Genomic interval engineering of mice identifies a novel modulator of triglyceride production.

To accelerate the biological annotation of novel genes discovered in sequenced regions of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such genes. Here we describe the targeted deletion of a 450-kb region on mouse chromosome 11, which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities, including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation, and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepatic very-low density lipoprotein triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450-kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2, a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2, carnitine, and hepatic triglyceride production is of particular importance because of the clinical consequence of hypertriglyceridemia and the paucity of genes known to modulate triglyceride secretion.

Estrogen modulation of apolipoprotein(a) expression. Identification of a regulatory element.

Elevated plasma levels of the lipoprotein particle Lp(a) are a major risk factor for cardiovascular disease. Lp(a) plasma levels are determined by the level of expression of its characteristic protein component, apo(a). Apo(a) expression is modulated by several hormones, of which estrogens are the best known. The chromosomal region responsible for estrogen response was identified within an apo(a) enhancer located at approximately 26 kilobases from the apo(a) promoter. Although the estrogen-responsive unit contains a potential estrogen response element, binding of estrogen receptor-alpha to DNA was not necessary. The receptor, activated by bound estradiol, interacts through its transactivation domains with a transcription factor necessary for the function of the enhancer, preventing its binding to DNA.

Apolipoprotein(a) gene enhancer resides within a LINE element.

Apolipoprotein(a), (apo(a)), is the distinguishing protein portion of the lipoprotein(a) particle, elevated plasma levels of which are a major risk factor for cardiovascular disease. A search for enhancer elements that control the transcription of the apo(a) gene led to the identification of an upstream element that contains target binding sites for members of the Ets and Sp1 nuclear protein families. The enhancer element functions in either orientation to confer a greater than 10-fold increase in the activity of the apo(a) minimal promoter in cultured hepatocyte cells. Unexpectedly, the enhancer element is located within a LINE retrotransposon element, suggesting that LINE elements may function as mobile regulatory elements to control the expression of nearby genes.

Identification of a receptor mediating absorption of dietary cholesterol in the intestine.

Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.

Reconstitution and further characterization of the cholesterol transport activity of the small-intestinal brush border membrane.

The sterol (free and esterified cholesterol) transport activity of the small-intestinal brush border membrane was solubilized with the short-chain detergent diheptanoylphosphatidylcholine and reconstituted to an artificial membrane system (proteoliposomes). The resulting proteoliposomes were identified as unilamellar membrane vesicles ranging in size between 50 and 200 nm with a broad maximum at 70-110 nm. That the sterol transport protein was indeed incorporated into the lipid bilayer was shown by density gradient centrifugation on a Ficoll gradient: the proteoliposomes yielded a single band with an apparent density of 1.035 g/mL. By subjecting solubilized brush border membrane vesicles (BBMV) to gel filtration on Sephadex G-200 prior to reconstitution, a 7-fold enrichment of the sterol transport activity was achieved relative to the original BBMV. The experimental evidence presented lends strong support to the notion that the sterol transport protein is an integral protein of the brush border membrane which is anchored in the lipid bilayer by at least one hydrophobic domain. The active center(s) is (are) exposed to the external side of the membrane. Anchoring of this protein to the lipid bilayer by a glycosylphosphatidylinositol moiety is unlikely. The reconstituted proteoliposomes behaved very similarly to the original BBMV in terms of facilitated sterol uptake. Using these proteoliposomes, a hitherto unknown activity of the brush border membrane was discovered. Long-chain triacylglycerols can be taken up by this membrane as such and need not be hydrolyzed prior to absorption.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily.

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd approximately 0.37 microM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

The uptake of cholesterol at the small-intestinal brush border membrane is inhibited by apolipoproteins.

The uptake of free and esterified cholesterol at the brush border membrane is protein-mediated. Here we show that this sterol uptake is effectively inhibited by exchangeable serum apolipoproteins. Binding of the apolipoprotein to the brush border membrane mediates the inhibitory effect. Evidence is presented to show that the structural motif responsible for the inhibition is the amphipathic alpha-helix.

Comparison of cholesterol and sitosterol uptake in different brush border membrane models.

(I) There is little discrimination between cholesterol and the plant sterol sitosterol in the uptake at the brush border membrane (BBM). (II) This difference cannot account for the marked discrimination between cholesterol and sitosterol observed in the absorption of these two sterols by the small-intestinal epithelium. (III) This discrimination occurs during intracellular processing involving the esterification and incorporation into lipoprotein particles of the two sterols. This conclusion is based on a comparative study of sterol uptake by brush border membrane vesicles (BBMV) and sterol absorption by Caco-2 cells. (IV) The uptake of sitosterol by the BBM is energy-independent and facilitated in a manner analogous to cholesterol uptake [Thurnhofer, H., & Hauser, H. (1990a) Biochemistry 29, 2142-2148]. (V) The rate of cholesterol and sitosterol uptake by BBMV from both mixed bile salt micelles and small unilamellar vesicles (SUV) as the donor is directly proportional to the sterol content of the donor. (VI) The pseudo-first-order rate constants k1 for sterol uptake from SUV are independent of the sterol content up to 10-20 mol %. Above that, competition between the two sterols leads to a reduction of the k1 values.

A comparative study of sterol absorption in different small-intestinal brush border membrane models.

We reported previously that the absorption of cholesterol and long-chain cholesteryl esters by rabbit small-intestinal brush border membranes (BBMV) is protein-mediated (Thurnhofer, H., and H. Hauser. 1990. Biochemistry. 29:2142-2148; Compassi, S., M. Werder, D. Boffelli, F. E. Weber, H. Hauser, and G. Schulthess. 1995. Biochemistry. 34: 16473-16482). Evidence is presented for similar cholesterol transport activities in rabbit, pig, and human BBMV. As BBMV are subject to a number of limitations and the influence of these on sterol absorption is unknown, it is desirable to verify results obtained with this model system in other brush border membrane models more closely related to the in vivo situation. Sterol absorption in intact enterocytes parallels the absorption measured in BBMV, provided that both model systems are normalized to equal sucrase activity. The parallel behavior of the two brush border membrane models lends support to our previous conclusion that the brush border membrane takes up free and esterified cholesterol in a facilitated and energy-independent process. The absorption of sterols in small-intestinal segments mounted in the Ussing chamber is shown to be a complex process in which the diffusion of the bile salt micelles to the brush border membrane is rate-limiting. All brush border membrane models share the disadvantage of being unstable and subject to degradation. The seriousness of the problem increases apparently with the complexity of the model, i.e., in the order BBMV-->enterocytes-->intestinal segments. One main conclusion of this study is that no brush border membrane model is sufficient and satisfactory, therefore conclusive work in lipid absorption can never be based on a single brush border membrane model.

Cholesteryl ester absorption by small intestinal brush border membrane is protein-mediated.

This paper provides unambiguous evidence that brush border membrane vesicles (BBMV) routinely prepared from rabbit small intestine contain a protein that catalyzes the absorption of long-chain cholesteryl ester and ether. The protein is located on the lumenal side of the brush border membrane. The experiments demonstrate that cholesteryl oleate need not be hydrolyzed prior to its incorporation in the BBMV. Unexpectedly and surprisingly, the absorption kinetics of free and esterified cholesterol are very similar in small intestinal BBMV using mixed bile salt micelles and small unilamellar phospholipid vesicles as the donor. The water-soluble form of the protein responsible for this effect is released into the supernatant, probably by autoproteolysis, and catalyzes the exchange of both free and esterified cholesterol between two populations of small unilamellar phospholipid vesicles (SUV). The water-soluble form of the protein was partially purified by a two-step procedure involving gel filtration on Sephadex G-75 and anion-exchange chromatography on Mono Q, yielding a 50-fold increase in the specific activity of the protein. The resulting protein gave two bands on sodium dodecyl sulfate--10% polyacrylamide gel electrophoresis and was used to raise polyclonal antibodies in sheep. The IgG fraction of the sheep antisera blocked the cholesteryl oleate and cholesterol exchange between two populations of SUV mediated by the antigen. The same IgG fraction produced a partial inhibition of cholesterol absorption in small intestinal BBMV. We conclude from the data presented that, contrary to the general belief prevailing in the field of lipid digestion and absorption, long-chain cholesteryl esters may be taken up by the brush border membrane as such and need not be hydrolyzed prior to absorption. The actual contribution of this mechanism to the total absorption of long-chain cholesteryl esters is probably limited by the low solubility of these compounds in mixed bile salt micelles and lipid vesicles.

Absorption of monoacylglycerols by small intestinal brush border membrane.

The absorption of monoacylglycerol by small intestinal brush border membrane is a passive process, i.e., the movement of monoacylglycerol from small unilamellar phospholipid vesicles as donor particles through the aqueous medium and the incorporation into the outer monolayer of the lipid bilayer of the brush border membrane are passive processes involving diffusion of the lipid along a concentration gradient. Small unilamellar vesicles of egg phosphatidylcholine containing 1 mol% of radiolabeled hexadecylglycerol were used as donor, and rabbit small intestinal brush border membrane vesicles or intact enterocytes isolated from pig jejunum, as acceptor. Hexadecylglycerol was employed as a lipase-resistant model compound for monoacylglycerols. Both acceptor membranes behave similarly in terms of hexadecylglycerol absorption: the kinetics of hexadecylglycerol absorption are biphasic. The initial fast phase is due to the movement of hexadecylglycerol from the donor particle through the aqueous medium to the outer lipid monolayer of the acceptor membrane, and the second slow phase probably involves the flip-flop motion of hexadecylglycerol from the outer to the inner monolayer of the acceptor membrane. The values for the pseudo-first-order rate constants of the initial fast phase for hexadecylglycerol absorption are relatively large and primarily determined by the high solubility (cmc) of hexadecylglycerol in aqueous media. The pseudo-first-order rate constants depend linearly on the protein (lipid) concentration of the acceptor membrane, indicating that the on rate of the hexadecylglycerol into the brush border membrane is rate limiting. The mechanism of the hexadecylglycerol absorption involves mainly monomer diffusion and probably collision-induced transfer.

A theoretical method to predict DNA permutation gel electrophoresis from the sequence.

The gel electrophoretic permutation assays of DNA fragments experimentally investigated by different authors were theoretically reproduced using our theoretical model of sequence-dependent curvature. The general pattern of agreement obtained suggests that our method can be usefully adopted as an alternative to the experimental assay, in particular where the lack of a sufficient number of unique restriction sites in the fragment prevents the correct localization of the main bend site.

The curvature vector in nucleosomal DNAs and theoretical prediction of nucleosome positioning.

Using our model for predicting DNA superstructures from the sequence, the average distribution of the phases of curvature along the sequences of the set of the 177 nucleosomal DNAs investigated by Satchwell et al. (J. Mol. Biol. 191 (1986) 659) was calculated. The diagram obtained shows very significant features which allow the visualization of the intrinsic nucleosomal superstructure characterized by two quasi-parallel tracts of a flat left-handed superhelical turn connected by a left-handed inflection in a perpendicular direction; such a superstructure appears to be closely related to the nucleosome model of Travers and Klug (Phil. Trans. R. Soc. Lond. 317 (1987) 537). The nucleosomal curvature phase diagram was then adopted as a sensitive determinant for the nucleosome virtual positioning in DNAs via correlation function, obtaining a good agreement with the experimental mapping of SV40 regulatory region as recently investigated by Ambrose et al. (J. Mol. Biol. 209 (1989) 255). This analysis shows also the presence of a constant phase relation between the virtual nucleosome positions which suggests its possible implication in the nucleosome condensation in chromatin.