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Originally published In Press as doi:10.1074/jbc.M701742200 on April 3, 2007

J. Biol. Chem., Vol. 282, Issue 23, 17078-17089, June 8, 2007
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Inhibiting Proteasomal Degradation of Microsomal Triglyceride Transfer Protein Prevents CCl4-induced Steatosis*

Xiaoyue Pan{ddagger}, Farah N. Hussain{ddagger}, Jahangir Iqbal{ddagger}, Miriam H. Feuerman§, and M. Mahmood Hussain{ddagger}1

From the Departments of {ddagger}Anatomy and Cell Biology, Pediatrics, and §Biochemistry, SUNY Downstate Medical Center, Brooklyn, New York 11203

Received for publication, February 28, 2007 , and in revised form, April 3, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Carbon tetrachloride (CCl4) interferes with triglyceride secretion and causes steatosis, fibrosis, and necrosis. In mice, CCl4 decreased plasma triglyceride-rich lipoproteins, increased cellular lipids, and reduced microsomal triglyceride transfer protein (MTP) without diminishing mRNA levels. Similarly, CCl4 decreased apoB-lipoprotein production and MTP activity but had no effect on mRNA levels in primary enterocytes and colon carcinoma and hepatoma cells. CCl4 did not affect MTP synthesis but induced post-translational degradation involving ubiquitinylation and proteasomes in McA-RH7777 cells. By contrast, MTP inhibitor increased cellular lipids without affecting MTP protein. MTP was covalently modified when cells were incubated with 14CCl4. This modification was prevented by the inhibition of P450 oxygenases, indicating that CCl3· generated by these enzymes targets MTP for degradation. To determine whether inhibition of proteolysis could prevent CCl4 toxicity, mice were fed with CCl4 with or without lactacystin. Lactacystin increased ubiquitinylated MTP and prevented lipid accumulation in tissues. Thus, CCl4 induces post-translational degradation without affecting lipid transfer activity, whereas MTP antagonist inhibits lipid transfer activity without causing its destruction. These studies identify MTP as a major target of CCl4 and its degradation as a novel mechanism involved in the onset of steatosis, suggesting that inhibition of proteolysis may prevent some forms of steatosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatic accumulation of triglycerides (steatosis) is a major complication associated with obesity, insulin resistance, and alcoholic and nonalcoholic fatty liver disease (1). This is attributed to increased lipogenesis and decreased beta-oxidation followed by lipid peroxidation and mitochondrial dysfunction (1). Although benign, steatosis can advance to steatohepatitis, fibrosis, and cirrhosis.

Carbon tetrachloride (CCl4) is a colorless liquid that is nonflammable and volatile, with a distinct odor. In the 19th century it was used as an anesthetic. In the first 25 years of the 20th century its toxicity was firmly established (2). CCl4 was banned in the United States as a dry-cleaning agent in 1970 because of its hepatotoxicity. It is, however, used in the synthesis of refrigerants and production of semiconductors, as a fumigant, and in the processing of fats, oils, and rubber (3). Industrial emission is the major source of CCl4 in the air. It is found in low levels in ambient air and water (3).

CCl4 is rapidly metabolized by mixed function cytochrome P450 oxygenases of the endoplasmic reticulum (ER)2 resulting in the generation of CCl3· (4, 5). Adduct formation between CCl3· and DNA is believed to initiate hepatic cancer (5). CCl3· can also react with oxygen to form CCl3OO· (4, 5). CCl3OO· initiates a chain reaction leading to lipid peroxidation, changes in membrane permeability, and loss of calcium homeostasis. In addition, tissue lipid levels increase (see below). The initial cellular injury subsequently takes the form of inflammation (5, 6), and the chronic effects of CCl4 exposure include fatty degeneration, fibrosis, hepatocellular death, and carcinogenicity (5).

After oral administration, CCl4 concentrates in the liver. Early biochemical changes are obvious in the first couple of hours, and tissue necrosis is visible after 5 h. Around 12 h, central zone necrosis is evident (5). CCl4 decreases secretion of triglycerides as part of very low density lipoproteins and increases hepatic triglyceride by 195% within 3 h in rats (7). Thus, a distinctive feature of CCl4 toxicity is the rapidity of triglyceride accumulation in the liver due to a failure in their secretory mechanisms (2, 4, 5). This failure has been largely attributed to unspecified functional impairment in the Golgi apparatus (5), and the need to study the possible effects of chlorinated methanes on the molecular events involved in triglyceride secretion has been emphasized (4).

Triglyceride secretion requires assembly and secretion of lipoproteins by the liver and intestine, which is critically dependent on apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) (8–10). ApoB serves as a structural protein, and MTP present in the ER is a required chaperone for the assembly of triglyceride-rich lipoproteins. MTP transfers several lipids, and its phospholipid transfer activity is sufficient for lipoprotein production (11). In addition, MTP physically associates (12) with the N terminus of apoB (13). Thus, MTP is believed to bind and transfer lipids to nascent apoB forming primordial lipoprotein particles (8, 9). Expression and ablation of MTP affects triglyceride secretion indicating that it is rate-limiting (14, 15). Recently, MTP has been shown to be involved in the lipidation (16) of CD1d, glycolipid antigen-presenting molecule, and in the maturation of NKT cell development (17). Moreover, MTP levels have been inversely correlated with hepatosteatosis in individuals infected with type III hepatitis C virus (18). Decreased MTP levels have also been suggested to initiate alcoholic steatosis in rats (19). MTP inhibitors are known to cause steatosis (20, 21), but the role MTP plays in the development of other forms of steatosis is unknown. Here we show that CCl4 induces post-translational degradation of MTP and causes steatosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Rabbit anti-mouse apoB48/apoB100 (Biodesign, K23300R), goat anti-human apoAI and apoB (Biodesign), rabbit anti-protein disulfide isomerase (anti-PDI, Stressgen, SPA-890), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Chemicon, MAB374), anti-MTP (BD Transduction Laboratories, 612022), rabbit anti-ubiquitin (Abcam, Inc. ab7780), [35S]methionine (35S-Easy tag) and Solvable (PerkinElmer Life Sciences), [3H]glycerol (Amersham Biosciences), 14CCl4 (American Radiochemical), MG132 and ALLN (Sigma), and lactacystin (Kamiya Biomedical Co.) were purchased. Stock solutions of inhibitors were prepared in dimethyl sulfoxide (Me2SO). Dr. David Gordon, Bristol-Myers Squibb, kindly provided BMS197636. For in vivo experiments, CCl4 was mixed with olive oil and gavaged (1 µl/g body weight). For ex vivo and in vitro experiments, CCl4 was dissolved in Me2SO. Final concentrations of Me2SO did not exceed 0.25%.

Animals
C57Bl/6J mice (The Jackson Laboratory) were housed in an air-conditioned room at 22 °C with a 12-h lighting schedule and fed rodent chow. Male 10–12-week-old mice were used in this study. Food was removed the night before the experiments. Experimental mice received CCl4 in olive oil. Control mice received an equal volume of oil and saline. Blood was collected from the heart, and serum lipid levels were determined in plasma using commercial kits (Thermo Trace). Cholesterol and triglyceride in high density lipoproteins (HDL) were measured after the precipitation of apoB-lipoproteins with phosphotungstate magnesium chloride (22).

Cells
McA-RH777 cells (23) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine and 10% horse serum (Invitrogen). Primary enterocytes were isolated and cultured as described (24). HepG2 and Caco-2 cell were cultured as described previously (25, 26). Human apoB and apoAI secreted from cells were measured by enzyme-linked immunosorbent assay (23, 27).

Methods
MTP Activity Assays—To measure MTP activity, 100 µgof liver and intestine were homogenized in 1 ml of buffer K (1.0 mM EGTA, 1 mM Tris-HCl, and mM MgCl2) and centrifuged at 50,000 rpm for 1 h. The supernatant was used to measure MTP activity (28, 29). For in vitro activity assays, MTP purified from bovine liver or mice intestinal and liver microsomal proteins were incubated with different concentrations of olive oil or CCl4 in oil for 30 min in the presence of donor and acceptor vesicles. Changes in fluorescence due to lipid transfer by MTP were recorded and used to determine the effect of CCl4 on MTP activity.

Lipid Synthesis—To measure lipid synthesis and secretion, cells were labeled with 5 µCi/ml [3H]glycerol with or without CCl4 for 4 h. The media were collected, and the cells were washed twice with cold phosphate-buffered saline. Total lipids in the cells and medium were then extracted with chloroform-methanol (2:1, v/v). The organic solvent was evaporated, and the residues were dissolved in isopropanol and spotted on a thin layer chromatographic plate. Radiolabeled triglycerides and phospholipids were separated using hexanes/ethyl ether/acetic acid (80:20:2) as a developing solvent. Lipids were visualized by exposure to iodine vapors; bands corresponding to the marker lipids were scraped, and radioactivity was measured by liquid scintillation counting (26).

Metabolic Labeling and Immunoprecipitation—Cells were preincubated in methionine-free Dulbecco's modified Eagle's medium for 30 min and pulse-labeled with [35S]methionine (100–200 µCi/ml). For pulse-chase experiments, cells were incubated with [35S]methionine (200 µCi/ml) for 2 h in methionine-deficient media supplemented with 0.1% bovine serum albumin. Cells were washed and chased in Dulbecco's modified Eagle's medium containing 100 µM methionine in the presence and absence of CCl4 in Me2SO for different times. For immunoprecipitation, cell lysates were prepared in 50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 0.015% phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM EDTA, 1% sodium deoxycholate, 1% Triton, 1% SDS. Cell lysates were incubated at 70 °C for 15 min to ensure complete cell lysis and diluted with lysis buffer to achieve a SDS concentration of 0.1% (w/v) as reported (23, 30). Media were collected and centrifuged at 13,000 rpm for 10 min to remove cell debris. Supernatants (0.9 ml) were combined with 0.1 ml of 10x lysis buffer containing 1% SDS. Cell lysates and media were incubated with antibodies (1:100 dilution) for 2 h and then with 20 µl of protein A/G-Sepharose (10% solution) for an additional 2 h. Immunocomplexes were collected by centrifugation, washed three times with 10 mM Tris, pH 7.5, containing 0.1 M NaCl and 1% Triton, eluted in 50 µlof sample buffer, separated on SDS-polyacrylamide gel, and exposed to a phosphorimaging screen. Radioactivity in apolipoproteins was measured by liquid scintillation counting.

Western Blot Analysis—Proteins were separated under non-reducing conditions, transferred to nitrocellulose membranes, blocked for 2 h in 20 mM Tris, 137 mM NaCl, pH 7.5, containing 0.1% Tween 20 and 5% nonfat dry milk at room temperature. The blots were washed three times and incubated overnight at 4 °C in the same buffer containing 0.5% dry milk and a primary antibody (1:1000 dilution), washed, and then incubated with mouse horseradish peroxidase-conjugated secondary antibody (1:4000) in 1.0% skim milk for 1 h at room temperature. Immune reactivity was detected by chemiluminescence.

For subcellular fractionation experiments, cells were homogenized using a syringe with a 25-gauge needle (10 strokes) and centrifuged (500 x g, 10 min, 4 °C; Beckman GS-15R centrifuge). The postnuclear supernatant was then centrifuged (3,000 x g, 10 min, 4 °C) to get the heavy mitochondrial pellet. To isolate the microsomal fraction, the postmitochondrial supernatant was centrifuged at 100,000 x g for 1 h at 4 °C using a Beckman tabletop Ultracentrifuge TLA-110 rotor. The remaining supernatant was designated as the cytosolic fraction as reported previously (31, 32). The microsomal pellet and the supernatant proteins were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal anti-calnexin antibody (Stressgen; 1:2,000 dilution) or anti-MTP antibody (1:2,000 dilution).

To detect ubiquitin-conjugated proteins, cell or tissue homogenates were prepared in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS and incubated with protein A and anti-MTP antibodies for 2 h. Immunocomplexes were washed, resolved on SDS-polyacrylamide gels, and blotted onto polyvinylidene difluoride membranes. For detection of ubiquitinylated proteins, membranes were incubated with antibodies against ubiquitin or MTP. After 2 h, membranes were washed and incubated with the corresponding secondary antibody tagged with horseradish peroxidase, signals were detected by enhanced chemiluminescence (Amersham Biosciences).


Figure 1
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  		FIGURE 1.
Effect of CCl4 on plasma and tissue lipid levels in mice. Male C57BL/6J mice (10 weeks old, n = 6–10) were fasted (12 h) and gavaged at 09:30 h with a mixture (1 µl/gm body weight) of CCl4 and olive oil containing different indicated amounts of CCl4. Plasma, intestine, and liver were obtained after 4 h. Triglyceride (A) and cholesterol (B) levels in total plasma and apoB-lipoproteins were measured. In addition, lipids were extracted from the intestinal and liver tissue and used to measure triglyceride (C) and cholesterol (D). For the time course experiment, animals were gavaged with CCl4 (1 mg/kg) in olive oil, and plasma and tissues were collected at different times. Changes in plasma HDL and apoB-lipoprotein triglyceride (E) and cholesterol (F), as well as tissue triglycerides (G) and cholesterol (H), are plotted against time. Each point represents the mean ± S.D. *, p < 0.05; **, p < 0.01; significantly different from oil control group.

 
Analyses of Gene Expressions—Total tissue RNA was isolated from frozen tissues or cells using TRIzol (Invitrogen) (33, 34). RNA was treated with DNase I at 37 °C followed by inactivation at 75 °C for 5 min. cDNA was synthesized from total RNA using SuperScript First-strand Synthesis System (Invitrogen) and random hexamer primers. Real-time PCR measurements of individual cDNAs were performed in triplicate by TaqMan probe or SYBR Green methods using an ABI Prism 7000 (Applied Biosystems). The primer-probe sets used for MTP and 18 S rRNA were as follows: mouse MTP forward primer, 5'-ACG GCC ATT CCC ATT GTG-3'; reverse primer, 5'-GCC AGA GCT CCG AGA GAG AA-3'; fluorescence probe, 5'-(Fam)-GTC TTC CAG AGC CAC TGT AAA GGA TGTC-3'-(Tamra). 18 S rRNA forward primer, 5'-AGTCC CTTGCC CTTTG TACACA-3'; reverse primer, 5'-GATCC GAGGG CCTCA CTAAAC-3'; fluorescence probe, 5'-(Fam)-CGCCCGTCGCTACTACCGATTGGT-3'-(Tamra). Rat MTP forward primer, 5'-GTCACGATAACGGCTGTCAATG-3'; reverse primer, 5'-TGCCCTTGAAGAAGATGCTCTTCTC-3'; fluorescence probe, 5' (Fam) CGAAAATGCGGGTCAGCAGAGAGG-6TAM-RA3' (Tamra). GAPDH mRNA was used as an internal control (Applied Biosystems) (35).

Statistical Analysis—All determinations were made in triplicate or quadruplicate and reported as the mean ± S.D. Treatment groups were subjected to one-way analysis of variance and Bonferroni's test for multiple comparisons. Values of p < 0.05 were considered significant.


Figure 2
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  		FIGURE 2.
CCl4 reduces MTP in mice. Mice were gavaged with different concentrations of CCl4. After 4 h, MTP activity was measured in the intestine (A) and liver (B). In addition, MTP and GAPDH protein levels were measured in the intestine (C) and liver (D). To study time course of MTP loss, mice were gavaged with 1 mg/kg CCl4. MTP activity was measured at different indicated times in the intestine (E) and liver (F). Furthermore, MTP and GAPDH protein levels were determined by Western blot analysis in the intestine (G) and liver (H). MTP activity, MTP and GAPDH protein levels in the intestine and liver were determined using 100 µg of microsomal proteins. Each point represents the mean ± S.D. *, p < 0.05; **, p < 0.01; significantly different from oil control group.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CCl4 Decreases Plasma ApoB-lipoproteins and Increases Hepatic and Intestinal Lipid Levels—To study the acute effects of CCl4, mice were gavaged with saline, olive oil, or CCl4 in olive oil (Fig. 1). Olive oil had no effect on total plasma triglyceride (Fig. 1A) or cholesterol (Fig. 1B) levels compared with saline. In plasma, these lipids are carried on apoB-lipoproteins and HDL that mainly contain apoAI. Increasing amounts of CCl4 decreased plasma triglyceride and cholesterol (Fig. 1, A and B, total) mainly because of reductions in apoB-lipoproteins (Fig. 1A, 1B). Tissue analyses revealed that this treatment increased (~2-fold) intestinal as well as hepatic triglyceride (Fig. 1C) and cholesterol (Fig. 1D). These data indicate that CCl4 decreases plasma apoB-lipoproteins and increases tissue lipid levels.

Time course studies revealed maximum reductions in plasma triglycerides at 4 h (Fig. 1E, total). Subsequently, there was a trend toward an increase in their plasma levels. In fact, plasma lipids reverted to normal levels after 24 h (data not shown). CCl4 mainly decreased apoB-lipoprotein triglyceride (Fig. 1E, apoB-lipoproteins) without affecting HDL (Fig. 1E, HDL). Total plasma cholesterol levels also significantly decreased with time in CCl4-fed mice (Fig. 1F, total). Again, apoB-lipoprotein (Fig. 1F, apoB-lipoprotein) cholesterol levels were significantly reduced and HDL levels were unaffected (Fig. 1F, HDL). Tissue lipid analyses revealed that intestinal and hepatic triglyceride increased (~3-fold) up to 4 h and then decreased at 6 h (Fig. 1G). Similarly, hepatic and intestinal cholesterol levels increased until 4 h and started to decline later (Fig. 1H). These studies show that a single dose of CCl4 significantly decreases plasma apoB-lipoproteins and increases intestinal and hepatic triglyceride and cholesterol levels in a time-dependent fashion. Increases in liver triglyceride levels have been observed in CCl4-exposed animals (2, 4, 5); however, intestinal steatosis has not yet been appreciated.


Figure 3
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  		FIGURE 3.
Effect of CCl4 on in vitro MTP activity. A, purified MTP (1 µg) was incubated in triplicate with a mixture of donor and acceptor vesicles (Chylos, Inc.) and increasing amounts of CCl4 for 30 min, and the amounts of fluorescent triglycerides transferred were measured (28, 29). Liver (B) and intestinal (C) proteins (100 µg) were also incubated with increasing concentrations of CCl4 to study the effect of CCl4 on triglyceride transfer activity.

 


Figure 4
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  		FIGURE 4.
CCl4 reduces secretion of apoB-lipoproteins in primary enterocytes. A and B, mouse enterocytes were incubated with 200 µCi of [35S]methionine/cysteine in the presence of different indicated concentrations of CCl4 in Me2SO (52). After 4 h, media (A) and cells (B) were used to immunoprecipitate apoB and apoAI, resolved on SDS-PAGE, and exposed to phosphorimaging screens. Images from a representative experiment are shown (top panels). Bands from three independent experiments were quantified, and mean ± S.D. values were plotted as line graphs (bottom panels). C, enterocytes were radiolabeled with [35S]methionine for 4 h in the presence of various concentrations of CCl4. Cells were used for sequential precipitations of MTP and GAPDH. Bands corresponding to MTP and GAPDH from a representative experiment are shown. Quantitative data (mean ± S.D.) from three independent experiments are plotted as line graphs. D, enterocytes were exposed to different concentrations of CCl4 for 4 h and used to measure triglyceride transfer activity using 100 µg of protein. *, p < 0.05; **, p < 0.01, compared with controls.

 
CCl4 Decreases MTP in Mice—Because CCl4 had a major effect on plasma apoB-lipoproteins, we studied its effect on MTP. Mice were fed with increasing amounts of CCl4, and triglyceride transfer activity of MTP was measured after 4 h. Increasing CCl4 concentrations decreased MTP activity in the intestine (Fig. 2A) and liver (Fig. 2B). Western analyses showed significant decrease in MTP but no change in cytosolic marker GAPDH (Fig. 2, C and D). Time course studies showed that MTP activity decreased in the liver and the intestine with maximum reduction (~75%) evident at 4 h (Fig. 2, E and F). In addition, there was a time-dependent loss of protein in these tissues (Fig. 2, G and H). These studies indicate that CCl4 decreases MTP protein and activity in different tissues.


Figure 5
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  		FIGURE 5.
CCl4 decreases lipoprotein secretion and MTP activity in Caco-2 and HepG2 cells. A–D, human colon carcinoma Caco-2 cells were incubated with different concentrations of CCl4 for 4 h as described under "Experimental Procedures." For lipid secretion (A), cells also received [3H]glycerol (5 µCi/ml). Lipids were extracted from media and separated on thin layer plates, and bands corresponding to triglycerides (TG) and phospholipids (PL) were counted. The media were also used to measure apoB and apoAI by enzyme-linked immunosorbent assay (B). Cells were used to measure triglyceride transfer activity (C) and mRNA levels (D) by quantitative reverse transcriptase-PCR. E–H, human hepatoma HepG2 cells were incubated with different amounts of CCl4 for 4 h. For lipid analyses, these cells also received [3H]glycerol (E). Media were used for lipid extraction, as described in A and under "Experimental Procedures," and to measure apoB and apoAI by enzyme-linked immunosorbent assay (F). Cells were used to measure MTP activity (G) as well as mRNA quantifications (H). *, p < 0.05; **, p < 0.01, compared with controls.

 
To address whether CCl4 has a direct effect on MTP, we studied its effect on the purified and cellular MTP. CCl4 had no inhibitory effect on the lipid transfer activity of purified MTP (Fig. 3A). Furthermore, it did not alter hepatic and intestinal MTP activities (Fig. 3, B and C) excluding the possibility that CCl4 inhibits MTP.

CCl4 Decreases ApoB-lipoprotein Secretion and MTP Levels in Cultured Cells—To evaluate the mechanisms involved in decreases in plasma apoB-lipoproteins and tissue MTP in mice, we tested cultured cells for their response to CCl4. Mouse primary enterocytes were metabolically labeled with [35S]methionine for 4 h in the presence of increasing amounts of CCl4. Increasing concentrations of CCl4 decreased apoB secretion without affecting apoAI (Fig. 4A). Amounts of apoB were significantly lower in cells treated with higher concentrations of CCl4. However, this treatment had no effect on cellular apoAI levels (Fig. 4B). Next, we measured the amounts of radiolabeled MTP and GAPDH in cells treated with increasing CCl4 concentrations. This treatment significantly decreased cellular MTP without affecting GAPDH levels (Fig. 4C). We also measured MTP activity in enterocytes exposed to different CCl4 levels (Fig. 4D) and observed that MTP activity was reduced in cells exposed to higher levels of CCl4. These studies showed that CCl4 treatment decreases MTP but does not affect GAPDH levels. Moreover, it decreased apoB secretion without affecting apoAI secretion.

Studies were then performed in human colon carcinoma Caco-2 and hepatoma HepG2 cells (Fig. 5). CCl4 decreased secretion of lipids (Fig. 5A) and apoB (Fig. 5B) but had no effect on apoAI secretion in Caco-2 cells. Furthermore, it decreased cellular MTP activity (Fig. 5C). Analyses of mRNA levels revealed that CCl4 had no effect on apoB, apoAI, or MTP mRNA levels (Fig. 5D). Exposure of HepG2 to increasing amounts of CCl4 also resulted in decreased secretion of lipids (Fig. 5E) and apoB (Fig. 5F) consistent with other toxicity studies in these cells (36). In contrast, this treatment had no effect on apoAI secretion. Again, CCl4 decreased MTP activity (Fig. 5G) but had no effect on mRNA levels of apoB, apoAI, or MTP (Fig. 5H). These studies indicate that CCl4 specifically decreases apoB secretion by reducing MTP activity. Decrease in MTP was not due to changes in its mRNA levels, excluding transcriptional repression mechanisms.

Incubation of rat hepatoma McA-RH7777 cells with increasing concentrations of CCl4 significantly decreased triglyceride and phospholipid secretion (Fig. 6, A and B), increased cellular triglycerides, but not phospholipids and proteins (Fig. 6B), and reduced apoB secretion without affecting apoAI secretion (Fig. 6C) consistent with the inhibition of lipid secretion by CCl4 in primary rat hepatocytes (7, 37, 38). In these cells, MTP protein decreased, but protein disulfide isomerase (ER enzyme) and GAPDH remained unaltered (Fig. 6D). In contrast to its effects on protein levels, CCl4 had no effect on MTP mRNA levels eliminating the model in which changes in mRNA lead to differences in protein activities (Fig. 6E). In short, CCl4 reduces MTP activity in both intestinal and liver cells, decreases lipoprotein secretion, and increases intracellular triglyceride as it does in mice.

CCl4 Induces Post-translational Degradation of MTP Involving Ubiquitin and Proteasomes—The data presented thus far have indicated that CCl4 decreases MTP protein. Loss of protein with no change in mRNA levels could be because of reduced synthesis or enhanced post-translational degradation. To study the effect of CCl4 on MTP synthesis, McA-RH7777 cells were treated with CCl4 for 4 h and then radiolabeled for 5–15 min. MTP synthesis was similar in control and CCl4-treated cells (Fig. 7A). Moreover, the syntheses of apoB, apoAI, and GAPDH were not affected indicating that CCl4 does not affect protein synthesis. Next, we studied the fate of newly synthesized MTP. Cells were first labeled with [35S]methionine and then chased in the presence and absence of CCl4. In CCl4-treated cells, newly synthesized MTP disappeared faster than in control cells after 1 h of chase (Fig. 7B). Similarly, the disappearance of apoB was greater in CCl4-exposed cells compared with controls. In contrast, apoAI and GAPDH levels were not altered. Protein analyses in the media revealed that CCl4 inhibited apoB, but not apoAI, secretion. These studies indicate that CCl4 induces post-translational degradation of MTP. The decrease in apoB is consistent with the knowledge that inhibition of MTP reduces apoB secretion (39).


Figure 6
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  		FIGURE 6.
CCl4 decreases lipoprotein secretion and MTP activity in rat hepatoma cells. McA-RH7777 cells were treated with increasing concentrations of CCl4 dissolved in Me2SO (52) for 4 h and simultaneously labeled with either [3H]glycerol (5 µCi/ml) (A and B) or [35S]methionine (200 µCi/ml) (C and D). Lipids were extracted from media (A and B) and cells (B) and separated on thin layer plates; triglycerides (TG) and phospholipids (PL) were quantified (A) (22, 26). In B, counts present in control cells (0 mM) were normalized to 100%. C, cells were treated with different amounts of CCl4 for 4 h and metabolically labeled with [35S]methionine. Media were immunoprecipitated sequentially with anti-apoB and anti-apoAI antibodies and exposed to phosphorimaging screens, and bands were visualized. Data from a representative experiment are shown. Bands from three different experiments were quantified; data are presented as mean ± S.D. D, MTP, protein disulfide isomerase (PDI), and GAPDH were immunoprecipitated sequentially from cells, exposed, and quantified. E, cells were used for either MTP activity measurements or total RNA isolation. The MTP activity and MTP/18 S rRNA ratio in control cells were normalized to 100%. *, p < 0.05; ** p < 0.01, significantly different from control group.

 
Attempts were made to understand mechanisms involved in the post-translational degradation of MTP. The elimination of misfolded proteins and regulated enzymes involves retrograde translocation across the ER membrane and proteolysis by ubiquitin-proteasomes (40, 41). To determine whether proteasomes are involved in MTP degradation, McA-RH7777 cells were treated with inhibitors (lactacystin, MG132, ALLN) along with CCl4. Proteasomal inhibitors abolished CCl4-induced MTP degradation (Fig. 7C). Next, we studied the effect of proteasomal inhibitors on the post-translational degradation of MTP. Cells were radiolabeled with [35S]methionine and then chased in the media containing CCl4 with or without different inhibitors (Fig. 7D). Again, all of the inhibitors prevented post-translational degradation of MTP to different extents. We then asked whether the protected MTP retains its ability to transfer triglycerides. Cells were incubated with CCl4 in the presence and absence of lactacystin, and the amounts of MTP protein as well as its activity were measured (Fig. 7E). Incubation of cells with lactacystin restored MTP protein and activity (Fig. 7E). We then determined whether preincubation of cells with lactacystin would protect MTP from degradation. Preincubation of cells with lactacystin provided no protection against the loss of MTP protein and activity (Fig. 7F) underscoring the importance for its continuous presence to inhibit degradation. These data indicate that CCl4 induces MTP degradation involving proteasomes.


Figure 7
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  		FIGURE 7.
CCl4 induces post-translational degradation of MTP. A, McA-RH7777 cells were incubated with CCl4 for 4 h and then labeled in the presence and absence of 1 mM CCl4 with [35S]methionine for 5, 10, or 15 min. B, cells were labeled with [35S]methionine for 1 h and chased for 15, 60, or 120 min in the presence or absence of CCl4. Cells and media were used for immunoprecipitation. C, cells were treated with CCl4 with or without proteasomal inhibitors (53, 54) lactacystin (LAC,50 µM), MG132 (25 µM), or ALLN (100 µM) for 4 h and used for immunoprecipitation. DMSO, dimethyl sulfoxide. D, cells were labeled with [35S]methionine for 1 h and then chased in the presence of CCl4 along with inhibitors for 2 h. E, cells were treated or not with 1 mM CCl4 in the presence and absence of lactacystin for 4 h, and microsomal proteins were used for Western blotting and to measure MTP activity. F, cells were first pretreated with or without lactacystin for 30 min and then exposed to CCl4 in the presence and absence of lactacystin for 4 h. G, cells were incubated with or without CCl4 for 4 h, immunoprecipitated with anti-MTP antibodies (IP: MTP), and blotted with anti-ubiquitin antibodies (IB: anti-Ub). H, cells were treated as described in G in the presence and absence of lactacystin and used for immunoprecipitation. I, cells were treated with CCl4 in the presence and absence of inhibitors for 4 h, homogenized, centrifuged to separate microsomes and cytosol, and used to measure MTP activity as well as for Western analyses. J, cells were incubated with 5 µCi/ml 14CCl4 (1 mCi/mmol) for 4 h in the presence and absence of 10 µM ketoconazole (KTZ) or 50 µM lactacystin. Cells were used for sequential immunoprecipitation of MTP and GAPDH.

 
A key step in proteasomal degradation is the ubiquitinylation of proteins (40). CCl4 treatment enhanced ubiquitinylation of MTP (Fig. 7G), and the recovery of ubiquitinylated MTP increased in the presence of lactacystin (Fig. 7H) indicating that MTP degradation involves ubiquitinylation. To identify the site of degradation, MTP activity was measured in microsomes and cytosol. MTP was mainly present in microsomes (Fig. 7I). CCl4 decreased MTP in microsomes with no increase in the cytosol indicating no significant translocation to cytosol for disposal. Thus, CCl4-induced loss of MTP protein from microsomes involves ubiquitinylation and ER-associated proteasomal degradation.

Covalent Modification of MTP by CCl4—To understand how MTP is targeted for degradation, we hypothesized that CCl4 might covalently modify MTP. Incubation of cells with 14CCl4 resulted in its incorporation into MTP, and the amounts of modified MTP increased when cells were treated with lactacystin (Fig. 7J). Immunoprecipitation followed by exposure to phosphorimaging screens for 1 month did not reveal any incorporation of label in apoB, apoAI (data not shown), or GAPDH (Fig. 7J). Ketoconazole, an inhibitor of mixed function oxygenases (42), inhibited MTP modification (Fig. 7J) consistent with the understanding that bioactivation of CCl4 by these enzymes is required to inhibit lipid secretion by hepatocytes (7, 37, 38). We interpret these studies to suggest that CCl3· generated by cytochrome P450 oxygenases covalently attaches to MTP and that this modification targets MTP for proteasomal degradation.

MTP Inhibitor Does Not Induce Its Degradation—To determine whether MTP degradation is involved in other forms of steatosis, we compared the effect of CCl4 with an MTP antagonist, BMS197636 (43). Both CCl4 and BMS197636 decreased lipid secretion (Fig. 8A) and increased cellular triglyceride but not phospholipid levels (Fig. 8B). Cells treated with the inhibitor and CCl4 had significantly reduced MTP activity (Fig. 8C). CCl4 significantly reduced MTP protein, but the inhibitor did not (Fig. 8D). Thus, CCl4 and MTP antagonist cause steatosis by different mechanisms. MTP antagonists inhibit lipid transfer activity, whereas CCl4 induces protein degradation.


Figure 8
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  		FIGURE 8.
MTP antagonist does not induce its degradation. McA-RH7777 cells were incubated with Me2SO (DMSO, dimethyl sulfoxide) alone or Me2SO containing either 1 mM CCl4 or 1 µM BMS197636 for 4 h. Cells were labeled with [3H]glycerol, and lipids from media (A) and cells (B) were extracted and measured after separation. In another experiment, cells were treated with BMS197636 or CCl4 and used to measure MTP activity (C) and to visualize different proteins (D). *, p < 0.05; **, p < 0.01, compared with controls. TG, triglycerides; PL, phospholipids; PDI, protein disulfide isomerase.

 
Lactacystin Partially Prevents CCl4-induced Steatosis in Mice—To determine whether inhibition of proteolysis could prevent CCl4 toxicity, mice were first fed with saline or lactacystin. After 30 min, lactacystin-fed animals were gavaged with CCl4 with or without lactacystin (Fig. 9). MTP protein was decreased in the intestine and liver of CCl4-fed mice, and lactacystin partially protected the loss of MTP protein and activity in these tissues (Fig. 9, A and B). The ubiquitinylated MTP levels increased in the tissues of lactacystin-fed mice (Fig. 9, C–D). Furthermore, lactacystin partially restored MTP activity in these tissues (Fig. 9, E and F). Accumulation of triglyceride and cholesterol in the intestine (Fig. 9G) and liver (Fig. 9H) was prevented when mice were fed with CCl4 and lactacystin. Moreover, the decreases in plasma triglyceride (Fig. 9I) and cholesterol (Fig. 9J) associated with CCl4 toxicity were avoided by this treatment, mainly because of partial restoration of apoB100 and apoB48 lipoproteins (Fig. 9K). These studies indicate that proteasomal inhibitors may be useful in treating acute CCl4 toxicity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is known that the exposure to CCl4 induces cellular accumulation of triglycerides by unknown mechanisms. Here, we show that MTP degradation after short-term exposure to CCl4 is the major mechanism leading to the cellular accumulation of lipids. Furthermore, these studies demonstrate for the first time that CCl4 exposure results in covalent modification of MTP followed by its rapid and specific degradation involving ubiquitinylation and proteasomes (Fig 10). We also show that the prevention of MTP degradation by proteasomal inhibitors affords protection against CCl4 toxicity. Thus, covalent modification of MTP leading to its destruction is a key early and essential step in the onset of CCl4-induced steatosis.

Nothing is known about the mechanisms involved in the disposal of MTP under normal and pathologic conditions. MTP is believed to have a long half-life (44), and its levels are regulated mainly at the transcriptional level (45, 46). We recently showed that intestinal and hepatic MTP levels exhibit diurnal variations due to changes in transcription during 24 h.3 Here we show that the major mechanism involved in the loss of MTP by CCl4 is the induction of its degradation by ubiquitin-proteasome pathway. In contrast, MTP antagonist does not induce MTP degradation. Thus, only under certain conditions is MTP removed by proteasomes. It is likely that this mechanism may play a role in the onset of some forms of steatosis.

At this time we do not know the subcellular site of MTP degradation. Our data clearly show that MTP degradation is a post-translational process. Thus, it is not linked to the site of MTP mRNA translation. It is possible that MTP is modified and degraded at a specialized subcompartment that is involved in apoB-lipoprotein assembly or in a compartment that is enriched in P450 oxygenases.

Loss of MTP activity has been shown to result in the accumulation of lipids in the liver of hepatitis C virus-infected individuals (18). Furthermore, a significant correlation was observed between the decreases in MTP mRNA levels and the degree of steatosis in liver biopsies. In addition, several MTP antagonists have been shown to cause steatosis (20, 21). In contrast, short-term exposure to CCl4 causes loss of protein and accumulation of lipids in the cells. Thus, loss of MTP involving several mechanisms may lead to significant accumulation of lipids in the cells.

Our data show that CCl4-induced steatosis could be avoided by inhibiting proteasomal degradation of MTP. Inhibition of proteasomal degradation increases accumulation of the modified MTP (Figs. 7, 8, 9). This indicates that CCl4 modification does not inhibit its lipid transfer activity and that nondegraded, modified protein is able to transfer triglyceride and assist in its secretion avoiding steatosis. Thus, the mode of action of CCl4 is different from that of MTP antagonist. CCl4 induces protein degradation without affecting its lipid transfer activity, whereas MTP antagonists inhibit lipid transfer activity without causing its destruction.


Figure 9
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  		FIGURE 9.
Inhibition of MTP degradation prevents CCl4-induced steatosis. Mice (n = 3) were first gavaged with lactacystin (LAC, 10 mg/kg) or saline. After 30 min, saline-gavaged mice received either saline or CCl4 (1 mg/kg), and inhibitor-gavaged mice received CCl4 + lactacystin. Tissues and plasma were collected after 4 h. MTP and GAPDH proteins were analyzed by Western blotting of intestinal (A) and liver (B) proteins. Proteins were also immunoprecipitated (IP) and immunoblotted (IB) to detect the presence of ubiquitinylated MTP in the intestine (C) and liver (D). Intestinal (E) and hepatic (F) microsomal contents were used to measure MTP activity. Intestinal (G) and liver (H) tissues were also used to measure lipid levels. Plasma was used to measure total and apoB-lipoprotein triglyceride (I) and cholesterol (J). ApoB100, apoB48, and apoAI in plasma were visualized by Western analysis (K). *, p < 0.05; **, p < 0.01, compared with saline group.

 


Figure 10
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  		FIGURE 10.
A schematic diagram explaining the onset of steatosis involving MTP degradation by CCl4. CCl4· is converted to free radicals (CCl3 and Cl·) by cytochrome P450 oxygenases. MTP is covalently modified by CCl3·, ubiquitinylated, and degraded by proteasomes. This leads to increased accumulation of triglycerides and cholesterol in the tissues. If proteasomal degradation of MTP is inhibited by lactacystin, the CCl4 toxicity is averted in part because the protected MTP is able to assist in lipoprotein assembly and in the secretion of triglycerides.

 
Incubation of cells with 14CCl4 resulted in the covalent labeling of MTP (Fig. 7J). To explain this, we proposed that CCl4 is converted to CCl3· and these free radicals react with MTP (Fig 10). It has been demonstrated that CCl4 undergoes cleavage resulting in the generation of CCl3· and covalent modification of proteins and lipids (2, 4–6, 48). Significant evidence indicates that cytochrome P450 oxygenases play a key role in this process. For example, induction of these enzymes leads to increased hepatotoxicity, and their inhibition protects against CCl4 toxicity. Organisms with low activity, such as newborns of many species and chicken, are less sensitive to CCl4. Moreover, CYP2E1 knock-out mice are relatively more resistant to CCl4 hepatotoxicity compared with controls (49). We observed that inhibition of these oxygenases avoids MTP modification. Thus, it is likely that the generation of CCl3· by cytochrome P450 is a key step in MTP modification. Thus far, no specific protein modified by CCl3· has been identified, and it is assumed that it is probably a nonspecific process. Our identification that CCl4 covalently modifies MTP may lead the way to the identification of other proteins that are modified by CCl3·.

CYP2E1 has been recognized as the major enzyme involved in the bioactivation of CCl4 by the liver. However, the intestine is known to express several P450 oxygenases (50). The current understanding is that CYP2B1 or CYP2B2, and possibly CYP3A, can also metabolize CCl4 and form the trichloromethyl radical, CCl3· (5). More experiments are required to identify the enzyme involved in CCl4 bioactivation in the intestinal cells.

We do not know why MTP is so susceptible to CCl4 attack. It is possible that CCl4 acts like a lipid, a hydrophobic molecule. This is supported by the observations that the major molecules damaged by CCl4 are the membrane lipids. Another possible explanation is that the generation of CCl3· occurs near the membrane and thus membrane lipids are more prone to modification. Because MTP is a lipid transfer protein, it might attempt to transfer CCl3· from the membrane and during this process become modified and targeted for degradation. The preferential and rapid degradation of MTP after CCl4 exposure suggests that MTP may transfer as yet unidentified reactive species and needs further evaluation. If MTP is involved in the transfer of such reactive molecules, their identification might shed new light on the role of MTP in the assembly and secretion of apoB-lipoproteins and in the maintenance of membrane integrity.

Surveillance of the structural and functional integrity of proteins is of utmost importance to cells. Thus, we think that the modification of MTP after CCl4 exposure is sensed as deleterious and that cells target it for degradation. To our knowledge this is a novel mechanism explaining the toxicity involving environmental toxins. It remains to be determined whether proteasomal degradation of MTP might occur under other conditions such as exposure to other environmental toxins, oxidizing agents, or free radicals. It is possible that the degradation of proteins damaged by environmental toxins may be a reason for cytotoxicity. It is also likely that MTP degradation might be involved in the development of certain forms of steatosis such as those observed in hepatitis C virus infection (18) and alcoholic and nonalcoholic fatty liver, and it remains to be determined whether inhibiting protein degradation might provide protection against these forms.

Knowledge about the molecular processes involved in CCl4-induced hepatotoxicity has led to the use of antioxidants and mitogens to alleviate symptoms. Mitogens restore cellular methylation, and antioxidants preserve calcium homeostasis. In addition, antagonists of CYP450 have been shown to alleviate toxicity. On the other hand, compounds that induce cytochrome or delay tissue repair enhance CCl4 toxicity (4, 5). Here, we show that proteasomal inhibitors can also provide protection against CCl4-induced cytotoxicity. Proteasomal inhibitors are in clinical trial for the treatment of cancer (51). Perhaps they could also be evaluated for their efficacy in treating some forms of steatosis.

In summary, we have described the novel pathway that CCl4 employs to induce steatosis, showing that CCl4 exposure leads to covalent modification of MTP and its degradation by proteasomes. Inhibition of MTP degradation leads to prevention of cellular lipid accumulation. It remains to be determined whether the protection of MTP degradation would also avoid cellular necrosis and other pathologic indications associated with CCl4 toxicity. Moreover, these studies suggest that the loss MTP may be involved in the development of other forms of steatosis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL-64272 (to M. M. H.) and a postdoctoral fellowship from the American Heart Association, Heritage Affiliate (to X. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203. E-mail: mhussain{at}downstate.edu.

2 The abbreviations used are: ER, endoplasmic reticulum; ALLN, N-acetyl-Leu-Leu-norleucinal; ApoB, apolipoprotein B; Me2SO, dimethyl sulfoxide; GAPDH, glyceraldehyde phosphate dehydrogenase; MTP, microsomal triglyceride transfer protein; HDL, high density lipoprotein. Back

3 X. Pan and M. M. Hussain, submitted. Back


   ACKNOWLEDGMENTS
 
We acknowledge helpful discussions and technical guidance from Kamran Anwar and Kezhi Dai.



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