Artigo Acesso aberto Revisado por pares

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

2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês

10.1074/jbc.m309439200

ISSN

1083-351X

Autores

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

Tópico(s)

Lipid metabolism and disorders

Resumo

Lecithin-cholesterol acyltransferase deficiency is frequently associated with hypertriglyceridemia (HTG) in animal models and humans. We investigated the mechanism of HTG in the ldlr–/– × lcat–/– (double knockout (dko)) mice using the ldlr–/– × 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 LXRα (lxrα) 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. Lecithin-cholesterol acyltransferase deficiency is frequently associated with hypertriglyceridemia (HTG) in animal models and humans. We investigated the mechanism of HTG in the ldlr–/– × lcat–/– (double knockout (dko)) mice using the ldlr–/– × 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 LXRα (lxrα) 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. Hypertriglyceridemia (HTG) 1The 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. 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 (1Alexander C.M. Landsman P.B. Teutsch S.M. Haffner S.M. Diabetes. 2003; 52: 1210-1214Crossref PubMed Scopus (1261) Google Scholar). Known examples include diabetic dyslipidemia, insulin resistance, metabolic syndrome (2Ginsberg H.N. Am. J. Cardiol. 2003; 91: 29-39Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and familial combined hyperlipidemia (3Ayyobi A.F. McGladdery S.H. McNeely M.J. Austin M.A. Motulsky A.G. Brunzell J.D. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1289-1294Crossref PubMed Scopus (99) Google Scholar). 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 (4Gjone E. Scand. J. Clin. Lab. Invest. 33 Suppl. 1974; 137: 73-82Google Scholar, 5Frohlich J. McLeod R. Pritchard P.H. Fesmire J. McConathy W. Metabolism. 1988; 7: 3-8Abstract Full Text PDF Scopus (62) Google Scholar). Gene-targeted mice deficient in LCAT activities are associated with HTG in a gene dose-dependent manner (6Ng D.S. Francone O.L. Forte T.M. Zhang J. Haghpassand M. Rubin E.M. J. Biol. Chem. 1997; 272: 15777-15781Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Sakai N. Vaisman B.L. Koch C.A. Hoyt Jr., R.F. Meyn S.M. Talley G.D. Paiz J.A. Brewer Jr., H.B. Santamarina-Fojo S. J. Biol. Chem. 1997; 272: 7506-7510Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Transgenic mice (8Francone O.L. Gong E.L. Ng D.S. Fielding C.J. Rubin E.M. J. Clin. Invest. 1995; 96: 1440-1448Crossref PubMed Scopus (84) Google Scholar) and rabbits (9Hoeg J.M. Vaisman B.L. Demosky Jr., S.J. Meyn S.M. Talley G.D. Hoyt Jr., R.F. Feldman S. Berard A.M. Sakai N. Wood D. Brousseau M.E. Marcovina S. Brewer Jr., H.B. Santamarina-Fojo S. J. Biol. Chem. 1996; 271: 4396-4440Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) 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 (10Merkel M. Eckel R.H. Goldberg I.J. J. Lipid Res. 2002; 43: 1997-2006Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). In mice, homozygous LPL knock-out is perinatally lethal. Heterozygous LPL knock-out mice (lpl+/–) have been found to be moderately hypertriglyceridemic (11Marshall B.A. Tordjman K. Host H.H. Ensor N.J. Kwon G. Marshall C.A. Coleman T. McDaniel M.L. Semenkovich C.F. J. Biol. Chem. 1999; 274: 27426-27432Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 β-cells without any associated peripheral insulin resistance (11Marshall B.A. Tordjman K. Host H.H. Ensor N.J. Kwon G. Marshall C.A. Coleman T. McDaniel M.L. Semenkovich C.F. J. Biol. Chem. 1999; 274: 27426-27432Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (12Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3787) Google Scholar). Both insulin and liver X receptor α (LXRα) have been shown to be potent inducers of SREBP1 gene transcription (13Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Kimura S. Ishibashi S. Yamada N. Mol. Cell. Biol. 2001; 21: 2991-3000Crossref PubMed Scopus (434) Google Scholar). 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. LXRα 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 (14Ou J. Tu H. Shan B. Luk A. DeBose-Boyd R.A. Bashmakov Y. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6027-6032Crossref PubMed Scopus (406) Google Scholar, 15Field F.J. Born E. Murthy S. Mathur S.N. Biochem. J. 2002; 368: 855-864Crossref PubMed Scopus (62) Google Scholar) and mRNA stability (16Xu J. Teran-Garcia M. Park J.H. Nakamura M.T. Clarke S.D. J. Biol. Chem. 2001; 276: 9800-9807Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) 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 LXRα (17Laffitte B.A. Chao L.C. Li J. Walczak R. Hummasti S. Joseph S.B. Castrillo A. Wilpitz D.C. Mangelsdorf D.J. Collins J.L. Saez E. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5419-5424Crossref PubMed Scopus (420) Google Scholar) and SREBP1c (18Chakravarty K. Leahy P. Becard D. Hakimi P. Foretz M. Ferre P. Foufelle F. Hanson R.W. J. Biol. Chem. 2001; 276: 34816-34823Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 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. Animals—Breeding pairs of the ldlr–/–× lcat–/– double knock-out (dko) and their littermate ldlr–/– × lcat+/+ single knock-out (ko) mice were a kind gift from Dr. John Parks (19Furbee Jr., J.W. Francone O. Parks J.S. J. Lipid Res. 2002; 43: 428-437Abstract Full Text Full Text PDF PubMed Google Scholar). 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 (20Ng D.S. Maguire G.F. Wylie J. Ravandi A. Xuan W. Ahmed Z. Eskandarian M. Kuksis A. Connelly P.W. J. Biol. Chem. 2002; 277: 11715-11720Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 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 (21Forte T.M. Oda M.N. Knoff L. Frei B. Suh J. Harmony J.A. Stuart W.D. Rubin E.M. Ng D.S. J. Lipid Res. 1999; 40: 1276-1283Abstract Full Text Full Text PDF PubMed Google Scholar). Fast protein liquid chromatography (FPLC) fractionation on total plasma was performed on a Superose 6HR column (10 mm × 30 cm) (Amersham Biosciences) (22Ha Y.C. Barter P.J. J. Chromatogr. 1985; 341: 154-159Crossref PubMed Scopus (51) Google Scholar). 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. (24Jong M.C. Hofker M.H. Havekes L.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (431) Google Scholar). VLDL plus intermediate density lipoprotein (IDL) was isolated from pooled (n = 7) mouse plasma by ultracentrifugation at d < 1.019 g/ml (23Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6487) Google Scholar). 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 (25Wang C.S. Bass H.B. Downs D. Whitmer R.K. Clin. Chem. 1981; 27: 663-668Crossref PubMed Scopus (40) Google Scholar) 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 α (pparα) 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) (5′-ttcttacacgaccaccacca; 3′-gcgttgagcaccagagtgta), Angptl3 (XM_131498) (5′-attcaacaccggaaagatgg; 3′-tggagcatcattttggatga), CPT-1 (NM_013495) (5′-tccatgcataccaaagtgga; 3′-tcatcagtggcctcacagac), AXO (AF006688) (5′-ccgtctgcagcatcataaca; 3′-taaggcgccagtctgaaatc); GAPDH (NM008084) (5′-caaattcaacggcacagtca; 3′-ttgaagtcgcaggagacaac), PPARα (NM01144) (5′-ccagtactgccgttttcaca; 3′-cctctgcctctttgtcttcg), LXRα (AJ132601) (5′-ggatagggttggagtcagca; 3′-cttgccgcttcagtttcttc); PEPCK-1 (NM_011044) (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. 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 (19Furbee Jr., J.W. Francone O. Parks J.S. J. Lipid Res. 2002; 43: 428-437Abstract Full Text Full Text PDF PubMed Google Scholar). 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.Table IFasting plasma lipids and lipoproteins in ldlr-/- × lcat+/+ and ldlr-/- × lcat -/- miceT CholTGFCPLFC/CEmmldlr-/- × lcat -/- (n = 6)8.23 ± 1.08ap < 0.002.1.91 ± 0.59bp < 0.025.3.72 ± 0.52bp < 0.025.4.33 ± 0.59bp < 0.025.0.84 ± 0.18ap < 0.002.ldlr-/- × lcat+/+ (n = 4)11.65 ± 1.031.09 ± 0.182.81 ± 0.455.29 ± 0.470.32 ± 0.05a p < 0.002.b p < 0.025. Open table in a new tab In Vitro Lipolysis Assays—To assess the possibility that VLDL in the LCAT-deficient mice might be a poor substrate for lipolysis, we incubated the VLDL (d < 1.019 g/ml) fraction with bovine LPL and measured the rate of FFA release. Although the limited TG concentration range did not permit an accurate estimate of Vmax and Km, as seen in Fig. 2, the kinetic data suggest that VLDL-TG in the dko mice were more efficiently lipolyzed by exogenous LPL. PHLA—PHLA were determined in three pooled plasma samples (n = 3 animals/pool) each for both the dko mice and their ko controls (Fig. 3). Mean post-heparin LPL activity was reduced by 60.8% (15.87 ± 5.34 versus 40.42 ± 4.76 μm/ml/hr; p = 0.004) in the dko mice, whereas the PHLA were not significantly different (10.23 ± 0.61 versus 9.39 ± 2.41, μm/ml/h; p = 0.61). In Vivo TG Production Rate—Mean rates of plasma TG accumulation after Triton WR1339 injection are shown in Fig. 4. There was an 8.0-fold increase in the TG production rate seen in the dko mice compared with the ko control (p = 0.04). Hepatic mRNA Level Determinations—The hepatic mRNA levels for srebp-1, lipogenic enzymes acc-1, fas, and scd-1, as determined by semiquantitative RT-PCR, are shown in Fig. 5. Compared with their controls, the dko mice had a 1.9-fold increase (0.63 ± 0.05 versus 0.33 ± 0.05) in the mRNA level of the acc-1 gene (p = 0.00007). In case of the fas gene, the mRNA level of the ko control mice was essentially undetectable (0.02 ± 0.02), and that of the dko mice was 0.73 ± 0.43, leading to a 36-fold change (p = 0.026). The mRNA levels for the scd-1 gene were elevated 1.2-fold in the dko mice versus control (1.10 ± 0.04 versus 0.91 ± 0.09, respectively; p = 0.003). We also observed a significant 2.16-fold increase in the srebp-1 mRNA level in the dko mice versus control (0.26 ± 0.10 versus 0.12 ± 0.03, respectively; p = 0.039). The mRNA levels of the angptl3 gene were not significantly different between the two genotypes (0.67 ± 0.06 versus 0.69 ± 0.27; p = 0.88). The mRNA levels of lxrα were 1.13-fold elevated in the dko mice compared with control (1.04 ± 0.116 versus 0.92 ± 0.017, respectively; p = 0.050). The mRNA levels of the two pparα target genes, axo-1 and cpt-1, were minimally different between the two groups (1.17 ± 0.35 versus 1.06 ± 0.55; p = 0.01) and (0.87 ± 0.09 versus 0.85 ± 0.03; p = 0.54), respectively, whereas the pparα mRNA levels were 0.84-fold lower than the control (1.06 ± 0.12 versus 1.26 ± 0.08; respectively, p = 0.01). The hepatic expression of pepck-1 gene was found to be reduced 48.7% in the dko mice compared with the control (0.61 ± 0.15 versus 1.19 ± 0.39; p = 0.013). Fasting Insulin and Glucose Levels—The fasting insulin levels of the dko mice were found to be 42% lower than their ko controls (0.92 ± 0.41 versus 1.59 ± 0.55 ng/ml; p = 0.007). The mean fasting blood glucose levels in the dko mice were reduced 31% compared with the ko control (6.0 ± 1.12 mmol/liter versus 8.7 ± 1.02 mmol/liter, p = 0.01) (Fig. 6). The incremental glucose excursions (postchallenge glucose levels minus fasting glucose level in individual animals) over the course of 2 h after the glucose challenge were not significantly different between the two groups (data not shown). In this paper, we report that the LDLR/LCAT dko mice, compared with its LDLR ko controls, are hypertriglyceridemic and are associated with a combination of hepatic TG overproduction and selective impairment in post-heparin LPL activity. This represents the first report of hepatic TG overproduction in association with a monogenic low HDL syndrome. We also observed a concomitant up-regulation of the hepatic expression of the srebp-1 gene and a number of its target lipogenic genes in these dko mice compared with the ko control. In addition, we are also the first to report that the dko mice are more insulin-sensitive than the control mice, on the basis of a concomitant reduction in fasting plasma glucose and insulin levels. These changes in glucose homeostasis were further found to be associated with a significant down-regulation of hepatic pepck-1, the gene for the rate-limiting enzyme for hepatic gluconeogenesis. Although the pepck-1 gene has been shown to be a target gene to both SREBP-1c and its upstream transactivator LXRα (17Laffitte B.A. Chao L.C. Li J. Walczak R. Hummasti S. Joseph S.B. Castrillo A. Wilpitz D.C. Mangelsdorf D.J. Collins J.L. Saez E. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5419-5424Crossref PubMed Scopus (420) Google Scholar, 18Chakravarty K. Leahy P. Becard D. Hakimi P. Foretz M. Ferre P. Foufelle F. Hanson R.W. J. Biol. Chem. 2001; 276: 34816-34823Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), further characterization of two of the major metabolic phenotypes seen in the dko mice, namely the hepatic TG over-production and improvement in insulin sensitivity, suggests SREBP1c to be the most likely mechanistic link. HTG has been consistently documented in humans with the rare complete LCAT deficiency syndrome (4Gjone E. Scand. J. Clin. Lab. Invest. 33 Suppl. 1974; 137: 73-82Google Scholar, 5Frohlich J. McLeod R. Pritchard P.H. Fesmire J. McConathy W. Metabolism. 1988; 7: 3-8Abstract Full Text PDF Scopus (62) Google Scholar). In rodent models, there is an inverse relationship between fasting plasma TG levels and the murine lcat gene dose (6Ng D.S. Francone O.L. Forte T.M. Zhang J. Haghpassand M. Rubin E.M. J. Biol. Chem. 1997; 272: 15777-15781Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Furthermore, transgenic mouse and rabbit models with overexpression of the human LCAT gene have been found to have a modest reduction in fasting TG (8Francone O.L. Gong E.L. Ng D.S. Fielding C.J. Rubin E.M. J. Clin. Invest. 1995; 96: 1440-1448Crossref PubMed Scopus (84) Google Scholar, 9Hoeg J.M. Vaisman B.L. Demosky Jr., S.J. Meyn S.M. Talley G.D. Hoyt Jr., R.F. Feldman S. Berard A.M. Sakai N. Wood D. Brousseau M.E. Marcovina S. Brewer Jr., H.B. Santamarina-Fojo S. J. Biol. Chem. 1996; 271: 4396-4440Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). 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 (5Frohlich J. McLeod R. Pritchard P.H. Fesmire J. McConathy W. Metabolism. 1988; 7: 3-8Abstract Full Text PDF Scopus (62) Google Scholar, 26Blomhoff J.P. Holme R. Sauar J. Gjone E. Scand. J. Clin. Lab. Invest. Suppl. 1978; 150: 177-182Crossref PubMed Scopus (13) Google Scholar). 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 (27Maugeais C. Tietge U.J. Tsukamoto K. Glick J.M. Rader D.J. J. Lipid Res. 2000; 41: 1673-1679Abstract Full Text Full Text PDF PubMed Google Scholar, 28Mensenkamp A.R. Jong M.C. van Goor H. van Luyn M.J. Bloks V. Havinga R. Voshol P.J. Hofker M.H. van Dijk K.W. Havekes L.M. Kuipers F. J. Biol. Chem. 1999; 274: 35711-35718Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 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 (29Herz J. Qiu S.Q. Oesterle A. DeSilva H.V. Shafi S. Havel R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4611-4615Crossref PubMed Scopus (88) Google Scholar). 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 (30Grefhorst A. Elzinga B.M. Voshol P.J. Plosch T. Kok T. Bloks V.W. van der Sluijs F.H. Havekes L.M. Romijn J.A. Verkade H.J. Kuipers F. J. Biol. Chem. 2002; 277: 34182-34190Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). 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 (31Schumaker V.N. Adams G.H. J. Theor. Biol. 1970; 26: 89-91Crossref PubMed Scopus (56) Google Scholar) 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 (5Frohlich J. McLeod R. Pritchard P.H. Fesmire J. McConathy W. Metabolism. 1988; 7: 3-8Abstract Full Text PDF Scopus (62) Google Scholar, 32Wang C.S. Alaupovic P. Gregg R.E. 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