Hyperlipidemias: Havekes LM

Rensen PC, van Dijk KW, Havekes LM. · No affiliation provided · Arterioscler Thromb Vasc Biol. · Pubmed #16306435 links to  free full text

This publication has no abstract.

DeRuiter MC, Alkemade FE, Gittenberger-de Groot AC, Poelmann RE, Havekes LM, van Dijk KW. · Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands. · Curr Opin Lipidol. · Pubmed #18607178 No free full text.

Abstract: PURPOSE OF REVIEW: In the last 20 years, an increasing amount of epidemiological and pathological evidence has become available illustrating the relationship between an adverse in-utero environment and increased risk of vascular disease in the offspring. It is now generally accepted that epigenetic phenomena, such as either DNA methylation or chromatin modifications or both mediate the long-term memory and thus developmental programming of cells and tissues. RECENT FINDINGS: In utero, the placenta and fetus are exposed to the metabolic, antioxidant and pro-inflammatory and anti-inflammatory signals from the mother and will likely respond specifically. In the fetus, these responses may lead to permanent changes either in DNA methylation or chromatin modification or both and these changes may lead to increased atherosclerosis susceptibility in adulthood. However, the molecular mechanisms responsible for the translation of an adverse maternal environment into permanent epigenetic changes are poorly understood. SUMMARY: In this review, we briefly summarize the possible signals crossing the placental barrier and discuss the molecular mechanisms of epigenetic programming in the developing fetus leading to increased athero-susceptibility of the vessel wall.

van Dijk KW, Rensen PC, Voshol PJ, Havekes LM. · Department of Human Genetics, Leiden University Medical Center, PO Box 9503, 2000 RA Leiden, The Netherlands. · Curr Opin Lipidol. · Pubmed #15166778 No free full text.

Abstract: PURPOSE OF REVIEW: Apolipoprotein (apo)CIII and apoAV play an important role in triglyceride metabolism as evidenced by the unambiguous and opposing phenotypes of transgenic and knockout mouse models. In this review we discuss studies on the genetics, protein structure, and regulation of apoCIII and apoAV and compare their potential molecular mechanisms of action in triglyceride metabolism. We examine the hypothesis that apoCIII and apoAV synergistically affect triglyceride metabolism. RECENT FINDINGS: It has now been firmly established that variation in plasma triglyceride levels in a wide range of human populations is strongly associated with genetic variation at the chromosomal locus encoding both the APOC3 and APOA5 genes, the APOA1/C3/A4/A5 gene cluster. The close physical linkage of these genes and the frequent concurrence of genetic variants, however, complicate the assignment of specific metabolic defects to specific polymorphisms. Recent insight into the regulation of APOC3 and APOA5 gene expression and structural modeling studies on the apoAV protein have provided novel clues for the potential molecular mechanisms responsible for the effects of apoCIII and apoAV on triglyceride metabolism. SUMMARY: Hypertriglyceridemia is a major independent risk factor in the development of cardiovascular disease. Moreover, triglyceride-derived fatty acids are thought to play a key role in the development and progression of the metabolic syndrome. As modulators of triglyceride metabolism, apoCIII and apoAV are key players and potential therapeutic targets. However, little is known of their molecular mechanism and potential cooperativity. Rational therapeutic application will require the filling of this hiatus in our knowledge.

den Boer M, Voshol PJ, Kuipers F, Havekes LM, Romijn JA. · TNO Prevention and Health, Gaubius Laboratory Leiden, Leiden, The Netherlands. · Arterioscler Thromb Vasc Biol. · Pubmed #14715643 links to  free full text

Abstract: Epidemiological studies in humans, as well as experimental studies in animal models, have shown an association between visceral obesity and dyslipidemia, insulin resistance, and type 2 diabetes mellitus. Recently, attention has been focused on the excessive accumulation of triglycerides (TG) in the liver as part of this syndrome. In this review, important principles of the pathophysiological involvement of the liver in the metabolic syndrome obtained in rodent models are summarized. We focus on non-alcoholic causes of steatosis, because the animal experiments we refer to did not include alcohol as an experimental condition. In general, there is continuous cycling and redistribution of non-oxidized fatty acids between different organs. The amount of TG in an intrinsically normal liver is not fixed but can readily be increased by nutritional, metabolic, and endocrine interactions involving TG/free fatty acid (FFA) partitioning and TG/FFA metabolism. Several lines of evidence indicate that hepatic TG accumulation is also a causative factor involved in hepatic insulin resistance. Complex interactions between endocrine, metabolic, and transcriptional pathways are involved in TG-induced hepatic insulin resistance. Therefore, the liver participates passively and actively in the metabolic derangements of the metabolic syndrome. We speculate that similar mechanisms may also be involved in human pathophysiology.

de Beer F, Smelt AH, van Vark LC, Hoogerbrugge N, Havekes LM, Gevers Leuven JA. · TNO Prevention and Health, Gaubius Laboratory, Leiden University Medical Center, The Netherlands. · J Intern Med. · Pubmed #11908467 No free full text.

Abstract: OBJECTIVE: To investigate the short-term effect of treatment with tibolone on plasma lipid and lipoprotein levels in postmenopausal women with type III hyperlipoproteinemia (HLP). DESIGN AND INTERVENTION: Patients were randomized to receive, in a double-blind cross-over fashion, a fixed dose of tibolone, 2.5 mg once daily or placebo for 8 weeks. The two treatment periods were separated by a wash-out period of 6 weeks. At each visit body weight and blood pressure were determined. Before and after each treatment period, fasting venous blood samples were obtained from the patients for biochemical measurements. SETTING: The Leiden University Medical Center. SUBJECTS: Postmenopausal women with type III HLP (aged < or = 65 years) were recruited from the Lipid Clinics of the Leiden University Medical Center, the Amsterdam Medical Center, the Utrecht Medical Center and the University Hospital Rotterdam. Five out of 25 women with type III HLP were eligible to be included in the study. Four of the five included patients completed the study according to the protocol. One patient was excluded from blinded therapy because total cholesterol levels increased above 20 mmol L(-1). MAIN OUTCOME MEASURES: A significant reduction of plasma triglyceride, total cholesterol, VLDL cholesterol and VLDL triglyceride levels. RESULTS: Plasma triglyceride and total cholesterol levels decreased from 6.82 +/- 3.58 to 2.45 +/- 1.36 mmol L(-1) and from 13.53 +/- 3.64 to 6.61 +/- 2.03 mmol L(-1), respectively (both P < 0.05). The body mass index remained unchanged. The glycated haemoglobin percentage decreased significantly from 5.8 to 5.3%. Treatment with tibolone resulted in a profound reduction in plasma apolipoprotein E, VLDL cholesterol and VLDL triglyceride levels (mean reductions of 66, 77 and 70%, respectively, P < 0.05). CONCLUSIONS: Tibolone is a valuable adjuvant to current therapy in postmenopausal women with type III HLP.

de Man FH, de Beer F, van der Laarse A, Jansen H, Leuven JA, Souverijn JH, Vroom TF, Schoormans SC, Fruchart JC, Havekes LM, Smelt AH. · Department of Cardiology, Leiden University Medical Center, The Netherlands. · Atherosclerosis. · Pubmed #11164425 No free full text.

Abstract: Fibrates are regarded as drugs of choice in hypertriglyceridemia (HTG). Downregulation of apolipoprotein (apo) C-III gene expression and upregulation of lipoprotein lipase (LPL) gene expression have been suggested to explain the hypolipidemic action of fibrates. This study was designed to study the effects of bezafibrate therapy on very low density lipoprotein (VLDL) susceptibility to lipolysis, VLDL binding to the low density lipoprotein (LDL) receptor and postheparin LPL activities in patients with HTG. VLDL lipolysis was studied with heparan sulfate proteoglycan-bound LPL. Binding affinity of VLDL to the LDL receptor was determined in J774 cells with 125I-labeled control LDL. Eighteen HTG patients were randomized to receive, in a double-blind placebo-controlled cross-over fashion, 400 mg bezafibrate once daily for 6 weeks. In response to bezafibrate therapy, plasma triglyceride and apoC-III levels decreased by 69 and 42%, respectively. HTG VLDL was lipolyzed less efficiently compared to control VLDL, and lipolysis did not improve by bezafibrate therapy. VLDL binding affinity to the LDL receptor was comparable between the control group and HTG group, and did not change upon bezafibrate therapy. However, the post-heparin LPL activity in the HTG patients increased from 153 to 192 U/l (P = 0.025). A strong inverse relation was observed between the change in LPL activities and the change in triglyceride levels (r = -0.62, P = 0.006). In conclusion, the hypolipidemic action of bezafibrate therapy in HTG may be attributed to increased LPL activity, whereas VLDL susceptibility to lipolysis and LDL receptor binding are not affected.

Gerritsen G, van der Hoogt CC, Schaap FG, Voshol PJ, Kypreos KE, Maeda N, Groen AK, Havekes LM, Rensen PC, van Dijk KW. · Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. · J Lipid Res. · Pubmed #18263930 links to  free full text

Abstract: Apolipoprotein E2 (apoE2)-associated hyperlipidemia is characterized by a disturbed clearance of apoE2-enriched VLDL remnants. Because excess apoE2 inhibits LPL-mediated triglyceride (TG) hydrolysis in vitro, we investigated whether direct or indirect stimulation of LPL activity in vivo reduces the apoE2-associated hypertriglyceridemia. Here, we studied the role of LPL and two potent modifiers, the LPL inhibitor apoC-III and the LPL activator apoA-V, in APOE2-knockin (APOE2) mice. Injection of heparin in APOE2 mice reduced plasma TG by 53% and plasma total cholesterol (TC) by 18%. Adenovirus-mediated overexpression of LPL reduced plasma TG by 85% and TC by 40%. Both experiments indicate that the TG in apoE2-enriched particles is a suitable substrate for LPL. Indirect activation of LPL activity via deletion of Apoc3 in APOE2 mice did not affect plasma TG levels, whereas overexpression of Apoa5 in APOE2 mice did reduce plasma TG by 81% and plasma TC by 41%. In conclusion, the hypertriglyceridemia in APOE2 mice can be ameliorated by the direct activation of LPL activity. Indirect activation of LPL via overexpression of apoA-V does, whereas deletion of apoC-III does not, affect the plasma TGs in APOE2 mice. These data indicate that changes in apoA-V levels have a dominant effect over changes in apoC-III levels in the improvement of APOE2-associated hypertriglyceridemia.

Westerterp M, Berbée JF, Pires NM, van Mierlo GJ, Kleemann R, Romijn JA, Havekes LM, Rensen PC. · The Netherlands Organization for Applied Scientific Research-Quality of Life, Department of Biomedical Research, Gaubius Laboratory, Leiden, The Netherlands. · Circulation. · Pubmed #17967778 links to  free full text

Abstract: BACKGROUND: Lipopolysaccharide (LPS), which is released from gram-negative bacteria on multiplication or lysis, aggravates atherosclerosis in humans and rodents by inducing inflammation via toll-like receptors. Because apolipoprotein C-I (apoCI) enhances the LPS-induced inflammatory response in macrophages in vitro and in mice, we investigated the effect of endogenous apoCI expression on LPS-induced atherosclerosis in mice. METHODS AND RESULTS: Twelve-week-old apoe-/- apoc1-/- and apoe-/- apoc1+/+ mice received weekly intraperitoneal injections of LPS (50 microg) or vehicle for a period of 10 weeks, and atherosclerosis development was assessed in the aortic root. LPS administration did not affect atherosclerotic lesion area in apoe-/- apoc1-/- mice but increased it in apoe-/- apoc1+/+ mice. In fact, apoCI expression increased the LPS-induced atherosclerotic lesion area by 60% (P<0.05), concomitant with an increase in LPS-induced plasma levels of fibrinogen and E-selectin. This indicated that apoCI increased the LPS-induced inflammatory state, both systemically (ie, fibrinogen) and at the level of the vessel wall (ie, E-selectin). In addition, both macrophage-derived apoCI and HDL-associated apoCI increased the LPS-induced tumor necrosis factor-alpha response by macrophages in vitro. CONCLUSIONS: We conclude that apoCI is crucially involved in LPS-induced atherosclerosis in apoe-/- mice, which mainly relates to an increased inflammatory response toward LPS. We anticipate that apoCI plasma levels contribute to accelerated atherosclerosis development in individuals who have chronic infection.

de Haan W, van der Hoogt CC, Westerterp M, Hoekstra M, Dallinga-Thie GM, Princen HM, Romijn JA, Jukema JW, Havekes LM, Rensen PC. · Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, P.O. Box 2215, 2301 CE Leiden, The Netherlands. · Atherosclerosis. · Pubmed #17868678 No free full text.

Abstract: OBJECTIVE: In addition to lowering low-density lipoprotein (LDL)-cholesterol, statins modestly increase high-density lipoprotein (HDL)-cholesterol in humans and decrease cholesteryl ester transfer protein (CETP) mass and activity. Our aim was to determine whether the increase in HDL depends on CETP expression. METHODS AND RESULTS: APOE*3-Leiden (E3L) mice, with a human-like lipoprotein profile and a human-like responsiveness to statin treatment, were crossbred with mice expressing human CETP under control of its natural flanking regions resulting in E3L.CETP mice. E3L and E3L.CETP mice were fed a Western-type diet with or without atorvastatin. Atorvastatin (0.01% in the diet) reduced plasma cholesterol in both E3L and E3L.CETP mice (-26 and -33%, P<0.05), mainly in VLDL, but increased HDL-cholesterol only in E3L.CETP mice (+52%). Hepatic mRNA expression levels of genes involved in HDL metabolism, such as phospholipid transfer protein (Pltp), ATP-binding cassette transporter A1 (Abca1), scavenger receptor class B type I (Sr-b1), and apolipoprotein AI (Apoa1), were not differently affected by atorvastatin in E3L.CETP mice as compared to E3L mice. However, in E3L.CETP mice, atorvastatin down-regulated the hepatic CETP mRNA expression (-57%; P<0.01) as well as the total CETP level (-29%) and cholesteryl esters (CE) transfer activity (-36%; P<0.05) in plasma. CONCLUSIONS: Atorvastatin increases HDL-cholesterol in E3L.CETP mice by reducing the CETP-dependent transfer of cholesterol from HDL to (V)LDL, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool.

Alkemade FE, Gittenberger-de Groot AC, Schiel AE, VanMunsteren JC, Hogers B, van Vliet LS, Poelmann RE, Havekes LM, Willems van Dijk K, DeRuiter MC. · Department of Anatomy and Embryology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. · Arterioscler Thromb Vasc Biol. · Pubmed #17656671 links to  free full text

Abstract: OBJECTIVE: Maternal hypercholesterolemia is associated with a higher incidence and faster progression of atherosclerotic lesions in neonatal offspring. We aimed to determine whether an in utero environment exposing a fetus to maternal hypercholesterolemia and associated risk factors can prime the murine vessel wall to accelerated development of cardiovascular disease in adult life. METHODS AND RESULTS: To investigate the epigenetic effect in utero, we generated genetically identical heterozygous apolipoprotein E-deficient progeny from mothers with a wild-type or apolipoprotein E-deficient background. A significant increase in loss of endothelial cell volume was observed in the carotid arteries of fetuses of apolipoprotein E-deficient mothers, but fatty streak formation was absent. Spontaneous atherosclerosis development was absent in the aorta and carotid arteries in adult life. We unilaterally placed a constrictive collar around the carotid artery to induce lesion formation. In offspring from apolipoprotein E-deficient mothers, collar placement resulted in severe neointima formation in 9 of 10 mice analyzed compared with only minor lesion volume (2 of 10) in the progeny of wild-type mothers. CONCLUSIONS: We conclude that the susceptibility to neointima formation of morphologically normal adult arteries is already imprinted during prenatal development and manifests itself in the presence of additional atherogenic risk factors in adult life. Future research will concentrate on the mechanisms involved in this priming process, as well as on prevention strategies.

Moen CJ, Tholens AP, Voshol PJ, de Haan W, Havekes LM, Gargalovic P, Lusis AJ, van Dyk KW, Frants RR, Hofker MH, Rensen PC. · Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. · J Lipid Res. · Pubmed #17609525 links to  free full text

Abstract: The Hyplip2 congenic mouse strain contains part of chromosome 15 from MRL/MpJ on the BALB/cJ background. Hyplip2 mice show increased plasma levels of cholesterol and predominantly triglycerides (TGs) and are susceptible to diet-induced atherosclerosis. This study aimed at elucidation of the mechanism(s) explaining the hypertriglyceridemia. Hypertriglyceridemia can result from increased intestinal or hepatic TG production and/or by decreased LPL-mediated TG clearance. The intestinal TG absorption and chylomicron formation were studied after intravenous injection of Triton WR1339 and an intragastric load of olive oil containing glycerol tri[(3)H]oleate. No difference was found in intestinal TG absorption. Moreover, the hepatic VLDL-TG production rate and VLDL particle production, after injection of Triton WR1339, were also not affected. To investigate the LPL-mediated TG clearance, mice were injected intravenously with glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles. In Hyplip2 mice, the particles were cleared at a decreased rate (half-life of 25 +/- 6 vs. 11 +/- 2 min; P < 0.05) concomitant with a decreased uptake of emulsion TG-derived (3)H-labeled fatty acids by the liver and white adipose tissue. The increased plasma TG levels in Hyplip2 mice do not result from an enhanced intestinal absorption or increased hepatic VLDL production but are caused by decreased LPL-mediated TG clearance.

Zadelaar AS, Boesten LS, Jukema JW, van Vlijmen BJ, Kooistra T, Emeis JJ, Lundholm E, Camejo G, Havekes LM. · Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands. · Arterioscler Thromb Vasc Biol. · Pubmed #16931788 links to  free full text

Abstract: OBJECTIVE: We investigated whether the dual PPARalpha/gamma agonist tesaglitazar has anti-atherogenic effects in ApoE*3Leiden mice with reduced insulin sensitivity. METHODS AND RESULTS: ApoE*3Leiden transgenic mice were fed a high-fat (HF) insulin-resistance-inducing diet. One group received a high-cholesterol (HC) supplement (1% wt/wt; HC group). A second group received the same HC supplement along with tesaglitazar (T) 0.5 micromol/kg diet (T group). A third (control) group received a low-cholesterol (LC) supplement (0.1% wt/wt; LC group). Tesaglitazar decreased plasma cholesterol by 20% compared with the HC group; cholesterol levels were similar in the T and LC groups. Compared with the HC group, tesaglitazar caused a 92% reduction in atherosclerosis, whereas a 56% reduction was seen in the cholesterol-matched LC group. Furthermore, tesaglitazar treatment significantly reduced lesion number beyond that expected from cholesterol lowering and induced a shift to less severe lesions. Concomitantly, tesaglitazar reduced macrophage-rich and collagen areas. In addition, tesaglitazar reduced inflammatory markers, including plasma SAA levels, the number of adhering monocytes, and nuclear factor kappaB-activity in the vessel wall. CONCLUSIONS: Tesaglitazar has anti-atherosclerotic effects in the mouse model that go beyond plasma cholesterol lowering, possibly caused by a combination of altered lipoprotein profiles and anti-inflammatory vascular effects.

Henneman P, Schaap FG, Havekes LM, Rensen PC, Frants RR, van Tol A, Hattori H, Smelt AH, van Dijk KW. · Department of Human Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. · Atherosclerosis. · Pubmed #16777114 No free full text.

Abstract: OBJECTIVE: The recently discovered apoAV is hypothesized to affect triglyceride metabolism by stimulating the lipolysis of triglycerides in VLDL and chylomicrons. We set out to determine the association between increased serum TG levels, plasma apoAV levels, and polymorphism of the APOA5 gene, with specific emphasis on the APOA5 S19W variation. This mutation alters the endoplasmic reticulum signal peptide and is hypothesized to impair apoAV secretion into the circulation. METHODS AND RESULTS: Two haplotype-tagging APOA5 polymorphisms, APOA5 S19W and APOA5 -1131T>C and plasma apoAV levels were determined in a population of patients with severe hypertriglyceridemia (HTG). As compared to a random control population, the allele frequencies of the APOA5 S19W and -1131T>C rare variants were significantly increased in HTG patients. Furthermore, the HTG population exhibited markedly elevated plasma apoAV levels that were positively correlated with serum TG levels. Plasma apoAV levels were positively correlated with occurrence of the APOA5 S19W rare variant. CONCLUSIONS: The increased allele frequencies of the APOA5 S19W and -1131T>C rare variants in the HTG population are in agreement with previous reports. Our data show a positive correlation between apoAV and TG levels. Moreover the finding of a positive association between apoAV levels and the APOA5 S19W rare variant is in disagreement with the hypothesis that this variant is poorly secreted.

Westerterp M, de Haan W, Berbée JF, Havekes LM, Rensen PC. · Netherlands Organization for Applied Scientific Research-Quality of Life, Department of Biomedical Research, Gaubius Laboratory, 2301 CE Leiden, The Netherlands. · J Lipid Res. · Pubmed #16537968 links to  free full text

Abstract: Previous studies have shown that overexpression of human apolipoprotein C-I (apoC-I) results in moderate hypercholesterolemia and severe hypertriglyceridemia in mice in the presence and absence of apoE. We assessed whether physiological endogenous apoC-I levels are sufficient to modulate plasma lipid levels independently of effects of apoE on lipid metabolism by comparing apolipoprotein E gene-deficient/apolipoprotein C-I gene-deficient (apoe-/-apoc1-/-), apoe-/-apoc1+/-, and apoe-/-apoc1+/+ mice. The presence of the apoC-I gene-dose-dependently increased plasma cholesterol (+45%; P < 0.001) and triglycerides (TGs) (+137%; P < 0.001), both specific for VLDL. Whereas apoC-I did not affect intestinal [3H]TG absorption, it increased the production rate of hepatic VLDL-TG (+35%; P < 0.05) and VLDL-[35S]apoB (+39%; P < 0.01). In addition, apoC-I increased the postprandial TG response to an intragastric olive oil load (+120%; P < 0.05) and decreased the uptake of [3H]TG-derived FFAs from intravenously administered VLDL-like emulsion particles by gonadal and perirenal white adipose tissue (WAT) (-34% and -25%, respectively; P < 0.05). As LPL is the main enzyme involved in the clearance of TG-derived FFAs by WAT, and total postheparin plasma LPL levels were unaffected, these data demonstrate that endogenous apoC-I suffices to attenuate the lipolytic activity of LPL. Thus, we conclude that endogenous plasma apoC-I increases VLDL-total cholesterol and VLDL-TG dose-dependently in apoe-/- mice, resulting from increased VLDL particle production and LPL inhibition.

van der Hoogt CC, Berbée JF, Espirito Santo SM, Gerritsen G, Krom YD, van der Zee A, Havekes LM, van Dijk KW, Rensen PC. · The Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, P.O. Box 2215, 2301 CE Leiden, The Netherlands. · Biochim Biophys Acta. · Pubmed #16478678 No free full text.

Abstract: We have recently shown that the predominant hypertriglyceridemia in human apolipoprotein C1 (APOC1) transgenic mice is mainly explained by apoCI-mediated inhibition of the lipoprotein lipase (LPL)-dependent triglyceride (TG)-hydrolysis pathway. Since the very-low-density lipoprotein receptor (VLDLr) and apoCIII are potent modifiers of LPL activity, our current aim was to study whether the lipolysis-inhibiting action of apoCI would be dependent on the presence of the VLDLr and apoCIII in vivo. Hereto, we employed liver-specific expression of human apoCI by using a novel recombinant adenovirus (AdAPOC1). In wild-type mice, moderate apoCI expression leading to plasma human apoCI levels of 12-33 mg/dl dose-dependently and specifically increased plasma TG (up to 6.6-fold, P < 0.001), yielding the same hypertriglyceridemic phenotype as observed in human APOC1 transgenic mice. AdAPOC1 still increased plasma TG in vldlr(-/-) mice (4.1-fold, P < 0.001) and in apoc3(-/-) mice (6.8-fold, P < 0.001) that were also deficient for the low-density lipoprotein receptor (LDLr) and LDLr-related protein (LRP) or apoE, respectively. Thus, irrespective of receptor-mediated remnant clearance by the liver, liver-specific expression of human apoCI causes hypertriglyceridemia in the absence of the VLDLr and apoCIII. We conclude that apoCI is a powerful and direct inhibitor of LPL activity independent of the VLDLr and apoCIII.

den Boer MA, Berbée JF, Reiss P, van der Valk M, Voshol PJ, Kuipers F, Havekes LM, Rensen PC, Romijn JA. · Department of Endocrinology and Diabetes, Leiden University Medical Center, Leiden, The Netherlands. · Arterioscler Thromb Vasc Biol. · Pubmed #16269669 links to  free full text

Abstract: OBJECTIVE: The use of the HIV protease inhibitor ritonavir (RTV) is frequently associated with hypertriglyceridemia and lipodystrophy. The aim of our study was to determine the mechanism underlying the observed hypertriglyceridemia. METHODS AND RESULTS: Feeding female APOE*3-Leiden transgenic mice a Western-type diet supplemented with RTV (35 mg/kg per day) for 2 weeks resulted in a 2-fold increase in fasting plasma triglyceride (TG) levels, which was specific for very low-density lipoprotein (VLDL). RTV did not change the hepatic VLDL-TG production. Instead, RTV did increase the postprandial TG response to an oral fat load (area under the curve, 25.5+/-12.1 versus 13.8+/-6.8 mmol/L per hour in controls; P<0.05). Likewise, RTV hampered the plasma clearance of intravenously injected glycerol tri[3H]oleate-labeled VLDL-like emulsion particles (half time, 19.3+/-10.5 versus 5.0+/-1.3 minutes in controls; P<0.05) associated with a decrease of 44% in plasma lipoprotein lipase activity. Accordingly, RTV decreased the uptake of TG-derived fatty acids (FAs) into adipose tissue, as well as the uptake of albumin-bound FA. CONCLUSIONS: We conclude that RTV causes hypertriglyceridemia via decreased lipoprotein lipase-mediated clearance of VLDL-TG. In addition, RTV specifically impairs the uptake of FA in adipose tissue, which may contribute to the lipodystrophy that is frequently observed in HIV-infected subjects on antiretroviral therapy.

Kreeft AJ, Moen CJ, Porter G, Kasanmoentalib S, Sverdlov R, van Gorp PJ, Havekes LM, Frants RR, Hofker MH. · Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands. · Atherosclerosis. · Pubmed #16159597 No free full text.

Abstract: The mechanisms of diet induced hyperlipidemia and atherosclerosis have been widely studied by delineating the role of candidate genes in transgenic and gene targeted mouse models. However, diet induced hyperlipidemia represents a complex process determined by many lipid genes that is only partly understood. This study is aimed at delineating the events induced by dietary intervention in different mouse models at the level of gene expression using microarray analysis. The focus is on the liver as the organ primarily responding to diet, and crucial in determining plasma lipid levels. Firstly, the effect of the genotype was studied. Expression profiles of liver genes were compared between APOE3Leiden (E3L), APOE knockout (E-/-) and C57BL/6JIco (B6) mice using the Incyte GEM 2.03 array carrying 9552 genes. Several hundred differentially expressed genes were identified indicating that the genotype alone effects gene expression. Secondly, the response of E3L mice to high-fat feeding was investigated using a mild and severe high-fat diet (diet W and N, respectively). Diet W caused differential regulation of 200 genes, while diet N affected the expression of 788 genes in B6 and 1010 genes in E3L mice. Annotation of these genes using the Gene Ontology (GO) database showed that two major processes were strongly affected by genotype and diet, namely lipid metabolism and inflammation, the latter as determined by "immune/defense response and detoxification" processes. Many nuclear receptor target genes were differentially regulated, with the largest effects modulated by the severe high-fat diet N, leading to the suppression of genes involved in bile acid, sterol, steroid, fatty acid, and detoxification metabolism. Strikingly, a substantial part of these nuclear receptor target genes were commonly regulated during the different experimental conditions. The common regulation of many nuclear receptor target genes underlying lipid and detoxification processes as found in this study, suggest a defense mechanism involving many nuclear receptors to protect against the accumulation of toxic endogenous lipids and bile acids. These results further strengthen the close link between hyperlipidemia and inflammatory processes.

Trion A, de Maat MP, Jukema JW, van der Laarse A, Maas MC, Offerman EH, Havekes LM, Szalai AJ, Princen HM, Emeis JJ. · Department of Cardiology, Leiden University Medical Center, The Netherlands. · Arterioscler Thromb Vasc Biol. · Pubmed #15920036 links to  free full text

Abstract: OBJECTIVE: C-reactive protein (CRP) has been associated with risk of cardiovascular disease. It is not clear whether CRP is causally involved in the development of atherosclerosis. Mouse CRP is not expressed at high levels under normal conditions and increases in concentration only several-fold during an acute phase response. Because the dynamic range of human CRP is much larger, apolipoprotein E*3-Leiden (E3L) transgenic mice carrying the human CRP gene offer a unique model to study the role(s) of CRP in atherosclerosis development. METHODS AND RESULTS: Atherosclerosis development was studied in 15 male and 15 female E3L/CRP mice; E3L transgenic littermates were used as controls. The mice were fed a hypercholesterolemic diet to induce atherosclerosis development. Cholesterol exposure did not differ between E3L/CRP and E3L mice. Plasma CRP levels were on average 10.2+/-6.5 mg/L in male E3L/CRP mice, 0.2+/-0.1 mg/L in female E3L/CRP mice, and undetectable in E3L mice. Quantification of atherosclerosis showed that lesion area in E3L/CRP mice was not different from that in E3L mice. CONCLUSIONS: This study demonstrates that mildly elevated levels of CRP in plasma do not contribute to the development of early atherosclerosis in hypercholesterolemic E3L/CRP mice.

Gerritsen G, Rensen PC, Kypreos KE, Zannis VI, Havekes LM, Willems van Dijk K. · Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. · J Lipid Res. · Pubmed #15863838 links to  free full text

Abstract: Adenovirus-mediated overexpression of human apolipoprotein E (apoE) induces hyperlipidemia by stimulating the VLDL-triglyceride (TG) production rate and inhibiting the LPL-mediated VLDL-TG hydrolysis rate. Because apoC-III is a strong inhibitor of TG hydrolysis, we questioned whether Apoc3 deficiency might prevent the hyperlipidemia induced by apoE overexpression in vivo. Injection of 2 x 10(9) plaque-forming units of AdAPOE4 caused severe combined hyperlipidemia in Apoe-/- mice [TG from 0.7 +/- 0.2 to 57.2 +/- 6.7 mM; total cholesterol (TC) from 17.4 +/- 3.7 to 29.0 +/- 4.1 mM] that was confined to VLDL/intermediate density lipoprotein-sized lipoproteins. In contrast, Apoc3 deficiency resulted in a gene dose-dependent reduction of the apoE4-associated hyperlipidemia (TG from 57.2 +/- 6.7 mM to 21.2 +/- 18.5 and 1.5 +/- 1.4 mM; TC from 29.0 +/- 4.1 to 16.4 +/- 9.8 and 2.3 +/- 1.8 mM in Apoe-/-, Apoe-/-.Apoc3+/-, and Apoe-/-.Apoc3-/- mice, respectively). In both Apoe-/- mice and Apoe-/-.Apoc3-/- mice, injection of increasing doses of AdAPOE4 resulted in up to a 10-fold increased VLDL-TG production rate. However, Apoc3 deficiency resulted in a significant increase in the uptake of TG-derived fatty acids from VLDL-like emulsion particles by white adipose tissue, indicating enhanced LPL activity. In vitro experiments showed that apoC-III is a more specific inhibitor of LPL activity than is apoE. Thus, Apoc3 deficiency can prevent apoE-induced hyperlipidemia associated with a 10-fold increased hepatic VLDL-TG production rate, most likely by alleviating the apoE-induced inhibition of VLDL-TG hydrolysis.

Espirito Santo SM, Rensen PC, Goudriaan JR, Bensadoun A, Bovenschen N, Voshol PJ, Havekes LM, van Vlijmen BJ. · Netherlands Organization for Applied Scientific Research-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands. · J Lipid Res. · Pubmed #15772433 links to  free full text

Abstract: 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.

Davidov E, Clish CB, Oresic M, Meys M, Stochaj W, Snell P, Lavine G, Londo TR, Adourian A, Zhang X, Johnston M, Morel N, Marple EW, Plasterer TN, Neumann E, Verheij E, Vogels JT, Havekes LM, van der Greef J, Naylor S. · Beyond Genomics, Inc., Waltham, Massachusetts, USA. · OMICS. · Pubmed #15703476 No free full text.

Abstract: Multitiered quantitative analysis of biological systems is rapidly becoming the desired approach to study hierarchical functional interactions between proteins and metabolites. We describe here a novel systematic approach to analyze organisms with complex metabolic regulatory networks. By using precise analytical methods to measure biochemical constituents and their relative abundance in whole plasma of transgenic ApoE*3-Leiden mice and an isogenic wild-type control group, simultaneous snapshots of metabolic and protein states were obtained. Novel data processing and multivariate analysis tools such as Impurity Resolution Software (IMPRESS) and Windows-based linear fit program (WINLIN) were used to compare protein and metabolic profiles in parallel. Canonical correlations of the resulting data show quantitative relationships between heterogeneous components in the TG animals. These results, obtained solely from whole plasma analysis allowed us, in a rapid manner, to corroborate previous findings as well as find new events pertaining to dominant and peripheral events in lipoprotein metabolism of a genetically modified mammalian organism in relation to ApoE3, a key mediator of lipoprotein metabolism.

Berbée JF, van der Hoogt CC, Sundararaman D, Havekes LM, Rensen PC. · Netherlands Organization for Applied Scientific Research-Prevention and Health, Gaubius Laboratory, 2301 CE Leiden, The Netherlands. · J Lipid Res. · Pubmed #15576844 links to  free full text

Abstract: Studies in humans and mice have shown that increased expression of apolipoprotein C-I (apoC-I) results in combined hyperlipidemia with a more pronounced effect on triglycerides (TGs) compared with total cholesterol (TC). The aim of this study was to elucidate the main reason for this effect using human apoC-I-expressing (APOC1) mice. Moderate plasma human apoC-I levels (i.e., 4-fold higher than human levels) caused a 12-fold increase in TG, along with a 2-fold increase in TC, mainly confined to VLDL. Cross-breeding of APOC1 mice on an apoE-deficient background resulted in a marked 55-fold increase in TG, confirming that the apoC-I-induced hyperlipidemia cannot merely be attributed to blockade of apoE-recognizing hepatic lipoprotein receptors. The plasma half-life of [3H]TG-VLDL-mimicking particles was 2-fold increased in APOC1 mice, suggesting that apoC-I reduces the lipolytic conversion of VLDL. Although total postheparin plasma LPL activity was not lower in APOC1 mice compared with controls, apoC-I was able to dose-dependently inhibit the LPL-mediated lipolysis of [3H]TG-VLDL-mimicking particles in vitro with a 60% efficiency compared with the main endogenous LPL inhibitor apoC-III. Finally, purified apoC-I impaired the clearance of [3H]TG-VLDL-mimicking particles independent of apoE-mediated hepatic uptake in lactoferrin-treated mice. Therefore, we conclude that apoC-I is a potent inhibitor of LPL-mediated TG-lipolysis.

Kypreos KE, van Dijk KW, Havekes LM, Zannis VI. · Molecular Genetics, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA. · J Biol Chem. · Pubmed #15576362 links to  free full text

Abstract: To identify the residues in the carboxyl-terminal region 260-299 of human apolipoprotein E (apoE) that contribute to hypertriglyceridemia, two sets of conserved, hydrophobic amino acids between residues 261 and 283 were mutated to alanines, and recombinant adenoviruses expressing these apoE mutants were generated. Adenovirus-mediated gene transfer of apoE4-mut1 (apoE4 (L261A, W264A, F265A, L268A, V269A)) in apoE-deficient mice (apoE(-/-)) corrected plasma cholesterol levels and did not cause hypertriglyceridemia. In contrast, gene transfer of apoE4-mut2 (apoE4 (W276A, L279A, V280A, V283A)) did not correct hypercholesterolemia and induced mild hypertriglyceridemia. ApoE-induced hyperlipidemia was corrected by co-infection with a recombinant adenovirus expressing human lipoprotein lipase. Both apoE4 mutants caused only a small increase in hepatic very low density lipoprotein-triglyceride secretion. Density gradient ultracentrifugation analysis of plasma and electron microscopy showed that wild-type apoE4 and apoE4-mut2 displaced apoA-I from the high density lipoprotein (HDL) region and promoted the formation of discoidal HDL, whereas the apoE4-mut1 did not displace apoA-I from HDL and promoted the formation of spherical HDL. The findings indicate that residues Leu-261, Trp-264, Phe-265, Leu-268, and Val-269 of apoE are responsible for hypertriglyceridemia and also interfere with the formation of HDL. Substitutions of these residues by alanine provide a recombinant apoE form with improved biological functions.

Muurling M, van den Hoek AM, Mensink RP, Pijl H, Romijn JA, Havekes LM, Voshol PJ. · Netherlands Organization for Applied Scientific Research-Prevention and Health, Gaubius Laboratory, Leiden, The Netherlands. · J Lipid Res. · Pubmed #14523051 links to  free full text

Abstract: Obese obob mice with strong overexpression of the human apolipoprotein C1 (APOC1) exhibit excessive free fatty acid (FFA) and triglyceride (TG) levels and severely reduced body weight (due to the absence of subcutaneous adipose tissue) and skin abnormalities. To evaluate the effects of APOC1 overexpression on hepatic and peripheral insulin sensitivity in a less-extreme model, we generated obob mice with mild overexpression of APOC1 (obob/APOC1(+/-)) and performed hyperinsulinemic clamp analysis. Compared with obob littermates, obob/APOC1(+/-) mice showed reduced body weight (-25%) and increased plasma levels of TG (+632%), total cholesterol (+134%), FFA (+65%), glucose (+73%), and insulin (+49%). Hyperinsulinemic clamp analysis revealed severe whole-body and hepatic insulin resistance in obob/APOC1(+/-) mice and, in addition, increased hepatic uptake of FFA and hepatic TG content. Treatment of obob/APOC1(+/-) mice with rosiglitazone strongly improved whole-body insulin sensitivity as well as hepatic insulin sensitivity, despite a further increase of hepatic fatty acid (FA) uptake and a panlobular increase of hepatic TG accumulation. We conclude that overexpression of APOC1 prevents rosiglitazone-induced peripheral FA uptake leading to severe hepatic steatosis. Interestingly, despite rosiglitazone-induced hepatic steatosis, hepatic insulin sensitivity improves dramatically. We hypothesize that the different hepatic fat accumulation and/or decrease in FA intermediates has a major effect on the insulin sensitivity of the liver.

Kypreos KE, Li X, van Dijk KW, Havekes LM, Zannis VI. · Molecular Genetics, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118-2394, USA. · Biochemistry. · Pubmed #12924933 No free full text.

Abstract: Apolipoprotein E2, which has an R158 for C substitution, has reduced affinity for the LDL receptor and is associated with type III hyperlipoproteinemia in humans. Consistent with these observations, we have found that following adenovirus-mediated gene transfer, full-length apoE2 aggravates the hypercholesterolemia and induces hypertriglyceridemia in E-deficient mice and induces combined hyperlipidemia in C57BL/6 mice. Unexpectedly, the truncated apoE2-202 form that has an R158 for C substitution when expressed at levels similar to those of the full-length apoE2 normalized the cholesterol levels of E-deficient mice without induction of hypertriglyceridemia. The apoE2 truncation increased the affinity of POPC-apoE particles for the LDL receptor, and the full-length apoE2 had a dominant effect in VLDL triglyceride secretion. Hyperlipidemia in normal C57BL/6 mice was prevented by coinfection with equal doses of each, the apoE2 and the apoE2-202-expressing adenoviruses, indicating that truncated apoE forms have a dominant effect in remnant clearance. Hypertriglyceridemia was completely corrected by coinfection of mice with an adenovirus-expressing wild-type lipoprotein lipase, whereas an inactive lipoprotein lipase had a smaller effect. The findings suggest that the apoE2-induced dyslipidemia is not merely the result of substitution of R158 for C but results from increased secretion of a triglyceride-enriched VLDL that cannot undergo lipolysis, inhibition of LpL activity, and impaired clearance of chylomicron remnants. Infection of E(-)(/)(-)xLDLr(-)(/)(-) double-deficient mice with apoE2-202 did not affect the plasma cholesterol levels, and also did not induce hypertriglyceridemia. In contrast, apoE2 exacerbated the hypercholesterolemia and induced hypertriglyceridemia, suggesting that the LDL receptor is the predominant receptor in remnant clearance.