy on cholesterol biosynthesis in hepatocytes is due to intracellular or extracellular action of Hcy. At present, it is not fully understood why higher concentrations of Hcy are required for the in vitro experiments to induce the similar adverse effects observed in an animal model (21, 22, 34, 49). We speculate that such a discrepancy might be due to differences in the microenvironment (in vivo vs. in vitro), and/or the duration of cells exposed to homocysteine (weeks vs. hours or days).

Although hyperhomocysteinemia is regarded as an independent risk factor for the development of atherosclerosis, a high plasma concentration of Hcy is often associated with other risk factors. In the Hordaland Hcy study (30), it was found that elevated plasma Hcy concentration was associated with other cardiovascular risk factors, including elevated cholesterol level, male sex, old age, smoking, high blood pressure, and lack of exercise. However, this study did not provide evidence regarding causality between risk factors (30). A recent study demonstrated that hyperhomocysteinemia was associated with sudden death resulting from coronary atherosclerosis with fibrous plaques (5). The data demonstrated a difference in the types of plaque in men with hyperhomocysteinemia and those with hypercholesterolemia (5). In contrast to plaques found in patients with hypercholesterolemia, plaques found in hyperhomocysteinemia predominantly consisted of fibrous tissue with less lipid (5). These observations suggest that a more complex mechanism may be involved in the plaque formation associated with hyperhomocysteinemia. Although hyperhomocysteinemia is regarded as a risk factor for atherosclerosis, the exact mechanisms by which Hcy promotes the development of atherosclerotic lesions remain to be investigated.

In conclusion, Hcy-induced cholesterol biosynthesis and elevation of cholesterol levels in the plasma may contribute to hepatic as well as cardiovascular disorders associated with hyperhomocysteinemia. In diet-induced hyperhomocysteinemic rats, there was a significant increase in the plasma cholesterol concentrations compared with the controls. Chronic exposure of the vessel wall to such a moderate elevation of plasma cholesterol concentration together with hyperhomocysteinemia may lead to vessel injury over a prolonged period of time. The long-term effect of Hcy-induced hypercholesterolemia on the cardiovascular system remains to be investigated.


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This study was supported by grants from the Manitoba Medical Services Foundation and the Manitoba Health Research Council to K. O.


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Address for reprint requests and other correspondence: K. O, Laboratory of Integrative Biology, NCARM, St. Boniface Hospital Research Centre, R4032, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (E-mail: karmino{at}sbrc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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