Vertebral fracture efficacy during risedronate therapy in patients using proton pump inhibitors.

UNLABELLED: Recent evidence suggests that proton pump inhibitor (PPI) use may affect fracture risk, an important issue for patients being concurrently treated for osteoporosis. The results of our post hoc analysis showed that, regardless of PPI concomitant use, risedronate significantly reduced the risk of new vertebral fractures compared with placebo. INTRODUCTION: Recent evidence suggests that PPI use may affect fracture risk, an important issue for patients being concurrently treated for osteoporosis. Moreover, data suggest that concomitant use of PPIs may wane the anti-fracture effect of bisphosphonates. We explored the relationship between concomitant use of PPIs and incident vertebral fractures among patients treated with risedronate or placebo. Bone mineral density (BMD) and upper gastrointestinal (UGI) adverse events (AEs) were also assessed. METHODS: This study is a post hoc analysis of a subset of patients participating in three prospective, randomized, placebo-controlled clinical trials, with durations of up to 3 years, which evaluated the efficacy of risedronate in reducing fracture risk: Vertebral Efficacy with Risedronate Trial-MultiNational (VERT-MN); Vertebral Efficacy with Risedronate Trial-North America (VERT-NA); and the risedronate Hip Intervention Program (HIP). RESULTS: Total enrollment included 2,729 risedronate and 2,725 placebo patients. Concomitant acid-suppressing drugs were used by 8.8% of the total population (n = 482). Regardless of PPI concomitant use, risedronate significantly reduced the risk of new vertebral fractures compared with placebo (risk reduction: PPI users 57%, p = 0.009; PPI non-users 38%, p < 0.001). BMD increased with risedronate, independent of PPI use. PPI users were at a 2.5-fold greater risk of experiencing at least one UGI AE compared with non-users. CONCLUSIONS: Risedronate significantly reduced the risk of new vertebral fractures compared with placebo, regardless of PPI concomitant use.

Binding, internalization, and hydrolysis of low density lipoprotein in long-term lymphoid cell lines from a normal subject and a patient with homozygous familial hypercholesterolemia.

Long-term established human lymphoid cells were shown to possess high affinity cell surface receptors for low density lipoprotein (LDL), the major cholesterol-carrying protein in human plasma. Binding of LDL to these receptors was followed by internalization of the lipoprotein and hydrolysis of its protein and cholesteryl ester components. Cultured lymphocytes from a patient with the homozygous form of familial hypercholesterolemia lacked cell surface LDL receptors and therefore failed to take up and degrade the lipoprotein with high affinity. Cultured human lymphocytes should prove useful for further studies of: (a) the relation between cholesterol metabolism and cellular function and (b) the mechanism by which LDL binding at the cell surface leads to internalization of the lipoprotein.

Regulation of cholesterol synthesis by low density lipoprotein in isolated human lymphocytes. Comparison of cells from normal subjects and patients with homozygous familial hypercholesterolemia and abetalipoproteinemia.

The rate of cholesterol synthesis from [14C]acetate was low in circulating blood lymphocytes freshly isolated from 17 normal subjects and 4 subjects with homozygous FH. On the other hand, the rate of cholesterol synthesis was two to fourfold above normal in freshly isolated lymphocytes from two subjects with abetalipoproteinemia. When the lymphocytes from subjects with all three genotypes were incubated for 48-72 h in the absence of lipoproteins, the rate of cholesterol synthesis increased by 5-15-fold. The subsequent addition of plasma LDL, but not HDL, rapidly suppressed cholesterol synthesis in the lymphocytes from normal subjects. In contrast, lymphocytes from the FH homozygotes, which have been shown previously to be deficient in cell surface LDL receptors, were resistant to LDL-mediated suppression of cholesterol synthesis. In addition to its ability to suppress cholesterol synthesis after it had been elevated by incubation of the cells in the absence of lipoproteins, LDL was able to suppress the induction of the enhanced rate of sterol synthesis when added to normal lymphocytes immediately after their isolation from the bloodstream. In contrast to the former action of LDL, the latter action of LDL-i.e., the suppression of induction of sterol synthesis-also occurred to a limited extent in lymphocytes from FH homozygotes. However, the FH lymphocytes, but not the normal cells, could be made resistant to this action of LDL by inclusion in the incubation medium of lipoprotein-deficient serum (30 percent, vol/vol) plus HDL (1 mg protein/ml). Considered together with previous data demonstrating a deficiency of LDL receptors in freshly isolated lymphocytes from FH homozygotes, the current studies provide evidence in support of the hypothesis that the interaction of plasma LDL with its cell surface receptor serves to regulate cholesterol synthesis in human lymphocytes.

Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells.

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.

Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletion-bearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletion-bearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins.

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.

Metabolism of cationized lipoproteins by human fibroblasts. Biochemical and morphologic correlations.

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 mug/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.

Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice.

When expressed in livers of transgenic mice, the human low density lipoprotein (LDL) receptor is specifically targeted to the basolateral (sinusoidal) surface of hepatocytes as determined by immunofluorescence and immunoelectron microscopy. The COOH-terminal cytoplasmic domain of the receptor (residues 790-839) contains a signal for this targeting. A mutant receptor truncated at residue 812 was localized exclusively to the apical (bile canalicular) surface. A mutant receptor terminating at residue 829 showed the normal basolateral distribution, as did a receptor in which alanine was substituted for serine 833, which was previously shown to be a site for phosphorylation in vitro. These data localize the basolateral targeting signal to the 17-residue segment between residues 812 and 828. A 10-amino acid stretch within this segment shows a 4/10 match with a sequence within a previously identified basolateral sorting motif for the receptor for polymeric IgA/IgM in MDCK cells. The four shared residues are spaced at intervals of three, raising the possibility that they all face the same side of an alpha-helix. We conclude that this 10-amino acid stretch may contain a signal that directs certain proteins, including the LDL receptor and the polymeric IgG/IgM receptor, to the basolateral surface of polarized epithelia.

Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding.

A cDNA encoding a cytoplasmic oxysterol binding protein was expressed at high levels by transfection in animal cells. This protein binds oxysterols such as 25-hydroxycholesterol that regulate sterol metabolism by transcriptional and posttranscriptional effects. In the transfected cells, some of the oxysterol binding protein (OSBP) was distributed diffusely in the cytoplasm, and some was bound to small vesicles near the nucleus, as revealed by indirect immunofluorescence. Upon addition of 25-hydroxycholesterol, most of the OSBP became concentrated in large perinuclear structures that stained with lentil lectin, a protein that stains the Golgi apparatus. The structures that contained OSBP were disrupted by brefeldin A, confirming their identification as Golgi. A mutant OSBP lacking the COOH-terminal oxysterol binding domain localized to the Golgi spontaneously, suggesting that this domain normally occludes the domain that binds to the Golgi and that sterols relieve this occlusion. The previously noted potential leucine zipper sequence in OSBP was not required for Golgi localization, nor was it essential for homodimer formation. We conclude that OSBP is triggered to bind extrinsically to Golgi membranes when it binds oxysterols and speculate that this translocation may play a role in the transport, metabolism, or regulatory actions of oxysterols.

Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex.

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.

Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts.

The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti-receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface.

Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts.

Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 125I-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 125I-EGF both bound preferentially to coated pits at 4 degrees C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 125I-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but 125I-EGF bound to coated pits and was internalized just as in normal fibroblasts.

Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice.

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.

Expression of the familial hypercholesterolemia gene in heterozygotes: mechanism for a dominant disorder in man.

Studies in ctltured fibroblasts indicate that the primary genetic abnormality in familial hypercholesterolemia involves a deficiency in a cell surface receptor for low density lipoproteins (LDL). In normal cells, binding of LDL to this receptor regulates cholesterol metabolism by suppressing cholesterol synthesis and increasing LDL degradation. In cells from heterozygotes, a 60 percent reduction in LDL receptors leads to a concentration-dependent defect in regulation, so that attainment of equal rates of cholesterol synthesis and LDL degradation in normal and heterozygous cells requires a two- to threefold higher concentration of LDL in the heterozygote. The identification of this genetic regulatory defect in fibroblasts of heterozygotes makes available an in vitro system for studying the effects of a dominant mutation on gene expression in mammalian cells.

Retinal degeneration in choroideremia: deficiency of rab geranylgeranyl transferase.

Rab geranylgeranyl transferase (GG transferase) is a two-component enzyme that attaches 20-carbon isoprenoid groups to cysteine residues in Rab proteins, a family of guanosine triphosphate-binding proteins that regulate vesicular traffic. The mutant gene in human choroideremia, an X-linked form of retinal degeneration, encodes a protein that resembles component A of rat Rab GG transferase. Lymphoblasts from choroideremia subjects showed a marked deficiency in the activity of component A, but not component B, of Rab GG transferase. The deficiency was more pronounced when the substrate was Rab3A, a synaptic vesicle protein, than it was when the substrate was Rab1A, a protein of the endoplasmic reticulum. The data imply the existence of multiple component A proteins, one of which is missing in choroideremia.

Independent pathways for secretion of cholesterol and apolipoprotein E by macrophages.

Cholesterol-loaded macrophages secrete cholesterol and apolipoprotein E. The current studies show that this secretion occurs by two independent pathways. In the absence of serum, the cells secrete apolipoprotein E, but not cholesterol. In the presence of monensin (an inhibitor of protein secretion), the cells secrete cholesterol, but little apolipoprotein E. After secretion, apolipoprotein E and cholesterol associate with high-density lipoprotein to form a particle that can deliver cholesterol to the liver by receptor-mediated endocytosis. We conclude that apolipoprotein E does not function to remove cholesterol from macrophages but rather to participate in "reverse cholesterol transport."

Regulation of plasma cholesterol by lipoprotein receptors.

The lipoprotein transport system holds the key to understanding the mechanisms by which genes, diet, and hormones interact to regulate the plasma cholesterol level in man. Crucial components of this system are lipoprotein receptors in the liver and extrahepatic tissues that mediate the uptake and degradation of cholesterol-carrying lipoproteins. The number of lipoprotein receptors, and hence the efficiency of disposal of plasma cholesterol, can be increased by cholesterol-lowering drugs. Regulation of lipoprotein receptors can be exploited pharmacologically in the therapy of hypercholesterolemia and atherosclerosis is man.

Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit.

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 fail-safe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues.

The LDL receptor gene: a mosaic of exons shared with different proteins.

The multifunctional nature of coated pit receptors predicts that these proteins will contain multiple domains. To establish the genetic basis for these domains (LDL) receptor. This gene is more than 45 kilobases in length and contains 18 exons, most of which correlate with functional domains previously defined at the protein level. Thirteen of the 18 exons encode protein sequences that are homologous to sequences in other proteins: five of these exons encode a sequence similar to one in the C9 component of complement; three exons encode a sequence similar to a repeat sequence in the precursor for epidermal growth factor (EGF) and in three proteins of the blood clotting system (factor IX, factor X, and protein C); and five other exons encode nonrepeated sequences that are shared only with the EGF precursor. The LDL receptor appears to be a mosaic protein built up of exons shared with different proteins, and it therefore belongs to several supergene families.

Lesch-Nyhan syndrome: rapid detection of heterozygotes by use of hair follicles.

A method is described which permits rapid phenotypic diagnosis of the Lesch-Nyhan heterozygote by direct assay of hypoxanthine guanine phosphori-bosyltransferase activity in single hair follicles obtained from the scalp.