EBM Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stangl, G. I.
Right arrow Articles by Eder, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stangl, G. I.
Right arrow Articles by Eder, K.
Experimental Biology and Medicine 232:81-87 (2007)
© 2007 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Homocysteine Thiolactone-Induced Hyperhomocysteinemia Does Not Alter Concentrations of Cholesterol and SREBP-2 Target Gene mRNAs in Rats

Gabriele I. Stangl1, Kristin Weisse, Cornelia Dinger, Frank Hirche, Corinna Brandsch and Klaus Eder

Institute of Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, D-06108 Halle/Saale, Germany

1 To whom requests for reprints should be addressed at Institut für Ernährungswissen-schaften, Emil-Abderhalden-Strasse 26, D-06108 Halle/Saale, Germany. E-mail: gabriele.stangl{at}landw.uni-halle.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present rat study was conducted to test whether hyper-homocysteinemia induced by dietary homocysteine (Hcy) alters the cholesterol concentration in plasma and tissue and the gene expression of genes involved in cholesterol biosynthesis and uptake. Therefore, rats were fed 100 or 200 mg Hcy per kilogram body mass per day (Hcy100 group and Hcy200 group, respectively) as DL-homocysteine thiolactone, or an Hcy-free diet, which served as control, over 14 days. Rats from the Hcy100 group and the Hcy200 group had higher plasma Hcy concentrations (34.4 ± 4.6 and 69.4 ± 11.5 µM, respectively) than rats fed an Hcy-free diet (9.5 ± 1.7 µM). The concentration of Hcy in liver was 2.6 and 3.8 times higher, and in small intestine was 2.6 and 5.1 times higher, in the Hcy100 group and the Hcy200 group, respectively, than in control rats (P < 0.05). The concentrations of cholesterol in plasma, lipoproteins, liver, and small intestine and the relative mRNA concentrations of sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and low-density lipoprotein (LDL) receptor in liver and small intestine were not influenced by DL-homocysteine thiolactone supplementation. In conclusion, in view of the experimental conditions used here, increased plasma and tissue concentrations of Hcy do not alter cholesterol metabolism of liver and intestine.

Key Words: homocysteine • cholesterol • gene expression • liver • small intestine • rat


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Homocysteine (Hcy) is an intermediary product in methionine metabolism that can be either converted to cysteine by transsulfuration or methylated in a folate- and vitamin B12–dependent reaction to form methionine. Hyperhomocysteinemia is considered an independent risk factor for vascular disorders resulting from atherosclerosis (1–3). Several potential mechanisms for homocysteine-induced atherosclerosis have been proposed. These include, for example, decreased bioavailability and synthesis of endothelium-derived nitric oxide (4), the vascular production of superoxide (5), increased proliferation of smooth muscle cells (6), enhanced coagulability (7), and protein modification (8). Additionally, it has been suggested that Hcy also may contribute to atherosclerosis by lipid dysregulation. A few studies have found a positive correlation between the plasma concentrations of Hcy and the plasma concentrations of cholesterol in hyperhomocysteinemic patients (9, 10) and in experimental animals (11–14).

Different dietary animal models of hyperhomocysteinemia have been performed to study possible Hcy-mediated effects on lipid metabolism. Those animal models include the oral administration of a high-methionine diet for 2 to 10 weeks (12–14) or excessive methionine combined with inadequate amounts of folate, which lead to increased plasma Hcy concentrations (12). The findings from those experiments show that dietary intervention with excessive methionine stimulates genes involved in cholesterol biosynthesis, such as the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and the low-density lipoprotein (LDL) receptor, which is responsible for the uptake of LDL from plasma into cells via an activation of the sterol regulatory element-binding protein-2 (SREBP-2; Refs. 12–14), a transcription factor involved in gene expression of HMG-CoA reductase and LDL receptor (15–17). Hcy generated from excessive amounts of methionine has been suggested as the factor responsible for the alteration of cholesterol levels and SREBP-2 activation (12, 13).

However, results from other studies propose a direct effect of methionine on cholesterol metabolism, without any involvement of Hcy (18–21). For example, rabbits that were fed a diet enriched in methionine and lysine developed hypercholesterolemia within 3.5 weeks, although Hcy concentration was not increased (21). Additionally, another study has shown that a methionine-induced stimulation of N-methylation of phosphatidylethanolamine (PE) to form phosphatidylcholine (PC) could be responsible for the hypercholesterolemia which was observed in rats fed high-methionine diets for 2 weeks (20). Both research groups proposed that hypercholesterolemia induced by high dietary methionine concentrations might be due to a disturbed hepatic phospholipid metabolism via S-adenosylmethionine (SAM; Refs. 20, 21), the active form of methionine that reflects dietary methionine level (20, 22).

Based on those different findings, there is considerable uncertainty regarding whether Hcy plays a causal role in hypercholesterolemia. To shed more light onto the role of homocysteine in cholesterol metabolism we performed a study in which we administered rats a diet supplemented with homocysteine as DL-homocysteine thiolactone. If the action of excessive methionine on cholesterol metabolism is mediated primarily by homocysteine, one would expect that hyperhomocysteinemia induced by an administration of dietary homocysteine also alters the concentration of cholesterol and the expression of genes involved in cholesterol synthesis, as observed with a high-methionine or folate-deficient diet. For that purpose rats were fed either a diet containing two different amounts of homocysteine as DL-homocysteine thiolactone over 14 days or an Hcy-free diet serving as control. The dietary administration of DL-homocysteine thiolactone has been shown to be a successful experimental approach to increase the Hcy concentrations in plasma and liver and to study Hcy-induced atherogenic processes (23, 24). The concentrations of dietary homocysteine used for our study were chosen to increase the plasma and liver concentrations of Hcy to extents similar to those observed in animal models of hyperhomocysteinemia (12, 13) and to induce hyperhomocysteinemia within clinical relevant ranges. To assess the Hcy-increasing effect of exogenous Hcy, we determined the Hcy concentrations in plasma, liver, and small intestine and the ratio of SAM to S-adenosylhomocysteine (SAH) in liver, which is important for a wide variety of methylation reactions (25). Besides the concentration of cholesterol in plasma, liver, and small intestine, we determined the relative concentrations of mRNA coding for SREBP-2 and its target genes, such as HMG-CoA reductase and the LDL receptor in liver and small intestine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals.
Male 5-month-old Sprague-Dawley rats (n = 30) supplied by Charles River (Sulzfeld, Germany) with an initial body weight (±SD) of 464 ± 28 g were assigned randomly to three groups of 10 rats each. The rats of the Hcy100 group were fed daily Hcy as DL-homocysteine thiolactone (Sigma-Aldrich, Taufkirchen, Germany), which corresponded to an intake of about 100 mg Hcy per kg body mass; the rats of the Hcy200 group received a diet that corresponded to a daily intake of about 200 mg Hcy per kg body mass. The control group received a diet without homocysteine. The rats were kept individually in macrolon cages in a room maintained at a temperature of 23°C and 50%–60% relative humidity with lighting from 0600 to 1800 h. The experimental procedures described followed established guidelines for the care and handling of laboratory animals and were approved by the council of Saxony-Anhalt, Germany.

Experimental Diets.
All rats were fed a semi-synthetic diet according to the AIN-93M diet formulated for maintenance of adult rats (26), which contained the following ingredients (g/kg): cornstarch, 633; casein, 140; sucrose, 100; soybean oil, 40; cellulose, 40; mineral mixture, 35; vitamin mixture, 9; DL-methionine, 2.0; and choline chloride, 1.0. Vitamins and minerals were supplemented according to recommendations from the American Institute of Nutrition for rat diets (26). Folate, vitamin B12, and vitamin B6, which are involved in one-carbon metabolism, were added in amounts of 2 mg, 25 µg, and 6 mg, respectively, per kilogram diet. DL-homocysteine thiolactone was included in the diet at the expense of cellulose. The diet for rats from the Hcy100 group contained 2.8 g/kg DL-homocysteine thiolactone, and the diet for rats from the Hcy200 group contained 5.6 g/kg DL-homocysteine thiolactone. To avoid differences in food intake, each rat received 17 g food per day. This food amount was close to the energetic requirement for maintenance for adult rats (27). Intakes of 17-g diets containing 2.8 and 5.6 g/kg DL-homocysteine thiolactone correspond to daily intakes of approximately 100 and 200 mg Hcy per kilogram body mass, respectively, for each rat from the Hcy100 and Hcy200 group. During the whole experiment, all rats were fed equal amounts of diet daily. Diets were administered once daily at 0800 h. Water was freely available from nipple drinkers. The experimental diets were fed for 14 days.

Sample Collection.
After a feeding period of 14 days the rats were killed by decapitation under light anesthesia with diethyl ether. Rats were not fasted before killing, because food deprivation leads to a significant downregulation of the genes involved in cholesterol metabolism (28) that were to be measured in this study. Plasma was separated from heparinized whole blood by centrifugation at 1880 g for 10 mins at 4°C. Livers were excised, weighed, and immediately snap frozen in liquid nitrogen. Samples of liver for total RNA isolation were stored at –80°C. The whole small intestine was carefully rinsed with ice-cold saline and opened lengthwise. Enterocytes of the small intestine were isolated by scraping (29). Enterocytes that were scraped off the gut mucosa were then mixed, weighed, and snap frozen in liquid nitrogen. For total RNA isolation, aliquots of the enterocytes were stored at –80°C.

Plasma Lipoprotein Isolation.
Plasma lipoproteins were separated by stepwise ultracentrifugation (Mikro-Ultrazentrifuge; Sorvall Products, Bad Homburg, Germany) at 900,000 g at 4°C for 1.5 hrs. The lipoprotein fractions were collected on the basis of their densities ({rho}; kg/l) as described previously for rats (30): LDL: 1.006 kg/l < {rho} < 1.063 kg/l; and HDL: {rho} > 1.063 kg/l. Plasma densities were adjusted with sodium chloride and potassium bromide. The lipoprotein fractions used for cholesterol analysis were removed by suction.

Cholesterol Analysis.
Lipids from liver and small intestine were extracted with a mixture of n-hexane and isopropanol (3:2 [v/v]; Ref. 31). The concentration of cholesterol in liver and small intestine was determined in aliquots of the lipid extracts after evaporating the solvent mixture and dissolving the lipids in a mixture of Triton X-100 and chloroform (1:1 [v/v]; Ref. 32). Concentrations of total cholesterol in plasma and lipoprotein fractions, liver, and small intestine were determined using an enzymatic reagent kit (catalog no. 1.14830; VWR International, Darmstadt, Germany).

Analyses of Hcy, SAM, and SAH.
Total Hcy (free Hcy, D- and L-enantiomers, Hcy disulfides, and Hcy-cysteine mixed disulfides) was determined by high-performance liquid chromatography (HPLC; Ref. 33). For measurement of Hcy concentrations in liver and small intestine, aliquots of the tissue were thawed and homogenized in ice-cold phosphate-buffered saline. The concentrations of SAM and SAH in the liver were measured by HPLC (34). Samples of frozen liver were thawed and homogenized in 0.5 M ice-cold perchloric acid, and the homogenates were centrifuged at 16,000 g for 20 mins at 4°C. The resulting supernatants were applied to an HPLC column (Hypersil ODS 250 x 4 mm2, 5 µm; Agilent Technologies, Waldbronn, Germany). The mobile phase was a 100-mM potassium dihydrogen phosphate solution containing 10 mM sodium heptane sulphonate and 3% (v/v) methanol. The flow rate was 1.5 ml/min, and the elution was monitored at 254 nm.

Gene Expression Analysis.
For analysis of gene expression, total RNA was extracted from frozen liver samples using Trizol reagent (Invitrogen, Karlsruhe, Germany). RNA was quantified by A260, and its integrity was verified by agarose gel electrophoresis using ethidium bromide for visualization. Total RNA, oligo dT primer (Operon, Köln, Germany) and RevertAid M-MuLV reverse transcriptase (MBI Fermentas, St. Leon-Rot, Germany) were used for cDNA synthesis (Mastercycler Personal; Eppendorf, Hamburg, Germany). The concentration of cDNA was analyzed by real-time detection polymerase chain reaction (PCR; DNA Engine Opticon System; MJ Research Inc., Waltham, MA). The expression signal of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal control for normalization, because the expression of GAPDH that was obtained as the value of cycle threshold (C(T)) was not influenced by dietary treatment. PCR was carried out in a final volume of 20 µl reaction mixture containing 500 µM dNTP (Roth, Karlsruhe, Germany); 3.5 mM MgCl2, 1.25 U Taq DNA polymerase, and 4 µl of 5x buffer (all from Promega, Mannheim, Germany); 0.5 µl of 10xSybr Green I (Sigma-Aldrich, Taufkirchen, Germany); 2 µl first-strand cDNA; and 1.34 µl primer mix. The primer sequences were as follows: GAPDH (EC 1.2.1.12), up: 5'-GCA-TGG-CCT-TCC-GTG-TTC-C-3', low: 5'-GGG-TGG-TCC-AGG-GTT-TCT-TAC-TC-3'; SREBP-2, up: 5'-CCG-GTA-ATG-ATG-GGC-CAA-GAG-AAA-G-3', low: 5'-AGG-CCG-GGG-GAG-ACA-TCA-GAA-G-3'; HMG-CoA re-ductase (EC 1.1.1.34), up: 5'-AAG-GGG-CGT-GCA-AAG-ACA-ATC-3', low: 5'-ATA-CGG-CAC-GGA-AAG-AAC-CAT-AGT-3'; LDL receptor, up: 5'-AGA-ACT-GCG-GGG-CCG-AAG-ACA-C-3', low: 5'-AAA-CCG-CTG-GGA-CAT-AGG-CAC-TCA-3'. The DNA of GAPDH, SREBP-2, HMG-CoA reductase, and LDL receptor was amplified in cycles of 20 secs denaturation at 95°C, 30 secs annealing at primer-specific temperatures, and 40 secs elongation at 72°C. Fluorescence was measured at 72°C. A final melting curve guaranteed the authenticity of the target product.

Statistical Analysis.
Means of the three groups were compared by the Fisher’s multiple range test using Minitab Statistical Software (Minitab, State College, PA). Values in the text are means ± SD. Means were considered significantly different at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth and Liver Weight.
All rats with diet-induced hyperhomocysteinemia appeared normal. Final body weights of rats were not influenced by the dietary treatment and were similar to the body weights at the beginning of the experiment, because the amounts of diets offered to rats were close to the energetic requirement for maintenance (control group: 470 ± 27 g; Hcy100 group: 473 ± 20 g; Hcy200 group: 475 ± 33 g; n = 10). Body weight gains within the 14 days of experiment did not differ between the three groups of rats (control group: 7.0 ± 3.8 g; Hcy100 group: 10.3 ± 7.7 g; Hcy200 group: 10.1 ± 5.5 g; n =10). The relative liver weights were not different between the three groups of rats (control group: 2.92 ± 0.22 g/100 g of body wt; Hcy100 group: 2.96 ± 0.20 g/100 g of body wt; Hcy200 group: 2.84 ± 0.20 g/100 g of body wt; n =10).

Hcy, SAM, and SAH Concentrations.
Rats from the Hcy100 and the Hcy200 groups had markedly higher concentrations of total Hcy in plasma, liver, and small intestine than rats fed the Hcy-free control diet (Fig. 1Go). There was a dose-dependent increase of the plasma and tissue Hcy concentrations in the rats. The concentration of SAM in liver was not different between the three groups of rats (Table 1Go). The concentration of SAH and the ratio of SAH to SAM in liver were significantly higher in the Hcy200 group than in the control group. Rats from the Hcy100 group had intermediate values of SAH and ratio of SAH to SAM that were not different from those of the control group and the Hcy200 group.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		Figure 1. Concentration of total Hcy in plasma, liver, and small intestine of rats with a mean dietary intake of either 0 (control), 100 (Hcy100), or 200 (Hcy200) mg Hcy per kg body mass per day. Each bar represents means ± SD, n =10. Bars that are not indicated with the same superscript are significantly different by Fisher’s multiple range test, P < 0.05.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Concentrations of SAM and SAH and the Ratio of SAH to SAM in Liver of Rats with a Mean Intake of Either 0 (Control), 100 (Hcy100), or 200 (Hcy200) mg Hcy per kg Body Mass per Daya
 
Cholesterol Concentrations.
The concentration of cholesterol in plasma, LDL, HDL, liver, and small intestine did not differ between the rats fed the Hcy-supplemented diets and the rats fed the control diet without Hcy (Table 2Go).


View this table:
[in this window]
[in a new window]

  		Table 2. Concentration of Total Cholesterol in Plasma, Lipoproteins, Liver, and Small Intestine of Rats with a Mean Intake of Either 0 (Control), 100 (Hcy100), or 200 (Hcy200) mg Hcy per kg Body Mass per Daya
 
Relative mRNA Concentrations.
The relative mRNA concentrations of SREBP-2, HMG-CoA reductase, and LDL receptor in liver and small intestine also were not altered by the administration of dietary Hcy compared with the control rats (Fig. 2Go).


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		Figure 2. Relative mRNA concentrations of SREBP-2, HMG-CoA reductase, and LDL receptor in liver (A) and small intestine (B) of rats with a mean intake of either 0 (control), 100 (Hcy100), or 200 (Hcy200) mg Hcy per kg body mass per day. Concentrations were related to the reference gene GAPDH. Values of the control group were set 1. Each bar represents mean ± SD, n =10. There were no differences in the relative mRNA concentrations of the genes measured in liver and small intestine between the three groups of rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings from this rat experiment show that exogenously administered Hcy as DL-homocysteine thiolactone, which led to markedly increased concentrations of Hcy in plasma, liver, and small intestine, did not alter the cholesterol concentrations in plasma and tissues and the expression of the key genes involved in cholesterol biosynthesis and uptake. As plasma and liver cholesterol concentrations were not influenced, we assume that excretion of cholesterol via bile acids also was not altered by dietary DL-homocysteine thiolactone. The failure of Hcy to affect cholesterol concentration and the mRNA coding for SREBP-2 target genes is in contrast to previous findings with hyperhomocysteinemic rats (12, 13). Since hyperhomocysteinemia in those studies was not induced by the administration of dietary homocysteine but by an excess of dietary methionine, we suggest that the cholesterol dysregulation observed in those rodents (12, 13) was not driven by Hcy itself but by another factor related to excessive dietary methionine, inadequate amounts of folate, or other experimental conditions. Factors such as age of the animals, duration of the experiment, overnight fasting, or dietary factors other than methionine could be responsible for the discrepancies observed. Although it is quite unusual to use commercially available diets that contained undefined ingredients for nutrition studies as in the studies of Werstuck et al. (12) and Woo et al. (13), these authors suggest that Hcy is responsible for the activation of SREBP-2, a transcription factor that plays a critical role in the homeostatic regulation of cholesterol in the cell and is responsible for the observed cholesterol accumulation in the hyperhomocysteinemic rodents (12, 13). SREBP-2 is synthesized as inactive precursor bound to the endoplasmatic reticulum membranes, and activation of this membrane-bound transcription factor involves a two-step proteolytic cascade through which the SREBP-2 molecule is released from the endoplasmic reticulum membrane and obtains its mature form in the Golgi complex as a transcription factor. The active SREBP-2 enters the nucleus and binds to promoter or enhancer regions of genes involved in cholesterol homeostasis, such as LDL receptor and enzymes involved in sterol biosynthesis (35). Under the experimental conditions used in our study the relative mRNA concentration of SREBP-2 of animals fed DL-homocysteine thiolactone was not different from that in the control group. Additionally, we suggest that nuclear concentrations of SREBP-2 remained unaffected by dietary Hcy treatment, because the relative concentrations of mRNA coding for the SREBP-2 target genes such as HMG-CoA reductase and the LDL receptor were not significantly different from those of the control group. The failure of Hcy to affect the mRNA coding for SREBP-2 target genes such as HMG-CoA reductase and the LDL receptor in liver and small intestine confirms that Hcy by itself does not dysregulate the cholesterol metabolism, although the Hcy concentrations in plasma, liver, and small intestine of the Hcy-treated rats in our experiment were within pathophysiologic ranges already.

The dietary administration of DL-homocysteine thiolactone has been used already as a successful dietary model of hyperhomocysteinemia in a few previous studies (23, 24). Findings from our study show that plasma Hcy concentration dose dependently increased within 2 weeks as dietary Hcy rose. Additionally, the increase of circulating Hcy was accompanied by an increase of the concentrations of cellular Hcy in hepatocytes and enterocytes, and the concentration of cellular SAH in the Hcy200 group. Therefore, data from the present study indicate that the orally administered Hcy as DL-homocysteine thiolactone is an efficient model to induce hyperhomocysteinemia and to increase cellular levels of Hcy, and further show that Hcy as DL-homocysteine thiolactone is absorbed and transported efficiently through the bloodstream into solid tissues. However, analysis of Hcy in plasma, liver, and small intestine by HPLC did not differentiate between the D- and L-homocysteine. From a few studies it is known that the effects of Hcy on endothelial cells, such as chemokine expression (36) and formation of reactive oxygene species (37), could only be observed with L- but not D-homocysteine. Assuming the fact that D-homocysteine does not induce any effects, the remaining L-homocysteine concentrations in the animals fed DL-homocysteine thiolactone are suggested as being high enough and being within ranges in which Hcy had a hypercholesterolemic effect as observed in other rodent studies (12, 13). Although we observed an alteration of the hepatic SAH concentration in the Hcy200 group compared with the control group, the concentration of SAM was not influenced by dietary DL-homocysteine thiolactone. Assuming that the hepatic SAM concentration, which is suggested as playing a critical role in the formation of PC from PE and in turn for regulation of cholesterol metabolism, is responsible for the hypercholesterolemia observed in studies with high-methionine diets (20, 22), it could be possible that the failure of Hcy to affect cholesterol concentration in our study is due to the fact that SAM concentration in liver remained unchanged.

In addition to the rat experiments, there also exist data from cell culture studies in which the effects of Hcy on primary hepatocytes and HepG2 cells were tested on cholesterol synthesis by use of free L-Hcy or DL-Hcy in the incubation medium (12, 13). Consistent with their in vivo findings, both research groups have found that the intracellular cholesterol concentration increased when the cells were incubated with a medium that contains increasing concentrations of Hcy from 1 to 5 mM. However, one must not forget that these Hcy concentrations are 10- to 50-fold higher than the plasma Hcy concentrations observed in severe hyperhomocystemic patients with rare genetic disorders (38). Therefore, it is questionable whether the use of such extremely high concentrations of Hcy is an appropriate in vitro model to verify possible effects observed in vivo. However, the plasma Hcy concentrations induced in the present rat study were within clinical relevant ranges. Apart from the fact that complete deficiencies of cystathionine ß-synthase or 5,10-methylenetetrahydrofolate reductase are associated with plasma Hcy concentrations greater than 100 µM (39), moderately increased concentrations of plasma homocysteine ranging from 15 to 60 µM are quite prevalent in the general population (38) and can be caused by renal impairment (40); folate and vitamin B6 deficiencies, particularly in high-methionine states (41, 42); certain medications such as fibrates or anticonvulsives (43); or common polymorphisms in the methylene tetrahydrofolate reductase gene (44).

In conclusion, this study shows that hyperhomocysteinemia produced by feeding of DL-homocysteine thiolactone does not influence expression of genes involved in cholesterol homeostasis and also does not alter the concentration of cholesterol in plasma, circulating lipoproteins, liver, and intestine.

Received for publication May 16, 2006. Accepted for publication June 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham J. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 324:1149–1155, 1991.[Abstract]
  2. Refsum H, Ueland PM, Nygård O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med 49:31–62, 1998.[CrossRef][Medline]
  3. Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 338:1042–1050, 1998.[Free Full Text]
  4. Ungvari Z, Csiszar A, Bagi Z, Koller A. Impaired nitric oxide-mediated flow-induced coronary dilation in hyperhomocysteinemia. Am J Pathol 161:145–153, 2002.[Abstract/Free Full Text]
  5. Dayal S, Arning E, Bottiglieri T, Böger RH, Sigmund CD, Faraci FM, Lentz SR. Cerebral vascular dysfunction mediated by superoxide in hyperhomocysteinemic mice. Stroke 35:1957–1962, 2004.[Abstract/Free Full Text]
  6. Tsai JC, Wang H, Perrella MA, Yoshizumi M, Sibinga NE, Tan LC, Haber E, Chang TH, Schlegel R, Lee ME. Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells. J Clin Invest 97:146–153, 1996.[Medline]
  7. Durand P, Lussier-Cacan S, Blache D. Acute methionine load-induced hyperhomocysteinemia enhances platelet aggregation, thromboxane biosynthesis, and macrophage-derived tissue factor activity in rats. FASEB J 11:1157–1168, 1997.[Abstract]
  8. Jakuboswski H, Zhang L, Bardeguez A, Aviv A. Homocysteine thiolactone and protein homocysteinylation in human endothelial cells. Implications for atherosclerosis. Circ Res 87:45–51, 2000.[Abstract/Free Full Text]
  9. Olszewski AJ, McCully KS. Homocysteine content of lipoproteins in hypercholesterolemia. Atherosclerosis 88:61–68, 1991.[CrossRef][Medline]
  10. Nygård O, Vollset SE, Refsum H, Stensvold I, Tverdal A, Nordrehaug JE, Ueland PM, Kvåle G. Total plasma homocysteine and cardiovascular risk profile: the Hordaland homoysteine study. JAMA 274:1526–1533, 1995.[Abstract/Free Full Text]
  11. Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R, Malinow MR, Maeda N. Mice deficient in cystathionine beta-synthase: role of chemokine and adhesion molecules. Arterioscler Thromb Vasc Biol 22: 77–83, 2002.
  12. Werstuck GH, Lentz SR, Dayal S, Hossain GS, Sood SK, Shi YY, Zhou J, Maeda N, Krisans SK, Malinow MR, Austin RC. Homocysteine-induced endoplasmatic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways. J Clin Invest 107:1263–1273, 2001.[Medline]
  13. Woo CWH, Siow YL, Pierce GN, Choy PC, Minuk GY, Mymin D, O K. Hyperhomocysteinemia induces hepatic cholesterol biosynthesis and lipid accumulation via activation of transcription factors. Am J Physiol Endocrinol Metab 288:E1002–E1010, 2005.[Abstract/Free Full Text]
  14. Hirche F, Schröder B, Knoth B, Stangl GI, Eder K. Effect of dietary methionine on plasma and liver cholesterol concentrations in rats and expression of hepatic gene involved in cholesterol metabolism. Br J Nutr 95:879–888, 2006.[Medline]
  15. Vallett SM, Sanchez HB, Rosenfeld JM, Osborne TF. A direct role of sterol regulatory element binding protein in activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene. J Biol Chem 271:12247–12253, 1996.[Abstract/Free Full Text]
  16. Horton JD, Shimomura I, Brown MS, Hammer RE, Goldstein JL, Shimano H. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest 101:2331–2339, 1998.[Medline]
  17. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131, 2002.[CrossRef][Medline]
  18. Sugiyama K, Kanamori H, Akachi T, Yamakawa A. Amino acid composition of dietary proteins affects plasma cholesterol concentration through alteration of hepatic phospholipid metabolism in rats fed a cholesterol-free diet. J Nutr Biochem 7:40–48, 1996.
  19. Sugiyama K, Yamakawa A, Kumazawa A, Saeki S. Methionine content of dietary proteins affects the molecular species composition of plasma phosphatidylcholine in rats fed a cholesterol-free diet. J Nutr 127:600–607, 1997.[Abstract/Free Full Text]
  20. Sugiyama K, Kumazawa A, Zhou H, Saeki S. Dietary methionine level affects linoleic acid metabolism through phosphatidylethanolamine N-methylation in rats. Lipids 33:235–242, 1998.[CrossRef][Medline]
  21. Giroux I, Kurowska EM, Freeman DJ, Carroll KK. Addition of arginine but not glycine to lysine plus methionine-enriched diets modulated serum cholesterol and liver phospholipids in rabbits. J Nutr 129:1807–1813, 1999.[Abstract/Free Full Text]
  22. Shimada Y, Morita T, Sugiyama K. Increased response of liver microsomal {Delta}6-desaturase activity to dietary methionine in rats. Biosci Biotechnol Biochem 67:743–751, 2003.[Medline]
  23. Frauscher G, Karnaukhova E, Muehl A, Hoeger H, Lubec B. Oral administration of homocysteine leads to increased plasma triglycerides and homocysteic acid – additional mechanisms in homocysteine induced endothelial damage? Life Sci 57:813–817, 1995.[CrossRef][Medline]
  24. Hill CH, Mecham R, Starcher B. Fibrillin-2 defects impair elastic fiber assembly in a homocysteinemic chick model. J Nutr 132:2143–2150, 2002.[Abstract/Free Full Text]
  25. Schatz RA, Vunnam CR, Sellinger OZ. Species and tissue differences in the catabolism of S-adenosyl-L-homocysteine: a quantitative, chromatographic study. Life Sci 20:375–383, 1977.[Medline]
  26. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc Writing Committee on the reformulation of the AIN-76A rodent diet. J Nutr 123:1939–1951, 1993.[Abstract/Free Full Text]
  27. National Research Council. Nutrient Requirements of Laboratory Animals. Washington, DC: National Academy of Sciences, 1995.
  28. Horton JD, Bashmakov Y, Shimomura I, Shimano H. Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice. Proc Natl Acad Sci U S A 95:5987–5992, 1998.[Abstract/Free Full Text]
  29. Mottino AD, Hoffman T, Jennes L, Vore M. Expression and localization of multidrug resistant protein mrp2 in rat small intestine. J Pharmacol Exp Ther 293:717–723, 2000.[Abstract/Free Full Text]
  30. Giudetti AM, Beynen AC, Lemmens AG, Gnoni GV, Geelen MJH. Hepatic fatty acid metabolism in rats fed diets with different contents of C18:0, C18:1 cis and C18:1 trans isomers. Br J Nutr 90:887–893, 2003.[CrossRef][Medline]
  31. Hara A, Radin NS. Lipid extraction of tissues with a low toxicity solvent. Anal Biochem 90:420–426, 1978.[CrossRef][Medline]
  32. De Hoff JL, Davidson JH, Kritchevsky D. An enzymatic assay for determining free and total cholesterol in tissues. Clin Chem 24:433–435, 1978.[Abstract/Free Full Text]
  33. Vester B, Rasmussen K. High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum. Eur J Clin Chem Clin Biochem 29:549–554, 1991.[Medline]
  34. Sugiyama K, Akachi T, Yamakawa A. Eritadenine-induced alteration of hepatic phospholipids metabolism in relation to its hypocholesterolemic action in rats. J Nutr Biochem 6:80–87, 1995.
  35. Brown MS, Goldstein JL. A proteolytic pathway that controls the cholesterol content of membrane, cells, and blood. Proc Natl Acad Sci U S A 96:11041–11048, 1999.[Abstract/Free Full Text]
  36. Poddar R, Sivasubramanian N, DiBello PM, Robinson K, Jacobsen DW. Homocysteine induces expression of secretion of monocyte chemoattractant protein-1 and interleukin-8 in human aortic endothelial cells. Circulation 103:2717–2723, 2001.
  37. Weiss N. Mechanisms of increased vascular oxidant stress in hyperhomocysteinemia and its impact on endothelial function. Curr Drug Metabol 6:27–36, 2005.[CrossRef][Medline]
  38. Jacques PF, Rosenberg IH, Rogers G, Selhub J, Bowman BA, Gunter EW, Wright JD, Johnson CL. Serum total homocysteine concentrations in adolescent and adult Americans: results from the third national health and nutrition examination survey. Am J Clin Nutr 69:482–489, 1999.[Abstract/Free Full Text]
  39. Mudd SH, Finkelstein JD, Refsum H, Ueland PM, Malinow MR, Lentz SR, Jacobsen DW, Brattstrom L, Wilcken B, Wilcken DE, Blom HJ, Stabler SP, Allen RH, Selhub J, Rosenberg IH. Homocysteine and its disulfide derivatives: a suggested consensus terminology. Arterioscler Thromb Vasc Biol 20:1704–1706, 2000.[Free Full Text]
  40. Nerbass FB, Draibe SA, Feiten SF, Chiarello PG, Vannucchi H, Cuppari L. Homocysteine and its determinants in nondialyzed chronic kidney disease patients. J Am Diet Assoc 106:267–270, 2006.[Medline]
  41. Mudd SH, Matorin AI, Levy HL. Homocysteine thiolactone: failure to detect in human serum or plasma. Res Commun Chem Pathol Pharmacol 63:297–300, 1989.[Medline]
  42. Selhub J, Jacques PF, Wilson PWF, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 270:2693–2698, 1993.[Abstract/Free Full Text]
  43. Dierkes J, Westphal S. Effect of drugs on homocysteine concentrations. Semin Vasc Med 5:124–139, 2005.[CrossRef][Medline]
  44. Lentz SR, Haynes WG. Homocysteine: is it a clinically important cardiovascular risk factor? Clev Clin J Med 71:729–734, 2004.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stangl, G. I.
Right arrow Articles by Eder, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stangl, G. I.
Right arrow Articles by Eder, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS