Molecular genetics of human lipoprotein lipase deficiency

Lipoprotein lipase (LPL) hydrolysis the triglyceride core of circulating chylomicrons and very-low-density lipoprotein, and modulates the levels and lipid composition of low and high density lipoproteins. Worldwide, more than 20 mutations in the LPL gene have been identified in patients with familial LPL deficiency. Most of these mutations are clustered in the region encoded by exons 4, 5 and 6 which forms the proposed catalytic domain of LPL. In French Canadians who have the highest reported frequency for LPL deficiency, three common mutations in the LPL gene have been identified which account for approximately 97% of mutant genes in this group. Simple DNA-based tests for the detection of all these mutations have been developed for the screening for carriers of LPL deficiency. This will facilitate further studies of phenotypic expression in heterozygous carriers and assessment of the risk of atherosclerosis in these individuals.     

Glucocorticoid-related impairment in the metabolism of low density lipoprotein by human fibroblasts

The metabolism of radiolabelled 125I-low density lipoprotein (LDL) was studied in cultured human dermal fibroblasts to investigate potential mechanisms contributing to the accelerated development of cardiovascular disease in patients treated chronically with corticosteroids. Fibroblasts exposed for 48 hours to pooled lipoprotein-poor (d greater than 1.25 gm/ml) serum from glucocorticoid-treated patients showed an increased capacity to bind LDL (p less than .001) compared to cells incubated under identical conditions with pooled serum from controls. In addition, a significantly (p less than .001) reduced amount of soluble radioactive material appeared in the media indicating that exposure of fibroblasts to corticosteroid serum also impaired their capacity to degrade LDL. If this tendency of cultured cells to accumulate cholesterol-rich lipoprotein when exposed to constituents of serum from these patients is present in patients receiving long-term treatment with glucocorticoids, it might influence arterial lipid accumulation and accelerate their developing cardiovascular disease.     

Elucidation of xenobiotic metabolism pathways in human skin and human skin models by proteomic profiling

Background

Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin.

Methodology/Principal Findings

Label-free proteomic analysis of whole human skin (10 donors) was applied and analysed using custom-built PROTSIFT software. The results showed the presence of enzymes with a capacity for the metabolism of alcohols through dehydrogenation, aldehydes through dehydrogenation and oxidation, amines through oxidation, carbonyls through reduction, epoxides and carboxylesters through hydrolysis and, of many compounds, by conjugation to glutathione. Whereas protein levels of these enzymes in skin were mostly just 4–10 fold lower than those in liver and sufficient to support metabolism, the levels of cytochrome P450 enzymes were at least 300-fold lower indicating they play no significant role. Four epidermal models of human skin had profiles very similar to one another and these overlapped substantially with that of whole skin.

Conclusions/Significance

The proteomics profiling approach was successful in producing a comprehensive analysis of the biotransformation characteristics of whole human skin and various in vitro skin models. The results show that skin contains a range of defined enzymes capable of metabolising different classes of chemicals. The degree of similarity of the profiles of the in vitro models indicates their suitability for epidermal toxicity testing. Overall, these results provide a rational basis for explaining the fate of xenobiotics in skin and will aid chemical safety testing programmes.     

Role of genetics and drug metabolism in human cancer risk

The research field concerning responses to drugs having a hereditary basis is called 'pharmacogenetics'. At least 5 dozen pharmacogenetic polymorphisms have been described in clinical medicine; many are responsible for marked differences in genetic predisposition toward toxicity or cancer. Three are detailed here: the acetylation, the debrisoquine, and the AH locus polymorphism. All 3 are very common among the United States' population: 1 in 2 is a 'slow acetylator', 1 in 12 is a 'poor metabolizer' for more than 2 dozen commonly prescribed drugs in the debrisoquine panel, and the CYP1A1 and CYP1A2 (cytochromes P(1)450 and P(3)450) genes are highly inducible by cigarette smoke in 1 of 10 patients. Differences in xenobiotic metabolism between individuals in the same family can be greater than 200-fold, suggesting that occupationally hazardous chemicals, as well as prescribed drugs having a narrow therapeutic window, might cause strikingly dissimilar effects between patients of differing genotypes. Our ultimate goal is 'preventive toxicology', i.e. the development of simple, inexpensive, unequivocal and sensitive assays to predict individual risk of toxicity or cancer. These tests could help the individual in choosing a safer life style or place of work and might aid the physician in deciding which drug to prescribe.     

A new era of human population genetics

A growing resource of methicillin-resistant Staphylococcus aureus (MRSA) genomes uncovers intriguing phylogeographic and recombination patterns and highlights challenges in identifying the source of these phenomena.     

Characterization of lipid and lipoprotein metabolism in primary human hepatocytes

Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model.     

Induction of growth-related metabolism in human vascular smooth muscle cells by low density lipoprotein

Human vascular smooth muscle cells (hVSMC) rendered quiescent by maintenance under serum-free culture conditions for 48 h exhibited several metabolic responses, normally associated with proliferation, following exposure to low density lipoprotein (LDL). LDL induced a time- and dose- (half-maximally effective concentration, ED50 25.0 +/- 8 nM) dependent activation of S6 kinase which could be negated following pretreatment of hVSMC with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h. In myo-[3H]inositol-prelabeled hVSMC, LDL caused a rapid (maximum within 1 min) decrease in phosphatidylinositol 4,5-bisphosphate (35% p less than 0.001) and phosphatidylinositol 4-phosphate (20%, p less than 0.01) with a return to prestimulated levels within 5-10 min. LDL induced a concomitant increase in [3H]inositol phosphates for which the order of generation was inositol-tris greater than -bis greater than -mono phosphate and which reached threshold levels of significance (p less than 0.05) above control values within 1, 2, and 10 min, respectively. The effect of LDL on hVSMC phosphoinositide metabolism was dose-dependent (half-maximally effective concentration, ED50 32.1 +/- 5.0 nM). This concentration, like that for S6 kinase, approximates with the KD (5-21 nM) for high affinity binding of 125I-LDL to specific receptors (1.5 x 10(4) sites/cell) on hVSMC. LDL induced a rapid but transient translocation of protein kinase C from the cytosol to membranes as assessed using both immunoblotting and [3H] 4-beta-phorbol-12-13-dibutyrate-binding procedures. Exposure of quiescent hVSMC to LDL elevated intracellular pH (delta pH 0.30 +/- 0.03, p less than 0.001). Such alkalinization was prevented in the presence of Na+/K+ exchange inhibitors such as amiloride, dimethylamiloride, and ethylisopropylamiloride. In an investigation of the nuclear action of LDL, a time-dependent induction of both c-myc and c-fos was observed. Such LDL-induced expression of these nuclear proto-onco-genes was not detectable in protein kinase C down-regulated hVSMC. Nevertheless, in spite of the cascade of "growth-promotional" responses elicited by LDL in quiescent hVSMC, this lipoprotein alone (under serum-free conditions) was neither mitogenic in nuclear labeling experiments, nor could it support growth of hVSMC in culture. We demonstrate that LDL might function in a complementary/synergistic fashion with other weakly mitogenic (to VSMC) growth factors and suggest that activation of protein kinase C (vis à vis intrinsic tyrosine kinase characteristic of other growth factor receptors) may be crucial to the signal transduction pathway for LDL.     

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III, and bradykinin (BK) and converted ANG I to the active metabolite ANG(1–7). ANG III was converted to the novel ANG IV metabolite [des-Arg¹]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.     

Lipid peroxidation of low density lipoprotein by human endothelial cells modifies its metabolism in vitro

Low density lipoprotein (LDL) undergoes qualitative changes when incubated with endothelial cells. Changes in LDL induced by cultured human endothelial cells were associated with release of substances reacting to thiobarbituric acid; they were prevented by addition of EDTA. Modification of LDL by human endothelium, therefore, appears to involve lipid peroxidation. Proneness of LDL to this process was indicated by its occurrence, to a smaller extent, on incubation in the absence of endothelium. Lipid peroxidation of LDL altered its electrophoretic mobility. Modified LDL, but not native LDL, was readily catabolised by human macrophages. Conditioning by human endothelium increased the rate of fractional catabolism of LDL in rabbits. If lipid peroxidation of LDL takes place in vivo it may promote conversion of macrophages to lipid-laden foam cells in the developing atheromatous plaque.     

Effect of ethanol on lipoprotein metabolism in human and animal blood

The amount and composition of human and animal blood plasma lipoproteins have been studied as affected by low temperature and high doses of ethanol. Results are presented. A conclusion about antiatherogenic properties of low doses of ethanol based on the data of its effect on the system of blood lipoproteins cannot be considered completely proved. Reliable interpretation of the results of the ethanol effect on lipoproteins and atherogenesis processes needs the further study of mechanisms of the ethanol effect on the exchange and modification of the basic classes of lipoproteins and their subfractions, many their peculiarities being not studied yet.     

Functional expression of human and mouse low density lipoprotein receptors in hybridomas

Though a mouse.human-human heterohybridoma, N12-16.63, secreting an antitetanus toxoid human monoclonal antibody grew well in a serum-free medium, its high producing subclone N12-69 required SSGF-I, a low density lipoprotein (LDL) from swine serum, or human-LDL (h-LDL) for growth. The growth-promoting action of SSGF-I was caused by its lipid fraction, and SSGF-I could be replaced completely with cholesterol in the presence of bovine serum albumin (BSA). Thus, cell line N12-69 is a cholesterol auxotroph of the heterohybridoma. N12-69 cells express both mouse and human LDL receptors on the cell surface in a ratio of 1:4. SSGF-I bound to both receptors with the same binding affinity, and h-LDL was also take up by the same receptors, though the affinity constant of the receptors for SSGF-I was 1.5 times stronger than that for h-LDL. The growth of N12-69 cells was completely inhibited by the addition of dextran sulfate, which is known to inhibit the binding of LDL to LDL receptors, to an SSGF-I or h-LDL containing medium but was not inhibited at all when dextran sulfate was added to a serum-free medium supplemented with cholesterol and BSA. Furthermore, an anti-human LDL receptor monoclonal antibody partially inhibited the growth of N12-69 cells in an SSGF-I or h-LDL containing medium. These findings suggest that N12-69 cells express both biologically active mouse and human LDL receptors on their cell surfaces and that SSGF-I or h-LDL is taken up by the both receptors to be utilized as a cholesterol source for the growth.     

Molecular genetics of biotin metabolism: old vitamin, new science

Biotin is a water-soluble vitamin that participates as a cofactor in gluconeogenesis, fatty acid synthesis and branched chain amino acid catabolism. It functions as the carboxyl carrier for biotin-dependent carboxylases. Its covalent attachment to carboxylases is catalyzed by holocarboxylase synthetase. Our interest in biotin has been through the genetic disease, biotin-responsive multiple carboxylase deficiency, caused by deficient activity of holocarboxylase synthetase. As part of these studies, we made the unexpected findings that the enzyme also targets to the nucleus and that it catalyzes the attachment of biotin to histones. We found that patients with holocarboxylase synthetase deficiency have a much reduced level of biotinylated histones, yet the importance of this process is unknown. The dual nature of biotin, as the carboxyl-carrier cofactor of carboxylases and as a ligand of unknown function attached to histones, is an enigma that suggests a much more involved role for biotin than anticipated. It may change our outlook on the optimal nutritional intake of biotin and its importance in biological processes such as development, cellular homeostasis and regulation.     

Cellular metabolism of human plasma intermediate-density lipoprotein (IDL)

The cellular metabolism of human plasma intermediate-density lipoprotein (IDL) was investigated in cultured human skin fibroblasts and hepG-2 cell in the absence and presence of exogenous recombinant or plasmatic apo E-3. IDL (d 1.006-1.019 g/ml) and LDL (d 1.019-1.063 g/ml) were prepared by centrifugation from the plasma of apo E-3/3 or 4/3 normolipidemic human subjects. Without added apo E-3, IDL binding and cell association are similar or slightly reduced while their degradation is one third to one half. This results in degradation to binding ratios for IDL that are half those for LDL. Exogenous apo E-3 enhances binding, association and degradation of IDL by 50-150%, but the degradation to binding ratio remains low. Exogenous apo E-3 also increased the ability of IDL but not LDL, to down-regulate the incorporation of [14C]acetate to sterol by the cells. The optimal concentration of apo E-3 is 4 micrograms protein/10 micrograms IDL protein and at that concentration appreciable amounts of the apo E are found associated with the lipoprotein. Apo E-2 has no effect on the cellular metabolism of IDL and apo E-3 is not effective in receptor-negative human fibroblasts. Monoclonal antibodies that block apo E binding to B,E (LDL) receptor (1D7) abolish the cellular metabolism of IDL while antibodies against B-100 (4G3) are ineffective. In competitive binding experiments, IDL is slightly more effective than LDL in displacing 125I-LDL from receptors in hepG-2 cells and appreciably more effective than LDL when tested against 125I-IDL. Apo E-3 increases the capacity of IDL to compete with either 125I-LDL or 125I-IDL. Addition of apo E-3 also increases the binding affinity of IDL to hepG-2 receptors, with Kd values of 2.50, 0.93 micrograms protein/ml, respectively. The study demonstrates the essential role that functional apo E molecules play in the interaction of human IDL with cellular receptors. Yet, in spite of presence of apo E in IDL (2-3 molecules/particle) and enrichment of IDL with apo E-3 (to 4-5 molecules/particle) the proteolytic degradation of the lipoprotein by specific cellular receptor is similar to LDL.     

Reconstruction of pathways associated with amino acid metabolism in human mitochondria

We have used a bioinformatics approach for the identification and reconstruction of metabolic pathways associated with amino acid metabolism in human mitochondria. Human mitochondrial proteins determined by experimental and computational methods have been superposed on the reference pathways from the KEGG database to identify mitochondrial pathways. Enzymes at the entry and exit points for each reconstructed pathway were identified, and mitochondrial solute carrier proteins were determined where applicable. Intermediate enzymes in the mitochondrial pathways were identified based on the annotations available from public databases, evidence in current literature, or our MITOPRED program, which predicts the mitochondrial localization of proteins. Through integration of the data derived from experimental, bibliographical, and computational sources, we reconstructed the amino acid metabolic pathways in human mitochondria, which could help better understand the mitochondrial metabolism and its role in human health.     

Effects of phenothiazines on low density lipoprotein metabolism in cultured human fibroblasts

Treatment of cultured human fibroblasts with trifluoperazine or chlorpromazine resulted in a biphasic effect on low density lipoprotein (LDL) catabolism, depending upon the dose. At up to 10^?5 M, a marked increase in LDL binding, internalization and degradation was observed. This phenomenon took place within the first hours of incubation with the drugs, suggesting a direct effect on cell membrane physical characteristics, probably related to the lipophilic properties of phenothiazines. Concentrations above 2 × 10^?5 M resulted in a relative decrease in LDL binding and internalization, and in a dramatic decrease in LDL degradation, which may be related to an inhibition of calmodulin-dependent processes.     

Population genetics: A new apportionment of human diversity

A worldwide survey of polymorphic molecular markers shows that the human population is genetically homogeneous, in close agreement with evidence from quite different genes and traits.