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Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

  • Sonia M. S. Espirito Santo
    Affiliations
    Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands

    Department of General Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands
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  • Patrick C.N. Rensen
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands

    Department of General Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands
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  • Jeltje R. Goudriaan
    Affiliations
    Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands
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  • André Bensadoun
    Affiliations
    Division of Nutritional Sciences, Cornell University, Ithaca, NY
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  • Niels Bovenschen
    Affiliations
    Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands

    Department of Plasma Proteins, Sanquin Research at Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands
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  • Peter J. Voshol
    Affiliations
    Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands

    Department of Endocrinology and Metabolism, Leiden University Medical Center, Leiden, The Netherlands
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  • Louis M. Havekes
    Affiliations
    Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands

    Department of General Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands

    Departments of Cardiology, Leiden University Medical Center, Leiden, The Netherlands
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  • Bart J.M. van Vlijmen
    Footnotes
    Affiliations
    Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands
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  • Author Footnotes
    2 Present address of B. J. M. van Vlijmen: Department of Hematology, C2-R, Hemostasis and Thrombosis Research Center, Leiden University Medical Center, Leiden, The Netherlands.
Open AccessPublished:March 16, 2005DOI:https://doi.org/10.1194/jlr.C500007-JLR200
      The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR−/−) mice.
      Collectively, although VLDLR deficiency in LRP and LDLR−/− mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.
      The very low density lipoprotein receptor (VLDLR) is expressed in tissues active in fatty acid metabolism (i.e., heart, muscle, adipose) and macrophages (
      • Sakai J.
      • Hoshino A.
      • Takahashi S.
      • Miura Y.
      • Ishii H.
      • Suzuki H.
      • Kawarabayasi Y.
      • Yamamoto T.
      Structure, chromosome location, and expression of the human very low density lipoprotein receptor gene.
      ,
      • Takahashi S.
      • Kawarabayasi Y.
      • Nakai T.
      • Sakai J.
      • Yamamoto T.
      Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity.
      ). In vitro studies show that the VLDLR binds apolipoprotein E (apoE) but not apoB-100 and that LPL modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDLR and vice versa (
      • Niemeier A.
      • Gafvels M.
      • Heeren J.
      • Meyer N.
      • Angelin B.
      • Beisiegel U.
      VLDL receptor mediates the uptake of human chylomicron remnants in vitro.
      ,
      • Takahashi S.
      • Suzuki J.
      • Kohno M.
      • Oida K.
      • Tamai T.
      • Miyabo S.
      • Yamamoto T.
      • Nakai T.
      Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase.
      ). Frykman et al. (
      • Frykman P.K.
      • Brown M.S.
      • Yamamoto T.
      • Goldstein J.L.
      • Herz J.
      Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.
      ) generated VLDLR-deficient (VLDLR−/−) mice and showed that these mice present normal plasma lipoprotein levels when fed a chow diet. Interestingly, only when the TG metabolism was stressed either by a high-fat diet (HFD) or by cross-breeding on a background of obesity (ob/ob) or low density lipoprotein receptor (LDLR) deficiency (
      • Goudriaan J.R.
      • Tacken P.J.
      • Dahlmans V.E.
      • Gijbels M.J.
      • van Dijk K.W.
      • Havekes L.M.
      • Jong M.C.
      Protection from obesity in mice lacking the VLDL receptor.
      ,
      • Tacken P.J.
      • Teusink B.
      • Jong M.C.
      • Harats D.
      • Havekes L.M.
      • van Dijk K.W.
      • Hofker M.H.
      LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice.
      ) did VLDLR deficiency result in moderate accumulation of plasma TG-rich lipoproteins. In agreement with these observations, we recently demonstrated that the postprandial TG response after an oral fat load is strongly increased in VLDLR−/− mice (
      • Goudriaan J.R.
      • Espirito Santo S.M.
      • Voshol P.J.
      • Teusink B.
      • Willems van Dijk K.
      • van Vlijmen B.J.
      • Romijn J.A.
      • Havekes L.M.
      • Rensen P.C.
      The VLDL receptor plays a major role in chylomicron metabolism by enhancing LPL-mediated triglyceride hydrolysis.
      ). Hence, the VLDLR plays a role in TG-rich lipoprotein metabolism that becomes apparent only after severely stressing TG metabolism.
      The LDLR and the low density lipoprotein receptor-related protein (LRP) are two other major members of the LDLR family that act in concert in the hepatic clearance of plasma lipoproteins (
      • van Vlijmen B.J.
      • Rohlmann A.
      • Page S.T.
      • Bensadoun A.
      • Bos I.S.
      • van Berkel T.J.
      • Havekes L.M.
      • Herz J.
      An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins.
      ). Absence of the LDLR in mice results in the accumulation of LDL-sized lipoproteins in plasma, whereas LRP deficiency does not affect plasma lipoprotein levels (
      • Rohlmann A.
      • Gotthardt M.
      • Hammer R.E.
      • Herz J.
      Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.
      ,
      • Ishibashi S.
      • Brown M.S.
      • Goldstein J.L.
      • Gerard R.D.
      • Hammer R.E.
      • Herz J.
      Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.
      ). Strikingly, LRP deficiency on an LDLR−/− background in mice results in aggravated hyperlipidemia attributable to the accumulation of TG-rich lipoprotein remnants (
      • van Vlijmen B.J.
      • Rohlmann A.
      • Page S.T.
      • Bensadoun A.
      • Bos I.S.
      • van Berkel T.J.
      • Havekes L.M.
      • Herz J.
      An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins.
      ). This indicates that the role of LRP in lipoprotein metabolism in vivo is masked by the LDLR, the presence of which can apparently fully compensate for the absence of the LRP (
      • van Vlijmen B.J.
      • Rohlmann A.
      • Page S.T.
      • Bensadoun A.
      • Bos I.S.
      • van Berkel T.J.
      • Havekes L.M.
      • Herz J.
      An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins.
      ). Whether a similar interaction exists between the VLDLR and LDLR and/or LRP is not yet known.
      Because we hypothesized that the addition of the absence of the VLDLR to the absence of the LDLR and hepatic LRP would severely aggravate the hyperlipidemic phenotype of LDLR−/−/LRP mice, we generated unique VLDLR, LDLR, and conditional LRP triple-deficient mice. It appeared that VLDLR deficiency in LDLR−/− and LRP mice does not aggravate hyperlipidemia on a chow diet. However, we do show that the additional absence of the VLDLR does lead to an aggravated phenotype on stressing TG metabolism, either by high-fat feeding or by giving an intragastric olive oil bolus, although these results may be attributed to the cumulative effects of the phenotypes of the individual mice (i.e., LRP, LDLR−/−, and VLDLR−/−). In summary, LRPLDLR−/−VLDLR−/− triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.

      MATERIALS AND METHODS

      Transgenic animals and diet

      We cross-bred VLDLR−/− mice (
      • Goudriaan J.R.
      • Tacken P.J.
      • Dahlmans V.E.
      • Gijbels M.J.
      • van Dijk K.W.
      • Havekes L.M.
      • Jong M.C.
      Protection from obesity in mice lacking the VLDL receptor.
      ) with mice conditionally lacking the LRP on an LDLR−/− background (MX1Cre:LRPlox/lox LDLR−/−; referred to hereafter as LRPLDLR−/−) (
      • Rohlmann A.
      • Gotthardt M.
      • Hammer R.E.
      • Herz J.
      Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.
      ), yielding a unique triple-knockout mouse model that lacks (conditionally) LRP, LDLR, and VLDLR (MX1Cre:LRPlox/loxLDLR−/− VLDLR−/−; referred to hereafter as LRPLDLR−/−VLDLR−/−). Mice were genotyped by PCR analysis on tail tip DNA for the presence of the “floxed” LRP allele, the MX1Cre transgene, and the disrupted LDLR and VLDLR allele as described previously (
      • Tacken P.J.
      • Teusink B.
      • Jong M.C.
      • Harats D.
      • Havekes L.M.
      • van Dijk K.W.
      • Hofker M.H.
      LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice.
      ,
      • Rohlmann A.
      • Gotthardt M.
      • Hammer R.E.
      • Herz J.
      Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.
      ). For experiments, 8–10 week old male VLDLR−/− (n = 8), VLDLR+/+ (n = 8), LRPLDLR−/−VLDLR−/− (n = 10), and LRPLDLR−/−VLDLR+/+ (n = 10) mice were used. To induce LRP deficiency, all mice (including VLDLR−/− and VLDLR+/+) received three intraperitoneal injections of 250 μl of a 1 mg/ml solution of polyinosinic:polycytidylic ribonucleic acid (pI:pC; Sigma, St. Louis, MO) at 2 day intervals as described previously (
      • Rohlmann A.
      • Gotthardt M.
      • Hammer R.E.
      • Herz J.
      Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.
      ). PCR analysis for the presence of the disrupted LRP allele and immunoblot analysis with antibodies directed against the 85 kDa subunit of LRP from liver tissue were performed as described previously (
      • van Vlijmen B.J.
      • Rohlmann A.
      • Page S.T.
      • Bensadoun A.
      • Bos I.S.
      • van Berkel T.J.
      • Havekes L.M.
      • Herz J.
      An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins.
      ,
      • Espirito Santo S.M.
      • Pires N.M.
      • Boesten L.S.
      • Gerritsen G.
      • Bovenschen N.
      • Willems Van Dijk K.
      • Jukema J.W.
      • Princen H.M.
      • Bensadoun A.
      • Li W.P.
      • et al.
      Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol.
      ). Mice were kept for 12 weeks on a standard rat/mouse chow diet (SRM-A; Hope Farms, Woerden, The Netherlands), followed by 10 weeks of HFD containing 24% corn oil, 24% casein, 18% corn starch, and 6% cellulose (Hope Farms). Diet and water were given ad libitum to the animals. The institutional committee on animal welfare of Netherlands Organization for Applied Scientific Research-Quality of Life approved all animal experiments.

      Plasma analysis

      Blood was collected by tail bleeding into chilled paraoxon-coated capillaries (
      • Zambon A.
      • Hashimoto S.I.
      • Brunzell J.D.
      Analysis of techniques to obtain plasma for measurement of levels of free fatty acids.
      ) 4 weeks after induction of LRP deficiency either after 4 h of fasting from 7:00 to 11:00 AM or after overnight fasting from 5:00 PM to 7:00 AM. Plasma was isolated and assayed for total cholesterol, TG, and glucose levels using enzymatic kits C0534, 337-B, and 315-500 (Sigma Diagnostics, Deisenhofen, Germany) and for FFA using the enzymatic kit Nefa-C (Wako Chemicals GmbH, Neuss, Germany). Plasma lipid distribution over lipoproteins was determined by size fractionation using fast-performance liquid chromatography (
      • Espirito Santo S.M.
      • Pires N.M.
      • Boesten L.S.
      • Gerritsen G.
      • Bovenschen N.
      • Willems Van Dijk K.
      • Jukema J.W.
      • Princen H.M.
      • Bensadoun A.
      • Li W.P.
      • et al.
      Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol.
      ). Plasma concentrations of mouse apoB-48 and apoB-100, apoE, and apoA-I were determined by immunoblotting using mouse apolipoprotein-specific polyclonal rabbit antiserum as described previously (
      • Espirito Santo S.M.
      • Pires N.M.
      • Boesten L.S.
      • Gerritsen G.
      • Bovenschen N.
      • Willems Van Dijk K.
      • Jukema J.W.
      • Princen H.M.
      • Bensadoun A.
      • Li W.P.
      • et al.
      Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol.
      ). Plasma levels of HL activity, LPL activity, and LPL mass were determined after 4 h of fasting as described previously (
      • van Vlijmen B.J.
      • Rohlmann A.
      • Page S.T.
      • Bensadoun A.
      • Bos I.S.
      • van Berkel T.J.
      • Havekes L.M.
      • Herz J.
      An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins.
      ,
      • Zechner R.
      Rapid and simple isolation procedure for lipoprotein lipase from human milk.
      ).

      Postprandial TG response

      Mice were fasted for 4 h. After a basal blood sample was taken by tail bleeding, the animals received an intragastric load of 200 μl of olive oil. Subsequently, blood was drawn at the indicated times after olive oil administration. Plasma was isolated and TG levels were determined as described above and are presented as relative increases from time 0.

      Statistical analysis

      All data are presented as means ± SD. Data were analyzed using the Mann-Whitney U-test. P < 0.05 was regarded as statistically significant.

      RESULTS

      General parameters

      Treatment of LDLR−/−VLDLR+/+ and LDLR−/− VLDLR−/− mice with pI:pC resulted in the presence of the disrupted LRP allele in tail tip DNA and in the complete absence of LRP protein in liver membrane extracts (Fig. 1). Upon LRP inactivation, the LRPLDLR−/− VLDLR−/− mice appeared healthy and displayed no signs of abnormalities, but throughout their life span they had a slightly lower body weight compared with LRPLDLR−/−VLDLR+/+ mice (19.6 ± 1.8 g vs. 22.4 ± 0.8 g; P = 0.02). However, reduced body weight was also observed in VLDLR deficiency only compared with wild-type controls (18.7 ± 0.9 g vs. 23.8 ± 2.7 g; P = 0.001), which is in agreement with previous reports (
      • Frykman P.K.
      • Brown M.S.
      • Yamamoto T.
      • Goldstein J.L.
      • Herz J.
      Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.
      ,
      • Goudriaan J.R.
      • Tacken P.J.
      • Dahlmans V.E.
      • Gijbels M.J.
      • van Dijk K.W.
      • Havekes L.M.
      • Jong M.C.
      Protection from obesity in mice lacking the VLDL receptor.
      ).
      Figure thumbnail gr1
      Fig. 1Low density lipoprotein receptor-related protein (LRP) deficiency. Mice were treated with polyinosinic:polycytidylic ribonucleic acid, and LRP deficiency was assessed by the presence of the disrupted LRP allele by PCR analysis of tail tip DNA (upper panel) and by the absence of the 85 kDa subunit of the LRP protein in the liver (lower panel). LDLR, low density lipoprotein receptor; VLDLR, very low density lipoprotein receptor.

      Plasma lipids on a chow diet

      On a regular chow diet, single VLDLR deficiency resulted in increased plasma TG levels after an overnight fast (Table 1). Under these conditions, VLDLR deficiency did not affect plasma cholesterol, FFA, and glucose levels or plasma lipoprotein distribution (Fig. 2). The deletion of both LRP and LDLR (LRPLDLR−/−) elicited severe hyperlipidemia as a result of the accumulation of VLDL/LDL-sized lipoproteins. Interestingly, VLDLR deficiency on this LRPLDLR−/− background did not influence plasma lipids, glucose (Table 1), and lipoprotein profiles (Fig. 2). In addition, levels of plasma apoB-100 (90 ± 43% vs. 100 ± 23%; P = 0.818), apoB-48 (95 ± 20% vs. 100 ± 15%; P = 0.394), apoE (125 ± 12% vs. 100 ± 13%; P = 0.100), and apoA-I (102 ± 46% vs. 100 ± 29%; P = 0.589) were not altered in LRPLDLR−/−VLDLR−/− vs. LRP LDLR−/−VLDLR+/+ mice, respectively.
      TABLE 1Plasma lipid and glucose levels on a chow diet after overnight fasting
      Chow DietHigh-Fat Diet
      AnimalsCholesterolTGsFFAGlucoseCholesterolTGsFFAGlucose
      mM
      Wild-type background
      VLDLR+/+1.8 ± 0.20.3 ± 0.1n.d.n.d.2.9 ± 0.40.4 ± 0.3n.d.n.d.
      VLDLR−/−1.9 ± 0.31.2 ± 0.3
      P < 0.05.
      n.d.n.d.3.2 ± 0.31.1 ± 0.2
      P < 0.05.
      n.d.n.d.
      LRPLDLR−/− background
      VLDLR+/+32.4 ± 6.58.6 ± 1.91.3 ± 0.26.0 ± 1.432.4 ± 6.16.3 ± 2.51.1 ± 0.25.8 ± 0.9
      VLDLR−/−34.1 ± 3.68.9 ± 2.31.5 ± 0.55.6 ± 1.639.4 ± 7.412.5 ± 5.0
      P < 0.05.
      1.5 ± 0.36.5 ± 0.9
      LDLR, low density lipoprotein receptor; LRP, low density lipoprotein receptor-related protein; n.d., not determined; TG, triglyceride; VLDLR, very low density lipoprotein receptor. Values represent means ± SD of 8–10 mice per group.
      a P < 0.05.
      Figure thumbnail gr2
      Fig. 2Lipoprotein distribution. Plasma was obtained from mice on a wild-type background, VLDLR+/+ (A, E) and VLDLR−/− (B, F), and on a LRPLDLR−/− background, LRPLDLR−/−VLDLR+/+ (C, G) and LRPLDLR−/−VLDLR−/− (D, H), after 4 weeks of inducing LRP deficiency on a chow diet (upper panels) and after 10 weeks on a high-fat diet (lower panels) with overnight fasting. Lipoproteins in pooled plasma were size-fractionated by fast-performance liquid chromatography, and the plasma cholesterol (closed circles) and triglyceride (TG; open circles) contents of the individual fractions were determined.
      We next investigated the effect of VLDLR status in the presence or absence of the LDLR and LRP on plasma lipids after 4 h of fasting. As shown in Table 2, for both the wild-type and LRPLDLR−/− backgrounds, the effects of VLDLR deficiency on plasma lipid levels were comparable to those observed for the overnight fasting state. Single VLDLR−/− mice showed only modestly increased plasma TG levels and no effects on plasma cholesterol, FFA, and glucose levels (Table 2). Again, VLDLR deficiency on an LRPLDLR−/− background did not affect plasma lipids and glucose levels.
      TABLE 2Plasma lipids, glucose, and lipolytic enzymes on a chow diet after 4 h of fasting
      AnimalsCholesterolTGsFFAGlucoseHL ActivityLPL ActivityLPL Mass
      mMμmol FFA/h/mlng/ml
      Wild-type background
      VLDLR+/+2.2 ± 0.30.6 ± 0.2n.d.n.d.8.2 ± 1.321.0 ± 3.576.6 ± 3.1
      VLDLR−/−2.0 ± 0.31.3 ± 0.6
      P < 0.05.
      n.d.n.d.7.0 ± 0.817.1 ± 2.1
      P < 0.05.
      59.3 ± 7.9
      P < 0.05.
      LRPLDLR−/− background
      VLDLR+/+24.9 ± 5.66.4 ± 2.40.8 ± 0.210.5 ± 1.314.2 ± 2.239.5 ± 3.6180.6 ± 2.8
      VLDLR−/−19.8 ± 4.34.6 ± 1.70.7 ± 0.111.1 ± 1.414.6 ± 1.227.7 ± 4.1
      P < 0.05.
      151.6 ± 10.5
      P < 0.05.
      Values represent means ± SD of 8–10 mice per group.
      a P < 0.05.

      Plasma HL, LPL activity, and LPL mass

      Yagyu et al. (
      • Yagyu H.
      • Lutz E.P.
      • Kako Y.
      • Marks S.
      • Hu Y.
      • Choi S.Y.
      • Bensadoun A.
      • Goldberg I.J.
      Very low density lipoprotein (VLDL) receptor-deficient mice have reduced lipoprotein lipase activity. Possible causes of hypertriglyceridemia and reduced body mass with VLDL receptor deficiency.
      ) reported that the increase in plasma TG levels in VLDLR−/− mice is associated with reduced LPL activity in these mice. As shown in Table 2, single VLDLR−/− mice indeed showed a significant 19% decrease in LPL activity (P = 0.02) and a 23% decrease in LPL mass (P = 0.03) compared with control VLDLR+/+ mice. Interestingly, LPL activity and mass levels were ∼1.8-fold and 2.4-fold higher for mice on an LRPLDLR−/− background compared with mice on a wild-type background, respectively. The LRPLDLR−/−VLDLR−/− mice also had a significant 30% lower plasma LPL activity (P = 0.001) and a 16% decrease in plasma LPL mass (P = 0.04) compared with control LRPLDLR−/−VLDLR+/+ mice. HL activity was not affected upon deletion of the VLDLR on both wild-type and LRPLDLR−/− backgrounds. However, as for LPL activity, HL activity levels were increased ∼2-fold in mice on an LRPLDLR−/− background compared with mice on a wild-type background (Table 2).

      Postprandial TG response

      Stressing TG metabolism by forced feeding through an intragastric load of olive oil proved to be very effective at evoking a clear effect of VLDLR deficiency on plasma TG. Single VLDLR−/− mice had a strong increase in postprandial TG response compared with controls (Fig. 3A), as indicated by a 12-fold increased area under the curve (AUC) [218 ± 170 mM TG/h vs. 12 ± 5 mM TG/h (P = 0.01) for VLDLR−/− and VLDLR+/+, respectively], which is in agreement with our recent report (
      • Goudriaan J.R.
      • Espirito Santo S.M.
      • Voshol P.J.
      • Teusink B.
      • Willems van Dijk K.
      • van Vlijmen B.J.
      • Romijn J.A.
      • Havekes L.M.
      • Rensen P.C.
      The VLDL receptor plays a major role in chylomicron metabolism by enhancing LPL-mediated triglyceride hydrolysis.
      ). Likewise, LRPLDLR−/− VLDLR−/− mice had a strong increase in postprandial TG response compared with control LRPLDLR−/−VLDLR+/+ mice (Fig. 3B) [AUC = 411 ± 107 mM TG/h vs. 163 ± 85 mM TG/h (P = 0.002) for LRPLDLR−/−VLDLR−/− and LRPLDLR−/−VLDLR+/+, respectively]. The total TG response in VLDLR−/− and LRPLDLR−/−VLDLR−/− mice was similar, as indicated by a similar mean increase in AUC compared with their respective controls (206 and 248 mM TG/h, respectively). However, the TG response in the LRP LDLR−/−VLDLR−/− mice was remarkably prolonged. Whereas TG levels in VLDLR−/− mice reached baseline TG levels within 24 h after gavage, TG levels were still significantly increased at 48 h after gavage in LRPLDLR−/− VLDLR−/− mice, which is probably related to the high remnant levels in plasma that compete for the binding of the nascent chylomicrons to LPL.
      Figure thumbnail gr3
      Fig. 3Postprandial TG response. Overnight fasted VLDLR+/+ (closed squares) and VLDLR−/− (open squares) mice on a wild-type (A) or LRPLDLR−/− (B) background were given an intragastric bolus of 200 μl of olive oil. Blood samples were drawn at the indicated times after gavage. Plasma TG concentrations were determined and corrected for time 0 values. Values represent means ± SD of eight mice per group. * P < 0.05.

      Plasma lipids under HFD

      Finally, TG metabolism was assessed by challenge of mice with HFD (Table 1). As observed on a chow diet, VLDLR−/− mice had ∼3-fold higher plasma TG levels (P = 0.02) compared with wild-type mice, whereas plasma cholesterol was not different. Remarkably, on this diet, LRPLDLR−/− VLDLR−/− mice also displayed 2-fold higher plasma TG levels (P = 0.032) but no change in plasma cholesterol, FFA, and glucose compared with the control LRPLDLR−/− VLDLR+/+ mice. Thus, under high-fat feeding conditions, the role of the VLDLR in TG-rich lipoprotein metabolism becomes evident in the absence of the LRP and LDLR.

      DISCUSSION

      Studies in VLDLR−/− mice only revealed a role of this receptor specifically in postprandial TG-rich lipoprotein metabolism after severely stressing TG metabolism (
      • Frykman P.K.
      • Brown M.S.
      • Yamamoto T.
      • Goldstein J.L.
      • Herz J.
      Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.
      ,
      • Goudriaan J.R.
      • Tacken P.J.
      • Dahlmans V.E.
      • Gijbels M.J.
      • van Dijk K.W.
      • Havekes L.M.
      • Jong M.C.
      Protection from obesity in mice lacking the VLDL receptor.
      ,
      • Tacken P.J.
      • Teusink B.
      • Jong M.C.
      • Harats D.
      • Havekes L.M.
      • van Dijk K.W.
      • Hofker M.H.
      LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice.
      ,
      • Goudriaan J.R.
      • Espirito Santo S.M.
      • Voshol P.J.
      • Teusink B.
      • Willems van Dijk K.
      • van Vlijmen B.J.
      • Romijn J.A.
      • Havekes L.M.
      • Rensen P.C.
      The VLDL receptor plays a major role in chylomicron metabolism by enhancing LPL-mediated triglyceride hydrolysis.
      ). Interestingly, the role of the LRP in TG-rich lipoprotein metabolism is fully compensated for by the presence of the LDLR (
      • van Vlijmen B.J.
      • Rohlmann A.
      • Page S.T.
      • Bensadoun A.
      • Bos I.S.
      • van Berkel T.J.
      • Havekes L.M.
      • Herz J.
      An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins.
      ). We wondered whether a prominent role of the VLDLR would be similarly overtaken by the LDLR or LRP. Here, we show that the absence of a phenotype for VLDLR−/− mice with respect to TG-rich lipoprotein remnant levels is not attributable to backup activity of the LRP/LDLR pathway. Therefore, it seems reasonable to conclude that the contribution of VLDLR to the clearance of TG-rich lipoproteins is not rate-limiting under physiological circumstances but becomes apparent after stressing TG metabolism by high-fat feeding or giving a large TG bolus.
      Previously, we demonstrated that adenovirus-mediated overexpression of the LDLR family antagonist receptor-associated protein (RAP) in LRP/LDLR double-deficient mice elicits marked hyperlipidemia in addition to the preexisting hypercholesterolemia in these animals and decreases LPL activity (
      • van Vlijmen B.J.
      • Rohlmann A.
      • Page S.T.
      • Bensadoun A.
      • Bos I.S.
      • van Berkel T.J.
      • Havekes L.M.
      • Herz J.
      An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins.
      ). Because RAP binds to the VLDLR with high affinity (
      • Battey F.D.
      • Gafvels M.E.
      • FitzGerald D.J.
      • Argraves W.S.
      • Chappell D.A.
      • Strauss J.F.
      • Strickland D.K.
      The 39-kDa receptor-associated protein regulates ligand binding by the very low density lipoprotein receptor.
      ), we speculated that it was possible that a RAP-mediated inhibition of the VLDLR underlies the observed impaired LPL-mediated lipolysis and subsequent hypertriglyceridemia. However, because LRP LDLR−/−VLDLR−/− and LRPLDLR−/−VLDLR+/+ mice have comparable hyperlipidemia, we can now conclude that RAP-induced hypertriglyceridemia does not directly involve the VLDLR. This is further supported by our observation that adenovirus-mediated overexpression of RAP still elicits marked hypertriglyceridemia and decreases LPL activity in LRPLDLR−/−VLDLR−/− mice (data not shown).
      LRPLDLR−/−VLDLR+/+ and LRPLDLR−/−VLDLR−/− mice present higher plasma lipid levels after overnight fasting compared with 4 h of fasting, as observed for LDLR−/− VLDLR−/− mice (
      • Tacken P.J.
      • Teusink B.
      • Jong M.C.
      • Harats D.
      • Havekes L.M.
      • van Dijk K.W.
      • Hofker M.H.
      LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice.
      ). This effect is probably caused by an increased hepatic production of VLDL-TG, which is the primary source of FFA for peripheral tissues in the absence of chylomicrons in the fasted state.
      Yagyu et al. (
      • Yagyu H.
      • Lutz E.P.
      • Kako Y.
      • Marks S.
      • Hu Y.
      • Choi S.Y.
      • Bensadoun A.
      • Goldberg I.J.
      Very low density lipoprotein (VLDL) receptor-deficient mice have reduced lipoprotein lipase activity. Possible causes of hypertriglyceridemia and reduced body mass with VLDL receptor deficiency.
      ) and Goudriaan et al. (
      • Goudriaan J.R.
      • Espirito Santo S.M.
      • Voshol P.J.
      • Teusink B.
      • Willems van Dijk K.
      • van Vlijmen B.J.
      • Romijn J.A.
      • Havekes L.M.
      • Rensen P.C.
      The VLDL receptor plays a major role in chylomicron metabolism by enhancing LPL-mediated triglyceride hydrolysis.
      ) showed that impaired TG-rich lipoprotein catabolism in VLDLR−/− mice is associated with reduced LPL activity. This has been explained by reduced translocation of LPL over endothelial cells as related to the chaperone function of the VLDLR (
      • Obunike J.C.
      • Lutz E.P.
      • Li Z.
      • Paka L.
      • Katopodis T.
      • Strickland D.K.
      • Kozarsky K.F.
      • Pillarisetti S.
      • Goldberg I.J.
      Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor.
      ). Likewise, we now show that VLDLR deficiency also reduces LPL protein and activity on an LRPLDLR−/− background. In addition, the LRPLDLR−/− background resulted in increased LPL and HL activities in postheparin plasma, irrespective of VLDLR status. LPL and HL are both well-established ligands for LRP (
      • Herz J.
      • Strickland D.K.
      LRP: a multifunctional scavenger and signaling receptor.
      ), and plasma LPL mass is increased in LRP mice (
      • Espirito Santo S.M.
      • Pires N.M.
      • Boesten L.S.
      • Gerritsen G.
      • Bovenschen N.
      • Willems Van Dijk K.
      • Jukema J.W.
      • Princen H.M.
      • Bensadoun A.
      • Li W.P.
      • et al.
      Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol.
      ). However, as LPL activity levels are not affected in LRP mice (
      • Espirito Santo S.M.
      • Pires N.M.
      • Boesten L.S.
      • Gerritsen G.
      • Bovenschen N.
      • Willems Van Dijk K.
      • Jukema J.W.
      • Princen H.M.
      • Bensadoun A.
      • Li W.P.
      • et al.
      Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol.
      ), it is uncertain whether LRP deficiency contributes to increased LPL protein and activity levels in LRPLDLR−/− mice. Most likely, the increase in LPL is a direct consequence of the severe hyperlipidemia in LRPLDLR−/− mice, because both total HDL and LPL are also increased in genetically unrelated severely hypertriglyceridemic mice as a result of apoC-I expression (
      • Berbée J.F.
      • van der Hoogt C.C.
      • Sundararaman D.
      • Havekes L.M.
      • Rensen P.C.
      Severe hypertriglyceridemia in human APOC1 transgenic mice is caused by apoC-I-induced inhibition of LPL.
      ).
      The unique triple-deficient mouse model (LRPLDLR−/− VLDLR−/−) enabled us to conclude that the LRP/LDLR pathway does not mask a prominent role for the VLDLR in TG-rich lipoprotein metabolism. Apart from the LDLR family members, other mechanisms also have been identified that contribute to TG-rich lipoprotein uptake and degradation, such as LRP5 (
      • Magoori K.
      • Kang M.J.
      • Ito M.R.
      • Kakuuchi H.
      • Ioka R.X.
      • Kamataki A.
      • Kim D.H.
      • Asaba H.
      • Iwasaki S.
      • Takei Y.A.
      • et al.
      Severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E.
      ), apoB-48 receptor (
      • Brown M.L.
      • Ramprasad M.P.
      • Umeda P.K.
      • Tanaka A.
      • Kobayashi Y.
      • Watanabe T.
      • Shimoyamada H.
      • Kuo W.L.
      • Li R.
      • Song R.
      • et al.
      A macrophage receptor for apolipoprotein B48: cloning, expression, and atherosclerosis.
      ), LR11 (
      • Taira K.
      • Bujo H.
      • Hirayama S.
      • Yamazaki H.
      • Kanaki T.
      • Takahashi K.
      • Ishii I.
      • Miida T.
      • Schneider W.J.
      • Saito Y.
      LR11, a mosaic LDL receptor family member, mediates the uptake of apoE-rich lipoproteins in vitro.
      ), heparan sulfate proteoglycans (
      • Wilsie L.C.
      • Orlando R.A.
      The low density lipoprotein receptor-related protein complexes with cell surface heparan sulfate proteoglycans to regulate proteoglycan-mediated lipoprotein catabolism.
      ), and scavenger receptor class B type I (
      • Out R.
      • Kruijt J.K.
      • Rensen P.C.
      • Hildebrand R.B.
      • Vos P. De
      • Eck M. Van
      • van Berkel T.J.
      Scavenger receptor BI plays a role in facilitating chylomicron metabolism.
      ). Our LRPLDLR−/−VLDLR−/− mouse model serves as a unique tool to elucidate the contributions of these pathways in TG-rich lipoprotein clearance in the absence of the three quantitatively important main apoE-recognizing receptors. This will further advance our understanding of the mechanisms by which plasma levels of TG-rich lipoproteins are regulated in vivo.

      Acknowledgments

      This research was supported by the Royal Netherlands Academy of Arts and Sciences (fellowship to B.J.M.v.V.), the European Union (project QLK1-CT-1999-498), the Leiden University Medical Center (Gisela Thier Fellowship to P.C.N.R.), the Netherlands Organization for Scientific Research (Grant 903-39-192/194 and Netherlands Organization for Scientific Research-VIDI Grant 917.36.351 to P.C.N.R., Netherlands Organization for Scientific Research-VENI Grant 916.36.071 to P.J.V.), and the Netherlands Diabetes Foundation (Grant 96.604). In addition, the authors thank Marijke Voskuilen (Netherlands Organization for Applied Scientific Research-Quality of Life, Leiden, The Netherlands) for excellent technical assistance.

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