Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein.

Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr−/− mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile.

and nonHDL-C, and administered control ASO (50 mg/kg/ week), apoB ASO (50 mg/kg/week), or MTP ASO (50 mg/kg/ week) for periods ranging from 2 to 12 weeks while remaining on diet. For 12 week studies, mice received intraperitoneal injections of ASO once weekly for 6 weeks and then once every other week for the remainder of the study. Body weight was monitored throughout the study, with no signifi cant differences between control and treated mice observed in any of the studies. At the end of treatment period, animals were fasted for 4 h and euthanized for blood and tissue analysis.

Plasma chemistry and lipoprotein cholesterol analysis
Plasma lipids and aminotransferase concentrations were quantifi ed on an Olympus AU400e automated clinical chemistry analyzer (Melville, NY). HPLC analysis of LDLr Ϫ / Ϫ mice cholesterol distribution among VLDL-C, LDL-C, and HDL-C fractions was performed as described previously. Percent area under curve VLDL-C, LDL-C, and HDL-C was determined using Peakfi t™ v 4.12 (Seasolve Software®, Framingham MA) and then multiplied by TPC to determine VLDL-C, LDL-C, and HDL-C, respectively.

Histology
Oil Red O (ORO) staining on sections of snap-frozen liver and intestinal samples was performed as described (14). For quantitation of lipid droplets, digital 10× (1,700 × 1,100 m) images (four images per treatment group, with each image taken from a different mouse) of ORO-stained sections were analyzed with the ChromaColor™ software to identify all ORO-stained droplets. Those densitometric images were quantifi ed using Image Pro Plus™ software, which sorted the droplets into one of four sizes: 5-50 m 2 , 51-100 m 2 , 101-1,000 m 2 , or >1,000 m 2 , with the largest size class considered to be an artifact of the staining procedure.

Western blotting
Liver homogenates were prepared and immunoblotted as described. GAPDH or tubulin was used as a protein loading control for all blots. For the quantitation of hepatic proteins, two samples from each treatment group were run on two separate blots (n = 4 samples per treatment). Western blots were visualized, and band density was analyzed using the LI-COR Odyssey™ imaging system. Fluorescence of the bands of interest were normalized to load controls and expressed as mean fold change relative to control ASO. For small intestine, a single blot was analyzed (n = 2 samples per treatment).

Tissue lipid composition
Liver TG, free cholesterol, and total cholesterol were extracted and analyzed as described previously (15). Intestinal lipid was quantitated after fasting the mice for 4-5 h. Mice were then euthanized, and the small intestine from the pyloric valve to the cecum was collected and divided into three equal pieces. The luminal contents were fl ushed with PBS from the proximal section, and each section was cut longitudinally with scissors and fl attened, and the mucosal layer was scraped off with a glass slide. An aliquot of the cells was suspended in PBS. One-fi fth of the suspension was brought to 1 N NaOH for protein determination by the Lowry method, and three-fi fths of the lipid was extracted by the Bligh-Dyer method described previously. The extract was solubilized with 2% Triton ×100 in dH 2 O, and lipids were quantifi ed using colorimetric assays as described for the liver lipid extraction.

MTP activity assay
Aliquots of livers isolated from DIO mice treated with ASO for 6 weeks were snap frozen and sent to Chylos® Inc. (New York, VLDL into cholesteryl ester (CE)-rich LDL particles. Therefore, reduction of apoB and/or MTP lowers LDL levels by decreasing the number of particles secreted into the circulation. In contrast, statins lower LDL-C principally by enhancement of LDL receptor-mediated clearance of LDL particles from the circulation (3).
Pharmacological suppression of hepatic apoB production via antisense oligonucleotide (ASO) treatment has also led to reductions in VLDL-C and LDL-C and the amelioration of atherosclerosis in murine preclinical models (11,12). Furthermore, although a murine-specifi c apoB ASO reduced apoB production and secretion from the liver, hepatic steatosis and elevation of transaminases were not observed in these mice (11). In high fat-fed mice administered the apoB ASO, liver TG accumulation was shown to be reduced as a result of secondary, compensatory mechanisms that lead to reduced lipogenesis and increased fatty acid oxidation. Additionally, there were no effects on dietary fat absorption, chylomicron formation, or increases in intestinal TG accumulation due to limited ASO distribution to the small intestine.
Given the importance of MTP to VLDL production, we also wished to evaluate the effects of an inhibitor of MTP in C57BL/6 and LDLr Ϫ / Ϫ mice. However, experiments directly comparing the therapeutic reduction of apoB and MTP have not been possible because apoB is not targetable by small molecule approaches. Therefore, to perform such studies, we designed an ASO to murine MTP and directly compared its activity with that of an apoB antisense drug, ISIS 147764 (11), in preclinical pharmacology models.

Mice
Six week old male C57BL/6 or LDLr Ϫ / Ϫ (Jackson Laboratory) mice in cages (n = 3-5 per cage) on a 12-h light-dark cycle for the duration of the studies. All procedures and protocols were approved by an institutional animal care and use committee. C57BL/6 mice were fed chow or a high-fat diet (60% calories as fat; Research Diets D12492) (DIO model) for 3 weeks. After the diet acclimation period, mice were randomized based on TPC and nonHDL-C and then administered assigned ASOs in saline (5 mg/ml) by intraperitoneal injection at a dose of 25, 12.5, or 6.25 mg/kg twice weekly for 2 or 6 weeks. LDLr Ϫ / Ϫ mice were fed a standard chow diet or a Western diet (Harland Teklad Diet 88137) consisting of 42% of calories as fat and 0.15% cholesterol for 1 week. The mice were then bled, randomized based on TPC

Quantitative real-time PCR
Real-time PCR was performed as described (11). Sequences of the primer probe sets used are provided in supplementary Table I. All data were normalized to cyclophilin A mRNA levels and were expressed as percent of hepatic mRNA expression in control ASO-treated mice.

Statistical analysis
The majority of pharmacology experiments were performed in at least two independently performed studies with groups of 4 to 12 animals. All values are expressed as mean ± SEM. To determine statistical signifi cance, one-and two-way ANOVA analysis with Tukey's post hoc test was carried out using GraphPad Prism 5™ software with statistical signifi cance being set at p < 0.05.

RESULTS
Second-generation ASOs targeting murine apoB (ISIS 147764), whose pharmacology has been described in detail in two previous studies (11,12), or MTP (ISIS 144477), which in primary mouse hepatocytes reduced MTP mRNA NY), where MTP activity was quantitated as described previously (16).

Quantitation of TG and cholesterol absorption
DIO mice administered ASO for 6 weeks were fasted overnight and then received an intraperitoneal injection of poloxamer 407 at a dose of 1 mg/g body weight. One hour after injection, a baseline retro-orbital bleed was collected, and the mice were orally gavaged with 2.5 Ci of 3 H triolein in 200 l of olive oil. Animals were bled 90 and 180 min after gavage, and counts per minute (cpm) values were quantifi ed from 20 l of serum by liquid scintillation counting (LSC Instrument; Beckman). Cholesterol absorption in DIO mice treated with ASO for 6 weeks was quantitated using the dual fecal isotope method as described previously (17).

Hepatic ApoB and TG secretion
Hepatic and apoB secretion was carried out as previously described (18).

Analysis of hepatic fatty acid synthesis
Quantitation of hepatic fatty acid synthesis was carried out using the tritiated water method described previously (19). Signifi cantly different when compared with MTP ASO treatment. after 6 weeks of MTP ASO treatment showed that the reduction in activity ( Ϫ 71%) was consistent with the hepatic MTP mRNA and total protein reductions ( Fig. 2C ). Administration of the apoB ASO signifi cantly increased MTP activity by 25% when compared with control.
As expected, reduction of hepatic apoB and MTP led to signifi cant reductions in TPC in the DIO mice and LDLr Ϫ / Ϫ mice fed chow or Western diet ( Tables 1 and 2 ). In DIO mice ( Table 1 ), the reductions in TPC were due to reductions in nonHDL-C and HDL-C, a plasma lipid profi le that is similar to that observed in ApoB +/ Ϫ or MTP (20,21). In addition, neither apoB nor MTP ASO had any effect on plasma TG, which was not surprising given the relatively low plasma TG levels of this model. However, apoB and MTP ASO treatment signifi cantly decreased plasma TG in Western diet-fed LDLr Ϫ / Ϫ mice by Ϫ 69% and Ϫ 75%, respectively ( Table 2 ). More detailed evaluation of plasma samples taken from apoB and MTP ASO-treated LDLr Ϫ / Ϫ mice indicated that the TPC and plasma TG reductions were primarily due to decreases in apoB-containing lipoproteins; however, there was a significant decrease ( Ϫ 30%) in HDL-C in the chow-fed LDLr Ϫ / Ϫ mice ( Table 2 ). For VLDL, similar reductions were observed for apoB ASO ( Ϫ 58% for chow, Ϫ 82% for Western diet) and MTP ASO ( Ϫ 71% for chow, Ϫ 85% for Western diet) treatment. However, LDL-C was reduced to a greater extent by apoB ASO ( Ϫ 61% for chow, Ϫ 73% for Western in a dose-dependent manner (supplementary Fig. 1), were used for all studies. To compare the pharmacological effects of apoB and MTP ASOs on safety, effi cacy, and hepatic steatotic progression, three mouse models were used. Initially, ASOs were administered to chow-fed C57BL/6 mice twice weekly (12.5-50 mg/kg/week) for 2 or 6 weeks to assess their effi cacy and tolerability. To further investigate the effects on liver TG accumulation between apoB and MTP ASO treatment, the two compounds were administered to DIO mice, a model of hepatic steatosis, for 2 or 6 weeks. Finally, because the severe familial hypercholesterolemia patient population is the primary indication for apoB and MTP drugs, male LDLr Ϫ / Ϫ mice were fed chow or Western diets and administered ASOs at 50 mg/ kg/week for 6 weeks.
In all three mouse models, ASO administration led to signifi cant, dose-dependent suppression of hepatic target mRNA expression, reaching 70% to 90% reduction when compared with control ASO-treated animals at the high dose after 6 weeks of treatment ( Fig. 1 ). Consistent with the mRNA data, DIO and LDLr Ϫ / Ϫ mice treated with the apoB ASO-demonstrated reductions in hepatic target protein levels when compared with control ASO treatment ( Fig. 2 ). The MTP ASO-treated DIO and LDLr Ϫ / Ϫ reductions in hepatic target protein were also consistent with the degree of hepatic MTP mRNA suppression. Furthermore, measurement of hepatic MTP activity in DIO mice ApoB and MTP ASO administration reduced liver target protein expression in several murine models. A: DIO C57BL/6 mice were administered ASO (50 mg/kg/week) for 2 or 6 weeks. B: Chow-fed or Western diet-fed LDLr Ϫ / Ϫ were administered ASO for 6 weeks. Liver protein (50 g) was immunoblotted with polyclonal Abs raised against apoB, MTP, or GAPDH (load control). Numbers below bands are mean density (n = 4) relative to control ASO-treated mice. C: Quantitation of hepatic MTP activity (n = 5 per group) in DIO mice administered ASO for 6 weeks. # Signifi cantly different ( p < 0.05) when compared with control ASO.
Signifi cantly different when compared with MTP ASO treatment.
However, due to the weak and variable apoB48 protein bands, it is diffi cult to draw a distinct conclusion from the Western blot results. After 2 and 6 weeks of treatment, the MTP ASO led to signifi cant reductions of Ϫ 50% and Ϫ 59% in proximal SI MTP mRNA expression, respectively. Unlike the apoB ASO, the MTP ASO had little to no effect on intestinal MTP protein expression and increased apoB48 protein expression. The reduced effi cacy of the ASOs relative to liver was expected based on the observation in previous studies of limited distribution of ASO (22) to the intestine.
To determine if the reduction of intestinal apoB and MTP led to physiologic effects on lipid and sterol absorption, we measured TG and cholesterol absorption in DIO mice treated with ASOs for 6 weeks ( Fig. 3C ). Poloxamer 407-treated mice were administered a tritiated triolein gavage, and the appearance of radioactivity was quantitated over a 3 h period. ApoB ASO-treated animals had a modest, but signifi cantly reduced, secretion of tritiated triolein into the plasma ( Ϫ 30%) at the 3 h time point when compared with control ASO. The MTP ASO also showed a diet) versus MTP ASO ( Ϫ 40% for chow, Ϫ 42% for Western diet) administration. Therefore, in DIO and LDLr Ϫ / Ϫ mouse models, 6 weeks of treatment with the apoB or MTP ASOs led to signifi cant reductions in hepatic apoB or MTP protein that in turn led to decreased plasma TPC and, in a severely hyperlipidemic model, decreased plasma TG.
Suppression of intestinal expression of apoB or MTP can reduce lipid absorption and potentially lower plasma lipid levels by impairing intestinal secretion of apoB48containing chylomicron particles. To determine if the amelioration of dyslipidemia observed with ASO treatment was due to alterations in intestinal metabolism, lipid absorption ( Fig. 3 ) was quantifi ed. Dietary food intake and animal body weights were unaffected by apoB or MTP ASO treatment; nor was there evidence of steatorrhea/ loose stools (data not shown). Proximal intestinal apoB mRNA expression was unchanged after 2 weeks of apoB ASO treatment but was signifi cantly reduced by 62% after 6 weeks ( Fig. 3A ). Intestinal apoB48 protein also appeared to be reduced, albeit more modestly than the apoB mRNA expression, with 6 weeks of ASO treatment ( Fig. 3B ).  whereas MTP ASO showed a 3.9-fold increase in liver TG that was signifi cantly greater than the apoB ASO and control ASO treatments ( Table 3 ). As with the chow and DIO mice, liver CE was signifi cantly increased to a similar extent with apoB ASO and MTP ASO treatment, and hepatic free cholesterol concentrations did not change with either treatment (data not shown). Therefore, upon receiving a dietary challenge, DIO and LDLr Ϫ / Ϫ mice treated with the MTP ASO developed more extensive hepatic steatosis and liver injury, as evidenced by elevated transaminases, when compared with apoB ASO nonsignifi cant tendency ( Ϫ 25%) to reduce tritiated triolein when compared with the control ASO. Neither compound demonstrated a signifi cant effect on cholesterol absorption as measured by the dual fecal isotope method. Proximal intestine histology and lipid content were also analyzed. Consistent with minimal effects on total lipid absorption, there were no signifi cant changes in TG or total cholesterol concentrations when quantitated and compared with the control ASO-treated mice ( Fig. 3D ); nor were there observable increases in neutral lipid accumulation detected using H&E and ORO staining methods ( Fig. 3E ).
In two previous publications ( 11,12 ), no reductions were observed in intestinal apoB48 protein levels; nor were there any effects on lipid absorption observed after extended administration of the apoB ASO in the DIO and LDLr Ϫ / Ϫ mice used in those experiments. However, in this most recent study, a modest but signifi cant reduction in apoB48 levels was noted. Nonetheless, the apparent reduction in apoB48 protein levels did not produce increased intestinal lipid deposition or reduced cholesterol/TG absorption. This variation in the protein modulation may be attributed to differences in mouse progeny, dietary constituents, experimental protocols, or test reagents in studies performed over the last decade.
After demonstration that the apoB and MTP ASO were suffi ciently effi cacious, we evaluated the safety profi le of the two ASOs using a number of parameters ( Tables 1-3 ). Liver TG and CE concentrations were evaluated in chow-fed C57BL/6, DIO, and LDLr Ϫ / Ϫ mice. In chow-fed C57BL/6 mice, hepatic TG levels ( Table 3 ) were only increased by the MTP ASO at 6 weeks. Liver CE concentrations, however, were signifi cantly increased in apoB ASO-treated animals after 2 weeks and in apoB and MTP ASO-treated animals after 6 weeks. Hepatic free cholesterol concentrations were similar between all groups at both time points (data not shown). Despite these differences in liver TG accumulation, administration of apoB or MTP ASO to chowfed mice did not lead to changes in body weight progression or hepatic transaminases (data not shown), indicating that apoB and MTP ASO were well tolerated in the absence of a dietary challenge.
A markedly different safety profi le was observed in the face of a dietary challenge. Two weeks of high-dose (50 mg/kg/week) apoB or MTP ASO treatment in DIO mice led to a signifi cant 3.3-fold elevation in liver TG when Fig. 3. Intestinal effects of ApoB and MTP ASOs in DIO mice. A: Proximal intestine mRNA expression of targeted genes in 50 mg/kg/week control ASO-, apoB ASO-, and MTP ASO-treated (n = 5 per group) DIO animals were determined by quantitative PCR as described in Materials and Methods. B: One hundred micrograms of protein isolated from the proximal intestine was immunoblotted with polyclonal Abs raised against apoB, MTP, or GAPDH (load control). Numbers below bands are mean density (n = 2) relative to control ASO-treated mice. C: Quantitation of 3 H-triolein absorption in DIO mice administered ASO for 6 weeks was carried out as described in Materials and Methods. Cholesterol absorption was carried out in DIO mice administered ASO for 6 weeks using the dual fecal isotope method. D: Proximal intestine triglyceride and total cholesterol was quantitated as described in Materials and Methods. E: Images of ORO-stained snap frozen proximal intestinal sections from mice treated with ASO for 6 weeks. ORO-stained sections were counterstained with hematoxylin. Bars represent mean; error bars represent SEM. # Signifi cantly different ( p < 0.05) when compared with control ASO.
Signifi cantly different ( p < 0.05) when compared with MTP ASOtreated mice at same dosage. treatment. Using these models, we attempted to identify the factors that would lead to such differences in the development of steatosis observed between the two targets.
To determine if the discrepancies in hepatic TG accumulation between apoB and MTP ASO treatment were due to differences in VLDL secretion, apoB and TG accumulation was quantifi ed in ASO-treated mice administered a Triton WR1339/ 35 S methionine bolus ( Fig. 4 ). Our data showed that apoB and MTP ASO administration significantly reduced TG secretion to a similar extent in DIO and Western diet-fed LDLr Ϫ / Ϫ mice when compared with control. However, quantitation of 35 S apoB protein secretion in DIO mice demonstrated that, relative to MTP, the apoB ASO was a more potent inhibitor of apoB48 secretion at the 120 min and at the 180 min time points. The relative insensitivity of apoB48 to MTP suppression has been well documented in vitro and in vivo (23)(24)(25). Therefore, the differences in TG accumulation between apoB ASO and MTP ASO-treated mice were not due to differences in reduction of hepatic TG secretion.
Densitometric analysis of ORO-stained histological sections ( Fig. 5A, B ) was used to examine treatment differences in lipid droplet density and size distribution after 2 and 6 weeks of treatment. At 2 weeks ( Fig. 5C ), the total lipid droplet area (i.e., the ORO-stained area) of apoB and MTP ASO-treated livers showed similar signifi cant elevations when compared with the control ASO animals, consistent with the liver TG concentrations obtained from tissue extractions ( Table 3 ). Further analysis of lipid droplet size distribution demonstrated that a majority of the lipid droplets in all three ASO treatments were of small diameter (5-50 m 2 ). However, with MTP ASO treatment there was a signifi cant increase in the number of larger lipid droplets and in the percentage of total lipid area of larger lipid droplets (51-100 m 2 ) when compared with controls ( Fig. 5C ).
Analysis of livers from animals treated for 6 weeks revealed a more pronounced histological phenotype ( Fig. 5B ). In silico quantitation ( Fig. 5C ) showed that, much like liver TG concentrations, MTP ASO had signifi cantly more total lipid area when compared with apoB and control, whereas apoB ASO had a total lipid area that was intermediate between the control and MTP ASO. Much like the Based on microarray data and studies described previously (11,12), we had identifi ed PPAR ␥ as being regulated by apoB ASO. Quantitation of hepatic mRNA ( Table 4 ) revealed that, although there were no signifi cant differences compared with control ASO, 2 weeks of apoB ASO treatment led to signifi cant decreases in hepatic mRNA expression of PPAR ␥ and PPAR ␥ -regulated genes FSP27 and CD36 as well as the adipocyte-specifi c marker adipsin relative to the MTP ASO. Furthermore, apoB and MTP ASO treatment signifi cantly reduced hepatic SCD-1 mRNA expression, a key enzyme in the lipogenic pathway (27). After 6 weeks of apoB ASO treatment, DIO mice again showed signifi cant reductions in hepatic PPAR ␥ mRNA when compared with MTP ASO-treated mice ( Table 5 ). In contrast, the hepatic expression of PPAR ␥ and its target genes CD36 and FSP27, as well as the adipocyte-specifi c marker adipsin, which is more highly expressed with PPAR ␥ overexpression (28), were signifi cantly greater in MTP ASOtreated animals.
Because increased hepatic de novo lipogenesis can contribute to liver TG accumulation, we studied this synthetic process by evaluating transcriptional factors and fatty acid synthesis in mice treated with ASO for 6 or 12 weeks. The differences in liver TG concentrations after 12 weeks of treatment were similar to shorter-duration studies (4.3 mg/g liver wet weight [WW] with control ASO vs. 20.3 mg/g liver WW with apoB ASO vs. 81.7 mg/g liver WW with MTP ASO). DIO mice administered the highest dose of apoB or MTP ASO showed similar reductions in acetyl-coA carboxylase 1 (ACC1) mRNA expression ( Table 5 ), a pathway mRNA expression produced by apoB ASO relative to the MTP ASO.
Another potential contributing factor to hepatic steatosis is reduced fatty acid oxidation. An accepted in vivo biomarker for fatty acid oxidation is ␤ -hydroxybutyrate (3HB) ( 29 ). In DIO mice treated for 2 weeks, plasma 3HB levels were signifi cantly increased with apoB and MTP ASO treatment ( Table 1 ), indicating that these animals were compensating for elevated levels of hepatic TG levels by increasing fatty acid oxidation. However, after 6 weeks of treatment, 3HB levels remained elevated only with MTP ASO treatment, possibly because apoB ASO-treated mice no longer had elevated liver TG concentrations.

DISCUSSION
Given that antisense drugs share similar pharmacokinetic properties and distribute predominantly in the liver key enzyme involved in fatty acid synthesis. Furthermore, after 6 weeks of ASO treatment, hepatic AMPK ␣ 2 mRNA signifi cantly increased by 66% when compared with control. Hepatic ACC1 protein expression was also reduced by both drug treatments, whereas apoB ASO-treated animals showed more pronounced protein reduction relative to MTP ASO treatment ( Fig. 6 ) after 6 weeks of ASO treatment. When hepatic lipogenesis was quantifi ed after 6 and 12 weeks of treatment, this process tended to be reduced at both time points with the apoB ASO, but these changes did not achieve statistical signifi cance ( P = 0.28 and P = 0.14, respectively). MTP ASO treatment tended to suppress lipogenesis at 6 weeks ( P = 0.58) but had no effect after 12 weeks ( P = 0.96). Therefore, the differences in TG accumulation between apoB and MTP ASO treatment appear to be due in part to 1 ) the inability of MTP ASO-treated animals after long-term treatment to suppress lipogenesis, 2 ) the development of larger lipid droplets with the MTP ASO, and 3 ) reduced PPAR ␥ Signifi cantly different ( P < 0.05) when compared with MTP ASO treatment. AMPK ␣ mRNA levels were increased after apoB ASO treatment ( Table 5 ). An established method to detect hepatic ␤ oxidation is to evaluate 3HB, or ketone levels, in vivo (32,33). Using that technique, it appeared that 3HB was increased in apoB ASO-treated DIO mice after 2 weeks of treatment ( Table 1 ), when liver TG levels were elevated in comparison to controls ( Table 3 ), but after 6 weeks, when hepatic TG levels were similar to controls, 3HB levels returned to those observed in control ASO-treated mice. This was in contrast to MTP ASO-treated animals, which had elevated plasma TG and 3HB levels and at the highest administered dose and increased transaminase levels at both the 2 week and 6 week time points. Sustained fatty acid oxidation has been shown to increase reactive oxygen species, which, through many well documented pathways, including the peroxidation of polyunsaturated fatty acids, leads to enhanced oxidative stress, thus potentially contributing to the development of liver injury (30).
In MTP ASO-treated mice, many lipogenic genes were also down-regulated, with similar reductions as observed in apoB ASO-treated mice such as SCD-1, whereas less robust down-regulation was demonstrated for ACC1. Finally, AMPK ␣ levels were unaffected by MTP ASO treatment. Regardless of these reductions in lipogenic genes, de novo fatty acid synthesis, as determined using tritiated water, was not signifi cantly reduced in MTP-treated mice ( Fig. 6 ), perhaps due to more modest effects on ACC1 mRNA and protein. ACC1 is a key enzyme that catalyzes the synthesis of malonyl CoA and plays a pivotal role in de novo lipogenesis and mitochondrial fatty acid oxidation (34). These observations suggest that blocking VLDL secretion with an MTP or apoB ASO may trigger common metabolic pathways to compensate for increased hepatic TG deposition, including down-regulation of lipogenesis and increased ␤ -oxidation. These adaptive processes have also been observed in mice with a targeted apoB38.9 mutation (35) and in hepatocytes isolated from humans with familial hypobetalipoproteinemia (36).
Another potential contributor to the consistent hepatic steatosis observed in MTP mice but not in apoB ASOtreated mice relates to differential effects upon PPAR ␥regulated genes that are thought to be involved in lipid droplet formation and homeostasis. We observed key differences between apoB and MTP ASO treatment in lipid droplet size ( Fig. 5 ) and the expression of PPAR ␥ and FSP27 genes ( Tables 4 and 5 ) previously implicated in lipid droplet metabolism. Others have shown that FSP27, a lipid droplet associated protein, is integral to the PPAR ␥dependent development of hepatic steatosis in mice (37). In vitro evidence indicates that FSP27 overexpression results in TG accumulation and increased lipid droplet size in AML12 cells, primary hepatocytes (37), and COS cells due in part to decreases in fatty acid oxidation and TG turnover. Furthermore, recent in vivo experiments demonstrated rather than intestine, we performed head-to-head comparisons with target-specifi c antisense drugs to evaluate the metabolic consequences of hepatic apoB and MTP suppression. Our studies reveal that the ASO-mediated modulation of apoB or MTP mRNA resulted in similar reductions in their respective hepatic mRNA levels, with comparable concurrent suppression in hepatic TG secretion and serum lipid levels. Despite equivalent impairment of VLDL secretion, MTP reduction produced persistent increased hepatic TG accumulation that resulted in increased ALT elevations relative to the apoB ASO.
In earlier publications from our laboratory (11,12), we suggested that antisense suppression of apoB does not result in hepatic steatosis due to secondary compensatory changes in the expression of multiple enzymes involved in lipogenic and ␤ -oxidative pathways in the liver. Data presented here confi rm and extend those observations. When liver fat was quantifi ed in DIO mice administered the apoB ASO, there was an early, transient increase in hepatic TG levels observed after 2 weeks of treatment, but by 6 weeks, liver TG returned to levels present in control animals ( Table 3 ). A number of experiments using transcriptional profi ling and metabolomic analyses were performed to determine the physiological sequelae of apoB reduction. Those analyses revealed that apoB ASO treatment downregulated several key lipogenic genes, including SCD-1, ACC1, and PPAR ␥ . Consistent with these fi ndings, hepatic fatty acid synthesis, as determined by tritiated water analytical methods, demonstrated that de novo lipogenesis was decreased in vivo ( Fig. 6 ).
In addition to decreased fatty acid synthesis, another adaptive response to increased liver TG is to enhance oxidation of fatty acid stores (30). Extensive studies have shown that AMP-activated protein kinase ␣ (AMPK ␣ ) is a key energy sensor whose expression has been correlated with increased fatty acid oxidation and decreased lipogenesis (30, 31). As described previously and shown herein, sections from ASO-treated DIO mice (n = 4 per group) were analyzed as described in Materials and Methods. Bars represent mean; error bars represent SEM. # P < 0.05 when compared with control ASO.
Signifi cantly different ( P < 0.05) when compared with MTP ASO treatment. Values represent mean ± SEM (n = 5 per group). a Signifi cantly different ( P < 0.05) when compared with control ASO.
b Signifi cantly different ( P < 0.05) when compared with MTP ASO treatment.
lipid droplets, which causes accumulation of lipid within the droplet. In the MTP ASO-treated animals, hepatic FSP27 mRNA expression and lipid droplet size were increased relative to controls and apoB ASO-treated animals. It is possible that the elevated FSP27 expression in MTP ASO-treated animals is shunting the TG normally exported in VLDL particles away from the fatty acid oxidation pathway and into lipid droplets where the TG cannot be mobilized. Therefore, differential effects on both fatty acid synthesis and lipid droplet morphology may help explain the difference in liver TG levels observed between high dose apoB and MTP ASO treatment.
In summary, although apoB and MTP play central roles in hepatic lipoprotein secretion, pharmacological suppression of these proteins leads to distinct differences in metabolic sequelae. Although there were similar reductions in hepatic TG secretion after 6 weeks of ASO treatment, MTP ASO-treated mice accumulated signifi cantly more hepatic TG than apoB ASO-treated mice. Our data suggest that this is likely due to the complex, multiphasic interplay of factors including decreased lipogenesis and changes in lipid droplet metabolism, as exemplifi ed by reductions in hepatic lipid droplet size and FSP27 mRNA expression with apoB ASO treatment relative to MTP ASO treatment. Although we believe that the factors elucidated in this manuscript contribute to the phenotypes observed in mice after apoB and MTP ASO treatment, other metabolic processes may occur. Further work is necessary to defi ne the precise signaling mechanisms and potential alternate pathways that differentiate the effects of long-term reductions in apoB and MTP in liver metabolism and lipid homeostasis.  6. Effects of ApoB and MTP ASOs on fatty acid synthesis in DIO mice. A: Hepatic expression of ACC1 protein in DIO mice administered 50 mg/kg/week of ASO for 6 weeks. B: Hepatic fatty acid synthesis in DIO mice (n = 6-12 per group) administered 50 mg/kg/week of ASO for 6 weeks. C: Hepatic fatty acid synthesis in DIO mice (n = 5/group) administered 50 mg/kg/week of ASO for 12 weeks. Fatty acid synthesis was measured using the tritiated water method described in Materials and Methods. Bars represent mean; error bars represent SEM. Values represent mean ± SEM (n = 4 per group). a Signifi cantly different ( P < 0.05) when compared with control ASO. b Signifi cantly different ( P < 0.05) when compared with MTP ASO treatment.