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Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores −2.56 and −2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (−28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (−47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [14C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance.

Brown adipose tissue (BAT) produces heat by burning TGs that are stored within intracellular lipid droplets and need to be replenished by the uptake of TG-derived FA from plasma. It is currently unclear whether BAT takes up FA via uptake of TG-rich lipoproteins (TRLs), after lipolysis-mediated liberation of FA, or via a combination of both. Therefore, we generated glycerol tri[3H]oleate and [14C]cholesteryl oleate double-labeled TRL-mimicking particles with an average diameter of 45, 80, and 150 nm (representing small VLDL to chylomicrons) and injected these intravenously into male C57Bl/6J mice. At room temperature (21°C), the uptake of 3H-activity by BAT, expressed per gram of tissue, was much higher than the uptake of 14C-activity, irrespective of particle size, indicating lipolysis-mediated uptake of TG-derived FA rather than whole particle uptake. Cold exposure (7°C) increased the uptake of FA derived from the differently sized particles by BAT, while retaining the selectivity for uptake of FA over cholesteryl ester (CE). At thermoneutrality (28°C), total FA uptake by BAT was attenuated, but the specificity of uptake of FA over CE was again largely retained. Altogether, we conclude that, in our model, BAT takes up plasma TG preferentially by means of lipolysis-mediated uptake of FA.

The melanocortin system is an important regulator of energy balance, and melanocortin 4 receptor (MC4R) deficiency is the most common monogenic cause of obesity. We investigated whether the relationship between melanocortin system activity and energy expenditure (EE) is mediated by brown adipose tissue (BAT) activity. Therefore, female APOE*3-Leiden.CETP transgenic mice were fed a Western-type diet for 4 weeks and infused intracerebroventricularly with the melanocortin 3/4 receptor (MC3/4R) antagonist SHU9119 or vehicle for 2 weeks. SHU9119 increased food intake (+30%) and body fat (+50%) and decreased EE by reduction in fat oxidation (−42%). In addition, SHU9119 impaired the uptake of VLDL-TG by BAT. In line with this, SHU9119 decreased uncoupling protein-1 levels in BAT (−60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Finally, SHU9119-treated mice pair-fed to the vehicle-treated group still exhibited these effects, indicating that MC4R inhibition impairs BAT activity independent of food intake. These effects were not specific to the APOE*3-Leiden.CETP background as SHU9119 also inhibited BAT activity in wild-type mice. We conclude that inhibition of central MC3/4R signaling impairs BAT function, which is accompanied by reduced EE, thereby promoting adiposity. We anticipate that activation of MC4R is a promising strategy to combat obesity by increasing BAT activity.

Important players in triglyceride (TG) metabolism include the liver (production), white adipose tissue (WAT) (storage), heart and skeletal muscle (combustion to generate ATP), and brown adipose tissue (BAT) (combustion toward heat), the collective action of which determine plasma TG levels. Interestingly, recent evidence points to a prominent role of the hypothalamus in TG metabolism through innervating the liver, WAT, and BAT mainly via sympathetic branches of the autonomic nervous system. Here, we review the recent findings in the area of sympathetic control of TG metabolism. Various neuronal populations, such as neuropeptide Y (NPY)-expressing neurons and melanocortin-expressing neurons, as well as peripherally produced hormones (i.e., GLP-1, leptin, and insulin), modulate sympathetic outflow from the hypothalamus toward target organs and thereby influence peripheral TG metabolism. We conclude that sympathetic stimulation in general increases lipolysis in WAT, enhances VLDL-TG production by the liver, and increases the activity of BAT with respect to lipolysis of TG, followed by combustion of fatty acids toward heat. Moreover, the increased knowledge about the involvement of the neuroendocrine system in TG metabolism presented in this review offers new therapeutic options to fight hypertriglyceridemia by specifically modulating sympathetic nervous system outflow toward liver, BAT, or WAT.

Cholesteryl ester transfer protein (CETP) activity results in a proatherogenic lipoprotein profile. In cholestatic conditions, farnesoid X receptor (FXR) signaling by bile acids (BA) is activated and plasma HDL cholesterol (HDL-C) levels are low. This study tested the hypothesis that FXR-mediated induction of CETP contributes to this phenotype. Patients with cholestasis and high plasma BA had lower HDL-C levels and higher plasma CETP activity and mass compared with matched controls with low plasma BA (each P < 0.01). BA feeding in APOE3*Leiden transgenic mice expressing the human CETP transgene controlled by its endogenous promoter increased cholesterol within apoB-containing lipoproteins and decreased HDL-C (each P < 0.01), while hepatic CETP mRNA expression and plasma CETP activity and mass increased (each P < 0.01). In vitro studies confirmed that FXR agonists substantially augmented CETP mRNA expression in hepatocytes and macrophages dependent on functional FXR expression (each P < 0.001). These transcriptional effects are likely mediated by an ER8 FXR response element (FXRE) in the first intron. In conclusion, using a translational approach, this study identifies CETP as novel FXR target gene. By increasing CETP expression, FXR activation leads to a proatherogenic lipoprotein profile. These results have clinical relevance, especially when considering FXR agonists as emerging treatment strategy for metabolic disease and atherosclerosis.

Bile acid sequestrants (BASs) are cholesterol-lowering drugs that also affect hyperglycemia. The mechanism by which BASs exert these and other metabolic effects beyond cholesterol lowering remains poorly understood. The present study aimed to investigate the effects of a BAS, colestilan, on body weight, energy expenditure, and glucose and lipid metabolism and its mechanisms of action in high-fat-fed hyperlipidemic APOE*3 Leiden (E3L) transgenic mice. Mildly insulin-resistant E3L mice were fed a high-fat diet with or without 1.5% colestilan for 8 weeks. Colestilan treatment decreased body weight, visceral and subcutaneous fat, and plasma cholesterol and triglyceride levels but increased food intake. Blood glucose and plasma insulin levels were decreased, and hyperinsulinemic-euglycemic clamp analysis demonstrated improved insulin sensitivity, particularly in peripheral tissues. In addition, colestilan decreased energy expenditure and physical activity, whereas it increased the respiratory exchange ratio, indicating that colestilan induced carbohydrate catabolism. Moreover, kinetic analysis revealed that colestilan increased [3H]NEFA incorporation in biliary cholesterol and phospholipids and increased fecal lipid excretion. Gene expression analysis in liver, fat, and muscle supported the above findings. In summary, colestilan decreases weight gain and improves peripheral insulin sensitivity in high-fat-fed E3L mice by enhanced NEFA incorporation in biliary lipids and increased fecal lipid excretion.

Caspase-1 is known to activate the proinflammatory cytokines IL-1β and IL-18. Additionally, it can cleave other substrates, including proteins involved in metabolism. Recently, we showed that caspase-1 deficiency in mice strongly reduces high-fat diet-induced weight gain, at least partly caused by an increased energy production. Increased feces secretion by caspase-1-deficient mice suggests that lipid malabsorption possibly further reduces adipose tissue mass. In this study we investigated whether caspase-1 plays a role in triglyceride-(TG)-rich lipoprotein metabolism using caspase-1-deficient and wild-type mice. Caspase-1 deficiency reduced the postprandial TG response to an oral lipid load, whereas TG-derived fatty acid (FA) uptake by peripheral tissues was not affected, demonstrated by unaltered kinetics of [3H]TG-labeled very low-density lipoprotein (VLDL)-like emulsion particles. An oral gavage of [3H]TG-containing olive oil revealed that caspase-1 deficiency reduced TG absorption and subsequent uptake of TG-derived FA in liver, muscle, and adipose tissue. Similarly, despite an elevated hepatic TG content, caspase-1 deficiency reduced hepatic VLDL-TG production. Intestinal and hepatic gene expression analysis revealed that caspase-1 deficiency did not affect FA oxidation or FA uptake but rather reduced intracellular FA transport, thereby limiting lipid availability for the assembly and secretion of TG-rich lipoproteins. The current study reveals a novel function for caspase-1, or caspase-1-cleaved substrates, in controlling intestinal TG absorption and hepatic TG secretion.

Transintestinal cholesterol efflux (TICE) provides an attractive target to increase body cholesterol excretion. At present, the cholesterol donor responsible for direct delivery of plasma cholesterol to the intestine is unknown. In this study, we investigated the role of HDL in TICE. ATP-binding cassette protein A1 deficient (Abca1−/−) mice that lack HDL and wild-type (WT) mice were intravenously injected with chylomicron-like emulsion particles that contained radiolabeled cholesterol that is liberated in the liver and partly reenters the circulation. Both groups secreted radiolabeled cholesterol from plasma into intestinal lumen and TICE was unaltered between the two mouse models. To further investigate the role of HDL, we injected HDL with radiolabeled cholesterol in WT mice and Abca1−/−×Sr-b1−/− mice that lack HDL and are also unable to clear HDL via the liver. The intestines of both mice were unable to take up and secrete radiolabeled cholesterol from HDL via TICE. Although a generally accepted major player in the hepatobiliary route-based cholesterol excretion, HDL plays no significant role in TICE in mice.

Insulin signaling in the central nervous system (CNS) is required for the inhibitory effect of insulin on glucose production. Our aim was to determine whether the CNS is also involved in the stimulatory effect of circulating insulin on the tissue-specific retention of fatty acid (FA) from plasma. In wild-type mice, hyperinsulinemic-euglycemic clamp conditions stimulated the retention of both plasma triglyceride-derived FA and plasma albumin-bound FA in the various white adipose tissues (WAT) but not in other tissues, including brown adipose tissue (BAT). Intracerebroventricular (ICV) administration of insulin induced a similar pattern of tissue-specific FA partitioning. This effect of ICV insulin administration was not associated with activation of the insulin signaling pathway in adipose tissue. ICV administration of tolbutamide, a KATP channel blocker, considerably reduced (during hyperinsulinemic-euglycemic clamp conditions) and even completely blocked (during ICV administration of insulin) WAT-specific retention of FA from plasma. This central effect of insulin was absent in CD36-deficient mice, indicating that CD36 is the predominant FA transporter in insulin-stimulated FA retention by WAT. In diet-induced insulin-resistant mice, these stimulating effects of insulin (circulating or ICV administered) on FA retention in WAT were lost. In conclusion, in insulin-sensitive mice, circulating insulin stimulates tissue-specific partitioning of plasma-derived FA in WAT in part through activation of KATP channels in the CNS. Apparently, circulating insulin stimulates fatty acid uptake in WAT but not in BAT, directly and indirectly through the CNS.

Human data suggest that reconstituted HDL (rHDL) infusion can induce atherosclerosis regression. Studies in mice indicated that rHDL infusion adversely affects VLDL levels, but this effect is less apparent in humans. This discrepancy may be explained by the fact that humans, in contrast to mice, express cholesteryl ester transfer protein (CETP). The aim of this study was to investigate the role of CETP in the effects of rHDL on VLDL metabolism by using APOE*3-Leiden (E3L) mice, a well-established model for human-like lipoprotein metabolism. At 1 h after injection, rHDL increased plasma VLDL-C and TG in E3L mice, but not in E3L mice cross-bred onto a human CETP background (E3L.CETP mice). This initial raise in VLDL, caused by competition between rHDL and VLDL for LPL-mediated TG hydrolysis, was thus prevented by CETP. At 24 h after injection, rHDL caused a second increase in VLDL-C and TG in E3L mice, whereas rHDL had even decreased VLDL in E3L.CETP mice. This secondary raise in VLDL was due to increased hepatic VLDL-TG production. Collectively, we conclude that CETP protects against the rHDL-induced increase in VLDL. We anticipate that studies evaluating the anti-atherosclerotic efficacy of rHDL in mice that are naturally deficient for CETP should be interpreted with caution, and that treatment of atherogenic dyslipidemia by rHDL should not be combined with agents that aggressively reduce CETP activity.

Low-grade inflammation in different tissues, including activation of the nuclear factor κB pathway in liver, is involved in metabolic disorders such as type 2 diabetes and cardiovascular diseases (CVDs). In this study, we investigated the relation between chronic hepatocyte-specific overexpression of IkB kinase (IKK)-β and hypertriglyceridemia, an important risk factor for CVD, by evaluating whether activation of IKK-β only in the hepatocyte affects VLDL-triglyceride (TG) metabolism directly. Transgenic overexpression of constitutively active human IKK-β specifically in hepatocytes of hyperlipidemic APOE*3-Leiden mice clearly induced hypertriglyceridemia. Mechanistic in vivo studies revealed that the hypertriglyceridemia was caused by increased hepatic VLDL-TG production rather than a change in plasma VLDL-TG clearance. Studies in primary hepatocytes showed that IKK-β overexpression also enhances TG secretion in vitro, indicating a direct relation between IKK-β activation and TG production within the hepatocyte. Hepatic lipid analysis and hepatic gene expression analysis of pathways involved in lipid metabolism suggested that hepatocyte-specific IKK-β overexpression increases VLDL production not by increased steatosis or decreased FA oxidation, but most likely by carbohydrate-responsive element binding protein-mediated upregulation of Fas expression. These findings implicate that specific activation of inflammatory pathways exclusively within hepatocytes induces hypertriglyceridemia. Furthermore, we identify the hepatocytic IKK-β pathway as a possible target to treat hypertriglyceridemia.

Timely sensing of lipopolysaccharide (LPS) is critical for the host to fight invading Gram-negative bacteria. We recently showed that apolipoprotein CI (apoCI) (apoCI1–57) avidly binds to LPS, involving an LPS-binding motif (apoCI48–54), and thereby enhances the LPS-induced inflammatory response. Our current aim was to further elucidate the structure and function relationship of apoCI with respect to its LPS-modulating characteristics and to unravel the mechanism by which apoCI enhances the biological activity of LPS. We designed and generated N- and C-terminal apoCI-derived peptides containing varying numbers of alternating cationic/hydrophobic motifs. ApoCI1–38, apoCI1–30, and apoCI35–57 were able to bind LPS, whereas apoCI1–23 and apoCI46–57 did not bind LPS. In line with their LPS-binding characteristics, apoCI1–38, apoCI1–30, and apoCI35–57 prolonged the serum residence of 125I-LPS by reducing its association with the liver. Accordingly, both apoCI1–30 and apoCI35–57 enhanced the LPS-induced TNFα response in vitro (RAW 264.7 macrophages) and in vivo (C57Bl/6 mice). Additional in vitro studies showed that the stimulating effect of apoCI on the LPS response resembles that of LPS-binding protein (LBP) and depends on CD14/ Toll-like receptor 4 signaling. We conclude that apoCI contains structural elements in both its N-terminal and C-terminal helix to bind LPS and to enhance the proinflammatory response toward LPS via a mechanism similar to LBP.

The cholesteryl ester transfer protein (CETP) facilitates the bidirectional transfer of cholesteryl esters and triglycerides (TG) between HDL and (V)LDL. By shifting cholesterol in plasma from HDL to (V)LDL in exchange for VLDL-TG, CETP aggravates atherosclerosis in hyperlipidemic APOE*3-Leiden (E3L) mice. The aim of this study was to investigate the role of CETP in TG metabolism and high-fat diet-induced obesity by using E3L mice with and without the expression of the human CETP gene. On chow, plasma lipid levels were comparable between both male and female E3L and E3L.CETP mice. Further mechanistic studies were performed using male mice. CETP expression increased the level of TG in HDL. CETP did not affect the postprandial plasma TG response or the hepatic VLDL-TG and VLDL-apolipoprotein B production rate. Moreover, CETP did not affect the plasma TG clearance rate or organ-specific TG uptake after infusion of VLDL-like emulsion particles. In line with the absence of an effect of CETP on tissue-specific TG uptake, CETP also did not affect weight gain in response to a high-fat diet. In conclusion, the CETP-induced increase of TG in the HDL fraction of E3L mice is not associated with changes in the production of TG or with tissue-specific clearance of TG from the plasma.

Increased plasma cholesterol is a known risk factor for cardiovascular disease. Lipoprotein particles transport both cholesterol and triglycerides through the blood. It is thought that the size distribution of these particles codetermines cardiovascular disease risk. New types of measurements can determine the concentration of many lipoprotein size-classes but exactly how each small class relates to disease risk is difficult to clear up. Because relating physiological process status to disease risk seems promising, we propose investigating how lipoprotein production, lipolysis, and uptake processes depend on particle size. To do this, we introduced a novel model framework (Particle Profiler) and evaluated its feasibility. The framework was tested using existing stable isotope flux data. The model framework implementation we present here reproduced the flux data and derived lipoprotein size pattern changes that corresponded to measured changes. It also sensitively indicated changes in lipoprotein metabolism between patient groups that are biologically plausible. Finally, the model was able to reproduce the cholesterol and triglyceride phenotype of known genetic diseases like familial hypercholesterolemia and familial hyperchylomicronemia. In the future, Particle Profiler can be applied for analyzing detailed lipoprotein size profile data and deriving rates of various lipolysis and uptake processes if an independent production estimate is given.

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp−ldlr−/−vldlr−/− mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp−ldlr−/−vldlr−/− mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.

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.

The H2 allele of APOC1, giving rise to increased gene expression of apolipoprotein C-I (apoC-I), is in genetic disequilibrium with the APOE4 allele and may provide a major risk factor for Alzheimer's disease (AD). We found that apoC-I protein is present in astrocytes and endothelial cells within hippocampal regions in both human control and AD brains. Interestingly, apoC-I colocalized with β-amyloid (Aβ) in plaques in AD brains, and in vitro experiments revealed that aggregation of Aβ was delayed in the presence of apoC-I. Moreover, apoC-I was found to exacerbate the soluble Aβ oligomer-induced neuronal death. To establish a potential role for apoC-I in cognitive functions, we used human (h) APOC1+/0 transgenic mice that express APOC1 mRNA throughout their brains and apoC-I protein in astrocytes and endothelial cells. The hAPOC1+/0 mice displayed impaired hippocampal-dependent learning and memory functions compared with their wild-type littermates, as judged from their performance in the object recognition task (P = 0.012) and in the Morris water maze task (P = 0.010). ApoC-I may affect learning as a result of its inhibitory properties toward apoE-dependent lipid metabolism. However, no differences in brain mRNA or protein levels of endogenous apoE were detected between transgenic and wild-type mice. In conclusion, human apoC-I expression impairs cognitive functions in mice independent of apoE expression, which supports the potential of a modulatory role for apoC-I during the development of AD.

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[3H]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[3H]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 3H-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.

In addition to efficiently decreasing VLDL-triglycerides (TGs), fenofibrate increases HDL-cholesterol levels in humans. We investigated whether the fenofibrate-induced increase in HDL-cholesterol is dependent on the expression of the cholesteryl ester transfer protein (CETP). To this end, APOE*3-Leiden (E3L) transgenic mice without and with the human CETP transgene, under the control of its natural regulatory flanking regions, were fed a Western-type diet with or without fenofibrate. Fenofibrate (0.04% in the diet) decreased plasma TG in E3L and E3L.CETP mice (−59% and −60%; P < 0.001), caused by a strong reduction in VLDL. Whereas fenofibrate did not affect HDL-cholesterol in E3L mice, fenofibrate dose-dependently increased HDL-cholesterol in E3L.CETP mice (up to +91%). Fenofibrate did not affect the turnover of HDL-cholesteryl ester (CE), indicating that fenofibrate causes a higher steady-state HDL-cholesterol level without altering the HDL-cholesterol flux through plasma. Analysis of the hepatic gene expression profile showed that fenofibrate did not differentially affect the main players in HDL metabolism in E3L.CETP mice compared with E3L mice. However, in E3L.CETP mice, fenofibrate reduced hepatic CETP mRNA (−72%; P < 0.01) as well as the CE transfer activity in plasma (−73%; P < 0.01). We conclude that fenofibrate increases HDL-cholesterol 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.

Mice that overexpress human apolipoprotein C-I (apoC-I) homozygously (APOC1+/+ mice) are protected against obesity and show cutaneous abnormalities. Although these effects can result from our previous observation that apoC-I inhibits FFA generation by LPL, we have also found that apoC-I impairs the uptake of a FFA analog in adipose tissue. In this study, we tested the hypothesis that apoC-I interferes with cellular FFA uptake independent of LPL activity. The cutaneous abnormalities of APOC1+/+ mice were not affected after transplantation to wild-type mice, indicating that locally produced apoC-I prevents lipid entry into the skin. Subsequent in vitro studies with apoC-I-deficient versus wild-type macrophages revealed that apoC-I reduced the cell association and subsequent esterification of [3H]oleic acid by ∼35% (P < 0.05). We speculated that apoC-I binds FFA extracellularly, thereby preventing cell association of FFA. We showed that apoC-I was indeed able to mediate the binding of oleic acid to otherwise protein-free VLDL-like emulsion particles involving electrostatic interaction. We conclude that apoC-I binds FFA in the circulation, thereby reducing the availability of FFA for uptake by cells. This mechanism can serve as an additional mechanism behind the resistance to obesity and the cutaneous abnormalities of APOC1+/+ mice.

The impact of apolipoprotein C-I (apoC-I) deficiency on hepatic lipid metabolism was addressed in mice in the presence or the absence of cholesteryl ester transfer protein (CETP). In addition to the expected moderate reduction in plasma cholesterol levels, apoCIKO mice showed significant increases in the hepatic content of cholesteryl esters (+58%) and triglycerides (+118%) and in biliary cholesterol concentration (+35%) as compared with wild-type mice. In the presence of CETP, hepatic alterations resulting from apoC-I deficiency were enforced, with up to 58% and 302% increases in hepatic levels of cholesteryl esters and triglycerides in CETPTg/apoCIKO mice versus CETPTg mice, respectively. Biliary levels of cholesterol, phospholipids, and bile acids were increased by 88, 77, and 20%, respectively, whereas total cholesterol, HDL cholesterol, and triglyceride concentrations in plasma were further reduced in CETPTg/apoCIKO mice versus CETPTg mice. Finally, apoC-I deficiency was not associated with altered VLDL production rate. In line with the previously recognized inhibition of lipoprotein clearance by apoC-I, apoC-I deficiency led to decreased plasma lipid concentration, hepatic lipid accumulation, and increased biliary excretion of cholesterol. The effect was even greater when the alternate reverse cholesterol transport pathway via VLDL/LDL was boosted in the presence of CETP.

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.

CD36 is involved in high-affinity peripheral FFA uptake. CD36-deficient (cd36−/−) mice exhibit increased plasma FFA and triglyceride (TG) levels. The aim of the present study was to elucidate the cause of the increased plasma TG levels in cd36−/− mice. cd36−/− mice showed no differences in hepatic VLDL-TG production or intestinal [3H]TG uptake compared with wild-type littermates. cd36−/− mice showed a 2-fold enhanced postprandial TG response upon an intragastric fat load (P < 0.05), with a concomitant 2.5-fold increased FFA response (P < 0.05), suggesting that the increased FFA in cd36−/− mice may impair LPL-mediated TG hydrolysis. Postheparin LPL levels were not affected. However, the in vitro LPL-mediated TG hydrolysis rate as induced by postheparin plasma of cd36−/− mice in the absence of excess FFA-free BSA was reduced 2-fold compared with wild-type plasma (P < 0.05). This inhibition was relieved upon the addition of excess FFA-free BSA. Likewise, increasing plasma FFA in wild-type mice to the levels observed in cd36−/− mice by infusion prolonged the plasma half-life of glycerol tri[3H]oleate-labeled VLDL-like emulsion particles by 2.5-fold (P < 0.05).We conclude that the increased plasma TG levels observed in cd36−/− mice are caused by decreased LPL-mediated hydrolysis of TG-rich lipoproteins resulting from FFA-induced product inhibition of LPL.

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 × 109 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.

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.

Hepatic VLDL and glucose production is enhanced in type 2 diabetes and associated with hepatic steatosis. Whether the derangements in hepatic metabolism are attributable to steatosis or to the increased availability of FA metabolites is not known. We used methyl palmoxirate (MP), an inhibitor of carnitine palmitoyl transferase I, to acutely inhibit hepatic FA oxidation and investigated whether the FAs were rerouted into VLDL secretion and whether this would affect hepatic glucose production. After an overnight fast, male APOE3*Leiden transgenic mice received an oral dose of 10 mg/kg MP. Administration of MP led to an 83% reduction in plasma β-hydroxybutyrate (ketone body) levels compared with vehicle-treated mice (0.47 ± 0.07 vs. 2.81 ± 0.16 mmol/l, respectively; P < 0.01), indicative of impaired ketogenesis. Plasma FFA levels were increased by 32% and cholesterol and insulin levels were decreased by 17% and 50%, respectively, in MP-treated mice compared with controls. MP treatment led to a 30% increase in liver triglyceride (TG) content. Surprisingly, no effect on hepatic VLDL-TG production was observed between the groups at 8 h after MP administration. In addition, the capacity of insulin to suppress endogenous glucose production was unaffected in MP-treated mice compared with controls.In conclusion, acute inhibition of FA oxidation increases hepatic lipid content but does not stimulate hepatic VLDL secretion or reduce insulin sensitivity.

Fasting readily induces hepatic steatosis. Hepatic steatosis is associated with hepatic insulin resistance. The purpose of the present study was to document the effects of 16 h of fasting in wild-type mice on insulin sensitivity in liver and skeletal muscle in relation to 1) tissue accumulation of triglycerides (TGs) and 2) changes in mRNA expression of metabolically relevant genes. Sixteen hours of fasting did not show an effect on hepatic insulin sensitivity in terms of glucose production in the presence of increased hepatic TG content. In muscle, however, fasting resulted in increased insulin sensitivity, with increased muscle glucose uptake without changes in muscle TG content. In liver, fasting resulted in increased mRNA expression of genes promoting gluconeogenesis and TG synthesis but in decreased mRNA expression of genes involved in glycogenolysis and fatty acid synthesis. In muscle, increased mRNA expression of genes promoting glucose uptake, as well as lipogenesis and β-oxidation, was found.In conclusion, 16 h of fasting does not induce hepatic insulin resistance, although it causes liver steatosis, whereas muscle insulin sensitivity increases without changes in muscle TG content. Therefore, fasting induces differential changes in tissue-specific insulin sensitivity, and liver and muscle TG contents are unlikely to be involved in these changes.

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.

The VLDL receptor (VLDLr) is involved in tissue delivery of VLDL-triglyceride (TG)-derived FFA by facilitating the expression of lipoprotein lipase (LPL). However, vldlr−/− mice do not show altered plasma lipoprotein levels, despite reduced LPL expression. Because LPL activity is crucial in postprandial lipid metabolism, we investigated whether the VLDLr plays a role in chylomicron clearance. Fed plasma TG levels of vldlr−/− mice were 2.5-fold increased compared with those of vldlr+/+ littermates (1.20 ± 0.37 mM vs. 0.47 ± 0.18 mM; P < 0.001). Strikingly, an intragastric fat load led to a 9-fold increased postprandial TG response in vldlr−/− compared with vldlr+/+ mice (226 ± 188 mM/h vs. 25 ± 11 mM/h; P < 0.05). Accordingly, the plasma clearance of [3H]TG-labeled protein-free chylomicron-mimicking emulsion particles was delayed in vldlr−/− compared with vldlr+/+ mice (half-life of 12.0 ± 2.6 min vs. 5.5 ± 0.9 min; P < 0.05), with a 60% decreased uptake of label into adipose tissue (P < 0.05). VLDLr deficiency did not affect the plasma half-life and adipose tissue uptake of albumin-complexed [14C]FFA, indicating that the VLDLr facilitates postprandial LPL-mediated TG hydrolysis rather than mediating FFA uptake.We conclude that the VLDLr plays a major role in the metabolism of postprandial lipoproteins by enhancing LPL-mediated TG hydrolysis.

The low-density lipoprotein receptor (LDLR) is the first described receptor for apolipoprotein E (apoE). We hypothesize that the absence of the LDLR, similar to the absence of apoE, results in impaired learning and memory processes. Six-month-old homozygous Ldlr−/− and wild-type littermates (Ldlr+/+), maintained on a standard lab chow diet, were used. Unlike humans, Ldlr−/− mice, under these conditions, do not develop atherosclerosis. The results of the Morris water escape task revealed an impaired spatial memory in the Ldlr−/− mice in comparison with Ldlr+/+ mice. Also in a T-maze task, the working memory performance of the Ldlr−/− mice was impaired. Furthermore, Ldlr−/− mice, in comparison with Ldlr+/+ mice, display a decreased number of synaptophysin-immunoreactive presynaptic boutons in the hippocampus CA1. In conclusion, the results show in mice deficiency for the LDLR results in impaired hippocampal-dependent memory functions. A decrease in the number of presynaptic boutons may underlay these behavioral alterations. Therefore, the LDLR may be an important receptor for apoE in the central nervous system.

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.

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36−/−) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36−/− and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36−/− compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36−/− mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36−/− mice compared with CD36+/+ mice (110.9 ± 12.0 and 68.9 ± 13.6 μg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%.Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36−/− mice.

Familial dysbetalipoproteinemia associated with the apolipoprotein E2 (APOE2) genotype is a recessive disorder with low penetrance. We have investigated whether additional expression of full-length APOE3, APOE4, or a truncated variant of APOE4 (APOE4-202) can reduce APOE2- associated hyperlipidemia. This was achieved using adenovirus-mediated gene transfer to mice transgenic for human APOE2 and deficient for endogenous Apoe (APOE2.Apoe−/− mice). The hyperlipidemia of APOE2.Apoe−/− mice was readily aggravated by APOE3 and APOE4 overexpression. Only a very low dose of APOE4 adenovirus was capable of reducing the serum cholesterol and triglyceride (TG) levels. Expression of higher doses of APOE4 was associated with an increased VLDL-TG production rate and the accumulation of TG-rich VLDL in the circulation. In contrast, a high dose of adenovirus carrying APOE4-202 reduced both the cholesterol and TG levels in APOE2.Apoe−/− mice. Despite the absence of the C-terminal lipid-binding domain, APOE4-202 is apparently capable of binding to lipoproteins and mediating hepatic uptake. Moreover, overexpression of APOE4-202 in APOE2.Apoe−/− mice does not aggravate their hypertriglyceridemia. These results extend our previous analyses of APOE4-202 expression in Apoe−/− mice and demonstrate that apoE4-202 functions even in the presence of clearance-defective apoE2.Thus, apoE4-202 is a safe and efficient candidate for future therapeutic applications.

Previous studies with hypertriglyceridemic APOC3 transgenic mice have suggested that apolipoprotein C-III (apoC-III) may inhibit either the apoE-mediated hepatic uptake of TG-rich lipoproteins and/or the lipoprotein lipase (LPL)-mediated hydrolysis of TG. Accordingly, apoC3 knockout (apoC3−/−) mice are hypotriglyceridemic. In the present study, we attempted to elucidate the mechanism(s) underlying these phenomena by intercrossing apoC3−/− mice with apoE−/− mice to study the effects of apoC-III deficiency against a hyperlipidemic background. Similar to apoE+/+ apoC3−/− mice, apoE−/−apoC3−/− mice exhibited a marked reduction in VLDL cholesterol and TG, indicating that the mechanism(s) by which apoC-III deficiency exerts its lipid-lowering effect act independent of apoE. On both backgrounds, apoC3−/− mice showed normal intestinal lipid absorption and hepatic VLDL TG secretion. However, turnover studies showed that TG-labeled emulsion particles were cleared much more rapidly in apoC3−/− mice, whereas the clearance of VLDL apoB, as a marker for whole particle uptake by the liver, was not affected. Furthermore, it was shown that cholesteryl oleate-labeled particles were also cleared faster in apoC3−/− mice. Thus the mechanisms underlying the hypolipidemia in apoC3−/− mice involve both a more efficient hydrolysis of VLDL TG as well as an enhanced selective clearance of VLDL cholesteryl esters from plasma.In summary, our studies of apoC3−/− mice support the concept that apoC-III is an effective inhibitor of VLDL TG hydrolysis and reveal a potential regulating role for apoC-III with respect to the selective uptake of cholesteryl esters.

Scavenger receptors, which include various classes, play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins, resulting in the massive accumulation of cholesteryl esters. Because macrophage-derived foam cells are considered to be an important feature in early atherogenesis, we investigated the role of scavenger receptor class A (SR-A) overexpression, especially on macrophages in lipoprotein metabolism and atherosclerosis. Bone marrow from human SR-A (MSR1)-overexpressing mice was transplanted into irradiated low density lipoprotein receptor knockout [LDLR(−/−)] mice. The transplantation resulted in an increase in total serum cholesterol (approximately 15 to 25%), especially in the VLDL fraction, when compared with LDLR(−/−) mice that were transplanted with bone marrow of wild-type littermates. Quantification of atherosclerotic lesions in the mice that were fed a “Western-type” diet for 3 months revealed that there were no differences in mean lesion area between LDLR(−/−) mice transplanted with MSR1 overexpressing and wild-type littermate bone marrow, despite increased scavenger receptor activity in vitro. The presence or absence of the LDLR in the transplanted bone marrow did not influence these results. In conclusion, introduction of MSR1-overexpressing bone marrow in LDLR(−/−) mice via bone marrow transplantation resulted in a slight increase in lipoprotein levels, but had no effect on the atherosclerotic lesion area, despite increased scavenger receptor activity in vitro. —Herijgers, N., M. P. J. de Winther, M. Van Eck, L. M. Havekes, M. H. Hofker, P. M. Hoogerbrugge, and T. J. C. Van Berkel. Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knockout mice. J. Lipid Res. 2000. 41: 1402–1409.

To investigate the quantitative requirement for apolipoprotein (apo) E in the clearance of lipoproteins via the non-low density lipoprotein (LDL) receptor mediated pathway, human APOE was overexpressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes (Apoe−/−· Ldlr−/−) using adenovirus-mediated gene transfer. We found that a low level of APOE expression, that was capable of reducing the hyperlipidemia in Apoe−/− mice, did not result in a reduction of the hyperlipidemia in Apoe−/−· Ldlr−/− mice. Surpisingly, a very high level of APOE expression also did not result in a reductionof hypercholesterolemia in Apoe−/−· Ldlr−/− mice, despite very high levels of circulating apoE (>160 mg/dl). Only a moderately high level of APOE expression resulted in a reduction of serum cholesterol level (from 35.2 ± 6.7 to 14.6 ± 2.3 mmol/l) and the disappearance of VLDL from the serum. Moreover, the very high level of APOE expression resulted in a severe hypertriglyceridemia in Apoe−/−· Ldlr−/− mice and not Apoe−/− mice (25.7 ± 8.9 and 2.2 ± 1.8 mmol/l, respectively). This hypertriglyceridemia was associated with an APOE-induced increase in the VLDL triglyceride production rate and an inhibition of VLDL-triglyceride lipolysis. We conclude from these data that, for efficient clearance, the non-LDL receptor-mediated pathway requires a higher level of APOE expression as compared to the LDL receptor, but is more sensitive to an APOE-induced increase in VLDL production and inhibition of VLDL-triglyceride lipolysis.—van Dijk, K. W., B. J. M. Van Vlijmen, H. B. van't Hof, A. van der Zee, S. Santamarina-Fojo, T. J. C. van Berkel, L. M. Havekes, and M. H. Hofker. In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess APOE. J. Lipid Res. 1999. 40: 336–344.

Studying the effects of dietary fish oil on VLDL metabolism in humans is subject to both large intra- and interindividual variability. In the present study we therefore used hyperlipidemic apolipoprotein (APO) E*3-Leiden mice, which have impaired chylomicron and very low density lipoprotein (VLDL) remnant metabolism, to study the effects of dietary fish oil on serum lipids and VLDL kinetics under highly standardized conditions. For this, female APOE*3-Leiden mice were fed a fat- and cholesterol-containing diet supplemented with either 0, 3 or 6% w/w (i.e. 0, 6, or 12% of total energy) of fish oil. Fish oil-fed mice showed a significant dose-dependent decrease in serum cholesterol (up to -43%) and triglyceride levels (up to -60%), mainly due to a reduction of VLDL (-80%). LDL and HDL cholesterol levels were not affected by fish oil feeding. VLDL-apoB kinetic studies showed that fish oil feeding resulted in a significant 2-fold increase in VLDL-apoB fractional catabolic rate (FCR). Hepatic VLDL-apoB production was, however, not affected by fish oil feeding. VLDL-triglyceride turnover studies revealed that fish oil significantly decreased hepatic VLDL-triglyceride production rate (-60%). A significant increase in VLDL-triglyceride FCR was observed (+70%), which was not related to increased lipolytic activity. We conclude that APOE*3-Leiden mice are highly responsive to dietary fish oil. The observed strong reduction in serum very low density lipoprotein (VLDL) is primarily due to an effect of fish oil to decrease hepatic VLDL triglyceride production rate and to increase VLDL-apoB fractional catabolic rate.—van Vlijmen, B. J. M., R. P. Mensink, H. B. van 't Hof, R. F. G. Offermans, M. H. Hofker, and L. M. Havekes. Effects of dietary fish oil on serm lipids and VLDL kinetics in hyperlipidemic apolipoprotein E*3-Leiden transgenic mice. J. Lipid Res. 1998. 39: 1181–1188.

The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models.The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart.The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p

IntroductionThe role of glucocorticoids in atherosclerosis development is not clearly established. Human studies show a clear association between glucocorticoid excess and cardiovascular disease, whereas most animal models indicate an inhibitory effect of glucocorticoids on atherosclerosis development. These animal models, however, neither reflect long-term glucocorticoid overexposure nor display human-like lipoprotein metabolism.AimTo investigate the effects of transient and continuous glucocorticoid excess on atherosclerosis development in a mouse model with human-like lipoprotein metabolism upon feeding a Western-type diet.MethodsPair-housed female APOE*3-Leiden.CETP (E3L.CETP) mice fed a Western-type containing 0.1% cholesterol for 20 weeks were given corticosterone (50 µg/ml) for either 5 (transient group) or 17 weeks (continuous group), or vehicle (control group) in the drinking water. At the end of the study, atherosclerosis severity, lesion area in the aortic root, the number of monocytes adhering to the endothelial wall and macrophage content of the plaque were measured.ResultsCorticosterone treatment increased body weight and food intake for the duration of the treatment and increased gonadal and subcutaneous white adipose tissue weight in transient group by +35% and +31%, and in the continuous group by +140% and 110%. Strikingly, both transient and continuous corticosterone treatment decreased total atherosclerotic lesion area by -39% without lowering plasma cholesterol levels. In addition, there was a decrease of -56% in macrophage content of the plaque with continuous corticosterone treatment, and a similar trend was present with the transient treatment.ConclusionIncreased corticosterone exposure in mice with human-like lipoprotein metabolism has beneficial, long-lasting effects on atherosclerosis, but negatively affects body fat distribution by promoting fat accumulation in the long-term. This indicates that the increased atherosclerosis observed in humans in states of glucocorticoid excess may not be related to cortisol per se, but might be the result of complex indirect effects of cortisol.

BACKGROUND: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established. METHODOLOGY AND PRINCIPAL FINDINGS: We generated mouse Znf202 expression vectors, the functionality of which was established in several in vitro systems. Next, effects of adenoviral znf202 overexpression in vivo were determined in normo- as well as hyperlipidemic mouse models. Znf202 overexpression in mouse hepatoma cells mhAT3F2 resulted in downregulation of members of the Apoe/c1/c2 and Apoa1/c3/a4 gene cluster. The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified. Adenoviral Znf202 transfer to Ldlr-/- mice resulted in downregulation of apoe, apoc1, apoa1, and apoc3 within 24 h after gene transfer. Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated. At 5 days post-infection, the expression of the aforementioned genes was normalized, but mice had developed severe hepatosteatosis accompanied by hypercholesterolemia and hypoalphalipoproteinemia. A much milder phenotype was observed in wildtype mice after 5 days of hepatic Znf202 overexpression. Interestingly and similar to Ldl-/- mice, HDL-cholesterol levels in wildtype mice were lowered after hepatic Znf202 overexpression. CONCLUSION/SIGNIFICANCE: Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels.

ObjectiveNiacin potently lowers triglycerides, mildly decreases LDL-cholesterol, and largely increases HDL-cholesterol. Despite evidence for an atheroprotective effect of niacin from previous small clinical studies, the large outcome trials, AIM-HIGH and HPS2-THRIVE did not reveal additional beneficial effects of niacin (alone or in combination with laropiprant) on top of statin treatment. We aimed to address this apparent discrepancy by investigating the effects of niacin without and with simvastatin on atherosclerosis development and determine the underlying mechanisms, in APOE*3Leiden.CETP mice, a model for familial dysbetalipoproteinemia (FD).Approach and resultsMice were fed a western-type diet containing cholesterol without or with niacin (120 mg/kg/day), simvastatin (36 mg/kg/day) or their combination for 18 weeks. Similarly as in FD patients, niacin reduced total cholesterol by -39% and triglycerides by -50%, (both PConclusionNiacin decreases atherosclerosis development mainly by reducing nonHDL-cholesterol with modest HDL-cholesterol-raising and additional anti-inflammatory effects. The additive effect of niacin on top of simvastatin is mostly dependent on its nonHDL-cholesterol-lowering capacities. These data suggest that clinical beneficial effects of niacin are largely dependent on its ability to lower LDL-cholesterol on top of concomitant lipid-lowering therapy.

ObjectiveCentral neuropeptide Y (NPY) administration stimulates food intake in rodents. In addition, acute modulation of central NPY signaling increases hepatic production of very low-density lipoprotein (VLDL)-triglyceride (TG) in rats. As hypertriglyceridemia is an important risk factor for atherosclerosis, for which well-established mouse models are available, we set out to validate the effect of NPY on hepatic VLDL-TG production in mice, to ultimately investigate whether NPY, by increasing VLDL production, contributes to the development of atherosclerosis.Research design and methodsMale C57Bl/6J mice received an intracerebroventricular (i.c.v.) cannula into the lateral (LV) or third (3V) ventricle of the brain. One week later, after a 4 h fast, the animals received an intravenous (i.v.) injection of Tran(35)S (100 µCi) followed by tyloxapol (500 mg/kg body weight; BW), enabling the study of hepatic VLDL-apoB and VLDL-TG production, respectively. Immediately after the i.v. injection of tyloxapol, the animals received either an i.c.v. injection of NPY (0.2 mg/kg BW in artificial cerebrospinal fluid; aCSF), synthetic Y1 receptor antagonist GR231118 (0.5 mg/kg BW in aCSF) or vehicle (aCSF), or an i.v. injection of PYY3-36 (0.5 mg/kg BW in PBS) or vehicle (PBS).ResultsAdministration of NPY into both the LV and 3V increased food intake within one hour after injection (+164%, pConclusionIn mice, as opposed to rats, acute central administration of NPY increases food intake without affecting hepatic VLDL production. These results are of great significance when extrapolating findings on the central regulation of hepatic VLDL production between species.

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation, airways obstruction and emphysema, and is a risk factor for cardiovascular disease (CVD). However, the contribution of these individual COPD components to this increased risk is unknown. Therefore, the aim of this study was to determine the contribution of emphysema in the presence or absence of pulmonary inflammation to the increased risk of CVD, using a mouse model for atherosclerosis. Because smoke is a known risk factor for both COPD and CVD, emphysema was induced by intratracheal instillation of porcine pancreatic elastase (PPE).MethodsHyperlipidemic APOE*3-Leiden mice were intratracheally instilled with vehicle, 15 or 30 µg PPE and after 4 weeks, mice received a Western-type diet (WTD). To study the effect of emphysema combined with pulmonary inflammation on atherosclerosis, mice received 30 µg PPE and during WTD feeding, mice were intranasally instilled with vehicle or low-dose lipopolysaccharide (LPS; 1 µg/mouse, twice weekly). After 20 weeks WTD, mice were sacrificed and emphysema, pulmonary inflammation and atherosclerosis were analysed.ResultsIntratracheal PPE administration resulted in a dose-dependent increase in emphysema, whereas atherosclerotic lesion area was not affected by PPE treatment. Additional low-dose intranasal LPS administration induced a low-grade systemic IL-6 response, as compared to vehicle. Combining intratracheal PPE with intranasal LPS instillation significantly increased the number of pulmonary macrophages and neutrophils. Plasma lipids during the study were not different. LPS instillation caused a limited, but significant increase in the atherosclerotic lesion area. This increase was not further enhanced by PPE.ConclusionThis study shows for the first time that PPE-induced emphysema both in the presence and absence of pulmonary inflammation does not affect atherosclerotic lesion development.

Background/aimsBrown adipose tissue (BAT) dissipates energy stored in triglycerides as heat via the uncoupling protein UCP-1 and is a promising target to combat hyperlipidemia and obesity. BAT is densely innervated by the sympathetic nervous system, which increases BAT differentiation and activity upon cold exposure. Recently, Bone Morphogenetic Protein 7 (BMP7) was identified as an inducer of BAT differentiation. We aimed to elucidate the role of sympathetic activation in the effect of BMP7 on BAT by treating mice with BMP7 at varying ambient temperature, and assessed the therapeutic potential of BMP7 in combating obesity.Methods and resultsHigh-fat diet fed lean C57Bl6/J mice were treated with BMP7 via subcutaneous osmotic minipumps for 4 weeks at 21 °C or 28 °C, the latter being a thermoneutral temperature in which sympathetic activation of BAT is largely diminished. At 21 °C, BMP7 increased BAT weight, increased the expression of Ucp1, Cd36 and hormone-sensitive lipase in BAT, and increased total energy expenditure. BMP7 treatment markedly increased food intake without affecting physical activity. Despite that, BMP7 diminished white adipose tissue (WAT) mass, accompanied by increased expression of genes related to intracellular lipolysis in WAT. All these effects were blunted at 28 °C. Additionally, BMP7 resulted in extensive 'browning' of WAT, as evidenced by increased expression of BAT markers and the appearance of whole clusters of brown adipocytes via immunohistochemistry, independent of environmental temperature. Treatment of diet-induced obese C57Bl6/J mice with BMP7 led to an improved metabolic phenotype, consisting of a decreased fat mass and liver lipids as well as attenuated dyslipidemia and hyperglycemia.ConclusionTogether, these data show that BMP7-mediated recruitment and activation of BAT only occurs at subthermoneutral temperature, and is thus likely dependent on sympathetic activation of BAT, and that BMP7 may be a promising tool to combat obesity and associated disorders.

Physical activity (PA) is a main determinant of total energy expenditure (TEE) and has been suggested to play a key role in body weight regulation. However, thus far it has been challenging to determine what part of the expended energy is due to activity in freely moving subjects. We developed a computational method to estimate activity related energy expenditure (AEE) and resting metabolic rate (RMR) in mice from activity and indirect calorimetry data. The method is based on penalised spline regression and takes the time dependency of the RMR into account. In addition, estimates of AEE and RMR are corrected for the regression dilution bias that results from inaccurate PA measurements. We evaluated the performance of our method based on 500 simulated metabolic chamber datasets and compared it to that of conventional methods. It was found that for a sample time of 10 minutes the penalised spline model estimated the time-dependent RMR with 1.7 times higher accuracy than the Kalman filter and with 2.7 times higher accuracy than linear regression. We assessed the applicability of our method on experimental data in a case study involving high fat diet fed male and female C57Bl/6J mice. We found that TEE in male mice was higher due to a difference in RMR while AEE levels were similar in both groups, even though female mice were more active. Interestingly, the higher activity did not result in a difference in AEE because female mice had a lower caloric cost of activity, which was likely due to their lower body weight. In conclusion, TEE decomposition by means of penalised spline regression provides robust estimates of the time-dependent AEE and RMR and can be applied to data generated with generic metabolic chamber and indirect calorimetry set-ups.

ObjectiveIn addition to improve glucose intolerance, recent studies suggest that glucagon-like peptide-1 (GLP-1) receptor agonism also decreases triglyceride (TG) levels. The aim of this study was to evaluate the effect of GLP-1 receptor agonism on very-low-density lipoprotein (VLDL)-TG production and liver TG metabolism.Experimental approachThe GLP-1 peptide analogues CNTO3649 and exendin-4 were continuously administered subcutaneously to high fat diet-fed APOE*3-Leiden transgenic mice. After 4 weeks, hepatic VLDL production, lipid content, and expression profiles of selected genes involved in lipid metabolism were determined.ResultsCNTO3649 and exendin-4 reduced fasting plasma glucose (up to -30% and -28% respectively) and insulin (-43% and -65% respectively). In addition, these agents reduced VLDL-TG production (-36% and -54% respectively) and VLDL-apoB production (-36% and -43% respectively), indicating reduced production of VLDL particles rather than reduced lipidation of apoB. Moreover, they markedly decreased hepatic content of TG (-39% and -55% respectively), cholesterol (-30% and -55% respectively), and phospholipids (-23% and -36% respectively), accompanied by down-regulation of expression of genes involved in hepatic lipogenesis (Srebp-1c, Fasn, Dgat1) and apoB synthesis (Apob).ConclusionGLP-1 receptor agonism reduces VLDL production and hepatic steatosis in addition to an improvement of glycemic control. These data suggest that GLP-receptor agonists could reduce hepatic steatosis and ameliorate dyslipidemia in patients with type 2 diabetes mellitus.

Causes and consequences of the complex changes in lipids occurring in the metabolic syndrome are only partly understood. Several interconnected processes are deteriorating, which implies that multi-target approaches might be more successful than strategies based on a limited number of surrogate markers. Preparations from Chinese Medicine (CM) systems have been handed down with documented clinical features similar as metabolic syndrome, which might help developing new intervention for metabolic syndrome. The progress in systems biology and specific animal models created possibilities to assess the effects of such preparations. Here we report the plasma and liver lipidomics results of the intervention effects of a preparation SUB885C in apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE*3Leiden.CETP) mice. SUB885C was developed according to the principles of CM for treatment of metabolic syndrome. The cannabinoid receptor type 1 blocker rimonabant was included as a general control for the evaluation of weight and metabolic responses.ApoE*3Leiden.CETP mice with mild hypercholesterolemia were divided into SUB885C-, rimonabant- and non-treated control groups. SUB885C caused no weight loss, but significantly reduced plasma cholesterol (-49%, p

Lipids are known to play crucial roles in the development of life-style related risk factors such as obesity, dyslipoproteinemia, hypertension and diabetes. The first selective cannabinoid-1 receptor blocker rimonabant, an anorectic anti-obesity drug, was frequently used in conjunction with diet and exercise for patients with a body mass index greater than 30 kg/m(2) with associated risk factors such as type II diabetes and dyslipidaemia in the past. Less is known about the impact of this drug on the regulation of lipid metabolism in plasma and liver in the early stage of obesity.We designed a four-week parallel controlled intervention on apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE*3Leiden.CETP) transgenic mice with mild overweight and hypercholesterolemia. A liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometric approach was employed to investigate plasma and liver lipid responses to the rimonabant intervention. Rimonabant was found to induce a significant body weight loss (9.4%, p

BackgroundThe epsilon2, epsilon3, and epsilon4 alleles of the apolipoprotein E gene (APOE) encode three isoforms, apoE2, E3, and E4, respectively. The apoE isoforms circulate in different plasma concentrations, but plasma concentrations of the same isoform also differ between individuals. Whereas the isoforms have been associated with cardiovascular disease, the relation between plasma apoE levels and cardiovascular disease is unknown.Methods and findingsWe assessed APOE genotypes, plasma levels of apoE, cardiovascular risk factors, and mortality in a population-based sample of 546 individuals aged 85 y who participated in the Leiden 85-plus Study and were prospectively followed for specific causes of death for 5 y. Participants in the highest tertile of apoE levels suffered a twofold-increased risk of cardiovascular mortality (hazard ratio compared to lowest tertile, 2.08; 95% confidence interval [CI], 1.30 to 3.33). Among the 324 participants with the epsilon3epsilon3 genotype, the hazard from cardiovascular disease was threefold increased (highest versus lowest tertile 3.01; 95% CI 1.60 to 5.66), with similar estimates for men and women. Other causes of death were not increased significantly. Plasma levels of apoE in epsilon3epsilon3 participants were positively correlated with total cholesterol (p < 0.001), low-density lipoprotein cholesterol (p < 0.001) and triglycerides (p < 0.001) and negatively with high-density lipoprotein cholesterol levels (p = 0.010). Adjustment for plasma lipids did not change the hazard ratios, whereas interaction was absent. The risk associated with high levels of apoE, however, was strongest in participants from the lowest tertile of C-reactive protein (CRP) levels and absent in those from the highest tertile (p(interaction) < 0.001). Among participants from the lowest tertile of CRP levels, those with a high apoE levels had a significantly steeper increase in CRP than those with low apoE levels (p = 0.020). Similar cardiovascular mortality risks as in epsilon3epsilon3 participants were found in epsilon2 and epsilon4 carriers.ConclusionsIn old age, high plasma apoE levels precede an increase of circulating CRP and strongly associates with cardiovascular mortality, independent of APOE genotype and plasma lipids.