The gene for human apolipoprotein E (apo-E) was selected from a library of cloned genomic DNA by screening with a specific cDNA hybridization probe, and its structure was characterized. The complete nucleotide sequence of the gene as well as 856 nucleotides of the 5' flanking region and 629 nucleotides of the 3' flanking region were determined. Analysis of the sequence showed that the mRNA-encoding region of the apo-E gene consists of four exons separated by three introns. In comparison to the structure of the mRNA, the introns are located in the 5' noncoding region, in the codon for glycine at position -4 of the signal peptide region, and in the codon for arginine at position +61 of the mature protein. The overall lengths of the apo-E gene and its corresponding mRNA are 3597 and 1163 nucleotides, respectively; a mature plasma protein of 299 amino acids is produced by this gene. Examination of the 5' terminus of the gene by S1 nuclease mapping shows apparent multiple transcription initiation sites. The proximal 5' flanking region contains a "TATA box" element as well as two nearby inverted repeat elements. In addition, there are four Alu family sequences associated with the apo-E gene: an Alu sequence located near each end of the gene and two Alu sequences located in the second intron. This knowledge of the structure permits a molecular approach to characterizing the regulation of the apo-E gene.Apolipoprotein E (apo-E) is a component of various classes of plasma lipoproteins in all mammals that have been studied (for review, see refs. 1 and 2). It is a single chain polypeptide (Mr, 34,000) of 299 amino acids (3) that is synthesized initially with an 18-residue signal peptide that is removed cotranslationally (4,5). The amino acid sequence as well as the mRNA nucleotide sequence are known for both the human (3, 6) and rat (7) species. The major site of synthesis is the liver, but relatively abundant levels of apo-E mRNA have been detected in many extrahepatic tissues, including the brain and the adrenals (8). In addition, apo-E is produced by mouse peritoneal macrophages, as well as human monocytederived macrophages (9).A major function of apo-E is its mediation of the cellular uptake of specific lipoproteins through an interaction with apo-B,E(LDL) receptors on extrahepatic and hepatic cell surfaces and with distinct hepatic apo-E receptors (for review, see ref. 10). The receptor binding domain of human apo-E has been determined to be an arginine-and lysine-rich region in the vicinity of residues 140 and 160 (11,12). Variant forms of apo-E with single amino acid substitutions in this region show decreased receptor binding activity (13)(14)(15) and are associated with type III hyperlipoproteinemia and accelerated cardiovascular disease (for review, see refs. 16 and 17). Apolipoprotein E with normal receptor binding activity is found in two common isoforms, the E3 and E4 phenotypes, with either cysteine or arginine, respectively, at residue position 112 (13).Because of the central role that apo-E plays i...

To investigate the biochemical mechanism underlying the effect of sterol deprivation on longevity in Caenorhabditis elegans, we treated parent worms (P0) with 25-azacoprostane (Aza), which inhibits sitosterol-to-cholesterol conversion, and measured mean lifespan (MLS) in F2 worms. At 25 M (ϳEC 50 ), Aza reduced total body sterol by 82.5%, confirming sterol depletion. Aza (25 M) treatment of wild-type (N2) C. elegans grown in sitosterol (5 g/ml) reduced MLS by 35%. Similar results were obtained for the stress-related mutants daf-16(mu86) and gas-1(fc21). Unexpectedly, Aza had essentially no effect on MLS in the stress-resistant daf-2(e1370) or mitochondrial complex II mutant mev-1(kn1) strains, indicating that Aza may target both insulin/IGF-1 signaling (IIS) and mitochondrial complex II. Aza increased reactive oxygen species (ROS) levels 2.7-fold in N2 worms, but did not affect ROS production by mev-1(kn1), suggesting a direct link between Aza treatment and mitochondrial ROS production. Moreover, expression of the stress-response transcription factor SKN-1 was decreased in amphid neurons by Aza and that of DAF-28 was increased when DAF-6 was involved, contributing to lifespan reduction.Sterols are important molecules involved in membrane organization, hormone production, and signal processing. Because Caenorhabditis elegans lacks a de novo sterol biosynthesis pathway, it requires dietary cholesterol or plant sterols (e.g. sitosterol) that can be converted to cholesterol or its most abundant sterol, 7-dehydrocholesterol (1, 2) (Fig. 1A). Sterol depletion experiments have revealed that a restriction in the sterol supply caused by inadequate nutrition produces serious defects in development and reduces the longevity of C. elegans (3-7). For example, sterol starvation leads to an increase in embryonic lethality (7) and a decrease in lifespan (ϳ40%) in wild-type N2 worms (6). Most of the sterol depletion phenotype also occurs when worms are grown in the presence of sitosterol as a sterol nutrient and 25-azacoprostane (Aza), 2 an inhibitor of sterol C24-reductase and dealkylation of desmosterol during its conversion to cholesterol (8). Using proteomic approaches, our laboratory has previously shown that defects in development and growth in C. elegans caused by Aza treatment are associated with changes in the abundance of many proteins (3). The major proteins influenced by Aza treatment were the lipoproteins VIT-2 and VIT-6 and their putative receptors RME-2 and LRP-1, which were previously known to respond to sterols (3). Recently, the endogenous ligands of DAF-12 have been discovered to be 3-keto bile acid-like steroids, called ⌬ 4 -and ⌬ 7 -dafachronic acids (9). Dafachronic acids and related metabolites regulate longevity and stress resistance (9 -11). DAF-36, a Rieske-like oxygenase, and DAF-9, a cytochrome P450 enzyme, produce dafachronic acid ligands that activate the DAF-12 nuclear receptor (9). It was also reported that methyltransferase STRM-1 modifies sterol substrates for the synthesis of dafachronic acid (...

In Caenorhabditis elegans, slow fat consumption has been suggested to contribute to the extension of the survival rate during nutritionally adverse conditions. Here, we investigated the potential role of pyruvate dehydrogenase kinase (PDHK)-2, the C. elegans homolog of mammalian PDK, effects on fat metabolism under nutritional conditions. PDHK-2 was expressed at low levels under well-fed conditions but was highly induced during long-term starvation and in the dauer state. This increase in pdhk-2 expression was regulated by both DAF-16 and NHR-49. Dauer-specific induction of PDHK-2 was abolished upon entry into the post-dauer stage. Interestingly, in the long-term dauer state, stored fat levels were higher in daf-2(e1370);pdhk-2 double mutants than in daf-2(e1370), suggesting a positive relationship between PDHK-2 activity and fat consumption. PDHK-2 deficiency has been shown to lead to greater preservation of residual fats, which would be predicted to contribute to survival during the dauer state. A test of this prediction showed that the survival rates of daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants were higher than that of daf-2(e1370), suggesting that loss of either the ATP-binding domain (tm3075) or branched chain keto-acid dehydrogenase kinase domain (tm3086) of PDHK-2 leads to reduced fat consumption and thus favors increased dauer survival. This attenuated fat consumption in the long-term dauer state of C. elegans daf-2 (e1370);pdhk-2 mutants was associated with concomitant down-regulation of the lipases ATGL (adipose triglyceride lipase), HSL (hormone-sensitive lipase), and C07E3.9 (phospholipase). In contrast, PDHK-2 overexpression in wild-type starved worms induced lipase expression and promoted abnormal dauer formation. Thus, we propose that PDHK-2 serves as a molecular bridge, connecting fat metabolism and survival under nutritionally adverse conditions in C. elegans.