Acyl-Coenzyme A:Cholesterol Acyltransferase-2 (ACAT-2) Is Responsible for Elevated Intestinal ACAT Activity in Diabetic Rats
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《动脉硬化血栓血管生物学》
From the Department of Medical Biochemistry (M.H., M.S., K.F., Y.S., H.H., A.M., S.H.), the First Department of Internal Medicine (M.H., Y.S.), and the Department of Cell Pathology (Y.K., M.T.), Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan; the Department of Analytical Chemistry (H.H.), School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan; and the Department of Biochemistry (A.M.), Showa University School of Medicine, Tokyo, Japan.
ABSTRACT
Objective— Diabetes-induced dyslipidemia is seen in streptozotocin-induced diabetic rats. This is caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. Because two ACAT isozymes (ACAT-1 and ACAT-2) were identified, in the present study we determined which ACAT isozyme was involved in the elevated intestinal ACAT activity in diabetic rats.
Methods and Results— We cloned a full-length cDNA of rat ACAT-2. Its overexpression in ACAT-deficient AC29 cells demonstrated that the ACAT activity is derived from the cloned cDNA, and a 45-kDa protein of rat ACAT-2 cross-reacts with an anti-human ACAT-2 antibody. The tissue distribution of rat ACAT-2 mRNA revealed its restricted expression to liver and small intestine. Immunohistochemical analyses using an anti-human ACAT-2 antibody demonstrated that ACAT-2 is localized in villus–crypt axis of rat small intestine. The intestinal ACAT activity in diabetic rats was significantly immunodepleted by an anti–ACAT-2 antibody but not by an anti–ACAT-1 antibody. Finally, intestinal ACAT-2 in diabetic rats significantly increased at both protein and mRNA levels as compared with that in control rats.
Conclusions— Our data demonstrate that ACAT-2 isozyme is responsible for the increased intestinal ACAT activity of diabetic rats, suggesting an important role of ACAT-2 for dyslipidemia in diabetic patients.
Diabetic rats exhibit dyslipidemia caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. We determined which ACAT isozyme (ACAT-1 or ACAT-2) was involved in the elevated intestinal ACAT activity in diabetic rats. We demonstrated an important role of ACAT-2, implicating its involvement in dyslipidemia in diabetic patients.
Key Words: acyl-coenzyme A:cholesterol acyltransferase-2 ? diabetic rats ? diabetes-induced dyslipidemia ? cholesterol absorption ? atherosclerosis
Introduction
Acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT) plays an important role in regulation of plasma cholesterol. ACAT, an intracellular enzyme located in the rough endoplasmic reticulum, catalyzes cholesteryl esters from cholesterol and fatty acyl-CoA.1 Physiological roles of ACAT so far clarified are intestinal cholesterol absorption, hepatic assembly of very-low-density lipoprotein (VLDL), and cholesterol storage in steroidogenic tissues for steroid hormone biosynthesis,2 whereas it plays a pathological role in the formation of macrophages-derived foam cells in atherosclerotic lesions.3
Diabetes-related vascular disorders such as coronary heart disease (CHD) are serious diabetic complications and the major cause of death in diabetic patients.4,5 One of the major causes of these diseases is diabetes-induced dyslipidemia,6–8 in which intestinal cholesterol absorption plays a significant role.9–11 Recently, 3 studies with streptozotocin (STZ)-induced diabetic rats have clarified that an elevated intestinal ACAT activity is responsible for diabetes-induced dyslipidemia.12–14 Jiao et al12 first showed that the intestinal ACAT activity was significantly elevated, which was normalized by insulin treatment. Maechler et al13 made a similar observation and showed that the elevated plasma cholesterol level is reduced by administration of an ACAT inhibitor (CL-277082). Kusunoki et al14 further clarified that the increased plasma triglyceride level as well as lymphatic absorption of total cholesterol (TC) and triglyceride were reduced by an ACAT inhibitor (F-1394). Two ACAT isozymes (ACAT-1and ACAT-2) were identified in mammals.15 However, it remains unclear which ACAT isozyme is responsible for the elevated intestinal ACAT activity in diabetic rats. This issue was solved in the present study. Our results indicate an important role of ACAT-2 in diabetes-induced dyslipidemia.
Methods
Expression of Rat ACAT-2 in ACAT-Deficient Chinese Hamster Ovary Cells
To express rat ACAT-2 cDNA (online Figure IA, please see http://atvb.ahajournals.org) in cells, rat ACAT-2 cDNA was inserted into a pcDNA3.1(+) vector (Invitrogen). The 5'-RACE polymerase chain reaction (PCR) product inserted into pGEM-T was subcloned into a SacI/SmaI site of pSP72 (Promega) to introduce an EcoRI site. Both the 5'-RACE–PCR product in pSP72 and the 3'- RACE–PCR product in pGEM-T were subcloned into the EcoRI site of the pcDNA3.1(+). The plasmid containing full open reading frame of rat ACAT-2 in a proper sequence (pcDNA3.1/rACAT-2) was confirmed by sequencing on both strands and used in the following cellular studies. ACAT-deficient Chinese hamster ovary cells (AC29)16 were transfected with 4 μg/mL pcDNA3.1/rACAT-2 and 20 μL/mL LIPOFECTAMINE 2000 reagent (Gibco-BRL) in OPTI-MEM (Gibco-BRL). Stable transfectants were selected during 2 weeks of incubation with medium containing 800 μg/mL of G418. Unless otherwise specified, cells were incubated at 37°C in 5% CO2.
Animals
Male Wistar rats (6 weeks old, weighing 200 to 220 g) purchased from Charles River Japan were fasted overnight before induction of diabetes. Diabetes was induced by a single intravenous injection of 60 mg/kg of STZ (Sigma) freshly dissolved in 50 mmol/L sodium citrate (pH 4.5) under light ether anesthesia. Age-matched rats were injected with an equal volume of citrate buffer to serve as controls. Two weeks after injection of STZ, animals were starved for 18 hours and euthanized under the ether anesthesia. Blood was collected to measure glucose, TC, and triglyceride using enzymatic kits (Wako). All animal procedures were in strict accordance with the guidelines of Center for Animal Resource and Development of Kumamoto University.
Reverse-Transcription PCR
The tissues removed from normal rats immediately frozen in liquid nitrogen and total RNA was extracted by using RNeasy Mini kit (Qiagen). The cDNA was synthesized using Ready-To-Go You-Prime First-Strand Beads (Amersham). A 650-bp fragment was amplified with sense (5'-AGCAAGAGTTCCCACCCACT-3') and antisense (5'-TCGAGAAAAACCAGA GGAGGT-3') primers for rat ACAT-1, whereas a 260-bp fragment was amplified with sense (5'-TACGCCCTGGAAAACGGAAA-3') and antisense (5'-GTCTGGTAGGGCAGAAGCAG-3') primers for rat ACAT-2. The PCR mixture included 1.5 mmol/L MgCl2, 200 nmol/L of each primer, and 2.5 U of Taq. Reverse-transcriptase PCR (RT-PCR) conditions were: a first step of denaturation at 94°C for 3 minutes followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, and extension at 72°C for 1.5 minutes, with a final elongation step at 72°C for 10 minutes. Glyceraldehydes-3-phosphate dehydrogenase (G3PDH) cDNA was amplified as an internal control as described previously.17 The products were electrophoresed on 1.0% agarose gel.
Northern Blot Analyses
A fragment of rat ACAT-2 cDNA inserted into pGEM-T was amplified and used for preparation of digoxygenin (DIG)-labeled antisense RNA probes according to the manufacture’s instructions (DIG Northern starter kit from Roche). For RNA protection, samples were immediately submerged in RNAlater RNA stabilization reagent (Qiagen) after harvesting. Total RNA was extracted from these samples using RNeasy Mini kit (Qiagen) and subjected to electrophoresis on 1.0% agarose gel containing formaldehyde, followed by blotting to a Hybond N+ nylon membrane (Amersham). The membrane was hybridized with the heat-denatured RNA probe (500 ng/mL) and further incubated for 30 minutes with anti-DIG antibody conjugated with alkaline phosphatase. ACAT-2 mRNA was visualized using CDP-Star (Roche) chemiluminescence substrate as described previously.18
Immunohistochemical Analysis of ACAT-2 in Rat Small Intestine
Small intestines from normal rats were removed and fixed in 4% paraformaldehyde at 4°C overnight, dehydrated through a graded series of ethanol and xylene, and embedded in paraffin. The paraffin-embedded tissue blocks were cut sequentially into 3-μm-thick sections and mounted on poly-L-lysine-coated slides. Deparaffinized sections were boiled for 10 minutes in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval. The sample was then incubated for 20 minutes with 5% normal donkey serum and sequentially reacted at 4°C overnight with 2 μg/mL of an anti-human ACAT-2 antibody as a primary antibody followed by an indirect immunoperoxidase method described previously.19
Assay for ACAT Activity
The cultured cells were homogenized with buffer A (50 mmol/L Tris-HCl and 1 mmol/L EDTA at pH 7.8 with protease inhibitors). The tissues were soaked in ice-cold buffer A and homogenized according to method described previously.14 The enzyme activity was determined by the reconstituted assay as described previously.20 Briefly, homogenates obtained from cultured cells and tissues were mixed with 4 mol/L KCl and 20% 3--1-propanesulfonate (CHAPS) in buffer A to obtain the final concentration of 1 mol/L and 2%, respectively. These samples (80 μg in 20 μL) were reconstituted with 140 μL of sodium taurocholate–cholesterol-phosphatidylcholine (PC) mixed micelles (0.2 as cholesterol/PC molar ratio). The enzyme reaction was initiated by adding 20 μL of 250 μmol/L of oleoyl-CoA (20 dpm/pmol), followed by incubation for 15 minutes at 37°C. Lipids were then extracted and the radioactive choresteryl oleate was determined by thin-layer chromatography.
Antibodies
The rabbit polyclonal antibodies against human ACAT-1 (DM10)21 and human ACAT-2 (DM54)22 were generous gifts from Dr T.Y. Chang (Dartmouth Medical School, Hanover, NH). DM10 cross-reacts with rat ACAT-1 protein.23
Immunodepletion Analyses
An immunodepletion analysis was performed as described previously.22 Briefly, homogenates obtained from cultured cells and tissues were solubilized with 1 mol/L KCl and 2% CHAPS. Each sample (0.9 mg in 450 μL) was incubated with 50 μL of buffer A containing 12 μg of an anti-human ACAT-1 antibody, an anti-human ACAT-2 antibody or nonimmune IgG, followed by constant shaking for 30 minutes at 4°C. After addition of 100 μL of protein A-Sepharose, each reaction mixture was incubated at 4°C overnight, followed by separation of supernatant and immunoprecipitate by centrifugation at 14 000g for 1 minute. Each supernatant was measured for ACAT activity by the reconstituted assay.20
Immunoblot Analyses
Homogenates obtained from cultured cells and tissues were solubilized with 10% sodium dodecyl sulfate (SDS). Each sample (200 μg) was electrophoresed on 10% SDS-polyacrylamide gel (SDS-PAGE), transferred to a polyvinylidene difluoride transfer membrane, and subjected to immunoblot analyses with an anti-human ACAT-1 antibody and an anti-human ACAT-2 antibody as described previously.22 Density of bands was measured with an Image Gauge software in LAS-1000plus (Fuji Film).
Statistical Analysis
Statistical differences were measured by Student t test. Pearson correlation coefficient (r) was used to estimate linear relationships between variables. Simple linear regression analysis was performed to determine the relationship of intestinal ACAT-2 and plasma lipids levels. P<0.05 was regarded as statically significant.
Results
Cloning and Sequence of Rat ACAT-2 cDNA and Its Expression in AC29 Cells
We cloned a full-length cDNA of rat ACAT-2 (GeneBank Accession No. AB101480), which contained an open reading frame of 1572 bp (Figure IA). Its deduced amino acid sequence predicts a 524-residue protein with a calculated molecular weight of 64 kDa, showing 83%, 85%, and 84% identity with human,24 mouse,25 and monkey ACAT-2,26 respectively (Figure IB). Next, rat ACAT-2 cDNA was transfected into AC29 cells to determine whether the cloned rat ACAT-2 cDNA coded for an enzymatically active protein. The ACAT activity of 5 stable transfectants (rat ACAT-2 cells) was 5- to 25-fold higher than that of 2 mock transfectants (Figure 1A). Immunoblot analyses with an anti-human ACAT-2 antibody demonstrated a single 45-kDa band in rat ACAT-2 cells but no band in mock transfectants (Figure 1B). These results indicate that the 45-kDa protein is derived from rat ACAT-2 cDNA expressed in AC29 cells, suggesting that rat ACAT-2 protein is also recognized by an anti-human ACAT-2 antibody.
Figure 1. Expression of rat ACAT-2 cDNA in AC29 cells (A, B). A, Five transfectants (rat ACAT-2 cells) obtained by transfecting rat ACAT-2 cDNA into ACAT-deficient CHO cells (AC29) and 2 mock transfectants were determined for the ACAT activity by reconstituted assay as described in Methods. Data represent the mean of 3 separate experiments with triplicate assays. Error bars represent SD. B, These cells were solubilized and electrophoresed on 10% SDS-PAGE, followed by subjection to immunoblot analyses with an anti-human ACAT-2 antibody as described in Methods. Data are representative of 3 independent experiments.
Tissue Distribution of Rat ACAT-2 mRNA and Intestinal Immunohistochemical Localization
The expression of ACAT-2 mRNA were examined by RT-PCR in small intestine, liver, adrenal gland, testis, brain, kidney, and spleen. Whereas the expression of ACAT-1 mRNA was positively detected in all tissues examined, the expression of ACAT-2 mRNA was limited to intestine and liver (Figure 2A). Furthermore, Northern blot analyses showed that ACAT-2 mRNA was detected in rat ACAT-2 cells, McA-RH7777 cells (rat hepatoma-derived cell line), small intestine and liver of normal rat, but not mock transfectants (Figure 2B). In addition, the level of ACAT-2 mRNA in small intestine was more abundant than that in liver. Immunohistochemical localization of rat small intestinal ACAT-2 with an anti-human ACAT-2 antibody showed that positive ACAT-2 stainings were detected diffusely in villus-crypt axis (Figure 2C and 2D).
Figure 2. Tissue distribution of ACAT-1 and ACAT-2 mRNA in rat (A, B) and immunohistochemical analyses of ACAT-2 in rat small intestine (C, D). A, cDNA prepared from normal rat tissues was subjected to RT-PCR analyses for ACAT-1 and ACAT-2 with G3PDH as a control as described in Methods. Data are representative of 3 independent experiments. B, Total RNA was extracted from AC29 (lane 1), rat ACAT-2 cells (lane 2), McA-RH7777 cells (lane 3), small intestine (lane 4), and liver (lane 5) of normal rat. ACAT-2 mRNA was analyzed by Northern blot analyses with a 18S ribosomal RNA band as an internal control. A representative result from 3 independent experiments is shown. C and D, Small intestines from normal rats were removed and fixed and deparaffinized sections were reacted with an anti-human ACAT-2 antibody as described in Methods. Magnification x100 (C) and x400 (D). Brown color indicates positive reaction with an anti-human ACAT-2 antibody.
Effect of Diabetic Conditions on Expression of Rat Intestinal ACAT-2
Fasting blood glucose levels in diabetic rats were 22.0 mmol/L at day 4 (4 days after STZ injection), whereas those in control rats remained <6.0 mmol/L. Plasma TC and triglyceride levels in diabetic rats at day 14 were increased 1.5-fold and 2.9-fold, respectively (Table). The intestinal ACAT activity of diabetic rats at day 14 was 2.2-fold higher than that of control rats (Figure 3A). In contrast, the hepatic ACAT activity was not changed in control and diabetic rats (35.1±2.3 versus 41.8±6.6 pmol/mg protein/minute, respectively; NS). Therefore, it is likely that hepatic ACAT does not contribute to diabetes-induced dyslipidemia. Immunodepletion analyses revealed that an anti–ACATn-1 antibody significantly reduced the activity of rat ACAT-1 transfectants (rat ACAT-1 cells),23 but had no effect on the enzyme activity of rat ACAT-2 cells. Similarly, an anti–ACAT-2 antibody completely reduced the enzyme activity of rat ACAT-2 cells but had no effect on rat ACAT-1 cells (Figure 3B). Under these conditions, an anti–ACATn-2 antibody reduced the ACAT activity by >70% of the total intestinal activity of control and diabetic rats, whereas an anti–ACATn-1 antibody did not affect it at all (Figure 3B). Furthermore, we made immunoblot analyses to examine whether the diabetes-induced increase in small intestinal ACAT activity was caused by an increase in ACAT-2 but not ACAT-1. The immunoblot analyses using an anti–ACAT-2 antibody revealed at least 2 bands in rat small intestine: the upper band and the slightly lower band. The amount of intestinal ACAT-2 at protein levels in diabetic rats at day 14 was 2-fold higher than that in the intestine of control rats (Figure 4B). Under these conditions, parallel immunoblot analyses with an anti–ACATn-1 antibody failed to reveal a distinct band (data not shown), indicating that the major intestinal ACAT isozyme both in normal and diabetic rats was ACAT-2. In addition, the expression of intestinal ACAT-2 was significantly correlated with the level of plasma TC (r=0.56, P<0.01) and triglyceride (r=0.57, P<0.01) (Figure 5).
Glucose, Total Cholesterol, and Triglyceride in Normal and Diabetic Rats
Figure 3. Intestinal ACAT activity of normal and diabetic rats (A) and immunodepletion assay (B). A, Homogenates obtained from small intestine of normal (control) and diabetic (DM) rats were solubilized and determined for the ACAT activity as described in Methods. Data represent the mean of diabetic rats (n=16) and control rats (n=8). Error bars represent SD. *P<0.005 compared with control. B, Homogenates obtained from small intestine of normal rats (control) and diabetic rats (DM), and from rat ACAT-1 overexpressed cells (rat ACAT-1 cells), and rat ACAT-2 overexpressed cells (rat ACAT-2 cells) were solubilized. Each sample was incubated with a nonimmune rabbit IgG (white bars), an anti-human ACAT-1 antibody (hatched bars), an anti-human ACAT-2 antibody (latticed bars), or the combined mixture of an anti-human ACAT-1 antibody and an anti-human ACAT-2 antibody (black bars), followed by separation of supernatant and immunoprecipitate with protein A-Sepharose. Each supernatant was measured for ACAT activity as described in Methods.
Figure 4. Effect of diabetic conditions on expression of rat intestinal ACAT-2. A, Homogenates obtained from small intestine of 2 normal rats (control) and 4 diabetic rats (DM) were solubilized. Each sample was electrophoresed on 10% SDS-PAGE and subjected to immunoblot analyses with an anti-human ACAT-2 antibody as described in Methods. Data are representative of 4 independent experiments. B, Densitometric quantification of anti-human ACAT-2 antibody reactive bands was measured as described in Methods. Data represent the mean of diabetic rats (n=16) and control rats (n=8). Error bars represent SD. *P<0.005 compared with control. C, Total RNA was extracted from small intestine of normal rats (control) and diabetic rats (DM) and ACAT-2 mRNA was detected by Northern blot analyses. D, Densitometric scanning of (C). The intensity of a 18S ribosomal RNA band was used as an internal control to normalize the intensity of ACAT-2 mRNA subspecies. Data represent the mean of diabetic rats (n=9) and control rats (n=6). Error bars represent SD. *P<0.05 compared with control.
Figure 5. Correlation between intestinal ACAT-2 and plasma lipids. A, Plasma TC levels. B, Plasma triglyceride levels. Plasma levels of TC and triglyceride were determined in 16 diabetic rats (black circles) and 8 control rats (white circles). The regression equations in (A) and (B) are r=0.56 (P<0.01) and r=0.57 (P<0.01), respectively.
Finally, we made Northern blot analyses to demonstrate whether the increased intestinal ACAT-2 protein in diabetic rat was caused by increased ACAT-2 mRNA level. Northern blot analyses could detect a single distinct transcript in small intestine and liver (Figures 2B and 4C). On quantitative analyses based on densitometry of this transcript, ACAT-2 mRNA level were increased by 1.8-fold in diabetic rats (Figure 4C and 4D).
Discussion
ACAT plays an important role in cholesterol absorption from intestines9 and recent reports using ACAT inhibitors demonstrated that the intestinal ACAT activity is responsible for dyslipidemia in diabetic rats.12–14 Two ACAT isozymes (ACAT-1and ACAT-2) were identified in mammals,15 and it is important to determine which ACAT isozyme is responsible for the increased intestinal ACAT activity in diabetic rats. The results of the present study have clarified an important role of intestinal ACAT-2 for diabetes-induced dyslipidemia at a transcriptional level.
In parallel with an elevated intestinal ACAT activity, both plasma TC and triglyceride levels were increased in the diabetic rats (Table). The increased TC levels seems to result from an increase in intestinal absorption of dietary cholesterol caused by elevated intestinal ACAT activity, whereas the mechanism of the increased plasma triglyceride levels is not clear. The effect of intestinal ACAT on intestinal triglyceride absorption is not known. Microsomal triglyceride transfer protein (MTP) was reported to play a crucial role in the intestinal chylomicron formation as well as hepatic VLDL assembly.27,28 Triglyceride as well as cholesteryl esters are essential substrates for MTP.27 The increased cholesterol absorption in the diabetic rats induced mainly by upregulation of intestinal ACAT activity might also increase the intestinal MTP activity and then enhance intestinal chylomicron synthesis, thus inducing hypertriglyceridemia in the diabetic rats. In fact, recent reports showed that intestinal MTP expression in diabetes was higher than controls in both rabbits and rats.29,30
Recent studies revealed new molecules involved in the intestinal cholesterol absorption; ATP-binding cassette half-transporters ABCG5/ABCG831,32 and Niemann-Pick C1–like 1 protein (NPC1L1).33,34 ABCG5/ABCG8 are highly expressed in the liver and intestine.35 Mutations in the human genes encoding ABCG5/ABCG8 resulted in sitosterolemia with a reduced biliary secretion as well as an enhanced intestinal absorption of plant sterols (sitosterol, campesterol),36–38 suggesting an important role of ABCG5/ABCG8 in hepatobiliary cholesterol secretion and intestinal cholesterol absorption. In STZ-induced diabetic rats, both intestinal and hepatic ABCG5/ABCG8 expressions were reduced at a mRNA level that contributed to a reduction of hepatobiliary cholesterol secretion and an increased plant sterols concentrations.39 Therefore, it is possible that both the decreased intestinal ABCG5/ABCG8 and the increased intestinal ACAT-2 contribute to diabetes-induced dyslipidemia. NPC1L1 localized in jejunal enterocytes was demonstrated to be critical for intestinal cholesterol absorption.33 Ezetimibe, a selective inhibitor of intestinal cholesterol absorption,40 had no effect in NPC1L1-deficient mice, suggesting that NPC1L1 serves as an ezetimibe-dependent pathway for intestinal cholesterol absorption.33,34 NPC1L1-deficient mice had reduced intestinal uptake of plant sterols but did not change triglyceride absorption.33,34 It does not seem likely that NPC1L1 may contribute to hypertriglyceridemia induced under diabetic conditions.
Previous studies showed that intestinal ACAT activity was increased in diabetic rats, which was restored by insulin treatment, suggesting insulin deficiency might lead to the elevated intestinal ACAT activity under the diabetic condition.12,13 Furthermore, no significant differences in the intestinal ACAT activity could be detected between diabetic animals fed control chow and those fed the atherogenic diet.12 In vitro study with Caco-2 cells, human intestinal cell-derived cell line, showed that incubation of these cells with insulin significantly reduced the ACAT activity of these cells, suggesting an inhibitory effect of insulin on the intestinal ACAT activity.41 Our in vitro experiment with Caco-2 cells also resulted in reduction of ACAT activity by insulin but not high glucose (unpublished data). It is likely from these data that the intestinal ACAT activity is regulated by insulin but not by high glucose or diet.
Recent studies clarified the ACAT-2 promoter. Song et al identified the ACAT-2 promoter region whose function was increased during the differentiation of Caco-2 cells.42 In this connection, the expression of Cdx-2, a transcriptional regulator localized in nucleus, increased greatly during the differentiation of Caco-2 cells but was not detected in hepatoblastoma cell line HepG2 cells.43 Sequence analyses by computer showed that human, mouse, and rat ACAT-2 promoter region contained 4, 3, and 4 Cdx-2 elements, respectively. Electrophoretic mobility shift assay showed that Cdx-2 was able to efficiently bind to mouse and human ACAT-2 promoter regions, suggesting that Cdx-2 is a tissue-specific ACAT-2 regulator in intestinal cells.43 This could be the case under diabetic conditions because ACAT activity in intestine, but not in liver, was increased in diabetic rats (Figure 3A).
ACAT inhibition is emphasized as a potential anti-atherogenic strategy. However, results obtained from ACAT-1–deficient mice have not supported this notion unanimously.44–46 Animal experiments by Accad et al44 using ACAT-1–deficient mice bred either on apoE-deficient mice or LDL receptor-deficient mice revealed that deletion of ACAT-1 did not prevent these animals from development of atherosclerotic lesions. Yagyu et al45 using similar animal model systems reached the different conclusion that ACAT-1–deficiency reduces atherosclerotic lesions. However, Fazio et al46 showed that reconstitution of LDL receptor-deficient mice with ACAT-1–deficient macrophage resulted in a more significant increase in atherosclerotic lesions than LDL receptor-deficient mice reconstituted with wild-type macrophage. With these reports taken together, effectiveness of ACAT-1 inhibitors for anti-atherogenic therapy in humans remains controversial.
Although the expression of ACAT-1 is ubiquitous, the expression of ACAT-2 is restricted. Human ACAT-2 was expressed in intestine and mice ACAT-2 in intestine and liver,22 and rat ACAT-2 was shown to localize in intestine and liver in the present study. ACAT-2–deficient mice were completely resistant to diet-induced hypercholesterolemia.47 On further gene knockout of apoE, atherosclerotic lesions of these mice were significantly reduced when compared with those of apoE-deficient mice.48 These animal studies revealed that complete depletion of ACAT-2 prevents mice from atherosclerosis-prone hyperlipidemia, suggesting an importance of ACAT-2–derived intestinal and hepatic ACAT activity in diet-induced hypercholesterolemia.
Development of ACAT inhibitors has faced difficulty, caused mainly by adrenotoxicity, which might be induced by ACAT-1 suppression,49,50 and isozyme-specific ACAT inhibitors are expected to be a potential target. In this connection, results of the present study and previous ones using ACAT-2–deficient mice47,48 suggest the possibility that ACAT-2–specific inhibitors are beneficial as an anti-atherosclerotic drug by normalization of diabetes-induced dyslipidemia.
In conclusion, the cloning of rat ACAT-2 cDNA with its characterization have provided convincing evidence that the increased intestinal ACAT activity in diabetic rats is caused largely by a significant upregulation of ACAT-2 at both protein and mRNA levels but not ACAT-1, suggesting a major catalytic role of ACAT-2 in diabetes-induced dyslipidemia. Although results from animal studies with mice or rats may not be directly applicable to human, selective inhibition for ACAT-2 might ameliorate diabetes-induced dyslipidemia in human and even cardiovascular disease, because intestinal cholesterol absorption is reported to be significantly higher in diabetic patients with CHD than those without CHD.10
Acknowledgments
We thank Drs Ta-Yuan Chang and Catherine C.Y. Chang at Department of Biochemistry, Dartmouth Medical School, for the generous gift of DM10, DM54, and AC29 cells and helpful discussions. This work was supported in part by a Grant-in Aid for Scientific Research (B: 13470228 to S.H. and C: 14571101 to A.M.) from Japan Society for the Promotion of Science (JSPS), and by the Research Grant for Cardiovascular Diseases 13-4 from the Ministry of Health, Labor and Welfare.
References
Chang TY, Chang CC, Lin S, Yu C, Li BL, Miyazaki A. Roles of acyl-coenzyme A: cholesterol acyltransferase-1 and -2. Curr Opin Lipidol. 2001; 12: 289–296.
Chang TY, Chang CC, Cheng D. Acyl-coenzyme A: cholesterol acyltransferase. Annu Rev Biochem. 1997; 66: 613–638.
Miyazaki A, Sakashita N, Lee O, Takahashi K, Horiuchi S, Hakamata H, Morganelli PM, Chang CC, Chang TY. Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages. Arterioscler Thromb Vasc Biol. 1998; 18: 1568–1574.
Resnick HE, Howard BV. Diabetes and cardiovascular disease. Annu Rev Med. 2002; 53: 245–267.
Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med. 1998; 339: 229–234.
Turner RC, Millns H, Neil HA, Stratton IM, Manley SE, Matthews DR, Holman RR. Risk factors for coronary artery disease in non-insulin dependent diabetes mellitus: United Kingdom Prospective Diabetes Study (UKPDS: 23). BMJ. 1998; 316: 823–828.
Marks JB, Raskin P. Cardiovascular risk in diabetes: a brief review. J Diabetes Complications. 2000; 14: 108–115.
Haffner SM. Diabetes, hyperlipidemia, and coronary artery disease. Am J Cardiol. 1999; 83: 17F–21F.
Wilson MD, Rudel LL. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol. J Lipid Res. 1994; 35: 943–955.
Gylling H, Miettinen TA. Cholesterol absorption and lipoprotein metabolism in type II diabetes mellitus with and without coronary artery disease. Atherosclerosis. 1996; 126: 325–332.
Miettinen TA, Gylling H, Tuominen J, Simonen P, Koivisto V. Low synthesis and high absorption of cholesterol characterize type 1 diabetes. Diabetes Care. 2004; 27: 53–58.
Jiao S, Matsuzawa Y, Matsubara K, Kihara S, Nakamura T, Tokunaga K, Kubo M, Tarui S. Increased activity of intestinal acyl-CoA: cholesterol acyltransferase in rats with streptozocin-induced diabetes and restoration by insulin supplementation. Diabetes. 1988; 37: 342–346.
Maechler P, Wollheim CB, Bentzen CL, Niesor E. Role of the intestinal acyl-CoA: cholesterol acyltransferase activity in the hyperresponse of diabetic rats to dietary cholesterol. J Lipid Res. 1992; 33: 1475–1484.
Kusunoki J, Aragane K, Kitamine T, Kozono H, Kano K, Fujinami K, Kojima K, Chiwata T, Sekine Y. Postprandial hyperlipidemia in streptozotocin-induced diabetic rats is due to abnormal increase in intestinal acyl coenzyme A: cholesterol acyltransferase activity. Arterioscler Thromb Vasc Biol. 2000; 20: 171–178.
Buhman KF, Accad M, Farese RV. Mammalian acyl-CoA: cholesterol acyltransferases. Biochim Biophys Acta. 2000; 1529: 142–154.
Cadigan KM, Heider JG, Chang TY. Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A: cholesterol acyltransferase activity. J Biol Chem. 1988; 263: 274–282.
Matsuda H, Hakamata H, Miyazaki A, Sakai M, Chang CC, Chang TY, Kobori S, Shichiri M, Horiuchi S. Activation of acyl-coenzyme A: cholesterol acyltransferase activity by cholesterol is not due to altered mRNA levels in HepG2 cells. Biochim Biophys Acta. 1996; 1301: 76–84.
Maung K, Miyazaki A, Nomiyama H, Chang CC, Chang TY, Horiuchi S. Induction of acyl-coenzyme A: cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D(3) or 9-cis-retinoic acid in undifferentiated THP-1 cells. J Lipid Res. 2001; 42: 181–187.
Sakashita N, Miyazaki A, Takeya M, Horiuchi S, Chang CC, Chang TY, Takahashi K. Localization of human acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in macrophages and in various tissues. Am J Pathol. 2000; 156: 227–236.
Chang CC, Lee CY, Chang ET, Cruz JC, Levesque MC, Chang TY. Recombinant acyl-CoA: cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner. J Biol Chem. 1998; 273: 35132–35141.
Chang CC, Huh HY, Cadigan KM, Chang TY. Molecular cloning and functional expression of human acyl-coenzyme A: cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J Biol Chem. 1993; 268: 20747–20755.
Chang CC, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S, Lee CY, Strom SC, Kashyap R, Fung JJ, Farese RV, Jr., Patoiseau JF, Delhon A, Chang TY. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem. 2000; 275: 28083–28092.
Matsuda H, Hakamata H, Kawasaki T, Sakashita N, Miyazaki A, Takahashi K, Shichiri M, Horiuchi S. Molecular cloning, functional expression and tissue distribution of rat acyl-coenzyme A: cholesterol acyltransferase. Biochim Biophys Acta. 1998; 1391: 193–203.
Oelkers P, Behari A, Cromley D, Billheimer JT, Sturley SL. Characterization of two human genes encoding acyl coenzyme A: cholesterol acyltransferase-related enzymes. J Biol Chem. 1998; 273: 26765–26771.
Cases S, Novak S, Zheng YW, Myers HM, Lear SR, Sande E, Welch CB, Lusis AJ, Spencer TA, Krause BR, Erickson SK, Farese RV, Jr. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization. J Biol Chem. 1998; 273: 26755–26764.
Anderson RA, Joyce C, Davis M, Reagan JW, Clark M, Shelness GS, Rudel LL. Identification of a form of acyl-CoA: cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J Biol Chem. 1998; 273: 26747–26754.
White DA, Bennett AJ, Billett MA, Salter AM. The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triacylglycerol transfer protein. Br J Nutr. 1998; 80: 219–229.
Wetterau JR, Zilversmit DB. Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues. Biochim Biophys Acta. 1986; 875: 610–617.
Gleeson A, Anderton K, Owens D, Bennett A, Collins P, Johnson A, White D, Tomkin GH. The role of microsomal triglyceride transfer protein and dietary cholesterol in chylomicron production in diabetes. Diabetologia. 1999; 42: 944–948.
Phillips C, Bennett A, Anderton K, Owens D, Collins P, White D, Tomkin GH. Intestinal rather than hepatic microsomal triglyceride transfer protein as a cause of postprandial dyslipidemia in diabetes. Metabolism. 2002; 51: 847–852.
Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, Hobbs HH. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest. 2002; 110: 671–680.
Yu L, Hammer RE, Li-Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC, Hobbs HH. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci U S A. 2002; 99: 16237–16242.
Altmann SW, Davis HR Jr, Zhu LJ, Yao X, Hoos LM, Tetzloff G, Iyer SP, Maguire M, Golovko A, Zeng M, Wang L, Murgolo N, Graziano MP. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science. 2004; 303: 1201–1204.
Davis HR, Zhu LJ, Hoos LM, Tetzloff G, Maguire M, Liu J, Yao X, Iyer SP, Lam MH, Lund EG, Detmers PA, Graziano MP, Altmann SW. Niemann-pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole body cholesterol homeostasis. J Biol Chem. 2004. In press.
Lu K, Lee MH, Yu H, Zhou Y, Sandell SA, Salen G, Patel SB. Molecular cloning, genomic organization, genetic variations, and characterization of murine sterolin genes Abcg5 and Abcg8. J Lipid Res. 2002; 43: 565–578.
Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J, Kwiterovich P, Shan B, Barnes R, Hobbs HH. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science. 2000; 290: 1771–1775.
Lee MH, Lu K, Hazard S, Yu H, Shulenin S, Hidaka H, Kojima H, Allikmets R, Sakuma N, Pegoraro R, Srivastava AK, Salen G, Dean M, Patel SB. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption. Nat Genet. 2001; 27: 79–83.
Lu K, Lee MH, Hazard S, Brooks-Wilson A, Hidaka H, Kojima H, Ose L, Stalenhoef AF, Mietinnen T, Bjorkhem I, Bruckert E, Pandya A, Brewer HB Jr, Salen G, Dean M, Srivastava A, Patel SB. Two genes that map to the STSL locus cause sitosterolemia: genomic structure and spectrum of mutations involving sterolin-1 and sterolin-2, encoded by ABCG5 and ABCG8, respectively. Am J Hum Genet. 2001; 69: 278–290.
Bloks VW, Bakker-Van Waarde WM, Verkade HJ, Kema IP, Wolters H, Vink E, Groen AK, Kuipers F. Down-regulation of hepatic and intestinal Abcg5 and Abcg8 expression associated with altered sterol fluxes in rats with streptozotocin-induced diabetes. Diabetologia. 2004; 47: 104–112.
van Heek M, Farley C, Compton DS, Hoos L, Davis HR. Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function. Br J Pharmacol. 2001; 134: 409–417.
Jiao S, Moberly JB, Cole TG, Schonfeld G. Decreased activity of acyl-CoA: cholesterol acyltransferase by insulin in human intestinal cell line Caco-2. Diabetes. 1989; 38: 604–609.
Song BL, Qi W, Yang XY, Chang CC, Zhu JQ, Chang TY, Li BL. Organization of human ACAT-2 gene and its cell-type-specific promoter activity. Biochem Biophys Res Commun. 2001; 282: 580–588.
Song BL, Qi W, Wang CH, Yang JB, Yang XY, Lin ZX, Li BL. Preparation of an anti-Cdx-2 antibody for analysis of different species Cdx-2 binding to acat2 promoter. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003; 35: 6–12.
Accad M, Smith SJ, Newland DL, Sanan DA, King LE, Jr., Linton MF, Fazio S, Farese RV Jr. Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA: cholesterol acyltransferase 1. J Clin Invest. 2000; 105: 711–719.
Yagyu H, Kitamine T, Osuga J, Tozawa R, Chen Z, Kaji Y, Oka T, Perrey S, Tamura Y, Ohashi K, Okazaki H, Yahagi N, Shionoiri F, Iizuka Y, Harada K, Shimano H, Yamashita H, Gotoda T, Yamada N, Ishibashi S. Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia. J Biol Chem. 2000; 275: 21324–21330.
Fazio S, Major AS, Swift LL, Gleaves LA, Accad M, Linton MF, Farese RV, Jr. Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages. J Clin Invest. 2001; 107: 163–171.
Buhman KK, Accad M, Novak S, Choi RS, Wong JS, Hamilton RL, Turley S, Farese RV Jr. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice. Nat Med. 2000; 6: 1341–1347.
Willner EL, Tow B, Buhman KK, Wilson M, Sanan DA, Rudel LL, Farese RV Jr. Deficiency of acyl CoA: cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 2003; 100: 1262–1267.
Dominick MA, McGuire EJ, Reindel JF, Bobrowski WF, Bocan TM, Gough AW. Subacute toxicity of a novel inhibitor of acyl-CoA: cholesterol acyltransferase in beagle dogs. Fundam Appl Toxicol. 1993; 20: 217–224.
Dominick MA, Bobrowski WA, MacDonald JR, Gough AW. Morphogenesis of a zone-specific adrenocortical cytotoxicity in guinea pigs administered PD 132301–2, an inhibitor of acyl-CoA: cholesterol acyltransferase. Toxicol Pathol. 1993; 21: 54–62.(Masaharu Hori; Maki Satoh)
ABSTRACT
Objective— Diabetes-induced dyslipidemia is seen in streptozotocin-induced diabetic rats. This is caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. Because two ACAT isozymes (ACAT-1 and ACAT-2) were identified, in the present study we determined which ACAT isozyme was involved in the elevated intestinal ACAT activity in diabetic rats.
Methods and Results— We cloned a full-length cDNA of rat ACAT-2. Its overexpression in ACAT-deficient AC29 cells demonstrated that the ACAT activity is derived from the cloned cDNA, and a 45-kDa protein of rat ACAT-2 cross-reacts with an anti-human ACAT-2 antibody. The tissue distribution of rat ACAT-2 mRNA revealed its restricted expression to liver and small intestine. Immunohistochemical analyses using an anti-human ACAT-2 antibody demonstrated that ACAT-2 is localized in villus–crypt axis of rat small intestine. The intestinal ACAT activity in diabetic rats was significantly immunodepleted by an anti–ACAT-2 antibody but not by an anti–ACAT-1 antibody. Finally, intestinal ACAT-2 in diabetic rats significantly increased at both protein and mRNA levels as compared with that in control rats.
Conclusions— Our data demonstrate that ACAT-2 isozyme is responsible for the increased intestinal ACAT activity of diabetic rats, suggesting an important role of ACAT-2 for dyslipidemia in diabetic patients.
Diabetic rats exhibit dyslipidemia caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. We determined which ACAT isozyme (ACAT-1 or ACAT-2) was involved in the elevated intestinal ACAT activity in diabetic rats. We demonstrated an important role of ACAT-2, implicating its involvement in dyslipidemia in diabetic patients.
Key Words: acyl-coenzyme A:cholesterol acyltransferase-2 ? diabetic rats ? diabetes-induced dyslipidemia ? cholesterol absorption ? atherosclerosis
Introduction
Acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT) plays an important role in regulation of plasma cholesterol. ACAT, an intracellular enzyme located in the rough endoplasmic reticulum, catalyzes cholesteryl esters from cholesterol and fatty acyl-CoA.1 Physiological roles of ACAT so far clarified are intestinal cholesterol absorption, hepatic assembly of very-low-density lipoprotein (VLDL), and cholesterol storage in steroidogenic tissues for steroid hormone biosynthesis,2 whereas it plays a pathological role in the formation of macrophages-derived foam cells in atherosclerotic lesions.3
Diabetes-related vascular disorders such as coronary heart disease (CHD) are serious diabetic complications and the major cause of death in diabetic patients.4,5 One of the major causes of these diseases is diabetes-induced dyslipidemia,6–8 in which intestinal cholesterol absorption plays a significant role.9–11 Recently, 3 studies with streptozotocin (STZ)-induced diabetic rats have clarified that an elevated intestinal ACAT activity is responsible for diabetes-induced dyslipidemia.12–14 Jiao et al12 first showed that the intestinal ACAT activity was significantly elevated, which was normalized by insulin treatment. Maechler et al13 made a similar observation and showed that the elevated plasma cholesterol level is reduced by administration of an ACAT inhibitor (CL-277082). Kusunoki et al14 further clarified that the increased plasma triglyceride level as well as lymphatic absorption of total cholesterol (TC) and triglyceride were reduced by an ACAT inhibitor (F-1394). Two ACAT isozymes (ACAT-1and ACAT-2) were identified in mammals.15 However, it remains unclear which ACAT isozyme is responsible for the elevated intestinal ACAT activity in diabetic rats. This issue was solved in the present study. Our results indicate an important role of ACAT-2 in diabetes-induced dyslipidemia.
Methods
Expression of Rat ACAT-2 in ACAT-Deficient Chinese Hamster Ovary Cells
To express rat ACAT-2 cDNA (online Figure IA, please see http://atvb.ahajournals.org) in cells, rat ACAT-2 cDNA was inserted into a pcDNA3.1(+) vector (Invitrogen). The 5'-RACE polymerase chain reaction (PCR) product inserted into pGEM-T was subcloned into a SacI/SmaI site of pSP72 (Promega) to introduce an EcoRI site. Both the 5'-RACE–PCR product in pSP72 and the 3'- RACE–PCR product in pGEM-T were subcloned into the EcoRI site of the pcDNA3.1(+). The plasmid containing full open reading frame of rat ACAT-2 in a proper sequence (pcDNA3.1/rACAT-2) was confirmed by sequencing on both strands and used in the following cellular studies. ACAT-deficient Chinese hamster ovary cells (AC29)16 were transfected with 4 μg/mL pcDNA3.1/rACAT-2 and 20 μL/mL LIPOFECTAMINE 2000 reagent (Gibco-BRL) in OPTI-MEM (Gibco-BRL). Stable transfectants were selected during 2 weeks of incubation with medium containing 800 μg/mL of G418. Unless otherwise specified, cells were incubated at 37°C in 5% CO2.
Animals
Male Wistar rats (6 weeks old, weighing 200 to 220 g) purchased from Charles River Japan were fasted overnight before induction of diabetes. Diabetes was induced by a single intravenous injection of 60 mg/kg of STZ (Sigma) freshly dissolved in 50 mmol/L sodium citrate (pH 4.5) under light ether anesthesia. Age-matched rats were injected with an equal volume of citrate buffer to serve as controls. Two weeks after injection of STZ, animals were starved for 18 hours and euthanized under the ether anesthesia. Blood was collected to measure glucose, TC, and triglyceride using enzymatic kits (Wako). All animal procedures were in strict accordance with the guidelines of Center for Animal Resource and Development of Kumamoto University.
Reverse-Transcription PCR
The tissues removed from normal rats immediately frozen in liquid nitrogen and total RNA was extracted by using RNeasy Mini kit (Qiagen). The cDNA was synthesized using Ready-To-Go You-Prime First-Strand Beads (Amersham). A 650-bp fragment was amplified with sense (5'-AGCAAGAGTTCCCACCCACT-3') and antisense (5'-TCGAGAAAAACCAGA GGAGGT-3') primers for rat ACAT-1, whereas a 260-bp fragment was amplified with sense (5'-TACGCCCTGGAAAACGGAAA-3') and antisense (5'-GTCTGGTAGGGCAGAAGCAG-3') primers for rat ACAT-2. The PCR mixture included 1.5 mmol/L MgCl2, 200 nmol/L of each primer, and 2.5 U of Taq. Reverse-transcriptase PCR (RT-PCR) conditions were: a first step of denaturation at 94°C for 3 minutes followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, and extension at 72°C for 1.5 minutes, with a final elongation step at 72°C for 10 minutes. Glyceraldehydes-3-phosphate dehydrogenase (G3PDH) cDNA was amplified as an internal control as described previously.17 The products were electrophoresed on 1.0% agarose gel.
Northern Blot Analyses
A fragment of rat ACAT-2 cDNA inserted into pGEM-T was amplified and used for preparation of digoxygenin (DIG)-labeled antisense RNA probes according to the manufacture’s instructions (DIG Northern starter kit from Roche). For RNA protection, samples were immediately submerged in RNAlater RNA stabilization reagent (Qiagen) after harvesting. Total RNA was extracted from these samples using RNeasy Mini kit (Qiagen) and subjected to electrophoresis on 1.0% agarose gel containing formaldehyde, followed by blotting to a Hybond N+ nylon membrane (Amersham). The membrane was hybridized with the heat-denatured RNA probe (500 ng/mL) and further incubated for 30 minutes with anti-DIG antibody conjugated with alkaline phosphatase. ACAT-2 mRNA was visualized using CDP-Star (Roche) chemiluminescence substrate as described previously.18
Immunohistochemical Analysis of ACAT-2 in Rat Small Intestine
Small intestines from normal rats were removed and fixed in 4% paraformaldehyde at 4°C overnight, dehydrated through a graded series of ethanol and xylene, and embedded in paraffin. The paraffin-embedded tissue blocks were cut sequentially into 3-μm-thick sections and mounted on poly-L-lysine-coated slides. Deparaffinized sections were boiled for 10 minutes in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval. The sample was then incubated for 20 minutes with 5% normal donkey serum and sequentially reacted at 4°C overnight with 2 μg/mL of an anti-human ACAT-2 antibody as a primary antibody followed by an indirect immunoperoxidase method described previously.19
Assay for ACAT Activity
The cultured cells were homogenized with buffer A (50 mmol/L Tris-HCl and 1 mmol/L EDTA at pH 7.8 with protease inhibitors). The tissues were soaked in ice-cold buffer A and homogenized according to method described previously.14 The enzyme activity was determined by the reconstituted assay as described previously.20 Briefly, homogenates obtained from cultured cells and tissues were mixed with 4 mol/L KCl and 20% 3--1-propanesulfonate (CHAPS) in buffer A to obtain the final concentration of 1 mol/L and 2%, respectively. These samples (80 μg in 20 μL) were reconstituted with 140 μL of sodium taurocholate–cholesterol-phosphatidylcholine (PC) mixed micelles (0.2 as cholesterol/PC molar ratio). The enzyme reaction was initiated by adding 20 μL of 250 μmol/L of oleoyl-CoA (20 dpm/pmol), followed by incubation for 15 minutes at 37°C. Lipids were then extracted and the radioactive choresteryl oleate was determined by thin-layer chromatography.
Antibodies
The rabbit polyclonal antibodies against human ACAT-1 (DM10)21 and human ACAT-2 (DM54)22 were generous gifts from Dr T.Y. Chang (Dartmouth Medical School, Hanover, NH). DM10 cross-reacts with rat ACAT-1 protein.23
Immunodepletion Analyses
An immunodepletion analysis was performed as described previously.22 Briefly, homogenates obtained from cultured cells and tissues were solubilized with 1 mol/L KCl and 2% CHAPS. Each sample (0.9 mg in 450 μL) was incubated with 50 μL of buffer A containing 12 μg of an anti-human ACAT-1 antibody, an anti-human ACAT-2 antibody or nonimmune IgG, followed by constant shaking for 30 minutes at 4°C. After addition of 100 μL of protein A-Sepharose, each reaction mixture was incubated at 4°C overnight, followed by separation of supernatant and immunoprecipitate by centrifugation at 14 000g for 1 minute. Each supernatant was measured for ACAT activity by the reconstituted assay.20
Immunoblot Analyses
Homogenates obtained from cultured cells and tissues were solubilized with 10% sodium dodecyl sulfate (SDS). Each sample (200 μg) was electrophoresed on 10% SDS-polyacrylamide gel (SDS-PAGE), transferred to a polyvinylidene difluoride transfer membrane, and subjected to immunoblot analyses with an anti-human ACAT-1 antibody and an anti-human ACAT-2 antibody as described previously.22 Density of bands was measured with an Image Gauge software in LAS-1000plus (Fuji Film).
Statistical Analysis
Statistical differences were measured by Student t test. Pearson correlation coefficient (r) was used to estimate linear relationships between variables. Simple linear regression analysis was performed to determine the relationship of intestinal ACAT-2 and plasma lipids levels. P<0.05 was regarded as statically significant.
Results
Cloning and Sequence of Rat ACAT-2 cDNA and Its Expression in AC29 Cells
We cloned a full-length cDNA of rat ACAT-2 (GeneBank Accession No. AB101480), which contained an open reading frame of 1572 bp (Figure IA). Its deduced amino acid sequence predicts a 524-residue protein with a calculated molecular weight of 64 kDa, showing 83%, 85%, and 84% identity with human,24 mouse,25 and monkey ACAT-2,26 respectively (Figure IB). Next, rat ACAT-2 cDNA was transfected into AC29 cells to determine whether the cloned rat ACAT-2 cDNA coded for an enzymatically active protein. The ACAT activity of 5 stable transfectants (rat ACAT-2 cells) was 5- to 25-fold higher than that of 2 mock transfectants (Figure 1A). Immunoblot analyses with an anti-human ACAT-2 antibody demonstrated a single 45-kDa band in rat ACAT-2 cells but no band in mock transfectants (Figure 1B). These results indicate that the 45-kDa protein is derived from rat ACAT-2 cDNA expressed in AC29 cells, suggesting that rat ACAT-2 protein is also recognized by an anti-human ACAT-2 antibody.
Figure 1. Expression of rat ACAT-2 cDNA in AC29 cells (A, B). A, Five transfectants (rat ACAT-2 cells) obtained by transfecting rat ACAT-2 cDNA into ACAT-deficient CHO cells (AC29) and 2 mock transfectants were determined for the ACAT activity by reconstituted assay as described in Methods. Data represent the mean of 3 separate experiments with triplicate assays. Error bars represent SD. B, These cells were solubilized and electrophoresed on 10% SDS-PAGE, followed by subjection to immunoblot analyses with an anti-human ACAT-2 antibody as described in Methods. Data are representative of 3 independent experiments.
Tissue Distribution of Rat ACAT-2 mRNA and Intestinal Immunohistochemical Localization
The expression of ACAT-2 mRNA were examined by RT-PCR in small intestine, liver, adrenal gland, testis, brain, kidney, and spleen. Whereas the expression of ACAT-1 mRNA was positively detected in all tissues examined, the expression of ACAT-2 mRNA was limited to intestine and liver (Figure 2A). Furthermore, Northern blot analyses showed that ACAT-2 mRNA was detected in rat ACAT-2 cells, McA-RH7777 cells (rat hepatoma-derived cell line), small intestine and liver of normal rat, but not mock transfectants (Figure 2B). In addition, the level of ACAT-2 mRNA in small intestine was more abundant than that in liver. Immunohistochemical localization of rat small intestinal ACAT-2 with an anti-human ACAT-2 antibody showed that positive ACAT-2 stainings were detected diffusely in villus-crypt axis (Figure 2C and 2D).
Figure 2. Tissue distribution of ACAT-1 and ACAT-2 mRNA in rat (A, B) and immunohistochemical analyses of ACAT-2 in rat small intestine (C, D). A, cDNA prepared from normal rat tissues was subjected to RT-PCR analyses for ACAT-1 and ACAT-2 with G3PDH as a control as described in Methods. Data are representative of 3 independent experiments. B, Total RNA was extracted from AC29 (lane 1), rat ACAT-2 cells (lane 2), McA-RH7777 cells (lane 3), small intestine (lane 4), and liver (lane 5) of normal rat. ACAT-2 mRNA was analyzed by Northern blot analyses with a 18S ribosomal RNA band as an internal control. A representative result from 3 independent experiments is shown. C and D, Small intestines from normal rats were removed and fixed and deparaffinized sections were reacted with an anti-human ACAT-2 antibody as described in Methods. Magnification x100 (C) and x400 (D). Brown color indicates positive reaction with an anti-human ACAT-2 antibody.
Effect of Diabetic Conditions on Expression of Rat Intestinal ACAT-2
Fasting blood glucose levels in diabetic rats were 22.0 mmol/L at day 4 (4 days after STZ injection), whereas those in control rats remained <6.0 mmol/L. Plasma TC and triglyceride levels in diabetic rats at day 14 were increased 1.5-fold and 2.9-fold, respectively (Table). The intestinal ACAT activity of diabetic rats at day 14 was 2.2-fold higher than that of control rats (Figure 3A). In contrast, the hepatic ACAT activity was not changed in control and diabetic rats (35.1±2.3 versus 41.8±6.6 pmol/mg protein/minute, respectively; NS). Therefore, it is likely that hepatic ACAT does not contribute to diabetes-induced dyslipidemia. Immunodepletion analyses revealed that an anti–ACATn-1 antibody significantly reduced the activity of rat ACAT-1 transfectants (rat ACAT-1 cells),23 but had no effect on the enzyme activity of rat ACAT-2 cells. Similarly, an anti–ACAT-2 antibody completely reduced the enzyme activity of rat ACAT-2 cells but had no effect on rat ACAT-1 cells (Figure 3B). Under these conditions, an anti–ACATn-2 antibody reduced the ACAT activity by >70% of the total intestinal activity of control and diabetic rats, whereas an anti–ACATn-1 antibody did not affect it at all (Figure 3B). Furthermore, we made immunoblot analyses to examine whether the diabetes-induced increase in small intestinal ACAT activity was caused by an increase in ACAT-2 but not ACAT-1. The immunoblot analyses using an anti–ACAT-2 antibody revealed at least 2 bands in rat small intestine: the upper band and the slightly lower band. The amount of intestinal ACAT-2 at protein levels in diabetic rats at day 14 was 2-fold higher than that in the intestine of control rats (Figure 4B). Under these conditions, parallel immunoblot analyses with an anti–ACATn-1 antibody failed to reveal a distinct band (data not shown), indicating that the major intestinal ACAT isozyme both in normal and diabetic rats was ACAT-2. In addition, the expression of intestinal ACAT-2 was significantly correlated with the level of plasma TC (r=0.56, P<0.01) and triglyceride (r=0.57, P<0.01) (Figure 5).
Glucose, Total Cholesterol, and Triglyceride in Normal and Diabetic Rats
Figure 3. Intestinal ACAT activity of normal and diabetic rats (A) and immunodepletion assay (B). A, Homogenates obtained from small intestine of normal (control) and diabetic (DM) rats were solubilized and determined for the ACAT activity as described in Methods. Data represent the mean of diabetic rats (n=16) and control rats (n=8). Error bars represent SD. *P<0.005 compared with control. B, Homogenates obtained from small intestine of normal rats (control) and diabetic rats (DM), and from rat ACAT-1 overexpressed cells (rat ACAT-1 cells), and rat ACAT-2 overexpressed cells (rat ACAT-2 cells) were solubilized. Each sample was incubated with a nonimmune rabbit IgG (white bars), an anti-human ACAT-1 antibody (hatched bars), an anti-human ACAT-2 antibody (latticed bars), or the combined mixture of an anti-human ACAT-1 antibody and an anti-human ACAT-2 antibody (black bars), followed by separation of supernatant and immunoprecipitate with protein A-Sepharose. Each supernatant was measured for ACAT activity as described in Methods.
Figure 4. Effect of diabetic conditions on expression of rat intestinal ACAT-2. A, Homogenates obtained from small intestine of 2 normal rats (control) and 4 diabetic rats (DM) were solubilized. Each sample was electrophoresed on 10% SDS-PAGE and subjected to immunoblot analyses with an anti-human ACAT-2 antibody as described in Methods. Data are representative of 4 independent experiments. B, Densitometric quantification of anti-human ACAT-2 antibody reactive bands was measured as described in Methods. Data represent the mean of diabetic rats (n=16) and control rats (n=8). Error bars represent SD. *P<0.005 compared with control. C, Total RNA was extracted from small intestine of normal rats (control) and diabetic rats (DM) and ACAT-2 mRNA was detected by Northern blot analyses. D, Densitometric scanning of (C). The intensity of a 18S ribosomal RNA band was used as an internal control to normalize the intensity of ACAT-2 mRNA subspecies. Data represent the mean of diabetic rats (n=9) and control rats (n=6). Error bars represent SD. *P<0.05 compared with control.
Figure 5. Correlation between intestinal ACAT-2 and plasma lipids. A, Plasma TC levels. B, Plasma triglyceride levels. Plasma levels of TC and triglyceride were determined in 16 diabetic rats (black circles) and 8 control rats (white circles). The regression equations in (A) and (B) are r=0.56 (P<0.01) and r=0.57 (P<0.01), respectively.
Finally, we made Northern blot analyses to demonstrate whether the increased intestinal ACAT-2 protein in diabetic rat was caused by increased ACAT-2 mRNA level. Northern blot analyses could detect a single distinct transcript in small intestine and liver (Figures 2B and 4C). On quantitative analyses based on densitometry of this transcript, ACAT-2 mRNA level were increased by 1.8-fold in diabetic rats (Figure 4C and 4D).
Discussion
ACAT plays an important role in cholesterol absorption from intestines9 and recent reports using ACAT inhibitors demonstrated that the intestinal ACAT activity is responsible for dyslipidemia in diabetic rats.12–14 Two ACAT isozymes (ACAT-1and ACAT-2) were identified in mammals,15 and it is important to determine which ACAT isozyme is responsible for the increased intestinal ACAT activity in diabetic rats. The results of the present study have clarified an important role of intestinal ACAT-2 for diabetes-induced dyslipidemia at a transcriptional level.
In parallel with an elevated intestinal ACAT activity, both plasma TC and triglyceride levels were increased in the diabetic rats (Table). The increased TC levels seems to result from an increase in intestinal absorption of dietary cholesterol caused by elevated intestinal ACAT activity, whereas the mechanism of the increased plasma triglyceride levels is not clear. The effect of intestinal ACAT on intestinal triglyceride absorption is not known. Microsomal triglyceride transfer protein (MTP) was reported to play a crucial role in the intestinal chylomicron formation as well as hepatic VLDL assembly.27,28 Triglyceride as well as cholesteryl esters are essential substrates for MTP.27 The increased cholesterol absorption in the diabetic rats induced mainly by upregulation of intestinal ACAT activity might also increase the intestinal MTP activity and then enhance intestinal chylomicron synthesis, thus inducing hypertriglyceridemia in the diabetic rats. In fact, recent reports showed that intestinal MTP expression in diabetes was higher than controls in both rabbits and rats.29,30
Recent studies revealed new molecules involved in the intestinal cholesterol absorption; ATP-binding cassette half-transporters ABCG5/ABCG831,32 and Niemann-Pick C1–like 1 protein (NPC1L1).33,34 ABCG5/ABCG8 are highly expressed in the liver and intestine.35 Mutations in the human genes encoding ABCG5/ABCG8 resulted in sitosterolemia with a reduced biliary secretion as well as an enhanced intestinal absorption of plant sterols (sitosterol, campesterol),36–38 suggesting an important role of ABCG5/ABCG8 in hepatobiliary cholesterol secretion and intestinal cholesterol absorption. In STZ-induced diabetic rats, both intestinal and hepatic ABCG5/ABCG8 expressions were reduced at a mRNA level that contributed to a reduction of hepatobiliary cholesterol secretion and an increased plant sterols concentrations.39 Therefore, it is possible that both the decreased intestinal ABCG5/ABCG8 and the increased intestinal ACAT-2 contribute to diabetes-induced dyslipidemia. NPC1L1 localized in jejunal enterocytes was demonstrated to be critical for intestinal cholesterol absorption.33 Ezetimibe, a selective inhibitor of intestinal cholesterol absorption,40 had no effect in NPC1L1-deficient mice, suggesting that NPC1L1 serves as an ezetimibe-dependent pathway for intestinal cholesterol absorption.33,34 NPC1L1-deficient mice had reduced intestinal uptake of plant sterols but did not change triglyceride absorption.33,34 It does not seem likely that NPC1L1 may contribute to hypertriglyceridemia induced under diabetic conditions.
Previous studies showed that intestinal ACAT activity was increased in diabetic rats, which was restored by insulin treatment, suggesting insulin deficiency might lead to the elevated intestinal ACAT activity under the diabetic condition.12,13 Furthermore, no significant differences in the intestinal ACAT activity could be detected between diabetic animals fed control chow and those fed the atherogenic diet.12 In vitro study with Caco-2 cells, human intestinal cell-derived cell line, showed that incubation of these cells with insulin significantly reduced the ACAT activity of these cells, suggesting an inhibitory effect of insulin on the intestinal ACAT activity.41 Our in vitro experiment with Caco-2 cells also resulted in reduction of ACAT activity by insulin but not high glucose (unpublished data). It is likely from these data that the intestinal ACAT activity is regulated by insulin but not by high glucose or diet.
Recent studies clarified the ACAT-2 promoter. Song et al identified the ACAT-2 promoter region whose function was increased during the differentiation of Caco-2 cells.42 In this connection, the expression of Cdx-2, a transcriptional regulator localized in nucleus, increased greatly during the differentiation of Caco-2 cells but was not detected in hepatoblastoma cell line HepG2 cells.43 Sequence analyses by computer showed that human, mouse, and rat ACAT-2 promoter region contained 4, 3, and 4 Cdx-2 elements, respectively. Electrophoretic mobility shift assay showed that Cdx-2 was able to efficiently bind to mouse and human ACAT-2 promoter regions, suggesting that Cdx-2 is a tissue-specific ACAT-2 regulator in intestinal cells.43 This could be the case under diabetic conditions because ACAT activity in intestine, but not in liver, was increased in diabetic rats (Figure 3A).
ACAT inhibition is emphasized as a potential anti-atherogenic strategy. However, results obtained from ACAT-1–deficient mice have not supported this notion unanimously.44–46 Animal experiments by Accad et al44 using ACAT-1–deficient mice bred either on apoE-deficient mice or LDL receptor-deficient mice revealed that deletion of ACAT-1 did not prevent these animals from development of atherosclerotic lesions. Yagyu et al45 using similar animal model systems reached the different conclusion that ACAT-1–deficiency reduces atherosclerotic lesions. However, Fazio et al46 showed that reconstitution of LDL receptor-deficient mice with ACAT-1–deficient macrophage resulted in a more significant increase in atherosclerotic lesions than LDL receptor-deficient mice reconstituted with wild-type macrophage. With these reports taken together, effectiveness of ACAT-1 inhibitors for anti-atherogenic therapy in humans remains controversial.
Although the expression of ACAT-1 is ubiquitous, the expression of ACAT-2 is restricted. Human ACAT-2 was expressed in intestine and mice ACAT-2 in intestine and liver,22 and rat ACAT-2 was shown to localize in intestine and liver in the present study. ACAT-2–deficient mice were completely resistant to diet-induced hypercholesterolemia.47 On further gene knockout of apoE, atherosclerotic lesions of these mice were significantly reduced when compared with those of apoE-deficient mice.48 These animal studies revealed that complete depletion of ACAT-2 prevents mice from atherosclerosis-prone hyperlipidemia, suggesting an importance of ACAT-2–derived intestinal and hepatic ACAT activity in diet-induced hypercholesterolemia.
Development of ACAT inhibitors has faced difficulty, caused mainly by adrenotoxicity, which might be induced by ACAT-1 suppression,49,50 and isozyme-specific ACAT inhibitors are expected to be a potential target. In this connection, results of the present study and previous ones using ACAT-2–deficient mice47,48 suggest the possibility that ACAT-2–specific inhibitors are beneficial as an anti-atherosclerotic drug by normalization of diabetes-induced dyslipidemia.
In conclusion, the cloning of rat ACAT-2 cDNA with its characterization have provided convincing evidence that the increased intestinal ACAT activity in diabetic rats is caused largely by a significant upregulation of ACAT-2 at both protein and mRNA levels but not ACAT-1, suggesting a major catalytic role of ACAT-2 in diabetes-induced dyslipidemia. Although results from animal studies with mice or rats may not be directly applicable to human, selective inhibition for ACAT-2 might ameliorate diabetes-induced dyslipidemia in human and even cardiovascular disease, because intestinal cholesterol absorption is reported to be significantly higher in diabetic patients with CHD than those without CHD.10
Acknowledgments
We thank Drs Ta-Yuan Chang and Catherine C.Y. Chang at Department of Biochemistry, Dartmouth Medical School, for the generous gift of DM10, DM54, and AC29 cells and helpful discussions. This work was supported in part by a Grant-in Aid for Scientific Research (B: 13470228 to S.H. and C: 14571101 to A.M.) from Japan Society for the Promotion of Science (JSPS), and by the Research Grant for Cardiovascular Diseases 13-4 from the Ministry of Health, Labor and Welfare.
References
Chang TY, Chang CC, Lin S, Yu C, Li BL, Miyazaki A. Roles of acyl-coenzyme A: cholesterol acyltransferase-1 and -2. Curr Opin Lipidol. 2001; 12: 289–296.
Chang TY, Chang CC, Cheng D. Acyl-coenzyme A: cholesterol acyltransferase. Annu Rev Biochem. 1997; 66: 613–638.
Miyazaki A, Sakashita N, Lee O, Takahashi K, Horiuchi S, Hakamata H, Morganelli PM, Chang CC, Chang TY. Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages. Arterioscler Thromb Vasc Biol. 1998; 18: 1568–1574.
Resnick HE, Howard BV. Diabetes and cardiovascular disease. Annu Rev Med. 2002; 53: 245–267.
Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med. 1998; 339: 229–234.
Turner RC, Millns H, Neil HA, Stratton IM, Manley SE, Matthews DR, Holman RR. Risk factors for coronary artery disease in non-insulin dependent diabetes mellitus: United Kingdom Prospective Diabetes Study (UKPDS: 23). BMJ. 1998; 316: 823–828.
Marks JB, Raskin P. Cardiovascular risk in diabetes: a brief review. J Diabetes Complications. 2000; 14: 108–115.
Haffner SM. Diabetes, hyperlipidemia, and coronary artery disease. Am J Cardiol. 1999; 83: 17F–21F.
Wilson MD, Rudel LL. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol. J Lipid Res. 1994; 35: 943–955.
Gylling H, Miettinen TA. Cholesterol absorption and lipoprotein metabolism in type II diabetes mellitus with and without coronary artery disease. Atherosclerosis. 1996; 126: 325–332.
Miettinen TA, Gylling H, Tuominen J, Simonen P, Koivisto V. Low synthesis and high absorption of cholesterol characterize type 1 diabetes. Diabetes Care. 2004; 27: 53–58.
Jiao S, Matsuzawa Y, Matsubara K, Kihara S, Nakamura T, Tokunaga K, Kubo M, Tarui S. Increased activity of intestinal acyl-CoA: cholesterol acyltransferase in rats with streptozocin-induced diabetes and restoration by insulin supplementation. Diabetes. 1988; 37: 342–346.
Maechler P, Wollheim CB, Bentzen CL, Niesor E. Role of the intestinal acyl-CoA: cholesterol acyltransferase activity in the hyperresponse of diabetic rats to dietary cholesterol. J Lipid Res. 1992; 33: 1475–1484.
Kusunoki J, Aragane K, Kitamine T, Kozono H, Kano K, Fujinami K, Kojima K, Chiwata T, Sekine Y. Postprandial hyperlipidemia in streptozotocin-induced diabetic rats is due to abnormal increase in intestinal acyl coenzyme A: cholesterol acyltransferase activity. Arterioscler Thromb Vasc Biol. 2000; 20: 171–178.
Buhman KF, Accad M, Farese RV. Mammalian acyl-CoA: cholesterol acyltransferases. Biochim Biophys Acta. 2000; 1529: 142–154.
Cadigan KM, Heider JG, Chang TY. Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A: cholesterol acyltransferase activity. J Biol Chem. 1988; 263: 274–282.
Matsuda H, Hakamata H, Miyazaki A, Sakai M, Chang CC, Chang TY, Kobori S, Shichiri M, Horiuchi S. Activation of acyl-coenzyme A: cholesterol acyltransferase activity by cholesterol is not due to altered mRNA levels in HepG2 cells. Biochim Biophys Acta. 1996; 1301: 76–84.
Maung K, Miyazaki A, Nomiyama H, Chang CC, Chang TY, Horiuchi S. Induction of acyl-coenzyme A: cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D(3) or 9-cis-retinoic acid in undifferentiated THP-1 cells. J Lipid Res. 2001; 42: 181–187.
Sakashita N, Miyazaki A, Takeya M, Horiuchi S, Chang CC, Chang TY, Takahashi K. Localization of human acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in macrophages and in various tissues. Am J Pathol. 2000; 156: 227–236.
Chang CC, Lee CY, Chang ET, Cruz JC, Levesque MC, Chang TY. Recombinant acyl-CoA: cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner. J Biol Chem. 1998; 273: 35132–35141.
Chang CC, Huh HY, Cadigan KM, Chang TY. Molecular cloning and functional expression of human acyl-coenzyme A: cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J Biol Chem. 1993; 268: 20747–20755.
Chang CC, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S, Lee CY, Strom SC, Kashyap R, Fung JJ, Farese RV, Jr., Patoiseau JF, Delhon A, Chang TY. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem. 2000; 275: 28083–28092.
Matsuda H, Hakamata H, Kawasaki T, Sakashita N, Miyazaki A, Takahashi K, Shichiri M, Horiuchi S. Molecular cloning, functional expression and tissue distribution of rat acyl-coenzyme A: cholesterol acyltransferase. Biochim Biophys Acta. 1998; 1391: 193–203.
Oelkers P, Behari A, Cromley D, Billheimer JT, Sturley SL. Characterization of two human genes encoding acyl coenzyme A: cholesterol acyltransferase-related enzymes. J Biol Chem. 1998; 273: 26765–26771.
Cases S, Novak S, Zheng YW, Myers HM, Lear SR, Sande E, Welch CB, Lusis AJ, Spencer TA, Krause BR, Erickson SK, Farese RV, Jr. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization. J Biol Chem. 1998; 273: 26755–26764.
Anderson RA, Joyce C, Davis M, Reagan JW, Clark M, Shelness GS, Rudel LL. Identification of a form of acyl-CoA: cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J Biol Chem. 1998; 273: 26747–26754.
White DA, Bennett AJ, Billett MA, Salter AM. The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triacylglycerol transfer protein. Br J Nutr. 1998; 80: 219–229.
Wetterau JR, Zilversmit DB. Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues. Biochim Biophys Acta. 1986; 875: 610–617.
Gleeson A, Anderton K, Owens D, Bennett A, Collins P, Johnson A, White D, Tomkin GH. The role of microsomal triglyceride transfer protein and dietary cholesterol in chylomicron production in diabetes. Diabetologia. 1999; 42: 944–948.
Phillips C, Bennett A, Anderton K, Owens D, Collins P, White D, Tomkin GH. Intestinal rather than hepatic microsomal triglyceride transfer protein as a cause of postprandial dyslipidemia in diabetes. Metabolism. 2002; 51: 847–852.
Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, Hobbs HH. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest. 2002; 110: 671–680.
Yu L, Hammer RE, Li-Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC, Hobbs HH. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci U S A. 2002; 99: 16237–16242.
Altmann SW, Davis HR Jr, Zhu LJ, Yao X, Hoos LM, Tetzloff G, Iyer SP, Maguire M, Golovko A, Zeng M, Wang L, Murgolo N, Graziano MP. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science. 2004; 303: 1201–1204.
Davis HR, Zhu LJ, Hoos LM, Tetzloff G, Maguire M, Liu J, Yao X, Iyer SP, Lam MH, Lund EG, Detmers PA, Graziano MP, Altmann SW. Niemann-pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole body cholesterol homeostasis. J Biol Chem. 2004. In press.
Lu K, Lee MH, Yu H, Zhou Y, Sandell SA, Salen G, Patel SB. Molecular cloning, genomic organization, genetic variations, and characterization of murine sterolin genes Abcg5 and Abcg8. J Lipid Res. 2002; 43: 565–578.
Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J, Kwiterovich P, Shan B, Barnes R, Hobbs HH. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science. 2000; 290: 1771–1775.
Lee MH, Lu K, Hazard S, Yu H, Shulenin S, Hidaka H, Kojima H, Allikmets R, Sakuma N, Pegoraro R, Srivastava AK, Salen G, Dean M, Patel SB. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption. Nat Genet. 2001; 27: 79–83.
Lu K, Lee MH, Hazard S, Brooks-Wilson A, Hidaka H, Kojima H, Ose L, Stalenhoef AF, Mietinnen T, Bjorkhem I, Bruckert E, Pandya A, Brewer HB Jr, Salen G, Dean M, Srivastava A, Patel SB. Two genes that map to the STSL locus cause sitosterolemia: genomic structure and spectrum of mutations involving sterolin-1 and sterolin-2, encoded by ABCG5 and ABCG8, respectively. Am J Hum Genet. 2001; 69: 278–290.
Bloks VW, Bakker-Van Waarde WM, Verkade HJ, Kema IP, Wolters H, Vink E, Groen AK, Kuipers F. Down-regulation of hepatic and intestinal Abcg5 and Abcg8 expression associated with altered sterol fluxes in rats with streptozotocin-induced diabetes. Diabetologia. 2004; 47: 104–112.
van Heek M, Farley C, Compton DS, Hoos L, Davis HR. Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function. Br J Pharmacol. 2001; 134: 409–417.
Jiao S, Moberly JB, Cole TG, Schonfeld G. Decreased activity of acyl-CoA: cholesterol acyltransferase by insulin in human intestinal cell line Caco-2. Diabetes. 1989; 38: 604–609.
Song BL, Qi W, Yang XY, Chang CC, Zhu JQ, Chang TY, Li BL. Organization of human ACAT-2 gene and its cell-type-specific promoter activity. Biochem Biophys Res Commun. 2001; 282: 580–588.
Song BL, Qi W, Wang CH, Yang JB, Yang XY, Lin ZX, Li BL. Preparation of an anti-Cdx-2 antibody for analysis of different species Cdx-2 binding to acat2 promoter. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003; 35: 6–12.
Accad M, Smith SJ, Newland DL, Sanan DA, King LE, Jr., Linton MF, Fazio S, Farese RV Jr. Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA: cholesterol acyltransferase 1. J Clin Invest. 2000; 105: 711–719.
Yagyu H, Kitamine T, Osuga J, Tozawa R, Chen Z, Kaji Y, Oka T, Perrey S, Tamura Y, Ohashi K, Okazaki H, Yahagi N, Shionoiri F, Iizuka Y, Harada K, Shimano H, Yamashita H, Gotoda T, Yamada N, Ishibashi S. Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia. J Biol Chem. 2000; 275: 21324–21330.
Fazio S, Major AS, Swift LL, Gleaves LA, Accad M, Linton MF, Farese RV, Jr. Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages. J Clin Invest. 2001; 107: 163–171.
Buhman KK, Accad M, Novak S, Choi RS, Wong JS, Hamilton RL, Turley S, Farese RV Jr. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice. Nat Med. 2000; 6: 1341–1347.
Willner EL, Tow B, Buhman KK, Wilson M, Sanan DA, Rudel LL, Farese RV Jr. Deficiency of acyl CoA: cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 2003; 100: 1262–1267.
Dominick MA, McGuire EJ, Reindel JF, Bobrowski WF, Bocan TM, Gough AW. Subacute toxicity of a novel inhibitor of acyl-CoA: cholesterol acyltransferase in beagle dogs. Fundam Appl Toxicol. 1993; 20: 217–224.
Dominick MA, Bobrowski WA, MacDonald JR, Gough AW. Morphogenesis of a zone-specific adrenocortical cytotoxicity in guinea pigs administered PD 132301–2, an inhibitor of acyl-CoA: cholesterol acyltransferase. Toxicol Pathol. 1993; 21: 54–62.(Masaharu Hori; Maki Satoh)