Hepatic Mttp deletion reverses gallstone susceptibility in L-Fabp knockout mice.

Previous studies demonstrated that L-Fabp KO mice are more susceptible to lithogenic diet (LD)-induced gallstones because of altered hepatic cholesterol metabolism and increased canalicular cholesterol secretion. Other studies demonstrated that liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) reduced LD-induced gallstone formation by increasing biliary phospholipid secretion. Here we show that mice with combined deletion (i.e., DKO mice) are protected from LD-induced gallstone formation. Following 2 weeks of LD feeding, 73% of WT and 100% of L-Fabp KO mice developed gallstones versus 18% of Mttp-LKO and 23% of DKO mice. This phenotype was recapitulated in both WT and L-Fabp KO mice treated with an Mttp antisense oligonucleotide (M-ASO). Biliary cholesterol secretion was increased in LD-fed L-Fabp KO mice and decreased in DKO mice. However, phospholipid secretion was unchanged in LD-fed Mttp-LKO and DKO mice as well as in M-ASO-treated mice. Expression of the canalicular export pump ABCG5/G8 was reduced in LD-fed DKO mice and in M-ASO-treated L-Fabp KO mice. We conclude that liver-specific Mttp deletion not only eliminates apical lipoprotein secretion from hepatocytes but also attenuates canalicular cholesterol secretion, which in turn decreases LD-induced gallstone susceptibility.

Here we show that DKO mice are indeed protected against LD-induced cholesterol gallstone formation, not, however, because of augmented biliary phospholipid secretion, but rather through alterations in canalicular cholesterol secretion.

Animals and diets
All animals were maintained in a C57BL/6 background and housed on a 12 h light-dark cycle in a full-barrier facility. Male mice (10- mice were injected with pI-pC as above and used as controls. Inhibition of hepatic Mttp expression was also accomplished by antisense oligonucleotide (ASO) injection: 8-week-old male C57BL/6 WT (Jackson Lab) and L-Fabp Ϫ / Ϫ mice were injected intraperitoneally twice a week with an ASO targeted against Mttp ASO (M-ASO) or a mice) was increased availability of phosphatidylcholine as a result of the block in VLDL secretion ( 8 ).
Recent studies have demonstrated that deletion of L-Fabp , the dominant lipid binding protein expressed in mammalian liver ( 12 ), renders mice highly susceptible to LD-induced gallstone formation, at least in part through augmented canalicular cholesterol secretion ( 13 ). Those observations, considered together with earlier fi ndings suggesting that hepatic L-Fabp and Mttp transcription are coordinately regulated ( 14 ), led us to ask whether increasing biliary phospholipid secretion as a result of hepatic Mttp deletion might offset increased canalicular cholesterol secretion in mice with combined deletion ( Mttp-LKO ,  Mice from four experimental groups were fed a LD for 2 weeks. Sera were collected after fasting overnight and cholesterol, triglyceride, and ALT were analyzed (see Materials and Methods). The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically signifi cant ( P < 0.05). TG, triglyceride; TC, cholesterol. 2 = many ChMCs ( у 3/high power fi eld); 3 = aggregated ChMCs; 4 = presence of "sandy" light-translucent stones or "solid" lightopaque stones ( 6,7 ). Gallbladder bile from mice fed LD for 4 weeks was used for cholesterol saturation index (CSI) calculation. Gallbladder bile cholesterol content was measured enzymatically after chloroform:methanol (2:1) extraction. Gallbladder bile phospholipids and bile acid content were analyzed enzymatically using a phospholipid C kit and total bile acids assay kit (BQ 092A-EALD; BQ Kits, San Diego, CA). Cholesterol saturation indices in gallbladder bile were calculated using published parameters ( 16 ).

Biliary lipid secretion
Mice were fed a LD for 2 weeks and anesthetized after an overnight fast. An external bile fi stula was established surgically and bile samples collected during the fi rst 15 min were used for gallstone analysis. Bile samples were collected for another 60 min and used for biliary lipid secretion analysis. Hepatic bile volume was determined gravimetrically assuming a density of 1 g·ml Biliary phospholipids, cholesterol, and total bile acid content were determined enzymatically. Biliary lipid secretion values are presented as nmol/min/g liver weight.

Gene expression analysis
Hepatic total RNA was extracted and cDNA prepared using an ABI high capacity cDNA reverse transcription kit with 1 g of total RNA. Real-time quantitative PCR (qPCR) used cDNAs from four animals per group and was performed in triplicate on an ABI Step-One-Plus sequence detection system using SYBR Green PCR Master Mix (Applied Biosystems) and primer pairs (provided on request) designed by Primer Express software (Applied Biosystems). Relative mRNA abundance is expressed as fold change compared with Mttp fl ox/fl ox mice, normalized to GAPDH. Hepatic membrane protein was prepared as described previously ( 17 ). In brief, a total of 100-200 mg liver was homogenized by polytron in 1.2 ml buffer A [250 mM sucrose, 2 mM MgCl 2 , and 20 mM Tris-HCL (pH 7.5)] containing protease inhibitors (complete protease inhibitor mixture; Roche Diagnostics). The crude preparation was centrifuged at 2,000 g for 10 min at 4°C. The nonspecifi c control (C-ASO) for 4-6 weeks as detailed in the fi gure and table legends (ISIS). LD feeding was started after 2 weeks of ASO injection and continued for a further 2-4 weeks as indicated in the fi gure and table legends. All animal protocols followed National Institutes of Health guidelines and were approved by the Washington University Animal Studies Committee (IACUC # A3381-01).

Serum, hepatic lipid, and alanine aminotransferase determinations
Animals were euthanized after an overnight fast. Liver and serum were collected and frozen at Ϫ 80°C until analyzed. Serum triglyceride and cholesterol were measured using reagents from Wako (Neuss, Germany), L-type triglyceride M kits (catalog numbers 465-09791 and 461-09891), and a cholesterol E kit (catalog number 439-17501). Hepatic lipid was extracted using chloroform:methanol (2:1) and triglyceride, cholesterol, free cholesterol, FFAs, or phospholipids were measured enzymatically with the indicated Wako reagent kits: L-type triglyceride M kit, cholesterol E kit, free cholesterol E kit (catalog number 435-35801), HR series NRFA-HR2 kits (catalog numbers 991-34891 and 995-34791), and phospholipid C kit (catalog number 433-36201 ). Serum alanine aminotransferase (ALT) was analyzed by Teco ALT set (catalog number A526-120; Teco Diagnostics, Anaheim, CA). Serum from four to fi ve mice per group was pooled and lipoproteins fractionated by fast-performance liquid chromatography using tandem Superose 6 columns. Cholesterol content in each lipoprotein fraction was analyzed using Wako enzymatic kits.

Gallstone analysis and gallbladder bile cholesterol saturation index
Animals were fed a LD for 2 or 4 weeks (as indicated in the fi gure legends) and euthanized after an overnight fast. Gallbladder bile was collected either from intact gallbladders or from the fi rst 15 min of bile collection following cannulation for biliary secretion studies. Fresh gallbladder bile was immediately analyzed by polarizing microscopy using a visual scale with the following criteria: 0 = absence of cholesterol monohydrate crystals (ChMCs), 1 = small number of ChMCs (<3/high power fi eld); Mice from four experimental groups were fed a chow diet. Hepatic lipids were extracted and analyzed (see Materials and Methods). Hepatic lipid content is expressed as mean ± SE ( g/mg protein), except FFA (nmol/mg protein). The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically signifi cant ( P < 0.05). TG, triglyceride; TC, cholesterol; FC, free cholesterol; CE, cholesterol ester; PL, phospholipids . Mice from four experimental groups were fed a LD (C, D) for 2 weeks. Hepatic lipids were extracted and analyzed (see Materials and Methods). Hepatic lipid content is expressed as mean ± SE ( g/mg protein), except FFA (nmol/mg protein). The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically signifi cant ( P < 0.05). TG, triglyceride; TC, cholesterol; FC, free cholesterol; CE, cholesterol ester; PL, phospholipids.

Statistical analysis
Statistical signifi cance was determined with one-way ANOVA for multiple group comparisons and t -testing as post hoc test for comparing different pairs using GraphPad Prism 4 software (GraphPad, San Diego, CA). Data are expressed as the mean ± SE unless otherwise noted. ( Fig. 1A ) revealed the expected reductions in serum lipid (cholesterol and triglyceride) levels and lipoprotein distribution in both L-Fabp -suffi cient and L-Fabp Ϫ / Ϫ backgrounds ( Table 1 , Fig. 1B ). In addition, liverspecifi c Mttp deletion completely prevented the increase in VLDL and LDL cholesterol following 2 weeks of LD feeding ( Fig. 1C , Table 2 ). There were no differences in ALT levels among the genotypes in chow-fed animals and while ALT levels increased in all groups fed the LD for 2 weeks, hepatic Mttp deletion was associated with lower ALT levels in both L-Fabp -suffi cient and L-Fabp Ϫ / Ϫ mice ( Tables 1, 2 ). We will return to the implications of the ALT fi ndings in a later section.

Alterations in serum and hepatic lipid profi le following deletion of hepatic Mttp in chow-fed and LD-fed
Hepatic lipid determinations revealed the expected increase ( 15 ) in triglyceride content following deletion of hepatic Mttp in chow-fed mice ( Table 3 ). Hepatic cholesterol content (both free and esterifi ed) increased with 2 weeks of LD feeding in all genotypes, but the magnitude of this increase was signifi cantly attenuated with hepatic Mttp deletion in both L-Fabp -suffi cient and L-Fabp Ϫ / Ϫ mice ( Table 4 ). In particular, there was a 2.6-fold increase in hepatic free cholesterol content in LD-fed L-Fabp Ϫ / Ϫ mice, compared with 1.5-to 1.8-fold increases in the other three genotypes ( Tables 3, 4 ). These fi ndings suggest that hepatic Mttp deletion modifi es the changes in hepatic cholesterol metabolism observed in LD-fed L-Fabp Ϫ / Ϫ mice.

Hepatic Mttp deletion reverses gallstone susceptibility in
Based on the fi ndings above and earlier observations suggesting that Mttp-LKO mice are protected against LD-induced gallstones ( 8 ), we asked whether hepatic Mttp deletion in L-Fabp Ϫ / Ϫ mice would attenuate gallstone formation after 2 or 4 weeks of LD feeding. The gross visual appearance of the gallbladder in the different genotypes ( Fig. 2A ) confi rmed this expectation even after 2 weeks of LD feeding, which was verifi ed by the absence of polarizing cholesterol crystals ( Fig.  2B ) and the reduced incidence of gallstones and lower gallstone score ( Fig. 2C ). We extended those fi ndings to 4 weeks of LD feeding [to replicate the timing used by Amigo et al. ( 8 )], which revealed virtually identical fi ndings (zero gallstone incidence with hepatic Mttp deletion in either L-Fabpsuffi cient or L-Fabp Ϫ / Ϫ background, data not shown) and also reduced the biliary CSI ( Fig. 2D ).

Hepatic Mttp deletion decreases biliary cholesterol secretion
In order to examine the mechanisms underlying the attenuated gallstone susceptibility in L-Fabp Ϫ / Ϫ mice following supernatant was collected and recentrifuged at 120,000 g for 45 min at 4°C. The membrane pellet was resuspended in buffer B [80 mM NaCl, 2 mM CaCl 2 , 1% Triton X-100, 50 mM Tris-HCL (pH 8), and protease inhibitors]. Membrane proteins (50 g) were separated on 8% SDS-PAGE gel, transferred to Immobilon®-P membrane (Milllipore, Billerica, MA), immunoblotted with 1:1,000 monoclonal mouse anti-mouse ABCG8 antibody (NBP1-71706; Novus Biological, Littleton, CO), and quantitated by Kodak Image software. found that LXR ␣ and Cyp4a14 mRNA abundance [the latter a downstream target of PPAR ␣ ( 21, 22 )] were reduced in DKO mice ( Fig. 3B ). We found no increase in mRNA abundance for markers of ER stress in the setting of Mttp deletion ( Fig. 3C ), fi ndings in keeping with previous observations in mice ( 23 ) and generally consistent with the fi ndings noted above which revealed ALT levels were lower in Mttp-LKO mice. In view of other work showing that the expression of Abcg5/g8 is regulated at the posttranscriptional level ( 24,25 ), we examined the expression of Abcg8 protein in the various genotypes as a surrogate marker of the heterodimeric canalicular transporter. Those fi ndings demonstrated reduced Abcg8 protein abundance in membranes prepared from LD-fed Mttp-LKO and DKO mice compared with Mttp fl ox/fl ox mice ( Fig. 3D, E ).

Short-term ASO-mediated hepatic Mttp knockdown attenuates LD-induced gallstone formation by reducing canalicular cholesterol secretion
We next sought to examine the changes associated with liver-specifi c Mttp knockdown in LD-fed mice with a complementary approach using ASO-mediated knockdown, as recently described ( 26 ). This approach confi rmed that Mttp knockdown (M-ASO) led to a visual reduction in gallstone incidence, even after 4 weeks of LD feeding ( Fig. 4A, B ), eliminated bile cholesterol crystals ( Fig. 4C ), and tended to reduce the biliary CSI ( Fig. 4D ).
Treatment with the M-ASO produced the expected decrease in serum cholesterol in both LD-fed WT and L-Fabp Ϫ / Ϫ mice ( Table 7 ). Hepatic lipid content determination revealed an increase in triglycerides and cholesterol esters in the M-ASO-treated animals, but no increase in free cholesterol ( Table 8 ). Turning to biliary lipid secretion measurements, we found a trend toward decreased cholesterol secretion in M-ASO-treated WT mice which became signifi cant in M-ASOtreated L-Fabp Ϫ / Ϫ mice ( Table 9 ). Biliary bile acid secretion hepatic Mttp deletion, we measured biliary lipid secretion in the different genotypes. The fi ndings reveal comparable secretion rates of cholesterol, phospholipids, and bile acids in chow-fed mice and no difference in bile fl ow ( Table 5 ), suggesting that canalicular lipid output is unchanged at baseline among the genotypes. Biliary lipid secretion increased in all the genotypes following LD feeding, as expected, with no changes in bile fl ow by genotype ( Table 6 ). In addition, the fi ndings revealed increased biliary cholesterol secretion in LD-fed L-Fabp Ϫ / Ϫ mice and further demonstrated that hepatic Mttp deletion led to decreased biliary cholesterol secretion in mice with combined L-Fabp deletion ( Table 6 ). There was a trend to reduced biliary cholesterol secretion following hepatic Mttp deletion alone, but this did not reach statistical signifi cance ( Table 6 ). However, we could not confi rm the earlier observation that hepatic Mttp deletion leads to increased biliary phospholipid secretion ( 8 ). Our fi ndings, by contrast, reveal that biliary phospholipid secretion, if anything, tended to be reduced following hepatic Mttp deletion ( Table 6 ). These results suggest that the protection against cholesterol gallstone formation mediated by hepatic Mttp deletion likely involves alterations in canalicular cholesterol secretion rather than phospholipid output.
We explored the possible mechanisms for altered canalicular cholesterol secretion following LD feeding using RNA profi ling of candidate transporter genes as a fi rst step in identifying the pathways involved. The fi ndings show decreased mRNA abundance of the intracellular cholesterol transporter Abcg1 ( 18 ) in Mttp-LKO and DKO mice and increased abundance of bile acid transporters Abcb11 ( 19 ) and Oatp4 ( 20 ) in DKO mice, but no changes in mRNA expression of the canalicular cholesterol effl ux transporter Abcg5/g8 or of Srb1 ( Fig. 3A ). We also observed no changes in mRNA abundance of transcriptional regulators of hepatic cholesterol fl ux, including FoxO1 and PPAR ␣ , and we Mice from four experimental groups were fed a chow diet. Bile samples were collected for 1 h. Biliary cholesterol, phospholipids, and bile acids were analyzed (see Materials and Methods). Flow rate is expressed as l/ min/g liver weight. Biliary lipid secretion is expressed as nmol/min/g liver weight. The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically signifi cant ( P < 0.05). Mice from four experimental groups were fed a LD for 2 weeks. Bile samples were collected for 1 h. Biliary cholesterol, phospholipids, and bile acids were analyzed (see Materials and Methods). Flow rate is expressed as l/ min/g liver weight. Biliary lipid secretion is expressed as nmol/min/g liver weight. The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically signifi cant ( P < 0.05).

Hepatic Mttp deletion abrogates gallstones in L-Fabp
؊ / ؊ mice 545 to limit free cholesterol availability and attenuate biliary cholesterol secretion in LD-fed mice ( 8 ). In addition, and critical to the mechanisms postulated, those authors found no decrease in biliary cholesterol secretion rates in LD-fed Mttp-LKO mice but rather demonstrated increased phospholipid secretion ( 8 ). Our fi ndings differ from those data ( 8 ) in several key respects. First, we observed that hepatic cholesterol ester content in LD-fed Mttp-LKO and DKO mice was signifi cantly reduced rather than increased as reported by those workers. Second, we observed that biliary was higher in L-Fabp Ϫ / Ϫ mice, but there was no effect of ASO treatment in either the WT or L-Fabp Ϫ / Ϫ background ( Table 9 ). In addition, there was no signifi cant effect of M-ASO treatment on biliary phospholipid secretion rates ( Table 9 ). These fi ndings reinforce the earlier conclusions that the protective effect of Mttp knockdown is largely mediated through alterations in canalicular cholesterol secretion. We next examined canalicular transporter expression, which revealed a signifi cant decrease in mRNA abundance for both Abcg5 and Abcg8 in M-ASO-treated mice but no change in Abcb4 or Abcb11 ( Fig. 5A ). There was also a small but signifi cant decrease in Srb1 mRNA expression in M-ASO-treated L-Fabp Ϫ / Ϫ mice and a trend to reduced mRNA abundance of Cyp4a14 ( Fig. 5A ). The decrease in Abcg5/g8 mRNA expression in M-ASO-treated mice was accompanied by decreased protein expression for Abcg8, particularly in L-Fabp Ϫ / Ϫ mice ( Fig. 5B, C ). These fi ndings support the conclusion that Mttp knockdown alters canalicular cholesterol secretion in conjunction with decreased expression of the cholesterol transporter Abcg5/g8.

DISCUSSION
The major observations from this study suggest that reducing the expression of hepatic Mttp, either by genetic deletion or by ASO-mediated knockdown, attenuates gallstone formation in both WT and L-Fabp Ϫ / Ϫ mice, through pathways that result in decreased biliary cholesterol secretion. The fi ndings suggest that the critical functions of Mttp in the export of hepatocyte lipid extend beyond a role in VLDL-dependent pathways of apical secretion and include the regulation of canalicular cholesterol secretion pathways. Several aspects of these fi ndings and conclusions merit additional discussion.
Previous studies demonstrated that liver-specifi c Mttp deletion protected mice from LD-induced gallstone formation ( 8 ). Those studies demonstrated compensatory alterations in hepatic cholesterol metabolism in Mttp-LKO mice including increased cholesterol ester content and decreased cholesterogenesis, which in turn was postulated Fig. 3. A, B: mRNA expression of genes related to cholesterol, phospholipid, and bile acid metabolism and transport. Hepatic mRNA was extracted from mice fed a LD for 2 weeks. mRNA expression of indicated gene was quantitated by real-time qPCR and the value expressed as fold change relative to Mttp f/f mice, which was defi ned as 1. C: mRNA expression of genes related to ER stress. The bars represent the mean ± SE (n = 4-8 per group). D, E: Protein expression of cholesterol transporter Abcg8. Membrane protein (50 g) from the indicated genotypes of mice was separated by 8% SDS-PAGE. Abcg8 expression was determined by Western blot and quantitated by Kodak Image software, with ␤ -actin as loading control. D: The representative image. E: The bar graph of relative Abcg8 abundance (mean ± SE of 4 mice per group) after normalizing expression to Mttp f/f mice (n = 4 per group). * P < 0.05. ester, which was consistent with a role for Mttp in relieving product inhibition of cholesterol esterifi cation by transferring neutral lipid ( 28 ). Our fi ndings are consistent with that report and show that hepatic cholesterol ester content decreases in Mttp-LKO and DKO mice following LD feeding ( Tables 1-4 ). However, we did not fi nd the same results in LD-fed mice treated with the Mttp-ASO. In those experiments, we found that hepatic cholesterol ester content increased in both WT and L-Fabp Ϫ / Ϫ mice when they were treated with the Mttp-ASO ( Tables 7-9 ). The reasons for this discrepancy are not immediately apparent because we achieved comparable reduction of hepatic Mttp expression (>80%) following knockdown and genetic deletion experiments (data not shown), but it remains possible that differences in ASO versus genetic deletion approaches may be at least partially responsible. It should be noted that the genetic backgrounds of the mice used for the ASO and genetic deletion experiments are not truly identical (even though the studies were performed in C57BL/6 mice), because the control mice used in the genetic deletion experiments contained a "fl oxed" allele (Mttp fl ox/fl ox ) and also received pI-pC injections over several days. As alluded to above, earlier studies in LD-fed Mttp-LKO mice also showed increased hepatic cholesterol ester content ( 8 ).
Our fi ndings suggest a plausible mechanism by which hepatic Mttp deletion leads to attenuated canalicular cholesterol secretion, specifi cally via alterations in the expression of the heterodimeric cholesterol transporter Abcg5/ g8, which represents one of the dominant pathways for canalicular cholesterol export ( 17,(29)(30)(31). The fi ndings suggest decreased expression of Abcg8 in LD-fed Mttp-LKO and DKO mice and also following Mttp-ASO treatment of WT and L-Fabp Ϫ / Ϫ mice. The fi ndings in the Mttp-ASO-treated mice revealed more robust evidence (compared with the genetic deletion experiments) for decreased expression of both Abcg5 and Abcg8 mRNA and also demonstrated decreased Abcg8 protein abundance. The explanation for this subtle difference may again lie in the liver-specifi c Mttp knockdown approaches, which we speculate may result in less adaptation or compensation. Previous studies have demonstrated that the Abcg5/g8 heterodimer is regulated by alterations in leptin signaling as well as by chemical chaperones, suggesting that posttranscriptional events may represent an important mode of functional regulation ( 25 ). In this scenario, we speculate that interactions of Abcg5/g8 with endolumenal ER cholesterol secretion rates were decreased in Mttp-LKO and DKO mice. Third, we did not observe increased biliary phospholipid secretion in Mttp-LKO and DKO mice as reported by those workers, but rather found (if anything) slightly reduced secretion rates. The sources of the discrepancies between the current report and those earlier fi ndings may reside in a combination of factors, including the genetic background of the mice [C57BL/6 as used in the current study versus a 50/50 mixed C57BL/6 and 129/ SvJae background as used in that report ( 8 )] and the different facilities, which conceivably could include differences in microbial communities. We evaluated different durations of LD feeding (2-4 weeks in the current study versus 4-8 weeks in those earlier studies) which yielded virtually identical outcomes, suggesting that the timing of LD exposure is not a factor.
The observation that biliary phospholipid secretion was unaltered in the setting of hepatic Mttp deletion should not be construed as implying that there is no effect of hepatic Mttp on hepatic phospholipid metabolism. Indeed, Hussain and colleagues defi ned both neutral and polar lipid transfer activities of Mttp based on the properties of human versus Drosophila proteins and have demonstrated distinct effects on whole body lipid metabolism and in the apical secretion of hepatic VLDLs ( 27 ). However, those studies did not address the question of whether canalicular phospholipid secretion is altered following adenoviral Mttp expression. In addition, we did not specifi cally examine the secretion rates of individual phospholipid species, which could conceivably represent yet another level of regulation of biliary cholesterol saturation.
Other studies in Mttp-LKO mice demonstrated increased hepatic free cholesterol with decreased cholesterol WT and L-Fabp Ϫ / Ϫ mice received either control-ASO or Mttp-ASO for four weeks and were fed a LD for the fi nal 2 weeks, while continuing ASO injection (n = 4-5 per group). Sera were collected after overnight fasting and serum lipids and ALT assayed. The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically signifi cant ( P < 0.05). TG, triglyceride; TC, cholesterol. Notwithstanding the observations above, it remains possible that other pathways are involved in Mttp-dependent modulation of canalicular cholesterol secretion. These include Srb1-dependent pathways of canalicular cholesterol secretion where a role for Abcg5/g8 independent cholesterol transport has been invoked ( 32,33 ). While we found no evidence to support such a role for Srb1 in the current study, the possibility merits further examination. An additional consideration is that liver-specifi c Mttp deletion might conceivably promote hepatic cholesterol mobilization through nonbiliary pathways (34)(35)(36), a possibility that will require evaluation. In summary, the current fi ndings demonstrate an expanded role for hepatic Mttp in the regulation of canalicular cholesterol secretion and illustrate the complexity in orchestrating its metabolic compartmentalization.
proteins may promote the functional dimerization and directed traffi cking of the heterodimeric cholesterol transporter. The earlier fi ndings of Graf et al. ( 30 ), coupled with more recent fi ndings alluded to above ( 25 ) raise the testable possibility that Mttp functions as a chaperone for Abcg5/g8 assembly within the hepatocyte, conceivably in a manner analogous to that described for its chaperone function in relation to the initial steps in the cotranslational processing of apoB ( 10 ). That particular suggestion will require further examination to resolve the role of Mttp in the posttranslational processing, assembly, and vectorial delivery of the Abcg5/g8 heterodimer. WT and L-Fabp Ϫ / Ϫ mice received either control-ASO or Mttp-ASO for four weeks and were fed a LD for the fi nal 2 weeks, while continuing ASO injection (n = 4-5 per group). Bile was collected and biliary lipid secretion determined and expressed as nmol/min/g liver weight. The difference between values associated with different superscript letters for the parameters indicated in each vertical column is statistically signifi cant ( P < 0.05). TC, cholesterol; PL, phospholipids; BA, bile acids.

Fig. 5.
A: mRNA expression of genes related to cholesterol, phospholipid, and bile acid metabolism and transport. Hepatic mRNA was extracted from mice treated with the indicated ASO for 4 weeks and fed a LD for the fi nal 2 weeks. The mRNA expression of indicated genes was quantitated by real-time qPCR and the value is expressed as fold change related to C-ASO-treated WT mice, which was defi ned as 1. The bars represent the mean ± SE (n = 4 per group). B, C: Protein expression of cholesterol transporter Abcg8. Membrane protein (50 g) from the above mice was separated by 8% SDS-PAGE. Abcg8 protein expression was determined by Western blot and quantitated by Kodak Image software, with ␤ -actin as loading control. B: A representative image. C: A bar graph of relative Abcg8 abundance (mean ± SE of 4 mice per group) after normalizing expression to C-ASO-treated WT mice. * P < 0.05.