Hepatic steatosis and very low density lipoprotein secretion: the involvement of apolipoprotein E

Triglycerides comprise the major storage form of energy in the mammalian body. Prior to their use as a source of energy, however, triglycerides must be hydrolyzed to yield fatty acids. The toxic potential of free fatty acids requires that release from their stores is carefully balanced by uptake and utilization. Most tissues have the capacity to synthesize triglycerides, but only two cell types, i.e. adipocytes and hepatocytes, are able to store it in appreciable amounts. Obviously, adipose tissue is by far the most important site of triglyceride storage in the (human) body. Fat storage and release by adipose tissue and liver are coordinately regulated in a way that, under normal conditions, ensures energy supply to peripheral tissues during consecutive periods of feeding and fasting while, at the same time, the potential toxicity of free fatty acids is minimized. In this scenario the liver provides storage capacity when the supply of fat exceeds the direct demands for maintenance of energy homeostasis and releases fat in a controlled fashion to the periphery when supply via other sources is limited. This release occurs in the form of very low density lipoproteins (VLDL): triglyceride-rich particles of 30–100 nm in diameter containing a single molecule of apolipoprotein (apo) B as its most important structural component. Secretion of VLDL by the liver is regulated by a variety of factors (see below) that collectively must ensure release of adequate amounts of triglycerides from the liver at the proper moments. A permanent imbalance between fatty acid influx, utilization and VLDL secretion will, eventually, lead to accumulation of triglycerides and cholesterylesters in hepatocytes, i.e. to hepatic steatosis. It is now well established that hepatic steatosis is frequently associated with metabolic syndromes such as obesity, insulin resistance and type II diabetes mellitus, independent from excessive alcohol intake which is another frequent cause of hepatic steatosis. Although clearly different in their metabolic origins, it is recognized that non-alcoholic fatty liver disease and alcoholic fatty liver disease share several features and, most importantly, that fat accumulation in the liver per se is not a benign condition. In the recent past, several reports have provided evidence that excess fat increases the vulnerability of hepatocytes to potential harmful effects of ‘metabolic stress’, for instance imposed by cytokines, viral infections, and oxidative agents. As a consequence, hepatic steatosis is now recognized as a factor predisposing to development of fibrosis and cirrhosis, as excellently reviewed by Day and James [1Day C.P. James O.F. Hepatic steatosis: innocent bystander or guilty party.Hepatology. 1998; 27: 1463-1466Crossref PubMed Scopus (360) Google Scholar, 2James O.F. Day C.P. Non-alcoholic steatohepatitis: a disease of emerging identity and importance.J Hepatol. 1998; 29: 495-501Abstract Full Text PDF PubMed Scopus (348) Google Scholar] and by Tilg and Diehl [[3]Tilg H. Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis.N Engl J Med. 2000; 343: 1467-1476Crossref PubMed Scopus (827) Google Scholar]. Prevention or attenuation of hepatic steatosis has been proposed as a ‘function’ of VLDL secretion. Whether or not this should be assigned as a function, there is accumulating evidence to show that steatosis does indeed develop in experimental models in which impaired VLDL formation is a primary event (see below). Conversely, clinical conditions associated with hepatic steatosis, as exemplified by type II diabetes, are frequently characterized by an increased hepatic production of VLDL. In this review, we will give an overview of current insight in the VLDL formation process and its relationship to hepatic steatosis. Special reference will be given to the role of apolipoprotein E herein. Nascent VLDL particles acquire this particular apolipoprotein during the assembly/secretion cascade [4Fazio S. Yao Z. McCarthy B.J. Rall Jr, S.C. Synthesis and secretion of apolipoprotein E occur independently of synthesis and secretion of apolipoprotein B-containing lipoproteins in HepG2 cells.J Biol Chem. 1992; 267: 6941-6945Abstract Full Text PDF PubMed Google Scholar, 5Fazio S. Yao Z. The enhanced association of apolipoprotein E with apolipoprotein B-containing lipoproteins in serum-stimulated hepatocytes occurs intracellularly.Arterioscler Thromb Vasc Biol. 1995; 15: 593-600Crossref PubMed Scopus (32) Google Scholar]. Recent studies from our laboratories [6Kuipers F. van Ree J.M. Hofker M.H. Wolters H. in ‘t Veld G. Havinga R. et al.Altered lipid metabolism in Apolipoprotein E-deficient mice does not affect cholesterol balance across the liver.Hepatology. 1996; 24: 241-247Crossref PubMed Google Scholar, 7Kuipers F. Jong M.C. Lin Y. van Eck M. Havinga R. Bloks V. et al.Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E-deficient hepatocytes.J Clin Invest. 1997; 100: 2915-2922Crossref PubMed Scopus (150) Google Scholar, 8Mensenkamp A.R. Jong M.C. van Goor H. van Luyn M.J.A. Bloks V. Havinga R. et al.Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver.J Biol Chem. 1999; 274: 35711-35718Crossref PubMed Scopus (112) Google Scholar, 9Mensenkamp A.R. van Luyn M.J.A. van Goor H. Bloks V. Apostel F. Greeve J. et al.Hepatic lipid accumulation, altered very low density lipoprotein formation and apolipoprotein E deposition in apolipoprotein E3-Leiden transgenic mice.J Hepatol. 2000; 33: 189-198Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Mensenkamp A.R. Teusink B. Baller J.F.W. Wolters H. Havinga R. Willems van Dijk K. et al.Mice expressing the mutant apolipoprotein E3Leiden gene only show impaired very-low-density lipoprotein secretion.Arterioscler Thromb Vasc Biol. 2001; 21: 1366-1372Crossref PubMed Scopus (15) Google Scholar, 11Kypreos K. van Dijk K.W. van de Zee A. Havekes L.M. Zannis V.I. Domains of apolipoprotein E contributing to triglyceride and cholesterol homeostasis in vivo. Carboxyl-terminal region 203-299 promotes hepatic very low density lipoprotein-triglyceride secretion.J Biol Chem. 2001; 276: 19778-19786Crossref PubMed Scopus (59) Google Scholar] as well as from others [12Huang Y. Ji Z.S. Brecht W.J. Rall Jr, S.C. Taylor J.M. Mahley R.W. Overexpression of apolipoprotein E3 in transgenic rabbits causes combined hyperlipidemia by stimulating hepatic VLDL production and impairing VLDL lipolysis.Arterioscler Thromb Vasc Biol. 1999; 19: 2952-2959Crossref PubMed Scopus (78) Google Scholar, 13Huang Y. Liu X.Q. Rall Jr, S.C. Taylor J.M. von Eckardstein A. Assmann G. Mahley R.W. Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia.J Biol Chem. 1998; 273: 26388-26393Crossref PubMed Scopus (174) Google Scholar, 14Tsukamoto K. Maugeais C. Glick J.M. Rader D.J. Markedly increased secretion of VLDL triglycerides induced by gene transfer of apolipoprotein E isoforms in apoE-deficient mice.J Lipid Res. 2000; 41: 253-259Abstract Full Text Full Text PDF PubMed Google Scholar] strongly indicate that apoE has a role in the regulation of hepatic VLDL-triglyceride secretion, in addition to its well-established functions in mediating lipoprotein uptake from the circulation and in the control of lipase-mediated hydrolysis of lipoprotein triglycerides. The plasticity of the liver in terms of triglyceride storage capacity is central in the physiology of energy supply during the feeding-fasting transitions that occur in normal life. It is of importance to realize that the hepatic triglyceride pool is not a static entity but is continuously going through series of hydrolysis–reesterification cycles which, in all likelihood, enables the liver to adjust the ‘metabolic lipid flow’ adequately in response to changes in metabolic needs of the body. It is now well documented that triglycerides are not incorporated ‘en bloc’ into VLDL particles from their cytosolic storage pools. Rather, triglyceride lipolysis followed by reesterification at the endoplasmic reticulum (ER) represent crucial steps in the VLDL assembly process. A detailed description of experimental evidence in favor of this complex sequence of events and its potential implications is beyond the scope of this review and has recently excellently been summarized by Gibbons et al. [[15]Gibbons G.F. Islam K. Pease R.J. Mobilisation of triacylglycerol stores.Biochim Biophys Acta. 2000; 1483: 37-57Crossref PubMed Scopus (247) Google Scholar]. Hepatic triglycerides may be derived from different sources. First, the liver is able to synthesize fatty acids from acetyl-CoA, which can be used for the formation of triglycerides. Recent data from human and rat studies have demonstrated that the contribution of de novo lipogenesis to the hepatic triglyceride content is relatively small, even when the process is stimulated by dietary or pharmacological means [[16]Parks E.J. Hellerstein M.K. Carbohydrate-induced hypertriacylglycerolemia: historical perspective and review of biological mechanisms.Am J Clin Nutr. 2000; 71: 412-433Crossref PubMed Scopus (433) Google Scholar]. The second source is provided by dietary triglycerides that enter the liver via the chylomicron remnant pathway during the postprandial phase after ingestion of a fat-containing meal. Quantitative data on the contribution of this pathway to liver triglyceride pools are scarce but, in view of the extremely rapid clearance of chylomicron remnants, it can be estimated to be substantial. Finally, the liver is exposed to high levels of free fatty acids that are released from adipose tissues during periods of fasting, in particular from those fat pads that directly drain the portal vein. During fasting, hormone-sensitive lipase (HSL) actively releases fatty acids from triglycerides that are stored in cytosolic lipid droplets of adipocytes. This response is triggered by changes in prevailing plasma concentrations of insulin and catecholamines that antagonistically control a number of intracellular processes regulating the activation of HSL and its access to the lipid droplets (see Ref. [[17]Holm C. Osterlund T. Laurell H. Conteras J.A. Molecular mechanisms regulating hormone-sensitive lipase.Annu Rev Nutr. 2000; 20: 365-393Crossref PubMed Scopus (343) Google Scholar] for review). Free fatty acids that are released from adipose tissue in excess of the requirements of direct oxidation enter, to a large extent, the liver for storage as triglycerides. The liver has an extremely high capacity for synthesis of triglycerides from free fatty acids and it is likely that, in the normal adult liver, plasma free fatty acids contribute the bulk of the hepatic triglyceride pool [[18]Sidossis L.S. Mittendorfer B. Walser D. Chinkes D. Wolfe R.R. Hyperglycemia-induced inhibition of splanchnic fatty acid oxidation increases hepatic triacylglycerol secretion.Am J Physiol. 1998; 275: E798-E805PubMed Google Scholar]. Their storage in the liver may be essential to neutralize the potential deleterious effects of free fatty acids: the impact of free fatty acid-mediated ‘lipotoxicity’, in particular in pancreatic β-cells, and the role of the fat-derived hormone leptin in its prevention has recently been highlighted by Unger et al. [[19]Unger R.H. Zou Y-T. Lipotoxicity of β-cells in obesity and in other causes of fatty acid spillover.Diabetes. 2001; 50: S118-S121Crossref PubMed Google Scholar]. The glycerol backbone of triglycerides may be derived from the phosphatidate pathway, in which glycerol-3-phosphate – an intermediate in the glycolytic pathway – is esterified with fatty acids to form triglycerides. A high glycolytic flux, for instance induced by intake of a high carbohydrate diet, may thus increase triglyceride availability and VLDL secretion. Cell and molecular biological aspects involved in the control of assembly and secretion of VLDL by the liver has received wide attention in the past decades. This interest, apart from the intrinsic mechanistic importance relevant to fundamental principles of lipid transport, lipid-protein interaction and membrane biology, is mainly driven by the fact that VLDL is the obligatory precursor of low density lipoproteins (LDL), the atherogenic properties of which need no further discussion. It now widely appreciated that hepatic overproduction of VLDL constitutes the metabolic basis of various hyperlipidemic states in humans, including familial combined hyperlipidemia [[20]Venkasetan S. Cullen P. Pacy P. Halliday D. Scott J. Stable isotopes show a direct relation between VLDL apoB overproduction and serum triglyceride levels and indicate a metabolically and biochemically coherent basis for familial combined hyperlipidemia.Arterioscler Thromb. 1993; 13: 1110-1118Crossref PubMed Google Scholar] and diabetes [[21]Cummings M.H. Watts G.F. Umpleby A.M. Hennessy T.R. Nauomova R. Slavin B.M. et al.Increased hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 in NIDDM.Diabetologia. 1995; 38: 959-967Crossref PubMed Scopus (124) Google Scholar]. Essential in our understanding of the relationships between VLDL secretion and hyperlipidemia, and therefore with the development of cardiovascular disease, is the fact that VLDL produced by the liver does not constitute a homogeneous population of particles but actually covers a wide variety of sizes. Large, relatively triglyceride-rich VLDL or VLDL1 are delipidated in plasma to smaller VLDL and finally, by remodeling via processes of lipolysis and neutral lipid exchange, to slowly metabolized, small LDL of relatively high density. Conversely, smaller VLDL or VLDL2 give rise to rapidly metabolized LDL. In vitro studies have shown a diminished receptor binding for apo B contained in small LDL, the net effect being prolongation of the circulation time of small LDL which also has been shown to be much more readily oxidized than larger particles. Recent studies in humans, applying novel stable isotope techniques, have revealed that hepatic production of VLDL1 and VLDL2 is differentially regulated: the first process is suppressed by insulin while the latter is not [[22]Malmstrom R. Packard C.J. Caslake M. Bedford D. Stewart P. Yki-Jarvinen H. et al.Effects of insulin and acipimox on VLDL1 and VLDL2 apolipoprotein B production in normal subjects.Diabetes. 1998; 47: 779-787Crossref PubMed Scopus (159) Google Scholar]. Most importantly, insulin is no longer capable to effectively suppress production of VLDL1 in patients with type II diabetes [[23]Malmstrom R. Packard C.J. Caslake M. Bedford D. Stewart P. Yki-Jarvinen H. et al.Defective regulation of triglyceride metabolism by insulin in the liver in NIDDM.Diabetologia. 1997; 40: 454-462Crossref PubMed Scopus (275) Google Scholar], which probably represents a key factor in development of the atherogenic lipid profile (elevated triglycerides, low high density lipoprotein (HDL), small dense LDL) associated with this condition. Insight into regulation of VLDL secretion, in terms of both particle number and size, has rapidly increased over the past years, although controversies about specific steps in the process still exist. A number of excellent review articles on the subject have been recently published [24Kang S. Davis R.A. Cholesterol and hepatic lipoprotein assembly and secretion.Biochim Biophys Acta. 2000; 1529: 223-230Crossref PubMed Scopus (66) Google Scholar, 25Olofsson S.O. Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr Opin Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (186) Google Scholar, 26Shelness G.S. Sellers J.A. Very-low-density lipoprotein assembly and secretion.Curr Opin Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (228) Google Scholar, 27Gordon D.A. Jamil H. Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly.Biochim Biophys Acta. 2000; 1486: 72-83Crossref PubMed Scopus (190) Google Scholar, 28Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J Lipid Res. 1997; 38: 1937-1953Abstract Full Text PDF PubMed Google Scholar]: only the essentials will be highlighted here (see Fig. 1) . The VLDL assembly/secretion pathway in hepatocytes shows an absolute requirement for apoB, an unique amphipathic protein that provides a ‘matrix’ for formation of a particle containing a neutral lipid core in the lumen of the ER, and for a lipid transfer protein called microsomal triglyceride transfer protein (MTP). The apoB molecule is translocated during its translation into the lumen of the rough ER, where its N-terminal part interacts with MTP complexed with protein disulfide isomerase (PDI). This interaction facilitates both the translocation process and the addition of neutral lipid to the nascent lipoprotein particle. In the absence of functional MTP or insufficient lipid availability, the translocation is blocked and the ‘translocation-arrested’ apoB molecule becomes rapidly conjugated with ubiquitin and is degraded in the cytoplasm by proteasomes. When apoB translocation proceeds, in a manner depending on the addition of lipids and chaperone-assisted folding of the protein, a particle containing its full complement of neutral lipids in its core may be either assembled as a concerted series of transformations involving protein folding and addition of lipid, or, alternatively, by a process consisting of several steps in which partially completed lipoprotein particles may exist as intermediates. Bulk triglycerides destined for secretion are located in the smooth ER where lipid ‘droplets’ are formed by the action of MTP. These droplets may fuse with the primordial apoB-containing lipoprotein in a process independent from MTP that occurs at the junction between rough and smooth ER. Improper folding of the protein and/or lipid addition can lead to the production of a particle that is degraded at a site in the secretory pathway distal of the ER. Thus, in spite of is absolute dependence on apoB, VLDL assembly/secretion is not regulated at the level of apoB gene expression but in fact by the balance between apoB entering the degradation or the secretory pathways. Although this appears an uneconomic way of regulation, the costs of futile synthesis and degradation of the huge apoB molecule are apparently outweighed by the advantages that allow the liver cell to rapidly adapt to changing conditions. There are several factors that control the eventual release of apoB-containing lipoproteins by the liver, amongst which MTP expression and availability of essential all lipid components. Thus, the process is stimulated by overexpression of MTP in the mouse [[29]Tietge U.J. Bakillah A. Maugeais C. Tsukamoto K. Hussain M. Rader D.J. Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B.J Lipid Res. 1999; 40: 2143-2149PubMed Google Scholar], appears to have a specific need for de novo synthesized phospholipids as surface material [[30]Verkade H.J. Fast D.G. Rusinol A.E. Scraba D.G. Vance D.E. Impaired biosynthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum.J Biol Chem. 1993; 268: 24990-24996Abstract Full Text PDF PubMed Google Scholar], and is to a certain extent governed by the rate of cholesterol biosynthesis and cholesteryl ester formation [24Kang S. Davis R.A. Cholesterol and hepatic lipoprotein assembly and secretion.Biochim Biophys Acta. 2000; 1529: 223-230Crossref PubMed Scopus (66) Google Scholar, 25Olofsson S.O. Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr Opin Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (186) Google Scholar, 26Shelness G.S. Sellers J.A. Very-low-density lipoprotein assembly and secretion.Curr Opin Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (228) Google Scholar]. Other factors involved, for instance the activity of cholesterol 7α-hydroxylase possibly leading to induction of the SREBP family of transcription factors [[31]Miyake J.H. Doung X.D. Strauss W. Moore G.L. Castellani L.W. Curtiss L.K. et al.Increased production of apolipoprotein B-containing lipoproteins in the absence of hyperlipidemia in transgenic mice expressing cholesterol 7alpha-hydroxylase.J Biol Chem. 2001; 276: 23304-23311Crossref PubMed Scopus (47) Google Scholar], have recently been identified. Hormonal (insulin) and dietary factors also potently affect VLDL production [24Kang S. Davis R.A. Cholesterol and hepatic lipoprotein assembly and secretion.Biochim Biophys Acta. 2000; 1529: 223-230Crossref PubMed Scopus (66) Google Scholar, 25Olofsson S.O. Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr Opin Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (186) Google Scholar, 26Shelness G.S. Sellers J.A. Very-low-density lipoprotein assembly and secretion.Curr Opin Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (228) Google Scholar]. In addition, there is now ample data to indicate that apoE also has a role in the regulation of VLDL-triglyceride secretion (see below). The human APOE gene is expressed in various organs, including liver, brain, adrenals and spleen, but not in the intestine, but the largest quantities of APOE mRNA are clearly present in the liver. The liver is also the major source of plasma apoE, where it is present as a constituent of chylomicrons, chylomicron remnants, VLDL, intermediate density lipoproteins (IDL) and certain HDL subclasses. ApoE functions as a ligand that mediates binding and uptake of lipoproteins by receptors that belong to the LDL receptor family and it binds to cell-surface heparan sulfate proteoglycans, a process that facilitates receptor-mediated uptake of remnant lipoproteins. Secondly, apoE modulates lipolytic activity towards circulating lipoproteins, an effect that has been attributed to displacement or masking by apoE of apoC-II, a co-factor of LPL, on the lipoprotein surface. The human APOE gene is located on chromosome 19 and encodes a mature protein of 34.2 kDa. ApoE contains two structural domains joined by a protease-sensitive hinge region. The LDL receptor binding site is located in the N-terminal domain, whereas the C-terminal part of the protein contains the lipid binding domain that mediates association with lipoproteins. The human APOE gene is polymorphic, with three common alleles (E*2, E*3, E*4) at a single locus, giving rise to six possible phenotypes (E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, E3/E4). The three alleles are distinguished by their Cys and Arg contents at two polymorphic sites: the E*3 allele (Cys112, Arg158) is the most common allele (70–85%), followed by E*4 (Arg112, Arg158; 12–18%) and E*2 (Cys112, Cys158; 3–12%). The different isoforms differ from each other with respect to their association with lipoproteins, affinity for the LDL receptor and interactions with heparan sulfate proteoglycans. Homozygosity for E*2 is associated with development of hyperlipidemia in some, but not in all subjects with the E2/E2 phenotype. In addition, a number of rare mutant APOE genes have been described that show a dominant mode of inheritance concerning the development of familial dysbetalipoproteinemia (remnant hyperlipidemia or type III hyperlipidemia), including APOE*3Leiden, which gives rise to a 7-amino-acid tandem repeat of residues 120–126 that interferes with receptor binding (see Ref. [[32]Mahley R.W. Huang Y. Rall Jr, S.C. Pathogenesis of type III hyperlipidemia (dysbetalipoproteinemia): questions, quandaries, and paradoxes.J Lipid Res. 1999; 40: 1933-1949Abstract Full Text Full Text PDF PubMed Google Scholar] for review of apoE genetics and function). Insight into the various functions of apoE in control of plasma lipoprotein levels and in atherogenesis has greatly benefited from the introduction of apoE-deficient and apoE overexpressing mouse models. For a detailed overview on these subjects, the reader is referred to a number of excellent reviews [33Plump A.S. Breslow J.L. Apoliporotein E and the apolipoprotein E-deficient mouse.Annu Rev Nutr. 1995; 15: 495-518Crossref PubMed Scopus (125) Google Scholar, 34Willems van Dijk K. Hofker M.H. Havekes L.M. Use of transgenic mice to study the role of apolipoprotein E in lipid metabolism and atherosclerosis.Int J Tissue React. 2000; 22: 49-58PubMed Google Scholar, 35Willems van Dijk K. Hofker M.H. Havekes L.M. Dissection of the complex role of apolipoprotein E in lipoprotein metabolism and atherosclerosis using mouse models.Curr Atheroscler Rep. 1999; 1: 101-107Crossref PubMed Scopus (21) Google Scholar]. Recent studies have revealed another ‘function’ of apoE, namely in control of intracellular lipid metabolism and VLDL production by the hepatocyte. Our laboratory has demonstrated that apoE-deficient mice develop a fatty liver when fed normal chow (Fig. 2A) and that triglycerides accumulate primarily in perivenous areas of the liver lobules under these conditions (Fig. 2B) [[6]Kuipers F. van Ree J.M. Hofker M.H. Wolters H. in ‘t Veld G. Havinga R. et al.Altered lipid metabolism in Apolipoprotein E-deficient mice does not affect cholesterol balance across the liver.Hepatology. 1996; 24: 241-247Crossref PubMed Google Scholar]. Subsequently [[7]Kuipers F. Jong M.C. Lin Y. van Eck M. Havinga R. Bloks V. et al.Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E-deficient hepatocytes.J Clin Invest. 1997; 100: 2915-2922Crossref PubMed Scopus (150) Google Scholar], we were able to show that VLDL-triglyceride secretion is severely impaired in vivo in chow-fed apoE-deficient mice (Fig. 3) , in perfused livers and in vitro in cultured hepatocytes from these animals. VLDL particles secreted by primary hepatocytes from apoE-deficient mice are smaller in size than those of control C57BL/6 mice, while the ability to synthesize triglycerides is not impaired. Transplantation of apoE expressing bone-marrow cells from wild-type mice into apoE-deficient mice resulted in normalization of plasma lipid levels, but did not normalize hepatic VLDL secretion [[7]Kuipers F. Jong M.C. Lin Y. van Eck M. Havinga R. Bloks V. et al.Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E-deficient hepatocytes.J Clin Invest. 1997; 100: 2915-2922Crossref PubMed Scopus (150) Google Scholar]. This experiment showed that impairment of hepatic VLDL-triglyceride secretion in apoE-deficient mice is not due to aspecific effects of hyperlipidemia but is actually related to the absence of apoE in hepatocytes. Introduction of the human APOE3 gene in apoE-deficient mice by transgenesis prevented the development of a fatty liver and normalized VLDL-triglyceride secretion [[8]Mensenkamp A.R. Jong M.C. van Goor H. van Luyn M.J.A. Bloks V. Havinga R. et al.Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver.J Biol Chem. 1999; 274: 35711-35718Crossref PubMed Scopus (112) Google Scholar]. Introduction of APOE3 by adenoviral gene transfer in apoE-deficient mice, leading to a transient hepatic overexpression, resulted in a gene dose-dependent increase in VLDL-triglyceride secretion, reaching rates of up to 500% of control values [[8]Mensenkamp A.R. Jong M.C. van Goor H. van Luyn M.J.A. Bloks V. Havinga R. et al.Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver.J Biol Chem. 1999; 274: 35711-35718Crossref PubMed Scopus (112) Google Scholar]. The regulatory role of apoE in the control of VLDL-triglyceride secretion has been confirmed by other groups. Huang et al. [12Huang Y. Ji Z.S. Brecht W.J. Rall Jr, S.C. Taylor J.M. Mahley R.W. Overexpression of apolipoprotein E3 in transgenic rabbits causes combined hyperlipidemia by stimulating hepatic VLDL production and impairing VLDL lipolysis.Arterioscler Thromb Vasc Biol. 1999; 19: 2952-2959Crossref PubMed Scopus (78) Google Scholar, 13Huang Y. Liu X.Q. Rall Jr, S.C. Taylor J.M. von Eckardstein A. Assmann G. Mahley R.W. Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia.J Biol Chem. 1998; 273: 26388-26393Crossref PubMed Scopus (174) Google Scholar] showed that overexpression of APOE3 in rabbits and mice leads to increased VLDL secretion as well as to a disturbed hydrolysis of these lipoproteins, resulting in the development of hyperlipidemia. Likewise, Tsukamoto et al. [[14]Tsukamoto K. Maugeais C. Glick J.M. Rader D.J. Markedly increased secretion of VLDL triglycerides induced by gene transfer of apolipoprotein E isoforms in apoE-deficient mice.J Lipid Res. 2000; 41: 253-259Abstract Full Text Full Text PDF PubMed Google Scholar] showed that hepatic overexpression of the human APOE2, E3, or E4 genes using adenoviral gene transfer results in increased VLDL-triglyceride and VLDL-apoB secretion in mice. Overexpression of the mutant human APOE*3Leiden gene in mice, on the other hand, is associated with development of steatosis and impaired VLDL-triglyceride secretion [9Mensenkamp A.R. van Luyn M.J.A. van Goor H. Bloks V. Apostel F. Greeve J. et al.Hepatic lipid accumulation, altered very low density lipoprotein formation and apolipoprotein E deposition in apolipoprotein E3-Leiden transgenic mice.J Hepatol. 2000; 33: 189-198Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Mensenkamp A.R. Teusink B. Baller J.F.W. Wolters H. Havinga R. Willems van Dijk K. et al.Mice expressing the mutant apolipoprotein E3Leiden gene only show impaired very-low-density lipoprotein secretion.Arterioscler Thromb Vasc Biol. 2001; 21: 1366-1372Crossref PubMed Scopus (15) Google Scholar]. Thus, in contrast to ‘normal’ APO