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Biomedical subjects

R E Gregg

Publications and source records attributed to R E Gregg.

At least 19 recordsLinked to original sources
PubMed1996

Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step.

The microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of the lipoproteins containing apolipoprotein B (apoB): very low density lipoproteins and chylomicrons. Evidence indicates that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poor primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles. To determine if either step is mediated by MTP, a series of clonal cell lines stably expressing apoB-53 and MTP was established in non-lipoprotein-producing HeLa cells. MTP activity in these cells was approximately 30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis. Despite having robust levels of triglyceride and phospholipid synthesis, these cell lines, as exemplified by HLMB53-59, secreted >90% of the apoB-53 on relatively lipid-poor particles in the density range of 1.063-1.21 g/ml. These results suggested that coexpression of MTP and apoB only reconstituted the first but not the second step in lipoprotein assembly. To extend this observation, additional studies were carried out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined. Treatment of these cells with the MTP photoaffinity inhibitor BMS-192951 before pulse labeling with [35S]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly. After a 30-min [35S]methioneine/cysteine pulse labeling and 120 min of chase, all of the nascent apoB-48 was observed to have a density of high density lipoproteins (1.063-1.21 g/ml), indicating that only the first step of lipoprotein assembly had occurred. The addition of oleic acid to the cell culture media activated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period. Inactivation of MTP after completion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion. Thus, inhibition of MTP did not hinder the addition of bulk core lipid to the primordial lipoprotein precursor particles, indicating that MTP is not required for the second step of apoB-48 lipoprotein assembly.

Animals↗
PubMed1996

An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

Animals↗
PubMed1995

A 30-amino acid truncation of the microsomal triglyceride transfer protein large subunit disrupts its interaction with protein disulfide-isomerase and causes abetalipoproteinemia.

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the multifunctional enzyme, protein disulfide-isomerase, and a unique large, 97 kDa, subunit. It is found as a soluble protein within the lumen of the endoplasmic reticulum of liver and intestine and is required for the assembly of very low density lipoproteins and chylomicrons. Mutations in MTP which result in an absence of MTP function have been shown to cause abetalipoproteinemia. Here, the gene encoding the MTP 97-kDa subunit of an abetalipoproteinemic subject, which we have previously demonstrated lacks MTP activity and protein (Wetterau, J. R., Aggerbeck, L. P., Bouma, M.-E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay, G., Rader, D. J., and Gregg, R. E. (1992) Science 258, 999-1001), was isolated and sequenced. A nonsense mutation, which predicts the truncation of the protein by 30 amino acids, was identified. To investigate if this apparently subtle change in MTP could explain the observed absence of MTP, protein disulfide-isomerase was co-expressed with either the normal or mutant MTP 97-kDa subunit in Sf9 insect cells using a baculovirus expression system. Although there were high levels of expression of both the normal and mutant forms of the MTP 97-kDa subunit, only the normal subunit was able to form a stable, soluble complex with protein disulfide-isomerase. These results indicate that the carboxyl-terminal 30 amino acids of the MTP 97-kDa subunit plays an important role in its interaction with protein disulfide-isomerase.

Abetalipoproteinemia↗
PubMed1995

Microsomal triglyceride transfer protein. Specificity of lipid binding and transport.

Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein that is required for the assembly and secretion of very low density lipoproteins by the liver and chylomicrons by the intestine. To further elucidate the nature of the lipid molecule binding and transport site on MTP, we have studied the relative rates at which MTP transports different lipid species. Assay conditions were chosen in which there were minimal changes in the physical properties of the substrate membranes so that transfer rates would reflect MTP-lipid interactions at a membrane surface. Lipid transport rates decreased in order of triglyceride > cholesteryl ester > diglyceride > cholesterol > phosphatidylcholine. Changes in the hydrophobic nature of a lipid molecule by the addition of a fatty acid, modulated the ability of MTP to transport it. Addition of one acyl chain from diglyceride to triglyceride, lysophosphatidylcholine to phosphatidylcholine, or cholesterol to cholesteryl ester increased the rate of MTP-mediated transport 10-fold. In contrast, the lipid transport rate was insensitive to the changes in the structure or charge of the polar head group on phospholipid substrates. Zwitterionic, net negative, or net positive charged phospholipid molecules were all transported at a comparable rate. The ability of MTP to transport lipids is strongly correlated to the binding of these lipids to MTP. Thus, MTP has a specific preference for binding and transporting nonpolar lipid compared with phospholipids, and within a class of lipid molecules, a decrease in polarity increases its tendency to be transported.

Animals↗
PubMed1995

In vitro myotoxicity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, pravastatin, lovastatin, and simvastatin, using neonatal rat skeletal myocytes.

Pravastatin, lovastatin, and simvastatin, drugs which lower cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, have been linked to skeletal myopathies in humans and rats. The myotoxicity of these three drugs was compared, after 48 hr exposure, in cultures of primary neonatal rat skeletal myotubes. Measurements included HMG CoA reductase activity ([14C]acetate incorporation into cholesterol), indicators of membrane damage (CPK, LDH, and AST), cell viability (mitochondrial dehydrogenase metabolism of MTT), protein synthesis ([3H]leucine incorporation), and energy status (ATP). All three drugs inhibited cholesterol synthesis to the same extent in rat hepatocytes (IC50s approximately 0.07 microM). Lovastatin- and simvastatin-induced inhibition of cholesterol synthesis in myotubes was unchanged compared to that of hepatocytes, but pravastatin was 85-fold less potent (IC50 = 5.9 microM). Protein synthesis and ATP levels were the most sensitive indicators of toxicity. Pravastatin (IC50 = 759 microM) was > 100-fold less inhibitory of protein synthesis than lovastatin (IC50 = 5.4 microM) or simvastatin (IC50 = 1.9 microM). Addition of mevalonic acid (the immediate product of the HMG CoA reductase reaction), as 100 microM mevalonic acid lactone, reversed the toxicity of all three drugs. Removal of serum for 24-72 hr did not alter the toxicity of any of the drugs compared to cultures containing 10% serum, suggesting that differences in protein binding did not account for the differences in toxicity of the drugs. These results indicate that pravastatin is less myotoxic than lovastatin or simvastatin in this in vitro system using neonatal rat skeletal muscle cells, and this differential toxicity is correlated with the selective decrease in inhibition of HMG CoA reductase by pravastatin in nonhepatic tissues.

Animals↗
PubMed1995

Dominant expression of type III hyperlipoproteinemia. Pathophysiological insights derived from the structural and kinetic characteristics of ApoE-1 (Lys146-->Glu).

Type III hyperlipoproteinemia is characterized by delayed chylomicron and VLDL remnant catabolism and is associated with homozygosity for the apoE-2 allele. We have identified a kindred in which heterozygosity for an apoE mutant, apoE-1 (Lys146-->Glu), is dominantly associated with the expression of type III hyperlipoproteinemia. DNA sequence analysis of the mutant apoE gene revealed a single-point mutation that resulted in the substitution of glutamic acid (GAG) for lysine (AAG) at residue 146 in the proposed receptor-binding domain of apoE. The pathophysiological effect of this mutation was investigated in vivo by kinetic studies in the patient and six normal subjects, and in vitro by binding studies of apoE-1 (Lys146-->Glu) to LDL receptors on human fibroblasts and to heparin. The kinetic studies revealed that apoE-1 (Lys146-->Glu) was catabolized significantly slower than apoE-3 in normals (P < 0.005). In the proband, the plasma residence times of both apoEs were substantially longer and the production rate of total apoE was about two times higher than in the control subjects. ApoE-1 (Lys146-->Glu) was defective in interacting with LDL receptors, and its ability to displace LDL in an in vitro assay was reduced to 7.7% compared with apoE-3. The affinity of apoE-1 (Lys146-->Glu) to heparin was also markedly reduced compared with both apoE-2 (Arg158-->Cys) and apoE-3. These abnormal in vitro binding characteristics and the altered in vivo metabolism of apoE-1 (Lys146-->Glu) are proposed to result in the functional dominance of this mutation in the affected kindred.

Adult↗
PubMed1994

Secretion of apolipoprotein B-containing lipoproteins from HeLa cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability.

To elucidate the role of the microsomal triglyceride transfer protein (MTP) in lipoprotein assembly, MTP and apolipoprotein B-53 (apoB 53; the N-terminal 53% of apoB) were expressed in HeLa cells. The results showed that apoB-53 could be expressed in HeLa cells with or without expression of MTP. In contrast, efficient secretion of apoB-53 required expression of MTP. Ultracentrifugal density flotation analysis showed that apoB-53 was secreted predominantly as a particle with the density of high density lipoprotein. An essentially identical apoB-53 particle density distribution was obtained after transient expression of apoB-53 in McArdle RH-7777 rat hepatoma cells. The mass of apoB-53 secreted was greater, and the flotation density was lower, from cells fed lipid, suggesting that apoB secretion in HeLa cells was regulated by lipid availability, similar to what has been described for lipoprotein-producing cell lines. These results indicate that MTP is necessary and sufficient to direct the regulated secretion of apoB-53 in HeLa cells.

Animals↗
PubMed1994

The molecular basis of abetalipoproteinemia.

Abetalipoproteinemia is a recessive genetic disease in humans characterized by the virtual absence of apolipoprotein (apo)B and apoB-containing lipoproteins in plasma. Microsomal triglyceride transfer protein (MTP), a resident lipid transfer protein within the endoplasmic reticulum of hepatocytes and enterocytes, has been shown to be absent in enterocytes from subjects with this disease. MTP is a heterodimer of a unique large subunit and protein disulfide isomerase. It has been demonstrated that the absence of MTP in abetalipoproteinemia is secondary to mutations in the gene for the large subunit of MTP. Thus, mutations in the gene for the large subunit of MTP are a cause of abetalipoproteinemia, which indicates that the MTP is a necessary component for the assembly and secretion of apoB-containing lipoproteins from the liver and intestine.

Abetalipoproteinemia↗
PubMed1993

In vivo metabolism of a mutant form of apolipoprotein A-I, apo A-IMilano, associated with familial hypoalphalipoproteinemia.

Apo A-IMilano is a mutant form of apo A-I in which cysteine is substituted for arginine at amino acid 173. Subjects with apo A-IMilano are characterized by having low levels of plasma HDL cholesterol and apo A-I. To determine the kinetic etiology of the decreased plasma levels of the apo A-I in these individuals, normal and mutant apo A-I were isolated, radiolabeled with either 125I or 131I, and both types of apo A-I were simultaneously injected into two normal control subjects and two subjects heterozygous for apo A-IMilano. In the normal subjects, apo A-IMilano was catabolized more rapidly than the normal apo A-I (mean residence times of 5.11 d for normal apo A-I vs. 3.91 d for apo A-IMilano), clearly establishing that apo A-IMilano is kinetically abnormal and that it has a shortened residence time in plasma. In the two apo A-IMilano subjects, both types of apo A-I were catabolized more rapidly than normal (residence times ranging from 2.63 to 3.70 d) with normal total apo A-I production rates (mean of 10.3 vs. 10.4 mg/kg per d in the normal subjects). Therefore, in the subjects with apo A-IMilano, the decreased apo A-I levels are caused by rapid catabolism of apo A-I and not to a decreased production rate, and the abnormal apo A-IMilano leads to the rapid catabolism of both the normal and mutant forms of apo A-I in the affected subjects.

Adult↗
PubMed1992

Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.

Abetalipoproteinemia is a human genetic disease that is characterized by a defect in the assembly or secretion of plasma very low density lipoproteins and chylomicrons. The microsomal triglyceride transfer protein (MTP), which is located in the lumen of microsomes isolated from the liver and intestine, has been proposed to function in lipoprotein assembly. MTP activity and the 88-kilodalton component of MTP were present in intestinal biopsy samples from eight control individuals but were absent in four abetalipoproteinemic subjects. This finding suggests that a defect in MTP is the basis for abetalipoproteinemia and that MTP is indeed required for lipoprotein assembly.

Abetalipoproteinemia↗
PubMed1992

In vivo metabolism of apolipoprotein A-I in a patient with homozygous familial hypercholesterolemia.

Familial hypercholesterolemia (FH), caused by a defect in the low density lipoprotein (LDL) receptor, results in high plasma concentrations of LDL cholesterol due to both overproduction and delayed catabolism of LDL. FH is also associated with significantly lower levels of plasma high density lipoprotein cholesterol and apolipoprotein (apo) A-I in both heterozygous and homozygous patients. However, the metabolic basis of the hypoalphalipoproteinemia in FH has not been elucidated. We investigated the kinetics of apo A-I in a homozygous FH patient and two normal control subjects by using endogenous labeling with a stable isotopically labeled amino acid. Study subjects were administered a primed constant infusion of 13C6-phenylalanine for 12 hours. Apolipoproteins were isolated from plasma drawn at selected time points and analyzed for their isotopic enrichment by gas chromatography-mass spectrometry. The fractional catabolic rate of apo A-I in the FH subject was found to be substantially increased (0.38 day-1) compared with that of the normal subjects (mean, 0.26 day-1). In addition, the apo A-I production rate was decreased in the FH subject (6.5 mg/kg.day-1) compared with the normal subjects (mean, 11.1 mg/kg.day-1). In conclusion, the low levels of high density lipoprotein cholesterol and apo A-I in this homozygous FH patient are due to the combined metabolic defects of increased apo A-I catabolism and decreased apo A-I production.

Apolipoprotein A-I↗
PubMed1992

Clinical, pathologic, and biochemical features of a cholesterol lipidosis accompanied by hyperlipidemia and xanthomas.

We describe the unique clinical and histopathologic features of a child with biochemical and immunocytochemical features of Niemann-Pick disease type C (NPC). Clinically, she was found to have multiple xanthomas of the upper aerodigestive tract with dysphagia and expressive language delay, splenomegaly, bony infarcts, and type IIb hyperlipidemia. Neurologic examination was otherwise normal. Microscopy revealed foam cells in her bone marrow, liver, tongue, tonsils, glottis, and in normal-appearing peritonsillar mucosa. Lipid analysis of a liver biopsy specimen showed a small increase in phospholipids, a twofold increase in sphingomyelin, a fivefold increase in cholesterol, and a marked (25-fold) increase in bis(monoacylglycerol) phosphate. Lysosomal acid hydrolase activities in cultured skin fibroblasts were nondiagnostic. Biochemical and immunocytochemical studies of cultured fibroblasts demonstrated lysosomal accumulation of unesterified LDL-derived cholesterol as well as delayed induction of homeostatic responses to endogenous cholesterol consistent with a diagnosis of NPC. Based upon these observations, we speculate that this patient could have a new phenotypic expression of NPC or represents a new cholesterol lipidosis biochemically resembling NPC. The chance occurrence of two separate lipid disorders seems less likely.

Biopsy↗
PubMed1992

Inhibition of fatty acid synthesis decreases very low density lipoprotein secretion in the hamster.

The hamster was developed as a model to study very low density lipoprotein (VLDL) metabolism, since, as is the case in humans, the hamster liver was found to synthesize apoB-100 and not apoB-48. The effect of inhibiting fatty acid synthesis on the hepatic secretion of VLDL triglyceride (TG) and apolipoprotein (apo) B-100 in this model was then investigated. In an in vivo study, hamsters were fed a chow diet containing 0.15% TOFA (5-tetradecyloxy-2-furancarboxylic acid), an inhibitor of acetyl-CoA carboxylase. After 6 days of treatment, plasma triglyceride and cholesterol levels were decreased by 30.2% and 11.6%, respectively. When the secretion of VLDL-TG by the liver was measured in vivo after injection of Triton WR 1339, TOFA treatment was found to decrease VLDL-TG secretion by 40%. In subsequent in vitro studies utilizing cultured primary hamster hepatocytes, incubation with 20 microM TOFA for 4 h resulted in 98% and 76% inhibition in fatty acid and triglyceride synthesis, respectively; VLDL-TG secretion was decreased by 90%. When hepatocytes were pulsed with [3H]leucine, incubation with TOFA resulted in a 50% decrease in the incorporation of radiolabel into secreted VLDL apoB-100. The results of this study indicate that inhibition of intracellular triglyceride synthesis decreases the secretion of VLDL-TG and apoB-100, and does not result in the secretion of a dense, triglyceride-depleted lipoprotein.

Animals↗
PubMed1992

In vivo metabolism of a mutant apolipoprotein, apoA-IIowa, associated with hypoalphalipoproteinemia and hereditary systemic amyloidosis.

Apolipoprotein (apo) A-I is the major protein constituent of plasma high density lipoproteins (HDL). A kindred has been identified in which a glycine to arginine mutation at residue 26 in apoA-I is associated with hypoalphalipoproteinemia and hereditary systemic amyloidosis. We isolated the mutant protein, termed apoA-IIowa, from the plasma of an affected subject and studied its in vivo metabolism compared to that of normal apoA-I in two heterozygous apoA-IIowa subjects and two normal controls. Normal and mutant apoA-I were radioiodinated with 131I and 125I, respectively, reassociated with autologous plasma lipoproteins, and simultaneously injected into all subjects. Kinetic analysis of the plasma radioactivity curves demonstrated that the mutant apoA-IIowa was rapidly cleared from plasma (mean fractional catabolic rate [FCR] 0.559 day-1) compared with normal apoA-I (mean FCR 0.244 day-1) in all four subjects. The FCR of normal apoA-I was also substantially faster in the heterozygous apoA-IIowa subjects (mean FCR 0.281 days-1) than in the normal controls (mean FCR 0.203 days-1). Despite the rapid removal from plasma of apoA-IIowa, the cumulative urinary excretion of its associated radioactivity after 2 weeks (44%) of the injected dose) was substantially less than that associated with normal apoA-I (78% of injected dose), indicating extravascular sequestration of radiolabeled apoA-IIowa.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Ultrasound guidance of laser atherectomy.

Intravascular ultrasound imaging catheters and forward firing laser atherectomy systems are both being used in the coronary vascular tree of man. When used in conjunction, ultrasound imaging may reduce the probability of perforation and dissection related to laser atherectomy. A novel system is presented which combines ultrasound imaging and laser atherectomy capabilities on the same catheter. The system incorporates a holmium YAG laser directed perpendicular to the long axis of the catheter along with a co-directional ultrasound sensor. Ultrasound images are assembled by computer using the angle of the catheter tip and its linear advance associated with each ultrasound echo signal. In vivo experimental results are presented.

Angioplasty, Laser↗
PubMed1991

Severe hyperlipidemia due to multiple factors in a child with nephrotic syndrome.

An unusual case of severe hypercholesterolemia and hypertriglyceridemia is described in a child with nephrotic syndrome. The severe hyperlipidemia in this patient was most likely induced by multiple interacting factors which included the metabolic abnormalities of nephrotic syndrome, steroid therapy, the underlying genetic predisposition of ApoE-2 homozygosity as well as diet and diuretic therapy. The result of these factors was an extremely severe type III hyperlipoproteinemia. The pathogenesis of hyperlipidemia in this setting is discussed.

Apolipoproteins E↗
PubMed1991

Isolation and characterization of an apoA-II-containing lipoprotein (LP-A-II:B complex) from plasma very low density lipoproteins of patients with Tangier disease and type V hyperlipoproteinemia.

Previous studies have shown that very low density lipoproteins (VLDL) from patients with Tangier disease are less effective as a substrate for human milk lipoprotein lipase (LPL) than VLDL from normal controls as assessed by measuring the first order rate constant (k1) of triglyceride hydrolysis. Tangier VLDL also has a higher content of apolipoprotein (apo) A-II than normal VLDL. To explore the possible relationship between the relatively high concentration of apoA-II in VLDL and low k1 values, Tangier VLDL were fractionated on an anti-apoA-II immunosorber. The retained fraction contained a newly identified triglyceride-rich lipoprotein characterized by the presence of apolipoproteins A-II, B, C-I, C-II, C-III, D, and E (LP-A-II:B:C:D:E or LP-A-II:B complex), whereas the unretained fraction consisted of previously identified triglyceride-rich apoB-containing lipoproteins free of apoA-II. In VLDL from patients with Tangier disease or type V hyperlipoproteinemia, the LP-A-II:B complex accounted for 70-90% and 25-70% of the total apoB content, respectively. The LP-A-II:B complexes had similar lipid and apolipoprotein composition; they were poor substrates for LPL as indicated by their low k1 values (0.014-0.016 min-1). In contrast, the apoA-II-free lipoproteins present in unretained fractions were effective substrates for LPL with k1 values equal to or greater than 0.0313 min-1. These results indicate that triglyceride-rich lipoproteins consist of several apoB-containing lipoproteins, including the LP-A-II:B complex, and that lipoprotein particles of similar size and density but distinct apolipoprotein composition also possess distinct metabolic properties.

Adult↗