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J. Biol. Chem., Vol. 279, Issue 9, 7636-7642, February 27, 2004

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Hypertriglyceridemia in Lecithin-cholesterol Acyltransferase-deficient Mice Is Associated with Hepatic Overproduction of Triglycerides, Increased Lipogenesis, and Improved Glucose Tolerance^*

Dominic S. Ng, Chunhui Xie, Graham F. Maguire, Xianghong Zhu, Francisca Ugwu, Eric Lam, and Philip W. Connelly

From the Department of Medicine, St. Michael's Hospital, Toronto, Ontario M5B 1A6, Canada

Received for publication, August 26, 2003 , and in revised form, December 9, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Lecithin-cholesterol acyltransferase deficiency is frequently associated with hypertriglyceridemia (HTG) in animal models and humans. We investigated the mechanism of HTG in the ldlr–/– x lcat–/– (double knockout (dko)) mice using the ldlr–/– x lcat+/+ (knock-out (ko)) littermates as control. Mean fasting triglyceride (TG) levels in the dko mice were elevated 1.75-fold compared with their controls (p < 0.002). Both the very low density lipoprotein and the low density lipoprotein/intermediate density lipoprotein fractions separated by fast protein liquid chromatography were TG-enriched in the dko mice. In vitro lipolysis assay revealed that the dko mouse very low density lipoprotein (d < 1.019 g/ml) fraction separated by ultracentrifugation was a more efficient substrate for lipolysis by exogenous bovine lipoprotein lipase. Post-heparin lipoprotein lipase activity was reduced by 61% in the dko mice. Hepatic TG production rate, determined after intravenous Triton WR1339 injection, was increased 8-fold in the dko mice. Hepatic mRNA levels of sterol regulatory element binding protein-1 (srebp-1) and its target genes acetyl-CoA carboxylase-1 (acc-1), fatty acid synthase (fas), and stearoyl-CoA desaturase-1 (scd-1) were significantly elevated in the dko mice compared with the ko control. The hepatic mRNA levels of LXR (lxr) and its target genes including angiopoietin-like protein 3 (angptl-3) in the dko mice were unchanged. Fasting glucose and insulin levels were reduced by 31 and 42%, respectively in the dko mice, in conjunction with a 49% reduction in hepatic pepck-1 mRNA (p = 0.014). Both the HTG and the improved fasting glucose phenotype seen in the dko mice are at least in part attributable to an up-regulation of the hepatic srebp-1c gene.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Hypertriglyceridemia (HTG)¹ is a risk marker of coronary heart disease, particularly when it is associated with low levels of high density lipoprotein cholesterol (HDL-C), insulin resistance, and other features of metabolic syndrome (1). Known examples include diabetic dyslipidemia, insulin resistance, metabolic syndrome (2) and familial combined hyperlipidemia (3). In most of these cases, HTG is a common cause of low plasma levels of HDL-C. On the other hand, it is not known whether primary low HDL-C states may contribute to HTG.

Lecithin-cholesterol acyltransferase (LCAT) plays a central role in reverse cholesterol transport. The primary lipoprotein defect in LCAT deficiency is severe HDL deficiency, and many such subjects are frequently moderately hypertriglyceridemic (4, 5). Gene-targeted mice deficient in LCAT activities are associated with HTG in a gene dose-dependent manner (6, 7). Transgenic mice (8) and rabbits (9) overexpressing human LCAT gene have been reported to have a modest reduction in fasting plasma triglycerides (TG). These observations suggest that LCAT may play an important role in modulating plasma TG levels, but the underlying mechanisms have not been explored.

Lipoprotein lipase (LPL) plays a critical role in the lipolysis of TG in both very low density lipoprotein (VLDL) and chylomicrons. Complete low density lipoprotein (LDL) deficiencies caused by genetic mutations of the LPL gene or its cofactor apoC-II are associated with severe chylomicronemia. Partial LPL deficiency in patients heterozygous for LPL mutations is variably associated with HTG, low HDL-C, and accelerated atherosclerosis (10). In mice, homozygous LPL knock-out is perinatally lethal. Heterozygous LPL knock-out mice (lpl+/–) have been found to be moderately hypertriglyceridemic (11). In this model, LPL was also found to participate in glucose homeostasis. The lpl+/– mice developed relative hypoglycemia in conjunction with an elevated fasting insulin, the latter attributable to an increased secretion of insulin by the pancreatic -cells without any associated peripheral insulin resistance (11). The mechanism for the insulin oversecretion in the lpl+/– mice remains obscure.

Hepatic overproduction of TG predisposes to HTG in mammals. Common causes of increased hepatic TG synthesis include increased free fatty acid flux into the hepatocytes, as seen in obesity, and increased hepatic de novo lipogenesis, as seen in alcohol or carbohydrate-induced HTG. Recent studies suggest that enzymes participating in hepatic fatty acid synthesis are coordinately regulated by sterol regulatory element-binding protein (SREBP) at the transcriptional level, predominantly by the two isoforms of the SREBP1 gene, SREBP1a and SREBP1c, with the latter being considerably more abundant in the liver (12). Both insulin and liver X receptor (LXR) have been shown to be potent inducers of SREBP1 gene transcription (13). Insulin may induce SREBP1 gene transcription directly even in insulin-resistant states, which may account for the common occurrence of the strong association between insulin resistance and HTG. LXR ligands, both endogenous and exogenous, have been shown to cause up-regulation of the SREBP1 gene by targeting directly through the LXR response element. SREBP1 activity may also be suppressed by polyunsaturated fatty acids (PUFA) at both the transcriptional (14, 15) and mRNA stability (16) levels. More intriguingly, a number of genes known to modulate hepatic gluconeogenesis, including the rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK), have been shown to be directly and negatively regulated by LXR (17) and SREBP1c (18). We investigated the possible mechanism of LCAT deficiency-associated HTG in the LDL receptor knock-out (ldlr–/–) mouse background. We also explored the possible association of HTG with altered glucose metabolism and insulin resistance in this mouse model.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Animals—Breeding pairs of the ldlr–/–x lcat–/– double knock-out (dko) and their littermate ldlr–/– x lcat+/+ single knock-out (ko) mice were a kind gift from Dr. John Parks (19). Housing and all brothersister matings were carried out at the Vivarium, St. Michael's Hospital. All animals were 4 months or older and fed a chow diet as described previously (20). Age- and gender-matched littermates were used for all experiments. All experimental procedures were approved by the Animal Care Committee at St. Michael's Hospital.

Plasma Lipid Analyses—Plasma lipid analyses were performed on mice 6–8 months of age. Plasma was obtained as described previously (21). Fast protein liquid chromatography (FPLC) fractionation on total plasma was performed on a Superose 6HR column (10 mm x 30 cm) (Amersham Biosciences) (22). Plasma and Superose fractions were analyzed on an RA-1000 (Bayer Diagnostics) using enzymatic assays for total cholesterol, TG, glycerol blank, free cholesterol, and phospholipid.

In Vitro Lipolysis Assay—The lipolysis assay was adapted from the published method by Jong et al. (24). VLDL plus intermediate density lipoprotein (IDL) was isolated from pooled (n = 7) mouse plasma by ultracentrifugation at d < 1.019 g/ml (23). The VLDL plus IDL fraction was incubated with 0.2 unit of bovine lipoprotein lipase (Sigma) at 37 °C in the presence of FFA-free 3% bovine serum albumin. The reaction was stopped by the addition of 0.1% Triton X-100 in KH₂PO₄ at pH 6.9.

FFA released from the reaction was measured using a commercially available enzymatic colorimetric kit (NEFA Wako Chemicals USA, Inc.). Four different concentrations of VLDL-TG in the range of 0.1–0.6 mM were used. FFA released/min/liter against blank (reaction stopped without addition of LPL) were obtained, and all data points were means of duplicates.

Post-heparin Lipase Activities (PHLA)—The assay of post-heparin total lipase and hepatic lipase activities was adapted from a previously published method (25) for mouse samples. Briefly, after an overnight fast, mice 6–8 months of age were injected individually with heparin (LEO Pharmaceuticals, Inc., Ontario) intraperitoneally at 100 IU/kg. Blood was drawn 30 min postinjection, and the plasma samples from three or four gender-mixed animals were pooled and grouped by genotypes. Analyses were done on three pools of animals for each genotype. Aliquots of 5, 10, and 25 µl of pooled plasma were used for total lipase and 10, 30, and 50 µl for hepatic lipase determinations. Protamine sulfate was used as LPL inhibitor for the hepatic lipase assay. Frozen normal human sera were used as activator. FFA were then extracted and analyzed as described above.

TG Production Rate—In vivo determination of the TG production rate was carried out in 8–10-month-old, gender-matched mice. Serial measurements of plasma TG over 90 min were determined after a single dose tail vein injection of Triton WR1339 at 500 mg/kg body weight at 15% w/v in saline.

mRNA Quantitation of Hepatic Genes in Lipid and Glucose Metabolism—Study mice were fasted overnight before sacrifice. Hepatic mRNA levels of acetyl-CoA carboxylase-1 (acc-1), fatty acid synthase (fas), SREBP1 (srebp-1), angiopoietin-like protein 3 (angptl3), stearoyl-CoA desaturase-1 (scd-1), acyl-CoA oxidase (axo), carnitine palmitoyltransferase-1 (cpt-1), and peroxisomal proliferator activator receptor (ppar) were measured using semiquantitative RT-PCR with GAPDH (gapdh) as internal standard. Total RNA was extracted using TriZol (Invitrogen) as per the manufacturer's suggested protocol, and the purified mRNA product was snap frozen at –86 °C until use. RT-PCR was performed using the Superscript One-step RT-PCR kit (Invitrogen). The number of cycles for RT-PCR was optimized for each gene by selecting the cycle number within the exponential phase. The RT-PCR program was initiated by heating the sample at 50 °C for 30 min, 94 °C for 2 min, followed by 24–28 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min. The PCR finished with heating at 72 °C for 5 min. Intensity of the PCR product for each gene of interest was determined using the Bio-Rad GS800 densitometer normalized to that of gadph under identical conditions. Primers for the genes are: ACC-1 (5'-gggacttcatgaatttgctg; 3'-gtcattaccatcttcattacctca), FAS (NM017332) (5'-ggctttggcctggaactg; 3'-gaaggctacacaagctccaaa), SREBP1 (NM004176) (5'-tcaacaaccaagacagtgacttcc; 3'-gttctcctgcttgagtttctggtt), SCD-1 (NM_009127 [GenBank] ) (5'-ttcttacacgaccaccacca; 3'-gcgttgagcaccagagtgta), Angptl3 (XM_131498) (5'-attcaacaccggaaagatgg; 3'-tggagcatcattttggatga), CPT-1 (NM_013495 [GenBank] ) (5'-tccatgcataccaaagtgga; 3'-tcatcagtggcctcacagac), AXO (AF006688 [GenBank] ) (5'-ccgtctgcagcatcataaca; 3'-taaggcgccagtctgaaatc); GAPDH (NM008084) (5'-caaattcaacggcacagtca; 3'-ttgaagtcgcaggagacaac), PPAR (NM01144) (5'-ccagtactgccgttttcaca; 3'-cctctgcctctttgtcttcg), LXR (AJ132601 [GenBank] ) (5'-ggatagggttggagtcagca; 3'-cttgccgcttcagtttcttc); PEPCK-1 (NM_011044 [GenBank] ) (5'-aagttgcccaagatcttcca; 3'-taagggaggtcggtgttggac).

Fasting Plasma Insulin and Glucose Tolerance Test—Study mice (n = 9 for both dko and age- and gender-matched ko control) at 4–6 months old were fasted overnight before we obtained plasma samples for the assay. Insulin level was determined using the Linco rat/mouse insulin enzyme-linked immunosorbent assay kit as per the manufacturer's protocol (Cedarlane Laboratories, Inc., Ontario). The glucose tolerance test was performed by an intraperitoneal injection of glucose at 1.125 g/kg and blood glucose monitored every 20 min for 2 h. The blood glucose level was determined using an Accu-Chek Compact glucometer (Roche Applied Science) from tail bleed samples.

Statistical Analyses—Comparison of group mean ± S.D. was by Student's t test. Pearson statistics were used to evaluate correlation among data sets using the GraphPad Prism software (GraphPad Software Inc., San Diego), and a two-tailed p value of less than 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Lipid and Lipoprotein Analyses—As seen in Table I, we observed a significant 1.75-fold increase in fasting plasma TG levels in the dko mice compared with its ko controls. As expected, the total plasma free cholesterol:CE ratio is significantly higher in the dko mice as described previously (19). Upon separation by FPLC, the cholesterol level was significantly higher in the VLDL fractions and moderately reduced in the LDL/IDL fractions in the dko mice (Fig. 1). Not surprisingly, cholesterol levels of the HDL fractions are extremely low in these mice. Analysis of fasting TG after separation by FPLC revealed that most of the excess TG seen in the dko mice was concentrated in the VLDL fractions, and the LDL and IDL particles in the dko mice were also moderately enriched in TG (Fig. 1). The TG content in HDL fractions was extremely low in both genotypes. The distribution of phospholipids was comparable with that of cholesterol for both genotypes.

View larger version (17K): [in this window] [in a new window]   FIG. 1. FPLC fasting lipid profile of pooled plasmas from ldlr–/–x lcat–/– (dko) (n = 3) and ldlr–/– x lcat+/+ (ko) mice (n = 3).

View larger version (15K): [in this window] [in a new window]   FIG. 2. In vitro lipolysis assay of VLDL (d < 1.019 g/ml) from pooled plasma from ldlr–/– x lcat–/– (dko) (n = 3) and ldlr–/– x lcat+/+ (ko) mice (n = 3) using bovine LPL (Sigma).

View larger version (13K): [in this window] [in a new window]   FIG. 3. PHLA assays of ldlr–/– x lcat–/– (dko) (n = 3 pools) and ldlr–/– x lcat+/+ (ko) (n = 3 pools). LPL activity is obtained from total PHLA – hepatic lipase (HL) activity.

View larger version (18K): [in this window] [in a new window]   FIG. 4. In vivo TG production rate after Triton WR1339 intravenous injection on both ldlr–/– x lcat–/– (dko) (n = 8) and ldlr–/– x lcat+/+ (ko) mice (n = 8).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Hepatic mRNA levels by semiquantitative RT-PCR after normalization to that of gapdh gene (n = 5 for each genotype). a, expressions of lxr, its dependent genes and lipogenic genes; b, expressions of ppar and its dependent genes.

View larger version (19K): [in this window] [in a new window]   FIG. 6. a, fasting insulin levels of both ldl–/– x lcat–/– (dko) (n = 8), hatched bar; and ldlr–/– x lcat+/+ (ko) control mice (n = 11) (open bar). b, hepatic mRNA levels of pepck-1 by semiquantitative RT-PCR after normalization to that of gapdh (n = 5 for each genotype). c, glucose tolerance test after an intraperitoneal injection of glucose at 1.125 g/kg of body weight on ldlr–/– x lcat–/– (dko) (n = 4) and ldlr–/– x lcat+/+ (ko) mice (n = 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HTG has been consistently documented in humans with the rare complete LCAT deficiency syndrome (4, 5). In rodent models, there is an inverse relationship between fasting plasma TG levels and the murine lcat gene dose (6). Furthermore, transgenic mouse and rabbit models with overexpression of the human LCAT gene have been found to have a modest reduction in fasting TG (8, 9). The mechanism by which LCAT modulates TG metabolism has not been fully addressed. Decreased PHLA were found in some but not all subjects with LCAT deficiency (5, 26). However, a possible role of hepatic TG overproduction has not been explored. Based on existing published data on LCAT-deficient mice, fasting HTG is most consistently seen in the LDLR/LCAT dko mice. Unlike the apoE knock-out mouse model, the LDLR ko mice develop significant hyperlipidemia without the confounding effect of the absence of apoE on hepatic TG production (27, 28). In the absence of the LDL receptors, the hepatic uptake and endocytosis of circulating apoB-48-enriched VLDL particles via the LDL receptor related protein are significantly attenuated but are sufficient to avoid their accumulation in the circulation (29). Recent studies in mice examining the metabolic effect of exogenous LXR agonists suggest that an increased hepatic TG production alone is inadequate to achieve a sustained HTG without the simultaneous presence of a lipoprotein clearance defect (30). These aforementioned considerations make the LDLR/LCAT dko mouse an attractive in vivo model for studying the altered TG metabolism in LCAT deficiency.

Our first observation was the excess plasma TG in the dko mice being distributed throughout all non-HDL FPLC fractions, but disproportionately so in the VLDL subfractions (Fig. 1). Although previous theoretical considerations (31) suggest that LCAT may participate in the lipolytic process, our in vitro lipolysis assay findings do not support this notion. The data, rather, suggest that the dko mouse VLDL appear to be more effective substrates for lipoprotein lipase. The increase in the in vitro lipolytic efficiency in the dko mouse VLDL could therefore be a reflection of the relative TG enrichment of these lipoprotein particles. On the other hand, our finding of a significant reduction in PHLA in the dko mice suggests that a lipolytic clearance defect may at least in part contribute to the observed fasting HTG seen in these mice.

Modest HTG has been reported in a number of severe low HDL syndromes including complete LCAT deficiency, Tangier disease, and apoA-I deficiency (5, 32, 33). However, the causative role of the primary low HDL defects in the development of HTG remains obscure. In rodent models, the apoA-I knock-out mice were found to be HTG in the human apolipoprotein B-100 transgenic background only when fed a Western-type diet (34), and the HTG was attributable to a reduced PHLA but not hepatic TG overproduction. To date, there has been no report of primary monogenic low HDL syndrome-associated HTG being attributable to a hepatic lipoprotein overproduction.

Of surprise is the novel observation that hepatic TG production rate is increased markedly in the dko mice (Fig. 4). We were therefore prompted to investigate the potential role of hepatic fatty acid synthesis in the enhanced TG production rate. We observed a coordinated up-regulation of the expression of three lipogenic genes, acc-1, fas, and scd-1, in the liver, all encoding for key enzymes in the lipogenic cascade. Recent studies by a number of laboratories have elucidated the role of SREBP1 and SREBP2 in the regulation of intracellular lipid metabolism. The two splice variants of the SREBP1 gene have both been implicated to play a major role in the transcriptional activation of lipogenic genes in a coordinated manner (35). The observed up-regulation of the srebp-1 gene in the dko mice may explain the observed up-regulation of the acc-1, fas, and scd-1 genes. The increase in TG production may therefore be, at least in part, attributable to an enhanced lipogenesis mediated by an increased production of SREBP1.

To explore the mechanism underlying the observed up-regulation of the srebp-1 gene, we considered the possible contributions by its three major modulators, namely, insulin, LXR, and PUFA (12). Insulin confers a direct transcriptional activation of the SREBP1c gene and an increase in the synthesis of the SREBP1c precursor (36). In rodents, this stimulatory effect persists even when many other target tissues were rendered extremely insulin-resistant. Consequently, the hyperinsulinemia that develops in the presence of peripheral insulin resistance will continue to promote accelerated hepatic lipogenesis, contributing to TG overproduction (37). Our unexpected observation of a modest reduction in fasting insulin in the dko mice effectively ruled out chronic fasting hyperinsulinemia as a cause of the up-regulation of the srebp-1c gene expression.

It has been well established that SREBP1 is one of the target genes for LXR, mediating the action of LXR on lipogenesis and glucose metabolism. Through transactivation of its target genes, ligand binding of LXR has also been shown to play important roles in lipid (SREBP1, FAS), glucose (PEPCK, glucose-6-phosphatase, PGC1, GLUT4), lipoprotein (LPL, CETP, Angptl-3), bile acid (CYP7) metabolism, and lipid flux (ABCA1, ABCG5/8) (38). A number of lipoprotein-modulating proteins, which include LPL, CETP, and Angptl-3, have recently been shown to be LXR-responsive at the transcriptional level through binding to the LXR response elements (39–41). Angptl-3 is a hepatically derived secretory protein with one of its major functions being an inhibitor of lipoprotein lipase. Treatment of mice with pharmacologic dose of T0901317, an LXR ligand, showed differential response in the target genes, including a 1.5-fold increase in the angptl3 mRNA level in conjunction with a 4-fold increase in srebp-1c. Therefore, the lack of up-regulation of the angptl-3 gene in the dko mice is suggestive of an absence of a significant LXR-mediated transactivation of target genes in the dko mouse liver compared with the ko control.

The nuclear receptor PPAR plays a key role in the transduction of genes crucial in the -oxidation pathway, which includes those encoding for acyl-CoA oxidase (axo) and carnitine palmitoyltransferase-1 (cpt-1). The fasting state in mice is associated with a significant induction of the hepatic ppar gene and its target genes including cpt-1 and axo, presumably as a means to channel the fatty acids to the -oxidation pathway for fuel generation, rather than to serve as substrate for TG synthesis. Recent studies have reported a mutually suppressive crosstalk effect between the PPAR and LXR pathways (42, 43). An increased LXR activation in the mouse liver has been found to be associated with a repression in the PPAR/retinoid X receptor -mediated transactivation in a dose-dependent manner, but the effect is substantially less dramatic than that of the srebp-1 gene. In the dko mice, neither the expression of the axo gene nor the cpt-1 gene showed a significant difference in expression as compared with the ko control. This finding further lends support to the notion of a lack of significant LXR-mediated effect in the dko mouse liver. The findings are also consistent with the notion that the majority of the newly synthesized fatty acids in the dko mice are channeled for TG synthesis and secretion despite a concomitant reduction in fasting insulin levels.

To evaluate further the role of SREBP1c (18) and possibly LXR (17, 44) in the altered glucose metabolism observed in the fasted dko mice, we examined the hepatic expression of the gene for PEPCK, a major enzyme mediating hepatic gluconeogenesis. Recent studies revealed that hepatic PEPCK gene can be directly down-regulated both by SREBP1c and LXR. On the other hand, in addition to the pepck-1 gene, administration of an exogenous LXR ligand to mice has been shown to result in a significant coordinated alteration of expression of other genes in the gluconeogenic pathway, namely pgc-1 and g6p, as well as glut4, the latter encoding for the major glucose transporter for uptake of glucose by the peripheral tissue in mice (17). We observed a 49% reduction in the mRNA levels of the pepck-1 gene in the dko mice, but no difference in the hepatic expression of g6p and pgc-1. We also compared fasting insulin and glucose tolerance tests between the dko mice and their ko controls. A simultaneous reduction in both fasting glucose and fasting insulin is consistent with a reduction in hepatic glucose production as the reduction in pepck-1 gene expression would suggest in conjunction with an improvement in insulin sensitivity. Meanwhile, a lack of alteration in the post challenge glucose excursion is suggestive of a lack of effect on peripheral glucose uptake. Taken together, our data on the dko mice are consistent with an srebp-1-mediated up-regulation of de novo fatty acid synthesis, and a simultaneous down-regulation of hepatic gluconeogenesis, the former in corroboration with the observed TG overproduction, and the latter with the observed improved insulin sensitivity.

Although the degree of reduction in PHLA in the dko mice is comparable with those observed in mice heterozygous for the LPL knock-out allele (lpl+/–), the reduced fasting glucose levels seen in the dko mice are unlikely to be secondary to the reduced LPL activity. In their study of the lpl+/– mice, the authors reported a concomitant increase in fasting insulin and lower fasting glucose (11). It was determined that the higher level of insulin was associated with an increased insulin secretion by the lpl+/– mouse islets, possibly related to a reduction in the LPL expression in pancreatic -cells (11), but not to peripheral insulin resistance. By contrast, the concomitant reduction in both fasting glucose and fasting insulin seen in the dko mice is more suggestive of an increase in insulin sensitivity, at least in the context of hepatic gluconeogenesis in the fasted state. These unique changes in glucose homeostasis in the dko mice further support the notion that the altered hepatic srebp-1 expression may be a key contributor to the dyslipidemia and glucose metabolism changes seen in the dko mice, whereas LXR plays little or no role.

PUFA have also been shown to suppress srebp-1c gene transcription through more than one mechanism. PUFA may exert their direct effects through acceleration of mRNA degradation (16) and through promoting intracellular hydrolysis of sphingomyelin (45). Several lines of experimental evidence also suggest that PUFA may down-regulate the hepatic expression of the SREBP1c gene indirectly through inhibition of the LXR transactivation (14, 15). However, the indirect inhibitory effect of PUFA on the LXR-mediated transactivation of SREBP1 has recently been questioned (46). In the present study, because the mRNA levels of lxr and a number of its target genes of both groups of mice are not significantly different, the up-regulation of the srebp-1 gene seen in the dko mice is more likely the result of a direct inhibitory effect of PUFA.

In their analysis of the fatty acid composition of CEs in circulating apoB lipoproteins in the dko mice, Furbee et al. (19) reported that there was a near 100% reduction in 20-carbon PUFA and a 40% reduction in linoleic acid contents in LDL in the LDLR/LCAT dko mice compared with the LDLR knock-out control. As stated earlier, although the VLDLs in the dko mice may experience a significant reduction in their rate of uptake, it is still conceivable that the circulating CEs from these apoB-containing lipoproteins is taken up by the liver through endocytosis and hydrolyzed in the lysosomes. The lysosome-derived FFA may then be made available for esterification and oxidation (47) and possibly participate in the modulation of the SREBP1 gene transcription. Therefore, our data are consistent with the notion that the altered fatty acid composition of the CE in the dko LDLs may confer a biological effect on srebp-1c mRNA in a manner similar to those of dietary PUFA.

The reduced PHLA observed in the dko mice has also been reported in LCAT-deficient humans (26). The mechanism for this association is not well understood. The in vivo lipolytic activity of LPL is regulated by a large variety of factors at the transcriptional, post-transcriptional, translational, and post-translational levels (10). The LPL gene promoter contains a number of sequence motifs that are putatively responsive to a variety of hormonal and transcription factors (50) including response elements for PPAR (51), SREBP1c (52, 53) and LXR (39). Despite this, much of the regulation of the enzyme protein occurs at the post-translational level (55). Insulin and dexamethasone have both been shown in vitro to up-regulate LPL activity markedly in adipocytes independently and synergistically, not only through the induction of LPL gene expression but quantitatively more importantly through post-transcriptional and post-translational regulations (56, 57). In the case of insulin, regulation through increased synthesis and release (58, 59) of the protein from adipocyte to the endothelial cell surface have also been reported. Physiological doses of insulin and dexamethasone increased heparin-releasable LPL activity up to 8-fold and LPL synthesis up to 5-fold (57). By comparison, recent in vitro data suggest that, under physiological conditions, the extent of induction through the SREBP1 pathway in adipocytes is modest (2–3-fold) (53). A significant reduction in PHLA has also been reported in apoA-I knock-out mice in human apoB transgenic background (34). Because of impaired delivery of HDL-C, both apoA-I (54) and LCAT knock-out mice (6) share the phenotype of a compensated hypoadrenalism, and, in the former case, a reduced basal cortisol level and impaired response to adrenocorticotropic hormone stimulation was observed. It is therefore conceivable that the observed lower level of fasting insulin in the dko mice synergizes with a relative hypocortisolism and possibly other yet unidentified factors to result in the net reduction in LPL activity. A more systematic analysis is needed to elucidate further the role of other modulating factors in the mechanism for the reduction in PHLA in the dko mice.

In humans, an elevated plasma TG, low HDL-C in association with hyperglycemia, and/or impaired glucose tolerance are common features of insulin resistance (48), and this metabolic profile is also a cardinal feature of the metabolic syndrome (49). Individuals with such metabolic phenotypes are at an increased risk of developing coronary heart disease (1). However, in the present murine model of LCAT deficiency, we are the first to report the finding of a unique combination of fasting HTG, primary low HDL state caused by LCAT deficiency and an improved hepatic insulin sensitivity. This metabolic profile is remarkably similar to the novel observations made recently in mice after treatment with LXR agonists (17). The latter effect of LXR was attributed to the down-regulation of several hepatic gluconeogenic genes in the liver, namely pepck-1, pgc-1, and g6p, as a direct effect of LXR. In the present study, the gene expression profile seen in the dko mice is more consistent with a direct effect of an up-regulation of SREBP1c action, which in turn may be, at least in part, a result of the alteration of the fatty acid composition of the LDL-CE content in the dko mice (19). This suggests that in the LDL receptor-deficient background, LCAT deficiency results in a metabolic phenotype characterized by a dissociation between hepatic VLDL overproduction/low HDL state and improved insulin sensitivity which is, at least in part, attributable to the up-regulation of hepatic SREBP1c.

In summary, we have reported a novel finding of combined hepatic lipoprotein overproduction and impaired PHLA in association with a monogenic defect in HDL metabolism, namely complete LCAT deficiency, in a murine model. More intriguingly, the observed hepatic TG overproduction is associated with a significant lowering of fasting insulin and glucose levels and a reduction in the hepatic pepck-1 gene expression. Our data strongly suggest a major role of an enhanced SREBP1c activation being responsible for the disparate changes in lipid and glucose metabolism. Furthermore, increased insulin sensitivity, seen in this particular dyslipidemic model, may also in part account for the lack of accelerated atherogenesis in complete LCAT deficiency, both in humans and in rodent models (20), despite a severe deficiency of plasma HDL levels.

	FOOTNOTES

 
^* This work was supported in part by Heart and Stroke Foundation of Ontario Grants-in-aid N4124 (to D. S. N.) and T4027 (to P. W. C.) and by the Canada Foundation for Innovation New Opportunities (to D. S. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Canadian Institute for Health Research New Investigator awardee. To whom correspondence should be addressed: St. Michael's Hospital West Annex 2-015, 38 Shuter St., Toronto, Ontario M5B-1A6, Canada. Tel.: 416-864-5197; Fax: 416-864-5584; E-mail: ngd{at}smh.toronto.on.ca.

¹ The abbreviations used are: HTG, hypertriglyceridemia; ACC, acyl-CoA carboxylase; Angptl3, angiopoietin-like protein 3; AXO, acyl-CoA oxidase; CE, cholesterol ester; CETP, cholesterol ester transfer protein; CPT, carnitine palmitoyltransferase; dko, double knock-out; FAS, fatty acyl synthase; FFA, free fatty acids; FPLC, fast protein liquid chromatography; g6p, glucose-6-phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL-C, high density lipoprotein cholesterol; IDL, intermediate density lipoprotein; ko, single knock-out; LCAT, lecithin-cholesterol acyltransferase; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; LPL, lipoprotein lipase; LXR, liver X receptor; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1, peroxisomal proliferator activator receptor gamma coactivator 1; PHLA, post-heparin lipase activity; PPAR, peroxisomal proliferator activator receptor; PUFA, polyunsaturated fatty acids; RT-PCR, reverse transcription-PCR; SCD, stearoyl-CoA desaturase; SREBP, sterol regulatory element-binding protein; TG, triglycerides; VLDL, very low density lipoprotein.

	ACKNOWLEDGMENTS

 
We acknowledge the excellent technical assistance of Anne Berndl, Patrick Hui, Yun Lam, and Maureen Lee. We also thank Dr. Khosrow Adeli for the critical review of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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