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2
*
Institute of Molecular Biology,
Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Hong Kong, China and
**
Peking Union Medical College (PUMC), Beijing, China
2To whom correspondence should be addressed. E-mail: mcllin{at}hkusua.hku.hk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • MTP • garlic • gene expression • HepG2 cells • Caco-2 cells • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Elevated levels of plasma apolipoprotein B (apoB)3
-containing lipoproteins lead to atherosclerosis and coronary heart disease. The microsomal triglyceride transfer protein (MTP) plays a pivotal role in the assembly and secretion of the apoB-containing lipoproteins, the VLDL in the liver and chylomicrons in the intestine. MTP exists in the lumen of the endoplasmic reticulum as a heterodimer with protein-disulfide isomerase and is involved in the transfer of triglycerides, cholesterol esters and phospholipids to newly synthesized apoB [reviewed in (1
), and references therein]. In human patients with abetalipoproteinemia, the absence of functional MTP results in a defective production of apoB-containing lipoproteins (2
,3
). Recent studies using liver-specific MTP overexpressing (4
) and knock-out mice (5
,6
) have demonstrated that MTP is rate-limiting for VLDL apoB secretion. These findings strongly suggest that the regulation of MTP plays an important role in modulating lipoprotein production in the liver and intestine. Consistent with this hypothesis, inhibiting MTP activity has been shown to cause a dose-dependent decrease in the secretion rate of apoB-containing lipoproteins in vitro in human hepatoma HepG2 (7
) and intestinal Caco-2 cells (8
). A similar phenomenon was observed in vivo in rodent models (7
), and in Watanabe-heritable hyperlipidemic rabbits, a model for human homozygous familial hypercholesterolemia (9
).
Studies conducted by us and others have shown that hepatic MTP mRNA levels are up-regulated in hamsters fed a high fat and/or high cholesterol diet (10
,11
), in streptozotocin-induced diabetic rats (1
, 12
) and in Otsuka Long-Evans Tokushima Fatty rats with visceral fat accumulation (13
). These rats have atherogenic plasma apoB-containing VLDL and LDL profiles. Evidence obtained from in vitro studies in HepG2 cells demonstrated that insulin (14
–16
) and ethanol (17
) inhibit, whereas sterols (15
,16
) stimulate the transcription of the MTP gene. It has also been reported that hepatic MTP gene expression is regulated by cytokines and endotoxins (18
). These observations suggest that MTP level is transcriptionally regulated by multiple signals under physiologic as well as pathologic conditions to control lipoprotein assembly and secretion.
For many years, garlic (Allium sativum) has been attributed to possess therapeutic features. The effects of garlic on serum lipid levels and on atherosclerosis have been investigated extensively [reviewed in (19
–22
), and references therein]. Because controversy continues concerning the lipid-lowering properties of different standardized garlic preparations, we chose to examine the effects of fresh garlic homogenate and fresh garlic supernatant on the expression of the MTP gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture media and LipofectAMINE were purchased from Life Technologies (Grand Island, NY). Fatty acid–free bovine serum albumin (BSA) was obtained from Sigma Chemical (St. Louis, MO). 
32P-ATP was supplied by DuPont New England Nuclear (Boston, MA).
Preparation of fresh garlic extract (FGE) and fresh garlic homogenate (FGH).
Garlic bulbs (average 60 g/bulb), purchased from a local supermarket, were cut into small pieces and homogenized with 2 volumes of extraction solvent containing equal volumes of dimethyl sulfoxide (DMSO) and water in a motor-driven Teflon-glass homogenizer on ice. The FGE was prepared from the supernatant by centrifugation of fresh garlic homogenates at 9000 x g for 20 min at 4°C followed by sterilization through filtration. The FGH for the in vivo study was freshly prepared by homogenizing 1 g of garlic in 2 mL of water in a motor-driven Teflon-glass homogenizer. Cooked garlic homogenate was made with garlic that had been boiled for 1 h in water before homogenization. The extraction process was quite reproducible because the FGE and FGH prepared at separate times consistently produced similar results in the in vitro cell cultures and the in vivo animal studies.
Cell culture conditions for HepG2 and Caco-2 cells, quantitation of apoB and apoA1 secretion rates and lactate dehydrogenase (LDH) levels in the media.
HepG2 and Caco-2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. In a typical experiment, cells were seeded into 6-well (35 mm) culture plates, allowed to grow to 80% confluency, and then incubated with 5 mL of either the control medium (DMEM supplemented with 30 g/L BSA plus an equal amount of DMSO as used in the experimental media) or experimental medium (control medium containing 30 g/L BSA plus FGE dissolved in DMSO) at 37°C for 6 h. At the end of the experiments, media were collected and the amount of apoB and apoA1 secreted into the media was determined by standard ELISA methods as described previously (14
). LDH activity was determined as described in Sigma Procedure No. 500. Cells were harvested by the trypsin EDTA method and used for protein determination and RNA extraction. Protein contents were measured by the method of Lowry et al. (23
).
DNA excess solution hybridization assay for the quantitation of human MTP and actin mRNA levels.
HepG2 and Caco-2 cells express MTP and have been used widely to study the regulation of apoB-containing lipoprotein secretion. Total cellular RNA was isolated by the guanidinium thiocyanate method (TRISOLV, Cinna Biotex Labs, Houston, TX). The relative levels of MTP and actin mRNA were determined by the DNA excess solution hybridization assays, using oligonucleotide probes complementary to human MTP large subunit cDNA and human actin (Oncogene Science, Cambridge, MA). The oligonucleotides were end-labeled with 3-P ATP and the assay carried out as described (14
). The results obtained were within the linear range of the assay (1–20% S1 nuclease resistance).
Animals and treatments.
Male Sprague-Dawley rats (190–210 g; Taconic Farms, Germantown, NY) consumed nonpurified diet (Standard Purina Diet no. 5001) ad libitum for 1 wk before the study. They were randomly divided into two groups (n = 6 per group). Food was withheld for 2 h before gavage with FGH (5 g/kg). Control rats were similarly treated with either water or a cooked FGH preparation. Three hours after dosing, the rats were killed and samples of the liver (
0.5 g) and the small intestine (60 cm proximal section) were removed, frozen immediately in liquid N2, and stored at -80°C until RNA was extracted. This 3-h time point was chosen because our previous study indicated that both intestinal and hepatic MTP mRNA levels are fully regulated 3 h after an oral dosing of treatment substances such as ethanol (17
). We chose to use 5 g of fresh garlic/kg body on the basis of our previous experience indicating that this concentration is well tolerated by mice (24
) and rats (unpublished data) and is sufficient to produce a significant therapeutic effect in protecting animals against acetaminophen-induced hepatotoxicity (24
). The animal study protocol was approved by the appropriate committee and complied with NIH guidelines (25
).
Statistical analysis.
All data are presented as means ± SD. Data were analyzed using one-way ANOVA, and significant differences between means were evaluated by the Bonferroni post-hoc test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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FGE decreased MTP mRNA level in HepG2 and Caco-2 cells (Table 1)
. Significant 15 and 39% reductions were detected in HepG2 and Caco-2 cells, respectively, with 3 g/L FGE. Maximal 72 and 59% reductions were observed with 6 g/L FGE. In addition, despite the marked suppression of MTP mRNA levels, FGE did not affect actin mRNA levels in HepG2 and Caco-2 cells.
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FGE (6 g/L) caused modest 27% (P = 0.05) and 32% (P < 0.05) inhibitions in apoB secretion in HepG2 and Caco-2 cell lines, respectively (Table 2)
. It had no effect on apoA1 secretion. The FGE exerted no cytotoxic effects on HepG2 and Caco-2 cells, as indicated by normal cell morphology, cellular protein content and release of LDH to the medium.
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Rats fed 5 g/kg FGH had a substantial reduction (P < 0.05) in intestinal MTP mRNA levels -46% and -43% relative to water and cooked garlic controls (Table 3)
. The hepatic MTP mRNA level, however, was not significantly affected. Actin mRNA levels were not affected in either liver or intestine (Table 3)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–29
). A sib-pair linkage analysis of adult female twins found significant linkage between the MTP gene and the plasma triglyceride level (26
). A recently discovered functional polymorphism in the promoter of the MTP gene (-493G/T) has been shown to affect the plasma concentration of LDL cholesterol and the VLDL distribution between large and small particle species in familial hypercholesterolemia (27
) and in healthy men (28
,29
). This polymorphism is also associated with biological surrogates of steatohepatitis in patients with Type II diabetes (30
). Taken together, these data suggest the importance of modest differences in MTP level in determining the phenotype as well as the rate of lipoprotein secretion. The present study suggests that fresh garlic extract contains active components that can inhibit the expression of the MTP gene in both the liver and intestinal cell lines. We detected only a modest effect of FGE on apoB secretion. This observation is consistent with our previous finding that due to the long half-life of MTP, a decrease in the MTP mRNA level will result in only modest decreases in either the MTP protein level or the apoB secretion rate. We also found that oral administration of fresh garlic led to the MTP inhibitory effect only in the intestine. It is possible that the active components in FGH either were metabolized and/or inactivated in the liver or before reaching liver, or were not transported out of the intestine to enter the blood circulation and reach the liver. However, we cannot exclude the possibility that the sampling of liver was not performed at the most optimal time point. It is also possible that the small amount of residue food in the intestine might have aided in the absorption of the active components, thus providing better conditions for the detection of MTP regulation in the intestine. It is also possible that the effect seen in the HepG2 cells may be influenced in part by the presence of DMSO in the cell culture medium, which may have stimulated the uptake of the active components into the cells.
Versatile classes of refined MTP inhibitors have been developed and shown to be effective lipid-lowering drugs in animal models. Some of these drugs are currently being used in clinical trials (31
–34
). However, most of these MTP inhibitors act on both the intestine and the liver and thus may cause fatty liver. They are also transported in circulating blood, which may cause toxicity and other undesirable side effects. These two disadvantages are major concerns regarding the further development of MTP inhibitors as drugs despite their potent lipid-lowering activity. Therefore, intestine-specific MTP inhibitors would circumvent these adverse effects. Results from our studies suggest that the active components in fresh garlic are potential intestine-specific MTP inhibitors. At present, several substances have been found to be able to reduce MTP mRNA levels, including insulin and small molecules such as ethanol. The active component in garlic that possesses this MTP inhibition activity is not known. Results from our study suggest that they are heat unstable.
In view of the in vivo results showing decreased intestinal MTP mRNA in rats gavaged with fresh garlic, and the in vitro results indicating a direct effect of fresh garlic extract on MTP gene expression in HepG2 and Caco-2 cells, we propose that long-term dietary intake of fresh garlic may influence blood lipid and lipoprotein profiles by affecting MTP-dependent microsomal lipid transport in the intestine and thus decreasing chylomicron assembly and secretion. Future research aimed at identifying the active components and elucidating the molecular mechanism of garlic-induced changes in MTP gene expression and lipoprotein production should yield novel therapeutic measures for prevention and treatment of hyperlipidemia.
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FOOTNOTES |
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3 Abbreviations: apoB, apolipoprotein B; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; FGE, fresh garlic extract; FGH, fresh garlic homogenate; LDH, lactate dehydrogenase; MTP, microsomal triglyceride transfer protein.
Manuscript received 21 August 2001. Initial review completed 10 September 2001. Revision accepted 11 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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