The ability of multicellular organisms to transport fat, in the form of lipoprotein particles, through the circulatory system is one of the most primordial functions. Although there are several distinct lipoprotein transport systems that vary with the phylogeny of the organism, they all use similar mechanisms for assembly, secretion and tissue targeting. The major emphasis of this report is to review the advances made in our knowledge concerning the development of regulatory processes involved in the assembly of apolipoprotein (apo)³ B-containing lipoproteins by the liver of mammals.

The functional basis for why lipoprotein transport systems initially evolved provides insights into the complexity of how this pathway works and is controlled. Simply put, lipoproteins provide a means through which energy in the form of water-insoluble fat can be distributed from its site of synthesis and absorption to specific tissues and cells. The bioavailability of energy stored as fat (~37 kJ/g) is significantly greater than that which can be stored as carbohydrate glycogen (~17 kJ/g). The evolutionary advancement of storing energy in the form of fat has provided organisms with a tremendous advantage in adapting to environmental and developmental changes. For example, hibernation is successful only if the organism has had the ability to build up large stores of fat (triglycerides) during periods when food supplies are plentiful. Migrating birds and insects rely mainly on the lipoprotein transport of fat from stores to muscles, abrogating the need to stop for nourishment. When laying eggs, insects, fish, amphibians, reptiles and birds display over three orders of magnitude increases in the transport of fat, from the liver to oocytes, in order to facilitate egg development. Increases in fat transport, whether it involves lipophorin, vitellogenin or apo B-containing lipoproteins, show a common induction of the expression of genes necessary for their individual biosynthetic, assembly and secretion steps. Moreover, when physiologic requirements dictate the need for increased lipoprotein synthesis, metabolic signals activate these coordinate processes via increased transcription of specific genes. This concept has long been appreciated by those interested in dissecting the molecular events necessary for transcriptional regulation of gene expression. Hormonal induction of vitellogenin synthesis in insects and birds was one of the first model systems used to advance our knowledge of transcriptional regulation (Dolphin et al. 1971).

LIPOPROTEIN STRUCTURE AND FUNCTION

VLDL ASSEMBLY REQUIRES APOLIPOPROTEIN B

The functional basis for apo B's large size has recently been uncovered in elegant experiments examining the genetic basis for hypobetalipoproteinemia in humans and developing in vitro models that faithfully express the hypobetalipoproteinemic phenotype in cultured cells (reviewed in Young 1990). This disorder is characterized by a genetic alteration of the apo B gene, resulting in either the synthesis of truncated forms of apo B that are too short to assemble lipoproteins or inactivation of the gene. Mutant apo B forms having a size smaller than 31% of the N-terminus of the maximal coding region transcribed by the apo B gene show an inability to assemble lipoproteins containing a neutral lipid core. Further analysis showed that the size of apo B and the density of the lipoprotein varied inversely (Spring et al. 1992, Yao et al. 1991).

STRUCTURAL MOTIFS IN APOLIPOPROTEIN B RESEMBLE SOME OF THOSE FOUND IN VITELLOGENIN

High resolution X-ray crystallographic analysis and ³¹P-nuclear magnetic resonance studies of the lipovitellogenin complex show that -sheets of vitellogenin surround a cavity believed to be filled with lipid (Raag et al. 1988). It has been proposed that as more lipid is added, the cavity expands by movement of the -sheets. Apolipoprotein B might associate with lipids in a similar manner, because VLDL also consists of a cavity filled with lipid and surrounded by apo B. Furthermore, the -sheet structures of apo B are thought to contribute to its association with lipid. If, like vitellogenin, apo B serves as the scaffolding for the lipid core, VLDL assembly might involve a process by which, as lipid is added, the surrounding apo B folds to accommodate a growing lipid core.

VLDL IS ASSEMBLED IN THE ENDOPLASMIC RETICULUM

MOVEMENT OF APOLIPOPROTEIN B OUT OF THE ENDOPLASMIC RETICULUM IS RATE LIMITING

APOLIPOPROTEIN B IS DEGRADED INTRACELLULARLY BY A PROCESS THAT CAN ACCOUNT FOR POST-TRANSCRIPTIONAL REGULATION OF SECRETION

TRANSLOCATION OF APOLIPOPROTEIN B REQUIRES A UNIQUE PROCESS THAT MAY GOVERN ITS METABOLIC FATE: VLDL ASSEMBLY OR DEGRADATION

To overcome the obstacles inherent in the translocation of apo B, gene products that are probably not required by other proteins are essential for translocating and secreting apo B (Du et al. 1994). Perhaps the most compelling data showing that apo B requires a unique process for translocation is the finding that CHO cells exhibit a complete inability to translocate apo B forms large enough to assemble VLDL (apo B53), but not truncated apo B15 (Thrift et al. 1992). Translocation-arrested apo B53 is by a process inhibitable by the cysteine active site protease inhibitor acetyl-leucine, leucine, nor-leucal (ALLN) (Thrift et al. 1992). Although apo B lacks any amphipathic -helices that are sufficiently long to be predicted to span a membrane bilayer, in CHO cells treated with ALLN, apo B assumes a stable trans-membrane orientation in the endoplasmic reticulum (Du et al. 1994). The segments responsible for translocation arrest were found to reside outside of the membrane-spanning domain (i.e., on resides lying on the cytoplasmic side of the endoplasmic reticulum) (Du et al. 1994). The inability of CHO cells to translocate apo B across the endoplasmic reticulum is likely to be the lack of expression in these cells of a gene product that is present in liver and intestine. Several lines of evidence indicate that this gene product is microsomal triglyceride transfer protein (MTP).

MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN TRANSFERS LIPID TO THE NASCENT LIPOPROTEIN PARTICLE AND IS REQUIRED FOR APOLIPOPROTEIN B TRANSLOCATION: ANALOGIES TO VITELLOGENIN AND INSECT LIPOPHORIN

Several lines of evidence indicate that MTP is essential for apo B translocation. First, in cells not expressing MTP, the translocation of apo B constructs having a size sufficient to assemble lipoproteins is completely blocked (Du et al. 1994). However, co-expression of MTP in COS and HeLa cells allows apo B to be translocated and secreted as a lipoprotein particle (Gordon et al. 1994, Leiper et al. 1994). Secondly, in the human recessive disorder abetalipoproteinemia, functional loss of MTP blocks the secretion of apo B by both the liver and intestine, the major tissue sites of MTP (Sharp et al. 1993, Shoulders et al. 1993, Wetterau et al. 1992). Recent studies suggest that the inability of the livers and intestines of abetalipoproteinemic patients to secrete apo B is due to a block in its translocation across the endoplasmic reticulum (Du et al. 1996). Variable expression of MTP and/or availability of lipid may account for the variable translocation of apo B and secretion as VLDL.

COORDINATED REGULATION OF VLDL ASSEMBLY AND SECRETION: ANALOGIES TO LIPOPHORIN AND VITELLOGENIN PATHWAYS

Birds produce both vitellogenin and hepatic VLDL. Both lipoprotein transport systems show rapid induction by estrogen (Dolphin et al. 1971). Similar to its induction of vitellogenin synthesis, estrogen also increases the hepatic production of VLDL in birds. Recent studies in our laboratory suggest that a similar coordinated cascade of induction of the genes required for mammalian VLDL assembly and secretion occurs. We propose that nutritional and metabolic needs for hepatic VLDL secretion are signaled by a steroid that is not involved in the signaling required for reproduction. As a result, the mammalian VLDL secretory pathway can respond in a more versatile manner, independent of sex-linked processes. Although the identity of this signal remains to be elucidated, itlike estrogenseems to be derived from the isoprenoid/steroid biosynthetic pathway. It is possible that this steroid signal has evolved from the estrogen signal.

CHOLESTYRAMINE INDUCES HEPATIC LIPOGENESIS AND VLDL ASSEMBLY AND SECRETION

Another unexpected feature of cholestyramine treatment is that it increases hepatic VLDL production (Einarsson et al. 1991). In some hyperlipidemic patients, there is a direct relationship between hepatic bile acid synthesis and VLDL triglyceride secretion (Angelin et al. 1978). Clearly, cholestyramine causes multiple changes in hepatic lipid and lipoprotein metabolism (summarized in Table 1): 1) induction of cholesterol-7-hydroxylase, 2) increased expression of the LDL receptor, 3) induction of cholesterol biosynthesis by increasing the transcription of the some of the genes regulating the isoprenoid/sterol biosynthetic pathway, 4) increased synthesis of all VLDL lipids (i.e., cholesterol, cholesterol esters, triglycerides and phospholipids; R. A. Davis, unpublished observation) and 5) increased secretion of VLDL. All of these events can be ascribed to the activation of a single transcription factor that has the ability to coordinately induce the genes responsible for hepatic VLDL assembly: sterol regulatory binding protein (SREBP). To explain how this might occur, I will review how cholesterol metabolism, lipogenesis, and VLDL assembly and secretion are linked together.

Table 1. Effect of cholestyramine and cholesterol-7-hydroxylase on the expression of hepatic genes and lipogenic processes1 [View Table]

The amount of cholesterol in a cell is precisely regulated (reviewed in (Brown and Goldstein 1986). When cellular cholesterol levels are sufficient to meet the anabolic and catabolic requirements, there is a coordinate repression of the transcription of most, if not all, gene products involved in the cholesterol biosynthetic pathway: HMG-CoA synthase (Gil et al. 1986), HMG-CoA reductase (Gil et al. 1986), farnesyldiphosphate synthase (Ashby and Edwards 1989) and squalene synthase (Jiang et al. 1993). There are additional post-transcriptional mechanisms regulating the activity of many of these enzymes, including phosphorylation, protein degradation and rate of mRNA translation. In mammals, the major pathway for cholesterol uptake is via the LDL receptor (Brown and Goldstein 1986). Like the cholesterol biosynthetic enzymes, expression of the LDL receptor is regulated transcriptionally by negative feedback signaled by cellular cholesterol levels. Recent findings suggest that distinct transcription factors, whose activity is sensitive to the "regulatory" pool of cholesterol, control the transcription of genes that produce enzymes required for sterol biosynthesis and the LDL receptor (Briggs et al. 1993, Wang et al. 1994). Initiation of de-repression of conditionally positive sterol regulatory element (SRE) containing promoters begins with the proteolytic cleavage of the cytosolic domain of SREBP, a unique class of transcription factors whose initial translation produces an integral membrane protein of the endoplasmic reticulum. Once cleaved into its mature form, the N-terminal domain of SREBP, in concert with other transcription factors, activates gene transcription.

Sterol regulatory binding protein also regulates the transcription of genes controlling fatty acid synthesis (Kim et al. 1995). Because the availability of all of the lipid components required for VLDL consists of either sterols or fatty acid esters (triglycerides and phospholipids), the cellular level of mature SREBP will coordinately regulate the availability of the individual lipids required for its assembly. Previous studies showed that adding oleic acid to the medium of cultured rat hepatocytes increases both triglyceride and phospholipid synthesis and secretion (Davis and Boogaerts 1982). The combined data suggest that the availability of fatty acids, produced in response to SREBP induction of acetyl-CoA carboxylase and fatty acid synthase may be all that is needed to increase VLDL glycerolipid synthesis.

THE SIGNAL MEDIATING SREBP PROCESSING

CHOLESTEROL-7--HYDROXYLASE DECREASES THE ABILITY OF CHOLESTEROL TO REPRESS SRE-GOVERNED GENES AND INCREASES THE SYNTHESIS OF ALL VLDL LIPIDS AND THE SECRETION OF VLDL APOLIPOPROTEIN B

7-Hydroxylase was stably expressed in two different cultured cell lines that do not normally express it: CHO cells expressing apo B and McArdle rat hepatoma cells. The results (Table 1) show that 7-hydroxylase increased the cellular abundance of mature SREBP, induced SRE-governed gene transcription, increased the synthesis of all VLDL lipid components and the secretion of apo B (data not shown). Furthermore, this increased expression of 7-hydroxylase also decreased the intracellular degradation of apo B (data not shown). These changes can be reversed by adding two "oxysterols": 7-oxo-cholesterol together with 25-hydroxycholesterol (data not shown).

The combined data suggest that a common metabolic signal, whose activity is attenuated by 7-hydroxylase, may control SREBP-governed gene transcription and VLDL assembly. This signal, like estrogen in lower species, can induce all of the processes required for lipoprotein secretion and tissue targeting. The major difference is that this metabolic signal is related to nutritional state and is not limited to reproduction, as is estrogen. This versatile metabolic signaling provides mammals a means to control lipid metabolism and transport (i.e., VLDL secretion) in a manner that is responsive to both nutritional status and reproduction needs. Identification of the signal regulating SREBP-governed gene transcription and VLDL secretion will provide important clues as to the evolutionary divergence of the vitellogenin lipid transport system. Moreover, if this regulator is found to be derived from the same sterol precursor as estrogen, this finding would provide new insights into the co-evolution of ancestral metabolic regulators and genes of a common primordial pathway.