Diabetes and gout: efficacy and safety of febuxostat and allopurinol.

AIM: Assess influences of demographics and co-morbidities of gout patients with or without diabetes on safety and efficacy of urate-lowering agents. METHODS: Post-hoc analysis of 312 diabetic and 1957 non-diabetic gout patients [baseline serum urate levels (sUA) ≥8.0 mg/dl] enrolled in a 6-month randomized controlled trial comparing urate-lowering efficacy (ULE) and safety of daily xanthine oxidase inhibitors (XOIs) febuxostat (40 mg or 80 mg) and allopurinol (200 mg or 300 mg). We compared baseline demographic, gout and co-morbid characteristics, ULE, and safety of XOI treatment in diabetic and non-diabetic gout patients. ULE was measured by the proportion of diabetic and non-diabetic patients in each treatment group achieving final visit sUA < 6.0 mg/dl. Safety was monitored throughout the trial. RESULTS: Diabetic gout patients were older, more frequently female, and had longer gout duration. Co-morbidities were more frequent among diabetic patients: cardiovascular disease; impaired renal function; hyperlipidemia; and obesity (body mass index >30 kg/m²) (p < 0.001 for all comparisons). Febuxostat 80 mg ULE exceeded that of febuxostat 40 mg or allopurinol (p < 0.050) at all levels of renal function, achieving sUA goal range in the majority of diabetic and non-diabetic patients. Diabetics and non-diabetics reported self-limiting diarrhoea and URIs as the most common adverse events. CONCLUSIONS: Despite higher co-morbidity rates in diabetic patients, febuxostat and allopurinol were safe in both groups at the doses tested. Febuxostat 80 mg achieved sUA <6.0 mg/dl more often than febuxostat 40 mg or allopurinol at commonly prescribed doses.

Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1-mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1-mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression.

Long-term analgesic effects of inescapable shock and learned helplessness.

Although exposure to inescapable shocks induced analgesia in rats, the analgesia was not manifest 24 hours later. A brief reexposure to shock, however, restored the analgesia. This reexposure to shock had an analgesic effect only if the rats had been shocked 24 hours previously. Further, long-term analgesic effects depended on the controllability of the original shocks and not on shock exposure per se. Implications of these results for learned helplessness and stress-induced analgesia are discussed.

A genetic analysis of neural progenitor differentiation.

Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.

Synthesis of very low density lipoproteins in the cockerel. Effects of estrogen.

The effect of estrogen on the synthesis of plasma very low density lipoproteins (VLDL) in the cockerel was studied both in vivo and in vitro. Synthesis was studied by immunoprecipitation techniques with antisera prepared against VLDL and a major VLDL protein. VLDL were isolated from the plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels by ultracentrifugal flotation at d 1.006 g/ml. After delipidation, the lipid-free proteins (apoproteins) were fractionated on Sephadex G-150 and DEAE-cellulose. Both the hen and the estrogen-treated cockerel VLDL were shown to contain an identical apoprotein with a mol wt of approximately 12,000; the apoprotein is designated fraction B. Reduction and S-carboxy-methylation of fraction B resulted in a reduction of the molecular weight by approximately one-half, indicating a dimer-monomer relationship. Antiserum prepared to the hen VLDL dimer protein gave precipitin lines of complete identity to both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density lipoproteins, and plasma; no precipitin line was formed with either hen or cockerel high density lipoproteins. After a single subcutaneous injection of diethylstilbestrol into the cockerel, plasma VLDL protein, cholesterol, and triglyceride increased, reaching a maximum 24--48 h after hormone administration. Liver slices from similarly treated animals were incubated in vitro in culture medium in the presence of [3H]lysine for 2 h. Immunoprecipitable radioactivity in VLDL increased within 2 h of diethylstilbestrol treatment and reached a maximum at 24 h; VLDL radioactivity returned to base-line levels by 72 h. At the peak of induction, newly synthesized VLDL represented 11% of the total soluble protein synthesized. When actinomycin-D (5 mg/kg) was administered simultaneously with estrogen, the induction of VLDL synthesis was totally inhibited. To determine whether the effect of estrogen on VLDL synthesis was mediated at the level of transcription, partially-purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA activity for fraction B was quantitated in a wheat germ translation system. Fraction B mRNA was found to increase from a low base-line value to a maximum 16-24 h after estrogen treatment, returning towards baseline values at 30 h. At the peak of induction, fraction B constituted 12% of the total protein synthesized. The kinetics of induction of fraction B mRNA activity in the cell-free translation system is very similar to that observed in liver slice experiments. This finding suggests that estrogen stimulates VLDL synthesis, at least partially, by enhancing the accumulation of the mRNA for one of their major apoproteins.

PIP: In vivo and in vitro studies were undertaken to investigate the mechanism of the induction of the synthesis of very low density lipoproteins (VLDL) by estrogens in the cockerel. VLCL were isolated from plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels. VLCL from both these groups contained an identical apoprotein (Fraction B) with a molecular weight of about 12,000. Reduction and S-carboxy-methylation of this fraction reduced its molecular weight by approximately 50%, thus indicating a dimer-monomer relationship. When antiserum was prepared against the hen VLDL dimer protein, completely identical precipitin lines were found for both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density proteins, and plasma. However, no precipitin line was formed with hen and cockerel high density lipoptoteins. A single sc injection of diethylstilbestrol (DES) into the cockerel increased levels of plasma VLDL protein, cholesterol, and triglyceride, with maximum values occurring 24-48 hours after injection. Immunoprecipitation of liver slices from similarly treated animals showed an increase of radioactivity of VLDL within 2 hours of injection. Values reached a maximum at 24 hours and returned to baseline levles by 72 hours. Newly synthesized VLDL comprised 11% of the total soluble protein synthesized during the period of peak values. Actinomycin-D (5 mg/kg), when administered simultaneously with the estrogen, completely inhibited the induction of VLDL synthesis. In another experiment, partially purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA acitivity for Fraction B was measured in a wheat germ translation system. Values for Fraction B mRNA reached a maximum 16-24 hours after estrogen-treatment and returned to baseline levels by 30 hours. Fraction B represented 12% of the total protein synthesized at the peak of induction. The results suggest that estrogen stimulates the synthesis of VLDL by enhancing the accumulationg of the mRNA of 1 of their major components.

Control of 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured human fibroblasts by very low density lipoproteins of subjects with hypertriglyceridemia.

Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from human normolipemic plasma, and the VLDL, the intermediate density lipoprotein (IDL), and LDL from patients with Type III hyperlipoproteinemic plasma were tested for their abilities to suppress the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase in cultured human fibroblasts from normal subjects and a Type III patient. Regulation of cholesterol synthesis in the fibroblasts of a patient with Type III hyperlipoproteinemia appears to be normal. VLDL from normal subjects, isolated by angle head ultracentrifugation (d < 1.006) or by gel filtration on BioGel A-5m, were about 5 times less effective than LDL in suppressing HMG-CoA reductase activity, based on protein content, in agreement with previous reports with normal fibroblasts. Zonal centrifugation of normal VLDL isolated by both methods showed that the VLDL contained IDL. Normal VLDL from the angle head rotor, refractionated by the zonal method, had little, if any, ability to suppress the HMG-CoA reductase activity in either normal or Type III fibroblasts. VLDL, IDL, and LDL fractionated by zonal ultracentrifugation from Type III plasma gave half-maximum inhibition at 0.2-0.5 mug of protein/ml, indistinguishable from the suppression caused by normal LDL. Type III VLDL did not suppress HMG-CoA reductase in mutant LDL receptor-negative fibroblasts. Zonally isolated VLDL obtained from one Type IV and one Type V patient gave half-maximal suppression at 5 and 0.5 mug of protein/ml, respectively. Molecular diameters and apoprotein compositions of the zonally isolated normal and Type III VLDL were similar; the major difference in composition was that Type III VLDL contained more cholesteryl esters and less triglyceride than did normal VLDL. The compositions and diameters of the Type IV and Type V VLDL were similar to normal VLDL. These findings show that the basic defect in Type III hyperlipoproteinemia is qualitatively different from the cellular defect found in familial hypercholesterolemia, since the regulation of HMG-CoA reductase activity is normal in Type III fibroblasts. The metabolic defect in hypertriglyceridemia is related to the triglyceriderich lipoproteins which, free of other lipoproteins, have an enhanced ability to interact with cultured fibroblasts to regulate HMG-CoA reductase activity. These studies suggest that, in hypertriglyceridemia, there is a mechanism for direct cellular catabolism of VLDL which is not functional for normal VLDL.

Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine.

Despite the use of hearing protection devices (HPDs) and engineering changes designed to improve workspaces, noise-induced hearing loss continues to be one of the most common and expensive disabilities in the US military. Many service members suffer acoustic trauma due to improper use of HPDs, sound levels exceeding the protective capacity of the HPDs, or by unexpected, injurious exposures. In these cases, there is no definitive treatment for the hearing loss. This study investigated the use of the pharmacological agents N-acetylcysteine and acetyl-L-carnitine after acoustic trauma to treat cochlear injury. N-Acetylcysteine is an antioxidant and acetyl-L-carnitine a compound that maintains mitochondrial bio-energy and integrity. N-Acetylcysteine and acetyl-L-carnitine, respectively, significantly reduced permanent threshold shifts and hair cell loss compared to saline-treated animals when given 1 and 4 h post-noise exposure. It may be possible to obtain a greater therapeutic effect using these agents in combination or at higher doses or for a longer period of time to address the secondary oxidative events occurring 7-10 days after acute noise exposure.

A Multinational Analysis of Mutations and Heterogeneity in PZase, RpsA, and PanD Associated with Pyrazinamide Resistance in M/XDR Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne's assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8-10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance.

Interaction of tryptic peptides of apolipoprotein B-100 with dimyristoylphosphatidylcholine.

Apolipoprotein B-100, the major protein constituent of human plasma low-density lipoproteins (LDL), was carboxyamidomethylated, digested with trypsin and the water-soluble tryptic peptides were coincubated with liposomes of dimyristoylphosphatidylcholine (DMPC). At 24.3 degrees C the peptides induced lipid solubilization as evidenced by optical clearing of the lipid-peptide mixture. Lipid-peptide complexes were isolated by density-gradient ultracentrifugation in KBr and had the following properties: DMPC/peptide ratio of 5.6 (w/w); buoyant density of 1.07-1.09 g/ml; discoidal morphology (51 +/- 4 X 260 +/- 28 A) as determined by electron microscopy; and molecular weight of 1.5 X 10(6) as determined by nondenaturing polyacrylamide gel electrophoresis. Compared to liposomes and sonicated vesicles of DMPC, the lipid-peptide complexes had a more rigid structure as assessed by fluorescence polarization. Whereas intact LDL had 42% alpha-helix and 15% beta-pleated sheet, the lipid-peptide complexes contained 70% alpha-helix and less than 5% beta-pleated sheet. The lipid-peptide complexes did not bind to the fibroblast high-affinity LDL receptor. These results show that specific regions in apolipoprotein B-100 which interact with phospholipid have an amphipathic character and may represent primary sites for lipid-protein interaction in LDL.

Exchange of phospholipids between unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and plasma very low density lipoproteins.

Purified phosphatidylcholine exchange protein from bovine liver was used to exchange [14C]dipalmitoyl phosphatidylcholine from sonicated vesicles to human plasma very low density lipoproteins (VLDL). The exchange of [14C]-dipalmitoyl phosphatidylcholine for VLDL phospholipids was temperature dependent and linear with respect to time and amount of exchange protein. In the absence of the exchange protein, less than 10% of the [14C]dipalmitoyl phosphatidylcholine was transferred. At an initial weight ratio of [14C]-dipalmitoyl phosphatidylcholine vesicles to VLDL phospholipid (1.2 mg) of 2.2, the exchange protein (14 microgram) replaced 55% of the VLDL phospholipids with [14C]dipalmitoyl phosphatidylcholine in 15 min; VLDL protein and cholesterol content were unaltered. From these studies we conclude that the exchange protein is a useful method to alter the phospholipid composition of VLDL under conditions such that there is minimal perturbation of the lipoprotein.

Lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine. Effect of lipid organization and apolipoprotein C-II on enzyme activity.

The effect of phospholipid organization on the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine was examined with sonicated vesicles and Triton X-100 or lysomyristoylphosphatidylcholine solubilized lipid. Triton X-100-dimyristoylphosphatidylcholine substrates were prepared at various ratios of detergent to phospholipid so as to produce lipid structures varying from bilayers to micelles. Apolipoprotein C-II, the activator protein for lipoprotein lipase, enhanced the rate of the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine for each substrate tested. Although the absolute rate of lipoprotein lipase catalysis was different for each, the factor (the ratio of lipoprotein lipase activity with apolipoprotein C-II to that without the activator protein) was nearly constant, with a value of approximately 16. We conclude that the enhancement of lipoprotein lipase activity by apolipoprotein C-II is independent of the physical form of the phospholipid substrate.

Interaction of lipoprotein lipase with phospholipid vesicles. Role of apolipoprotein C-II and heparin.

Lipoprotein lipase is bound to heparin-like molecules at the surface of capillary endothelial cells. For maximal activity, the enzyme requires apolipoprotein C-II, a protein constituent of triacylglycerol-rich lipoproteins. In this report, the interactions of apolipoprotein C-II, heparin and sonicated vesicles of dipalmitoylphosphatidylcholine with purified bovine milk lipoprotein lipase were studied by gel filtration on Bio-Gel A5m. In the presence of vesicles of dipalmitoylphosphatidylcholine (1 mg), lipoprotein lipase (25 micrograms) associated with phospholipids even in the absence of apolipoprotein C-II. With limited phospholipid (40 micrograms), the amount of enzyme which associated with lipid decreased in the presence of apolipoprotein C-II (20 micrograms). Human plasma apolipoprotein C-III, another protein constituent of triacylglycerol-rich lipoproteins, also caused a decrease in the amount of enzyme associated with phospholipid. These results suggest that apolipoprotein C-II does not increase the activity of the enzyme by facilitating its interaction with a lipid interface. In the absence of lipid, lipoprotein lipase and apolipoprotein C-II (molar ratio, 1 : 1) eluted from Bio-Gel A5m as two separate components. The interaction of heparin with lipoprotein lipase was studied using a specific [3H]heparin, which was isolated by affinity chromatography on immobilized lipoprotein lipase; the [3H]heparin eluted with 0.6 M NaCl. Specific [3H]heparin coeluted with lipoprotein lipase when the enzyme was associated with phospholipid; the [3H]heparin was released from the enzyme by 0.75 M NaCl.

Interaction of plasma apolipoproteins with lipid monolayers.

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...

Preparation and properties of immobilized lipoprotein lipase.

Purified bovine milk lipoprotein lipase has been covalently attached to CH-Sepharose with water-soluble carbodiimide. The immobilized enzyme retained enzymic activity and was stimulated 7-fold by the addition of human apolipoprotein C-II. Both [3H]heparin and 125I-labeled apolipoprotein C-II bound to the immobilized enzyme; unlabeled heparin and apolipoprotein C-II competed for binding of their respective labeled compounds. Apolipoprotein C-II did not compete for binding of [3H]heparin and vice versa. Human apolipoprotein C-III did not bind to the immobilized enzyme nor did it compete for apolipoprotein C-II binding. We conclude from these studies that both apolipoprotein C-II and heparin interact with immobilized lipoprotein lipase and that they have different binding sites.

Comparison of acyl-oxyester and acyl-thioester lipids as substrates for bovine milk lipoprotein lipase.

The dithioester analog of dihexanoylphosphatidylcholine, rac-1,2-S,S-dihexanoyl-3-phosphocholine-1,2-dimercapto-3-propanol was synthesized and compared to the corresponding acyl-oxyester lipid as a substrate for bovine milk lipoprotein lipase. The apparent maximal reaction velocity (Vmax) for dihexanoyldithiophosphatidylcholine was considerably lower than that for dihexanoylphosphatidylcholine (0.12 vs. 5.0 mumol product released/min per mg lipoprotein lipase, respectively). The apparent Km values were 1.9 and 4.0 mM, respectively. 3-Butyrylthio-1,2-dibutyryloxypropane was also compared to tributyrylglycerol as a substrate for lipoprotein lipase; hydrolysis of the acyl-thioester bond was insignificant when compared to the corresponding oxyester derivative. Apolipoprotein C-II, the activator protein of lipoprotein lipase for long-chain fatty acyl substrates, had no effect on the hydrolysis of either the thio- or oxyester short-chain substrates. The low lipoprotein lipase activity for the thioester substrates is discussed in relation to the structure of the lipid and the active site of the enzyme.

Lipoprotein lipase-catalyzed hydrolysis of tri[14C]oleoylglycerol in a phospholipid interface. A monolayer study.

The lipoprotein lipase-catalyzed hydrolysis of triacylglycerol was determined in a lipid monolayer containing egg phosphatidylcholine and tri[14C]oleoylglycerol. In the presence of purified bovine milk lipoprotein lipase and fatty acid-free albumin, the rate of hydrolysis of tri[14C]oleoylglycerol, as determined by the decrease in surface activity, was dependent upon enzyme concentration and was enhanced by the addition of apolipoprotein C-II, the activator protein for the enzyme. Increasing the triacylglycerol content of the phospholipid monolayer from 1 to 6 mol% (relative to phospholipid) enhanced the rate of catalysis in the presence and absence of apolipoprotein C-II. However, at low substrate concentrations (less than 4 mol% tri[14C]oleoylglycerol), the activation factor for apolipoprotein C-II was greater than at high (4-6 mol%) triacylglycerol concentrations. The addition of sphingomyelin to the phosphatidylcholine monolayer decreased lipoprotein lipase activity. Based on these monolayer studies, we conclude that lipoprotein lipase catalyzes the hydrolysis of triacylglycerol at a phospholipid interface and that the rate of catalysis is dependent on the lipid composition of the monolayer.

Human plasma lipid transfer protein catalyzes the speciation of high density lipoproteins.

The role of purified plasma lipid transfer protein complexes in determining the particle size distribution of human plasma high density lipoproteins (HDL) was examined in vitro. Incubation of HDL2 or HDL3, isolated from normolipemic subjects with very low density lipoproteins (VLDL) or VLDL-remnants and lipid transfer protein complex had little or no effect on HDL particle size. In contrast, HDL isolated from patients with hypertriglyceridemia, designated HDL3D, showed speciation of particle size distribution when incubated with VLDL-remnants and the transfer protein. Incubation of HDL3D with VLDL-remnants and lipid transfer complex resulted in the production of two particles of radius 4.3 and 3.7 nm; incubation with VLDL or in the absence of the transfer protein did not result in a redistribution of particle size. We suggest that the action of lipid transfer protein complex on triacylglycerol-rich lipoprotein remnants and HDL accounts for the low levels of HDL-cholesterol observed in subjects with severe hypertriglyceridemia.

Fatty acyl chain specificity of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II and its (56-79) synthetic fragment.

Mixed acyl chain phosphatidylcholine molecules in Triton N-101 micelles were employed as substrates for lipoprotein lipase to test which substrate acyl chain has the greatest effect on activation of the enzyme by apolipoprotein C-II. The phospholipase A1 activity of lipoprotein lipase was measured by pH-stat. The activation factor (lipoprotein lipase activity plus apolipoprotein C-II/activity minus apolipoprotein C-II) increased monotonically with apolipoprotein C-II concentration up to 1 microM apolipoprotein C-II at an enzyme concentration of 0.01 microM. The maximal activation factor for phosphatidylcholine substrate molecules with sn-2 acyl chain lengths of 14 averages 14.8. By contrast, for sn-2 acyl chain lengths of 16 the activation factor was 29.2. Varying the sn-1 acyl chain length had no significant effect on the activation factor. The chain-length dependence of the activation factor is similar with the apolipoprotein C-II peptide fragment comprising residues 56-79, which does not include the lipid-binding region of apolipoprotein C-II. These data are consistent with a model for activation of lipoprotein lipase in which residues 56-79 bind to lipoprotein lipase and alter the interaction of the sn-2 acyl chain of the phosphatidylcholine (PC) substrate or the lysoPC product within the activated state complex.

Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films.

The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer.

Comparison of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins and trioleoylglycerol/phosphatidylcholine-stabilized particles as substrates for lipoprotein lipase.

The effect of apolipoproteins C-II and C-III on the lipoprotein lipase-catalyzed hydrolysis of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins and particles of trioleoylglycerol stabilized with a phosphatidylcholine monolayer was investigated. For both triacylglycerol-rich lipoproteins and artificial lipid particles, maximal lipoprotein lipase activity occurred at a constant apolipoprotein C-II/phospholipid mol ratio of 2.0 X 10(-4) and was independent of particle size, indicating that the amount of apolipoprotein C-II bound to the surface of the substrate is important for enzyme activation. The effect of apolipoprotein C-II on lipoprotein lipase activity with apolipoprotein C-II-deficient lipoproteins as substrate was to decrease the apparent Michaelis constant (Kmapp) from 7.1 to 1.0 mM with minor changes on the apparent maximal velocity (Vmax) (22.2 mmol free fatty acid released/h per mg enzyme). In contrast, apolipoprotein C-II increased the apparent Vmax from 2.4 to 20.0 mmol free fatty acid/h per mg enzyme for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol/phospholipid particles with little change in Kmapp (1.0 mM). Addition of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins or high-density lipoproteins to trioleoylglycerol/phospholipid particles in the presence of apolipoprotein C-II inhibited lipoprotein lipase activity. Lipoprotein lipase activity was also inhibited by the addition of a large excess of lipid-free apolipoprotein C-III to the artificial particles. The decrease in lipoprotein lipase activity correlated with the amount of bound apolipoprotein C-II. We suggest that the reported discrepancies on the effect of apolipoproteins C-II and C-III on lipoprotein lipase catalysis is related to differences in substrates and to the amount of added apolipoproteins.