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A cDNA that expresses a receptor for very low density lipoprotein (VLDL) was isolated from a rabbit heart cDNA library and characterized. The deduced amino acid sequence of the cDNA revealed that the cDNA encodes a protein with stridng homology to the low density lipoprotein (LDL) receptor. (12) with poly(A)+ RNA isolated from normal rabbit heart. To exclude the rabbit LDL receptor, the entire pooled cDNA library was digested with Sal I and recircularized with T4 DNA ligase. The presence of a unique Sal I site in the rabbit LDL receptor cDNA (7) and the vector results in loss of any LDL receptor cDNAs after recircularization and retransformation. The resulting LDL-receptor-subtracted cDNA library was screened with the 1.9-kilobase Sma I-Sal I fragment from the rabbit LDL receptor cDNA (7)

A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca 2؉ in response to glucose, and thereby glucoseinduced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5؊͞؊ islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5؊͞؊ islets. Furthermore, exposure of LRP5؉͞؉ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that Wnt͞LRP5 signaling contributes to the glucose-induced insulin secretion in the islets.and LRP6 are coreceptors involved in the Wnt signaling pathway (1-6). The Wnt signaling pathway plays a pivotal role in embryonic development (7,8) and oncogenesis (9) through various signaling molecules including Frizzled receptors (10), recently characterized LRP5 and LRP6 (1-6), and Dickkopf proteins (4, 6). In addition, the Wnt signaling is also involved in adipogenesis by negatively regulating adipogenic transcription factors (Tcfs) (11). Although Wnt signaling has been characterized in both developmental and oncogenic processes, little is known about its function in the normal adult.Recent studies have revealed that loss of function mutations in the LRP5 gene cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (12). LRP5 is expressed in osteoblasts and transduces Wnt signaling via the canonical pathway, thereby modulating bone accrual development (12, 13). A point mutation in a ''propeller'' motif in LRP5 causes a dominant-positive high bone density by impairing the action of a normal antagonist of the Wnt pathway, Dickkopf, thereby increasing Wnt signaling (14,15). In addition, the human LRP5 gene is mapped within the region (IDDM4) linked to type 1 diabetes on chromosome 11q13 (16).In previous studies, we and others showed that LRP5 is highly expressed in many tissues, including hepatocytes and pancreatic beta cells (17,18). We also showed that LRP5 can bind apolipoprotein E (apoE) (18). This finding raises the possibility that LRP5 plays a role in the hepatic clearance of apoE-containing chylomicron remnants, a major plasma lipoprotein carrying diet-derived cholesterol.To evaluate the in vivo roles of LRP5, we generated LRP...

Using peptide sequences derived from bovine cardiac acetyl-CoA synthetase (AceCS), we isolated and characterized cDNAs for a bovine and murine cardiac enzyme designated AceCS2. We also isolated a murine cDNA encoding a hepatic type enzyme, designated AceCS1, identical to one reported recently (Luong, A., Hannah, V. C., Brown, M. S., and Goldstein, J. L. (2000) J. Biol. Chem. 275, 26458 -26466). Murine AceCS1 and AceCS2 were purified to homogeneity and characterized. Among C2-C5 short and medium chain fatty acids, both enzymes preferentially utilize acetate with similar affinity. The AceCS2 transcripts are expressed in a wide range of tissues, with the highest levels in heart, and are apparently absent from the liver. The levels of AceCS2 mRNA in skeletal muscle were increased markedly under ketogenic conditions. Subcellular fractionation revealed that AceCS2 is a mitochondrial matrix enzyme. [ 14 C]Acetate incorporation indicated that acetyl-CoAs produced by AceCS2 are utilized mainly for oxidation.

Deposition of the yolk mass components of chicken oocytes, very low density lipoprotein (VLDL) and vitellogenin (VTG), is mediated by a 95 kDa plasma membrane protein, termed VLDL/VTG receptor (VLDL/VTGR). Molecular characterization of the VLDL/VTGR revealed that it is a member of the LDLR gene superfamily, and harbours eight complement‐type, cysteine‐rich ligand binding repeats at the N‐terminus. This ligand binding domain structure is the hallmark of the recently discovered mammalian so‐called VLDLRs, whose true physiological function remains to be elucidated. Northern blot analysis revealed that this receptor is expressed almost exclusively in oocytes, with very much lower levels of hybridizing transcripts present in heart and skeletal muscle. Heterologous expression of the cloned receptor demonstrated its ability to bind both VLDL and VTG. The receptor gene is located on the avian sex chromosome Z, in agreement with the sex linkage of a single‐gene defect in animals that fail to reproduce because of the lack of expression of functional VLDL/VTGR. In situ hybridization analysis of oocytes suggested that VLDL/VTGR mRNA may relocalize during oocyte growth. Thus, the current study has identified and characterized the first non‐mammalian VLDLR. Its key role in avian reproduction and extremely high evolutionary conservation shed new light on VLDLR function in mammals, which also express the gene in ovaries.

We report herein the cDNA cloning of a novel rat acyl-CoA synthetase (ACS) that preferentially uses arachidonate and eicosapentaenoate. This newly identified ACS (designated ACS4) contains 670 amino acids and is 68% identical to rat ACS3, a previously characterized ACS that is highly expressed in brain. ACS4 was overproduced in Escherichia coli and the resulting enzyme was purified to homogeneity. The purified enzyme utilizes arachidonate and eicosapentaenoate most preferentially among C 8 -C 22 saturated fatty acids and C 14 -C 22 unsaturated fatty acids. Kinetic analyses revealed that the enzyme has a high affinity for arachidonate and eicosapentaenoate and low affinity for palmitate. ACS4 transcripts are detectable in a wide range of tissues, with the highest level in adrenal gland. Immunoreactivity to ACS4 was detected in the zona fasciculata and reticularis of adrenal gland, in the corpus luteum and stromal luteinized cells in ovary, and in the Leydig cells of testis.

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The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal with familial hypercholesterolemia, produces a mutant receptor for plasma low-density lipoprotein (LDL) that is not transported to the cell surface at a normal rate. Cloning and sequencing of complementary DNA's from normal and WHHL rabbits, shows that this defect arises from an in-frame deletion of 12 nucleotides that eliminates four amino acids from the cysteine-rich ligand binding domain of the LDL receptor. A similar mutation, detected by S1 nuclease mapping of LDL receptor messenger RNA, occurred in a patient with familial hypercholesterolemia whose receptor also fails to be transported to the cell surface. These findings suggest that animal cells may have failsafe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues.Receptors and other cell surface proteins follow a complex path from their sites of synthesis in the rough endoplasmic reticulum to their sites of function in the plasma membrane (1). The orderly progression of such proteins through the endoplasmic reticulum and the Golgi complex was elucidated through study of model proteins, such as those of lipid-enveloped viruses. The signals that direct this movement and the fail-safe mechanisms that prevent denatured proteins from reaching the cell surface are still largely unknown. One way to solve the problem is by studying mutant proteins that do not reach the cell surface. Such mutations have been created artificially through in vitro mutagenesis of genes encoding viral envelope proteins (2). A second approach is through study of naturally occurring mutations in which transport is blocked. In human and animal cells, the most informative group of these mutations occurs in the gene for the low density lipoprotein (LDL) receptor and gives rise to a genetic disease called familial hypercholesterolemia (FH) (3, 4).FH is an autosomal dominant disorder characterized by an elevation of cholesterol in plasma and severe atherosclerosis (3, 4). The disease is caused by defects in the cell surface receptor for LDL, which is a cholesterol transport protein (5). When the LDL receptor is defective, LDL cannot enter cells by receptor-mediated endocytosis and the lipoprotein accumulates in plasma, eventually producing atherosclerosis (3). Some mutations alter the receptor in such a way that it cannot move from the rough endoplasmic reticulum to the Golgi complex (6, 7). Such transport-deficient mutations have been observed frequently in humans with FH (5- WHHL rabbits are homozygous for a mutant allele that produces an LDL receptor that is of normal apparent molecular size but is transported to the cell surface at only one-tenth the normal rate (7). The newly synthesized receptor receives its normal complement of high mannose N-linked sugars and the core N-acetylgalactosamine of the O-linked sugars (5). However, these carbohydrate chains are not processed to their mature form, apparently because the mutant receptor does no...