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Am J Physiol Endocrinol Metab 290: E17-E25, 2006. First published August 16, 2005; doi:10.1152/ajpendo.00597.2004
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Coordinated alteration of hepatic gene expression in fatty acid and triglyceride synthesis in LCAT-null mice is associated with altered PUFA metabolism

Hui Song,1,* Liping Zhu,1,* Clive M. Picardo,2 Graham Maguire,2 Vincent Leung,2 Philip W. Connelly,1,2 and Dominic S. Ng1

1Department of Medicine, St. Michael's Hospital; and 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Submitted 20 December 2004 ; accepted in final form 10 August 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Complete lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with fasting hypertriglyceridemia (HTG). We recently reported that, in ldlr–/–xlcat–/– mice, fasting HTG is associated with hepatic triglyceride overproduction in association with an upregulation of the hepatic srebp1 gene and altered expression of its target genes in lipogenesis and gluconeogenesis. We further investigated the role of hepatic polyunsaturated fatty acid (PUFA) metabolism in the modulation of the lipid phenotypes. In the ldlr–/–xlcat–/– mice, using the ldlr–/–xlcat+/+ littermate as controls, the hepatic level of cholesterol esters (CE) were reduced by 61.0% whereas the 20:4-CE and 22:6-CE contents were each reduced by >80%. In contrast, the hepatic levels of 20:4- and 22:6-containing phospholipid (PL) species were either unchanged or mildly elevated. Similar alterations of the hepatic PUFA in CE and in PL were also observed in the lcat–/– mice compared with their wild-type controls. In ldlr–/–xlcat–/– mice, hepatic mRNA level was markedly reduced for {Delta}-6 desaturase (fads2) (70.2%) and acyl-CoA:cholesterol acyltransferase-2 (soat2) (57.0%). A similar pattern of gene expression change was also observed in the lcat–/– single-knockout mice. In contrast, the acyl-CoA:diacylglycerol acyltransferase-2 (dgat2) mRNA level was 1.7-fold upregulated in the double-knockout mice. In summary, we observed coordinated alterations in hepatic expression of the gene for fads2, soat2, and dgat2, resulting in a reduction in total hepatic PUFA pool and differentially in the PUFA-CE pool, in association with an increase in dgat2 gene expression for promoting triglyceride synthesis and secretion. Some of the phenotypes are not readily explained by known mechanisms and may represent novel regulatory pathways.

lecithin:cholesterol acyltransferase; hypertriglyceridemia; polyunsaturated fatty acids; phospholipids; diacylglycerol acyltransferase; acyl-coenzyme A:cholesterol acyltransferase; desaturases

HYPERTRIGYCERIDEMIA IS A RISK FACTOR for atherosclerosis in population studies (28). A modest hypertriglyceridemia is frequently observed in lecithin:cholesterol acyltransferase (LCAT)-deficient subjects, including those heterozygous for the LCAT deficiency syndrome (15). Similarly, hypertriglyceridemia has also been reported in a number of gene-targeted mouse models for LCAT deficiency (25, 27). However, the underlying mechanism for this association remains poorly understood. Most recently, by studying LCAT knockout (lcat–/–) mice bred into the LDL receptor knockout (ldlr–/–) background, we reported that the hypertriglyceridemia seen in these double knockout (DKO) mice was associated with not only reduced postheparin lipoprotein lipase activity but, more intriguingly, a marked hepatic overproduction of triglyceride (TG). Furthermore, we also observed a twofold upregulation of the mRNA for hepatic sterol response element-binding protein-1 (srebp1) and a coordinated upregulation of its program of target lipogenic genes, which includes acetyl-CoA carboxylase-1 (acc1), fatty acyl synthase (fas), and stearoyl-CoA desaturase-1 (scd1) (27). The gene for phosphoenolpyruvate carboxykinase (pepck), a target gene of SREBP1 and a rate-limiting enzyme for hepatic gluconeogenesis, is downregulated in these mice, in association with reduced fasting glucose and insulin levels. We effectively ruled out chronic insulin excess and liver X receptor-{alpha} (LXR{alpha}) activation as potential causes for the observed induction of the srebp1 gene. A recent study on the same ldlr–/–xlcat–/– mice by Furbee et al. (16) revealed a marked reduction in the polyunsaturated fatty acid-cholesterol ester (PUFA-CE), a reciprocal increase in the PUFA-phospholipid (PL) content, and a net decrease in the concentration of PUFA in the circulating apolipoprotein (apo)B-containing lipoproteins in both the ldlr–/–xlcat–/– and lcat–/– mice compared with their respective controls. Several lines of experimental evidence, both in vitro and in vivo, suggest an important role of PUFA in the regulation of the srebp1 gene (17). It is therefore of considerable interest to further investigate the relationship between lipoprotein-derived PUFA-CE and hepatic lipid metabolism. A reduction in the circulating pool of PUFA-CE could compromise the hepatic abundance of PUFA. Depending on the extent of endogenous de novo lipogenesis and PUFA biosynthesis, the latter from the essential fatty acid precursors, a depletion of hepatic PUFA, especially those of 20:4 n6 and 22:6 n3, could modulate the gene transcription activities for a variety of enzymes and transcription factors, including SREBP1 (17). Previous studies suggest that, under physiological conditions, tissue PUFA content is maintained in a narrow range by feedback regulation of the endogenous synthetic pathway, for which desaturation by the rate-limiting enzyme {Delta}6-desaturase represents the first step (24). The gene for {Delta}6-desaturase has been shown to be strongly regulated by a variety of nutritional and hormonal factors, many of which act through the inductive effect of SREBP1 (24).

In the liver, fatty acid moieties either from hydrolysis of CE or from endogenous synthesis are primarily converted to fatty acyl-CoA by the action of acyl-CoA synthetase (ACS) and are made available for PL, CE, and TG synthesis. Sequential actions of glycerol phosphate acyltransferase (GPAT) and acylglycerol phosphate acyltransferase (AGPAT) result in the formation of phosphatidic acid (PA), which can then be used directly as substrate for the synthesis of phosphatidylglycerol (PG), phosphatidylinositol (PI), and cardiolipin. In the endoplasmic reticulum (ER), PA is quantitatively the main substrate for the formation of diacylglycerol (DAG), which lies at the branch point between phosphatidylcholine (PC), phosphatidylethanolamine (PE), and TG synthesis (13).

In TG synthesis, diacylglycerol acyltransferase (DGAT) mediates the final step in the synthetic pathway. DGAT1 mRNA expression in humans is highest in adipose tissue and small intestine (9). dgat1 Knockout mice have higher metabolic rate, insulin- and leptin-sensitivity, and resistance to high-fat diet-induced weight gain (30, 11). DGAT2, cloned recently, is most abundant in liver in both humans and mice and plays a more important role in tissue TG synthesis, including the liver, as evidenced by a profound lipopenia in the dgat2 knockout mice (33). The regulation of both DGAT1 and DGAT2 remain poorly understood.

Acyl-CoA:cholesterol acyltransferase (ACAT) or sterol O-acyltransferase (SOAT) enzymes catalyze the synthesis of CE from free cholesterol and fatty acyl-CoAs. In hepatocytes, CE can be incorporated into apoB lipoproteins for secretion or remain as neutral lipid droplets. In perfused livers of African green monkeys, Carr et al. (8) showed that ACAT inhibition results in a decrease in secretion of CE and apoB. However, the effects of cellular CE synthesis on apoB secretion in cell culture models are conflicting (34, 36). The expression of the two isoforms of ACAT is species and tissue specific. In human livers, SOAT1 is the dominant form (6, 7). SOAT2 is expressed only in the liver and small intestine and is the major ACAT in the liver of adult nonhuman primates (1) and mice (9). The regulation of ACAT is not well understood (6), but SOAT2 mRNA has been shown to be regulated by n3 fatty acids (4) and sterol load (29).

By using both the ldlr–/–xlcat–/– and lcat–/– mice as models for LCAT deficiency and ldlr–/–xlcat+/+ and lcat+/+ as their respective controls, we investigated the alterations in abundance and in fatty acid compositions of PL and CE as well as the mRNA messages of the genes involved in the cellular trafficking of fatty acids for PL and TG synthesis. We observed that, in LCAT-deficient mice, there is a dramatic reduction in hepatic abundance of PUFA-CE, particularly of 20:4-CE and 22:6-CE in conjunction with near-normal levels of the same PUFA in PL species, suggestive of differential utilization of PUFA for PL and CE synthesis. These findings are further associated with changes in the expressions of a number of genes involved in lipid metabolism, which include downregulation of hepatic mRNA levels of {Delta}6-desaturase and soat2 and a concomitant upregulation of the the dgat2 mRNA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Wild-type C57Bl/6 mice were purchased from the Jackson Lab (Bar Harbor, ME). LCAT–/– mice were created in Dr. E. M. Rubin's laboratory (Lawrence Berkeley National Laboratory, Berkeley, CA) as reported previously (25) and had been backcrossed with C57Bl/6 females (Jackson Lab) for more than 7 generations in our lab. Breeding pairs of the ldlr–/–xlcat–/– DKO and their littermate ldlr–/–xlcat+/+ single knockout (KO) mice were kind gifts from Dr. John Parks (16) and propagated by brother-sister matings. All animals were 12 wk or older and fed a chow diet as described previously (26) and its composition is presented in Appendix 1. Age- and sex-matched littermates were used for all experiments. All experimental procedures were approved by the Animal Care Committee at St. Michael's Hospital.

Liver lipid extraction and lipid measurements: electrospray-mass spectrometry of tissue fatty acid compositions. Study mice were fasted overnight before they were euthanized. Livers were surgically removed from anesthetized mice, blotted to remove excess blood, and frozen in liquid nitrogen. Weighed portions of the liver (50–100 mg) were homogenized in a Dounce tissue grinder on ice, followed by a modified method for lipid extraction (3, 14). Briefly, minced liver tissue was homogenized in 2 ml of ice-cold chloroform-methanol-water (1:2:0.3, vol/vol/vol), containing 0.005% butylated hydroxytoluene and internal standards di-C15:0-glycerophosphocholine, di-C14:0-glycerophosphoethanolamine, C17:0-cholesteryl ester, and C13:0-lysoglycerophosphocholine. Standards were added so that the final concentration upon injection was 10, 10, 10, and 2.5 µg/ml, respectively. The solution was transferred to a glass vial and centrifuged at 3,000 g at 4°C for 20 min. The supernatant was collected and stored on ice. The pellet was re-extracted with 1.2ml of chloroform:methanol:water (1:2:0.8, vol/vol), containing 0.005% BHT, vortexed for 30 min and centrifuged as before. After centrifugation, the supernatants were pooled. Chloroform (0.9 ml) was added and mixed. An aliquot of the extraction solution was diluted 1:1 with 10 mM ammonium acetate in chloroform-methanol (1:1, vol/vol) and then injected in an ABI 4000 LC/ESI/MS/MS instrument for lipid analyses based on the method described by Duffin (14). Simultaneously, portions of the mouse liver were used for RNA extraction for semiquantitative RT-PCR (27).

Enzymatic assay of total lipids. Lipids were extracted as described, except for the addition of internal standards. They were redissolved in chloroform and 2% Triton X-100 (vol/vol). After the extract was dried under N2, the lipids were reconstituted in H2O. Total cholesterol, free cholesterol, TG, and TG blank levels were measured by enzymatic assays (Bayer, Tarrytown, NY). The TG level was corrected for free glycerol, determined from the TG blank.

mRNA quantitation of hepatic genes in lipid metabolism. Hepatic mRNA levels of dgat1 and dgat2, soat2, mitochondrial glycerol-3-phosphate acyltransferase (gpam), microsomal triglyceride transfer protein (mtp), and {Delta}6 desaturase (fads2) were analyzed by semiquantitative RT-PCR with gapdh as an internal standard as described previously (27). Briefly, total RNA was extracted using TrIzol (Invitrogen) per the manufacturer's suggested protocol. RT-PCR was performed using the Superscript One-Step RT-PCR Kit (Invitrogen). Intensity of the PCR product for each gene of interest was determined using the Bio-Rad GS800 densitometer normalized to that of gapdh under identical conditions. Primers used for these genes are as follows (forward and reverse, respectively): fads2 (5'-ttctcctcctgtcccacatc; 3'-tctttatgtccgggtccttg), soat2 (5'-tgtcacagaacagggcagag; 3'-tgcaacagcttgctttatgg), dgat1 (5'-gtcaaggccaaagctgtctc; 3'-ggtctccaaactgcagaagc), dgat2 (5'-ctctgtcacctggctcaaca; 3'-gatgcctccagacatcaggt), mtp (5'-gctggaaggcttaattgcag; 3'-tatcgctttctggctgaggt), gpam (5'-agatggtcaaggctgcaact; 3'-acgggtatgacgaggatgtc), hmgcr (HMG-CoA reductase gene; 5'-tggagatcatgtgctgcttc; 3'-gacccaaggaaaccttagcc) gapdh (5'-caaattcaacggcacagtca; 3'-ttgaagtcgcaggagacaac).

Western blot analysis of microsomal soat2 protein mass. Liver samples (50–150 mg) were homogenized in 3 ml of ice-cold buffer containing 0.1 mol/l K2HPO4, 0.25 mol/l sucrose, and 1 mmol/l EDTA, pH 7.4. A protease inhibitor cocktail (Sigma) was added to the buffer before homogenization. The homogenate was then centrifuged for 15 min at 12,000 g (4°C) to remove cell debris. The resulting supernatant was centrifuged for 60 min at 100,000 g. The microsomal pellet from this spin was resuspended in 0.1 mol/l K2HPO4 at pH 7.4 and immediately frozen at –80°C. Protein concentration in this microsomal preparation was determined by means of a bicinchoninic acid protein assay kit (Pierce). For Western blot analyses, 30 µg of microsomal protein were analyzed after suspension in protein solubilization buffer (120 mmol/l Tris, pH 6.8, 20% glycerol, 4% sodium dodecyl sulfate, and bromophenol blue). Dithiothreitol was added to a final concentration of 100 mmol/l, and samples were incubated at 37°C for 30 min. Proteins were separated by 12% SDS-PAGE, transferred to a nitrocellulose membrane, and Western blotted for SOAT using the polyclonal antibody to SOAT2 (Cayman Chemical, Ann Arbor, MI). SOAT signal was detected using the Aurora ECL system (ICN Biomedicals, Costa Mesa, CA).

Statistical analyses. Comparison of group means ± SD was by Student's t-test. Pearson statistics were used to evaluate correlation among data sets with the GraphPad Prism software (GraphPad Software, San Diego, CA), and a two-tailed P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of LCAT deficiency on total hepatic lipids. The hepatic levels of total cholesterol, free cholesterol, and TG in the ldlr–/–xlcat–/– mice, lcat–/– mice, and their respective controls are shown in Table 1. In the ldlr–/–xlcat–/– mice, we observed a 54.1% reduction in free cholesterol, a 61.0% reduction in CE, and a 31.7% reduction in TG contents compared with the ldlr–/–xlcat+/+ controls (Table 1). Likewise, in the lcat–/– mice, we observed a 57.9% reduction in free cholesterol, a 60.0% reduction in CE, and an 88.5% reduction in TG levels compared with their wild-type (lcat+/+) controls (Table 1).


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  		Table 1. Total hepatic lipid analyses

 
Effects of LCAT deficiency on PUFA content of hepatic CE species. The hepatic levels of CE-derived fatty acid species in the ldlr–/–xlcat–/– mice and their control ldlr–/–xlcat+/+ mice are shown in Table 2, top. Those for lcat–/– and their wild-type (lcat+/+) control mice are shown in Table 2, bottom. Compared with their controls, the ldlr–/–xlcat–/– mice showed a 92.9% reduction (0.042 ± 0.008 vs. 0.59 ± 0.12, P = 0.003) in hepatic level of 20:4-CE and an 82.6% reduction (0.062 ± 0.006 vs. 0.36 ± 0.097, P = 0.009) in the level of 22:6-CE. Similar dramatic reductions of the same PUFA species were also observed in the lcat–/– mice. As shown in Table 2, bottom, the lcat–/– mice showed a 98.1% reduction (0.0046 ± 0.0014 vs. 0.234 ± 0.05, P = 0.015) in 20:4-CE and a 92.3% reduction (0.017 ± 0.003 vs. 0.226 ± 0.066, P = 0.03) in 22:6-CE. Collectively, these data suggest that the dramatic reduction in hepatic 20:4-CE and 22:6-CE levels are attributable to LCAT deficiency with only minimal influence by the absence of LDL receptor.


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  		Table 2. Analysis of hepatic CE by ESI/MS/MS

 
In the LCAT-deficient mice, there was also a dramatic reduction in CE of intermediary PUFA in the synthetic pathway, namely 20:3-CE. Compared with their controls, ldlr–/–xlcat–/– mice showed a 68.6% reduction (0.018 ± 0.003 vs. 0.057 ± 0.021, P = 0.034), and the lcat–/– mice showed an 89.6% reduction (0.0017 ± 0.0011 vs. 0.017 ± 0.007, P = 0.054). Although the 16:1 and 18:1 fatty acids are direct products of the enzyme SCD1, the enzyme whose hepatic gene expression level was found to be upregulated in the ldlr–/–xlcat–/– mice (27), the 16:1-CE and 18:1-CE levels in the DKO mice were found to be significantly reduced. We observed a 43% reduction (0.59 ± 0.11 vs. 1.05 ± 0.21, P = 0.022) in the 16:1-CE and a 35% reduction (2.32 ± 0.29 vs. 3.54 ± 0.95, P = 0.007) in 18:1-CE. Similar reductions in hepatic levels of 16:1-CE and 18:1-CE, by 46.2 and 33.1%, respectively, were also observed in the lcat–/– mice, except that the latter did not achieve statistical significance. The total hepatic CE content, as estimated by the sum of all measured CE subspecies, was reduced by 42.7% in the ldlr–/–xlcat–/– mice compared with the ldlr–/–xlcat+/+ controls (9.10 ± 2.20 vs. 5.21 ± 0.58, P = 0.034). Similarly, the total CE content in the lcat–/– mice was also reduced by 47.6% (2.27 ± 0.61 vs. 4.33 ± 10.88, P = 0.001). Changes in the calculated total CE are in good agreement with those determined by the enzymatic method (Table 1).

Effects of LCAT deficiency on PUFA content of hepatic PL species. Hepatic concentrations of the PC species with various fatty acid moieties in the sn-1 and sn-2 positions of the ldlr–/–xlcat–/– mice and their ldlr–/–xlcat+/+ controls are shown in Fig. 1A. Unlike what was observed for CE, there is no dramatic change in hepatic concentration of any PC subspecies between the two genotypes, including those containing PUFA 20:4 and 22:6. The two most abundant 20:4-containing species, namely 16:0/20:4 and 18:0/20:4, are 12 and 32% more abundant in the ldlr–/–xlcat–/– mice than their ldlr–/–xlcat+/+ controls, with P values being 0.003 and 0.0002, respectively. Meanwhile, statistically significant differences have also been observed for 16:0/18:0, 16:0/18:1, and 18:1/18:2, showing reductions of 16, 13, and 22%, respectively. Total hepatic PC concentrations are not significantly different between the two genotypes. Similar changes were also noted for the lcat–/– mice compared with their wild-type controls (Fig. 1B). The total hepatic tissue PC concentrations of the lcat–/– mice are not significantly different from those of their wild-type controls (P = 0.26). The 20:4-containing PCs, namely, the 16:0/20:4 and 18:0/20:4 PCs, were respectively 18 and 28% higher in the lcat–/– than in the wild-type controls, but the differences did not achieve statistical significance (P = 0.056 and 0.06, respectively).



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  		Fig. 1. Analysis of hepatic phosphatidylcholine by ESI/MS/MS. A: ldlr–/–xlcat–/– vs. ldlr–/– mice (n = 4 each). B: lcat–/– (n = 7) vs. lcat+/+ (n = 6) mice. *P < 0.05; **P < 0.005.

 
Hepatic concentrations of the PE species with various fatty acid moieties in the sn-1 and sn-2 positions of the ldlr–/–xlcat–/– mice and their ldlr–/–xlcat+/+ controls are shown in Fig. 2A. The total hepatic PE concentrations were not significantly different between the ldlr–/–xlcat–/– and their ldlr–/–xlcat+/+ controls (5.27 ± 0.24 vs. 5.11 ± 0.14, P = 0.30). Likewise, the total hepatic tissue PE concentrations of the lcat–/– mice were also not significantly different from their wild-type controls (P = 0.46; Fig. 2B). Among the individual subspecies, in ldlr–/–xlcat–/– mice statistically significant differences have been observed for 16:0/18:0, 16:0/18:1, 18:0/18:1, 18:0/18:3, and 16:0/22:6 (27, 22, 20, and 25% reductions and a 12% increase, respectively; Fig. 2).



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  		Fig. 2. Analysis of hepatic phosphatidylethanolamine by ESI/MS/MS. A: ldlr–/–xlcat–/– vs. ldlr–/– mice (n = 4 each). B: lcat–/– (n = 7) vs. lcat+/+ (n = 6) mice. *P < 0.05.

 
Hepatic mRNA level determinations. The hepatic mRNA levels for {Delta}6 desaturase, the rate-limiting enzyme for the synthesis of PUFA, including 20:4 and 22:6 from their essential fatty acid precursors, were found to be 70.2% reduced (0.301 ± 0.041 vs. 1.015 ± 0.190, P < 0.0001) in the ldlr–/–xlcat–/– mice compared with their ldlr–/–xlcat+/+ controls (Fig. 3). Similarly, a 70% reduction (P < 0.0001) in the normalized hepatic {Delta}6 desaturase was also observed in the lcat–/– mice (Fig. 3A). The mRNA for the mitochondrial GPAT gene gpam was not significantly different between the two groups (1.92 ± 0.24 vs. 1.91 ± 0.10, P = 0.47). The soat2 mRNA levels were reduced by 57.0% in the ldlr–/–xlcat–/– mice (0.204 ± 0.086, P = 0.005) and a 57% reduction in the lcat–/– mice (P = 0.01; Fig. 3B). Of the two gene variants involved in the final esterification step of TG synthesis, the mRNA levels of dgat2 were 1.70-fold upregulated in the DKO mice (0.828 ± 0.139 vs. 0.488 ± 0.081, P = 0.0013), but the dgat1 mRNA levels were not significantly different (0.667 ± 0.151 vs. 0.737 + 0.157, P = 0.234). In the lcat–/– mice, there was no difference in the mRNA level for dgat2 (0.97-fold, P = 0.91) and there was a 1.4-fold increase in dgat1, which was not statistically significant (P = 0.38). On the other hand, the mtp gene mRNA level was unaffected in the DKO mice (1.090 ± 0.560 vs. 1.163 ± 0.502, P = 0.408) but was reduced by 42% in the lcat–/– mice (P = 0.01; Fig. 3).



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  		Fig. 3. Hepatic mRNA levels by semiquantitative RT-PCR after normalization to that of gapdh gene. Expression of genes in fatty acid metabolism, triglyceride synthesis, and neutral lipid transfer. A: ldlr–/–xlcat–/– (n = 5) vs. ldlr–/– (n = 6) mice. B: lcat–/– (n = 5) vs. lcat+/+ (n = 5) mice.

 
The hmgcr gene mRNA level was upregulated 1.78-fold in the DKO mice (1.94 ± 0.41 vs. 1.09 ± 0.52, P = 0.016). A similar 1.83-fold increase (P = 0.013) was observed in the lcat–/– mice. The srebp1 gene mRNA was upregulated 3.1-fold in lcat–/– mice (0.1226 ± 0.047 vs. 0.042 ± 0.03, P = 0.017), in agreement with the twofold upregulation in the DKO mice previously reported (27).

Hepatic microsomal SOAT2 protein levels. Western blot analysis showed a significant 45.0% reduction (P = 0.025) in SOAT2 protein mass in the ldlr–/–xlcat–/– mice and a similar 65.7% reduction in the lcat–/– mice (P = 0.04) compared with their respective controls (Fig. 4).



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  		Fig. 4. Hepatic SOAT2 mass by Western blot in ldlr–/–xlcat–/– vs. ldlr–/–xlcat+/+ mice and in lcat–/– vs. lcat+/+ mice. Equal amounts of total proteins were loaded in each lane.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we present novel observations of selective distribution of PUFA into complex lipids in the liver of LCAT-deficient mice in association with coordinated alterations of gene expressions. In both the ldlr–/–xlcat–/– and lcat–/– mice, we observed matching dramatic reductions in hepatic PUFA-CE but relatively preserved PUFA contents of PL species in association with an upregulation of hepatic srebp1 mRNA and alterations of the hepatic expression of genes involved in fatty acid metabolism and TG and PL biosynthesis. Although the hepatic srebp1 gene upregulation may in part be attributable to a reduction in the hepatic PUFA pool, alterations of the expression of a number of other genes pertinent to hepatic lipid metabolism observed in this study suggest presence of novel regulatory pathways. The metabolic pathways of interest to the present work are outlined in Fig. 5.



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  		Fig. 5. Integrative metabolic framework of the present study.

 
In a previous study (27), we reported that the hypertriglyceridemia seen in the ldlr–/–xlcat–/– mice is associated with a coordinated upregulation of the hepatic lipogenic genes and the gene for their upstream transcription factor SREBP1. A nearly threefold upregulation of the hepatic srebp1 gene has also been observed in the lcat–/– single-KO mice, suggestive of an effect directly attributable to LCAT deficiency. In the case of the ldlr–/–xlcat–/– mice, we also ruled out hyperinsulinemia and LXR{alpha} induction as causative factors (27). Several lines of experimental evidence support the inhibitory role of PUFA in the transcriptional regulation of SREBP1 and the lipogenic genes. In light of the previous report by Furbee et al. (16) of a marked reduction of PUFA-CE in the circulating apoB-containing lipoproteins in both the ldlr–/–xlcat–/– and lcat–/– mice, we further explored the putative mechanistic links between these lipoprotein compositional changes and hepatic TG overproduction by examining the hepatic lipid compositions and gene expression levels of their modulating enzymes. In ldlr–/–xlcat–/– mice, we observed a nearly complete depletion of both hepatic 20:4-CE and 22:6-CE moieties compared with the control group. On the contrary, the 20:4 and 22:6 contents in both hepatic PC and PE were either unchanged or only slightly elevated. To rule out the possibility that the changes in hepatic CE profile observed were a direct effect of the absence of LDL receptors, we measured the same hepatic lipid species in lcat–/– mice by use of age- and sex-matched lcat+/+ (wild-type) mice as controls. We observed similar dramatic reductions in 20:4-CE and 22:6-CE in the lcat–/– mice.

In addition to the dramatic reductions in PUFA-CE in the LCAT-deficient models, all other CE species were also found to be significantly reduced, albeit to lesser extents. The significant reductions in total hepatic CE contents as seen in both LCAT-deficient models may be the result of a combination of several factors. 1) Reduced availability of lipoprotein-derived cholesterol. In the case of the ldlr–/–xlcat–/– DKO mice, both LDL receptor-mediated uptake of LDL-cholesterol and scavenger receptor class B type I-mediated uptake of HDL-cholesterol are compromised. In the case of the lcat–/– single-KO mice, the low levels of apoB-containing lipoproteins and HDL also limit the availability of lipoprotein-derived cholesterol. The upregulation of hepatic hmgcr seen in both LCAT strains and the previously reported upregulation of ldlr gene in the lcat–/– single-KO mice (18) are both suggestive of a reduced regulatory pool of cholesterol in LCAT deficiency. This is consistent with the observed reductions in hepatic free cholesterol levels in both LCAT-deficient strains (Table 3). 2) Decreased SOAT2-mediated esterification. Our observed marked reductions in the mRNA level of hepatic soat2 gene expression (Fig. 3) and its protein levels are consistent with this notion. Several lines of experimental evidence suggest that regulation of hepatic soat2 gene plays a key role in defending its cellular cholesterol content (29, 32). Experimentally induced sterol depletion in rats resulted in a reduction in hepatic SOAT activity along with a reduction in CE content without a concomitant increase in neutral cholesterol ester hydrolase activity (32). The downregulation of soat2 in both LCAT deficient strains may therefore represent a cellular response to a relative depletion of sterols in the regulatory pool. 3) Increased utilization of CE for VLDL assembly and secretion. Although hepatic overexpression of SOAT has been shown to cause an increase in VLDL production and secretion in experimental animal models (31), we observed instead similar reductions in soat2 mRNA and SOAT2 protein mass in both strains of LCAT-deficient mice. In conjunction with the previous reports that PUFA incorporates poorly into TG, our data make the selective channeling of of PUFA from CE to TG synthesis an unlikely scenario.


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  		Table 3. Nutritional content of rodent chow Diet

 
Our observation of a significant downregulation of hepatic soat2 gene and the protein mass in both strains of LCAT-deficient mice raises the possibility of cross talk between cholesterol and fatty acid metabolism in LCAT deficiency. Several lines of experimental evidence support the notion that SOAT2 competes for the same pool of lipoprotein-derived fatty acids (19). A reduced SOAT2 activity would generally have provided more fatty acids for PL synthesis. Because PUFA have been shown to be relatively poor substrates for esterification by SOAT2 (10), the effect of a reduction in SOAT2 activity on the observed differential distribution of PUFA between PL and CE could be relatively minor.

In the ldlr–/–xlcat–/– DKO mice, in addition to our previously reported upregulation of hepatic srebp1 gene along with its downstream lipogenic genes (27), we also observed an upregulation of dgat2, the gene encoding for the hepatic isoform of DGAT for mediating the final step of TG synthesis. This observation may further account for the observed increase in hepatic TG production in these mice as previously reported (27). The concomitant observation of a reduced soat2 mRNA and the corresponding protein mass would support our previous observation that the secreted VLDL is relatively TG rich but CE poor (27). The regulation of dgat2 gene expression remains poorly understood (22). Increased expression of the gene for DGAT has been shown to promote VLDL secretion in the McA RH7777 cell line, but it also results in cellular accumulation of TG (20). In leptin-deficient (ob/ob) mice, a mouse model of diet-induced obesity and insulin resistance, both hepatic dgat1 and dgat2 activities are increased more than twofold compared with wild-type controls in conjunction with an upregulation of hepatic srebp1 gene (35) despite an absence of the sterol response element in their promoters. These mice also develop increased hepatic lipogenesis, accumulation of hepatic TG, and hepatic VLDL overproduction. Hepatic microsomal transfer protein (mtp) mRNA is also upregulated in this model (2). In the ldlr–/–xlcat–/– mice, despite the shared phenotypes of srebp1 upregulation and increased hepatic TG production, the hepatic mRNA level of mtp was unchanged from the ldlr–/–xlcat+/+ controls (Fig. 4) in conjunction with a significant reduction in hepatic TG content, further distinguishing the phenotypic profile in this model of LCAT-deficient mice from those seen in the insulin-resistant ob/ob models, indicative of distinct regulatory pathways.

In the lcat–/– single-KO mice, despite a comparable upregulation of the srebp1 gene in the liver, we observed a more dramatic reduction in the hepatic TG content, and we did not observe any change in either the dgat1 or dgat2 mRNA level. The observed increase in dgat2 mRNA in the ldlr–/–xlcat–/– DKO mice may therefore reflect a novel interaction between the absence of both the LDL receptor and the LCAT enzyme. Likewise, the reduction in hepatic TG content in the face of increased lipogenesis and TG production suggests a role for additional regulatory factors.

PUFA circulate mainly in the form of PL and CE, with those in TG and nonesterified fatty acid (NEFA) forming a minor component (35). The nearly complete absence of PUFA-CE in the apoB-containing lipoproteins in both ldlr–/–xlcat–/– and lcat–/– mice would likely impact on the hepatic abundance of PUFA as exogenous source. Previous studies have shown that PUFA-enriched diets lead to a suppression of the nuclear form of SREBP1, as well as a reduction in the srebp1 mRNA level, in association with a reduced expression of hepatic {Delta}6 desaturase (12), the latter probably through suppression of srebp1 (17). On the contrary, depletion of dietary PUFA results in induction of a panel of gene changes including {Delta}6 and {Delta}5 desaturases, the rate-limiting enzymes for the endogenous synthesis of PUFA. We are somewhat surprised by the observed significant reduction the mRNA levels of {Delta}6 desaturase in LCAT-deficient mice. In light of the important physiological role of {Delta}6 desaturase in providing the cellular PUFA through mediating the conversion from dietary essential fatty acids, this reduction may contribute in part to the selective dramatic reduction in 20:4-CE and 22:6-CE. The reason for the marked downregulation of the hepatic {Delta}6 desaturase mRNA in these mice, despite a coordinated upregulation of hepatic srebp1 gene (4) and its target lipogenic genes, remains obscure (21, 23). Other known causes of reduced {Delta}6 desaturase mRNA include insulin deficiency, but again likely through the suppression of srebp1. However, the observed modest reduction in fasting insulin in the ldlr–/–xlcat–/– mice (27) is unlikely to completely account for the significant reduction in {Delta}6 desaturase mRNA level. Glucagon, epinephrine, glucocorticoids, and mineralocorticoids have also been shown to downregulate {Delta}6 desaturase mRNA (32) but their role in the current scenario requires confirmation. The increased PL-PUFA in circulating apoB lipoproteins in the LCAT-deficient mice (16) may also play a role. However, a mechanism by which the PL pool of PUFA would selectively regulate {Delta}6 desaturase remains to be elucidated. The finding of the downregulation of {Delta}6 desaturase, therefore, may represent a potential novel regulatory pathway.

In summary, in our investigation of the underlying mechanism of hepatic TG overproduction in LCAT-deficient mice, we reported additional novel findings of a selective channeling of hepatic PUFA for PL biosynthesis at the expense of a markedly depleted PUFA-CE pool. A combined reduction in gene expression levels of soat2 and fads2 could account for the reduction in overall CE level and, more dramatically, the hepatic PUFA-CE level, respectively. These changes would in turn suggest an overall reduction in the unesterified PUFA pool, consistent with the notion of a reduced inhibitory potential on srebp1 mRNA turnover. The observed upregulation of the dgat2 gene in the ldlr–/–xlcat–/– DKO mice may act in concert with increased lipogenesis to promote TG synthesis and secretion. However, the marked reduction in hepatic TG content in both LCAT-deficient strains suggests the involvement of additional regulatory pathways. Likewise, there is no known direct inductive effect of SREBP1 on the upregulation of soat and the dgat genes or on the downregulation of the {Delta}6 desaturase gene. Collectively, our findings suggest the existence of novel regulatory pathways that are responsive to the quality, quantity, and tissue redistribution of hepatic PUFA.

APPENDIX 1


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants-in-aid from the Heart and Stroke Foundation of Ontario (nos. N4124 and N5189 to D. S. Ng and no. T4948 to P. W. Connelly).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. S. Ng, St. Michael's Hospital, West Annex 2-015, 38 Shuter St., Toronto, ON M5B 1A6, Canada (e-mail: ngd{at}smh.toronto.on.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* These authors contributed equally to the paper. Back


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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