Characteristics of lipid composition of blood serum lipoproteins in insulin resistance

The purpose of this investigation was to study lipid composition of apoB- and apoA-containing lipoproteines in conditions of insulin resistance pathology. It was shown that hypertriglycerolemia and hypercholesterolemia were determined by triglycerole and cholesterol surplus in the composition of both apoB- and apoA-containing lipoproteins. Phospholipid deficiency in the composition of all lipoproteins fractions were established. Possible mechanisms of formation and metabolism in circulation of blood lipoproteins with altered lipid composition in the blood flow at insulin resistance is discussed.     

Lipoprotein metabolism in patients with diabetes of type II

Changes in contents of blood serum lipoproteins (LP) and activity of enzymes of their transformation were investigated in patients with diabetes mellitus of type II. It was found out that mechanisms of increasing of apoB-containing LP in these patients were on the one hand in enhancing of the processes of their new-formation in the liver. On the other hand, retardation of their arrival to peripheral tissues might be observed, that as a whole resulted in continuous LPLD circulation in blood. Above-mentioned processes were associated with a reciprocal decreasing of the amount of all the apoA-containing LP fractions examined. To a certain extent mechanisms of disturbance of LP metabolism in the circulation bed were stipulated by changes of function of the enzyme catalyzing their transformation.     

Lipoprotein receptors and atherosclerosis

The whole lipoprotein spectrum of human plasma may be divided into atherosclerotic and anti-atherosclerotic lipoproteins. To the first class belong apolipoprotein (apo) B and some apoE-containing lipoproteins of the very-low-density (VLDL), intermediate-density (IDL) and low-density (LDL) lipoprotein fractions. Anti-atherosclerotic lipoproteins are apoA-containing high-density lipoproteins (HDL). Circulating plasma lipoproteins are catabolized mainly by specific cell surface receptors (R) which react with apoB and apoE (B/E-R), for apoE (E-R) or for apoA (HDL-R). Whereas the B/E-R and E-R are responsible for the cellular uptake of lipoproteins and their lipid load by various organs, HDL-R are thought to promote lipid (cholesterol) efflux. There is an additional class of lipoprotein receptors, the so called scavenger-R which are responsible for the removal of altered or degraded lipoproteins for the circulation. Under normal physiological conditions, the concerted action of these receptors warrants efficient lipoprotein turnover and direction into target organs. Derangements of this system, however, may lead to the deposition and accumulation of atherogenic lipids, notably free cholesterol (FC) and cholesteryl esters (CE) in arterial tissue causing atherosclerosis and cardiac death.     

急性酒精中毒合并中度创伤性脑损伤大鼠海马AQP4的表达

目的:探讨大鼠急性酒精中毒合并颅脑外伤后AQP4在海马区表达的变化.方法:健康成年雄性SD大鼠96只,随机分为4组:假手术组(N组)、急性酒精中毒组(A组)、中度创伤性脑损伤组(T组)和急性酒精中毒合并中度创伤性脑损伤(AT组).腹腔注射酒精(2.5g/kg),2h后以重物自由落体击打大鼠头部建立急性酒精中毒合并中度创伤性脑损伤(traumatic brain injury,TBI)动物模型.各组动物分别存活1、3、5、14天.免疫组化方法检测海马CA1区AQP4的表达.结果:AQP4阳性产物分布于胶质纤维和毛细血管壁,各实验组表达均高于N组.术后1天T组比AT组表达显著增高(P＜0.01),术后3天AT组比T组表达增高(P＜0.05),术后14天AT组比T组表达显著增高(P＜0.01).结论:大鼠急性酒精中毒合并颅脑外伤后晚期,海马CA1区AQP4表达增高,可能加重晚期继发性脑水肿,是急性酒精中毒合并颅脑外伤预后不良的原因之一.     

Effects of inmecarb on alcohol intake and the state of hepatic cytochrome P-450 system during various stages of experimental alcoholism in rats

The influence of the new anti-alcohol drug Inmecarb on the alcohol consumption as well as on activity of the liver cytochrome P-450 system was studied in rats during chronic alcohol intoxication in the free choice situation between water and 15% ethanol solution. It was shown that voluntary alcohol consumption of different duration (10 days to 8 months) does not change the activity of liver cytochrome P-450 system. Inmecarb treatment (40 mg/kg, i.p. twice a day) during 14 days resulted in decrease of alcohol consumption in rats. This effect was most pronounced in late stages of experimental alcoholism. Inmecarb decreases the cytochrome P-450 content and suppresses the activity of aniline hydroxylase in rats with different duration of voluntary alcohol intoxication, but most pronounced effect was observed during the late stages of experimental alcoholism.     

Ability of six different lipoprotein fractions to regulate the rate of hepatic cholesterogenesis in vivo.

Two in vivo assay procedures were used to study the inhibitory activity of cholesterol carried in three intestinal lymph and three serum lipoprotein fractions on the rate of cholesterol synthesis in the liver. In the first preparation, different lipoproteins were injected intravenously as a bolus into rats at the mid-light phase of the diurnal light cycle, following which they were killed 12 hours later in the mid-dark phase of the cycle. Using this assay, three intestinal lymph lipoprotein fractions of varying Sf values all produced a similar degree of inhibition which averaged approximately 11%/mg of cholesterol injected. The serum lipoprotein fractions caused only about one-third this amount of inhibition. Detailed analysis of events occurring within the liver during this 12-hour assay period revealed that there were marked differences in the rate of net cholesterol uptake into the liver and in the rate of new removal of cholesterol esters from the liver following injection of each of these different lipoprotein fractions. The amount of inhibition of sterol synthesis produced by any fraction was proportional to the product of the incremental increase in hepatic cholesterol ester content and the time over which this increase in esters occurred. In the second type of assay where the lipoprotein fractions were administered to the animals as a continuous intravenous infusion over 24 hours the largest increase in hepatic cholesterol ester content and the greatest inhibition of cholesterol synthesis was found with intestinal lipoproteins having Sf values larger than 8000. Intestinal lipoprotein fractions with lower Sf values and all serum lipoprotein fractions were significantly less effective in bringing about an increase in hepatic cholesterol ester content and in producing inhibition of cholesterol synthesis by the liver. These studies emphasize the primary role of cholesterol carried in lipoproteins of intestinal origin in regulating hepatic sterol synthesis. The inhibitory activity of these fractions appears to correlate with the ability of these lipoproteins to bring about a maximal increase in hepatic cholesterol ester content which, in turn, appears to relate to the capacity of these fractions to transfer cholesterol rapidly into the hepatocyte while, at the same time, slowing the rate of cholesterol mobilization from the liver.     

Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey.

The plasma of squirrel monkeys contains extremely low levels of very low density lipoproteins. The delipidated apoproteins from the different lipoprotein density classes of this species show a heterogeneity similar to that of man and the rat. The biosynthesis of the apoproteins of squirrel monkey lipoproteins was studied in fasted normal and Triton WR1339-treated animals. After intravenous injection of [3-H] leucine, maximal labeling of very low density lipoproteins occurred after 1 h, intermediate density lipoproteins (d 1.006--1.019) in 2 h, and low density lipoproteins after 3 h. At all times, however, low density lipoproteins had the greatest percentage of radioactivity. Polyacrylamide gel electrophoresis revealed that the apoprotein B moiety of very low density and intermediate density lipoproteins contained 62% and 81% of the total radioactivity in these lipoproteins whereas the fast-migrating peptides were minimally labeled. In monkeys injected with Triton WR1339, 70--80% of the radioactivity incorporated into d smaller than 1.063 lipoproteins was in very low density lipoproteins with only 10--15% in intermediate and low density lipoproteins. After injection of 3-H-labeled very low density lipoproteins and [14-C] leucine into Triton-treated monkeys, catabolism of 3-H-labeled very low density lipoprotein to intermediate and low density lipoproteins was small and was significantly less than corresponding values for the incorporation of [14-C] leucine. Thus, breakdown of very low density lipoproteins could not account for all the labeled apoprotein B present in the intermediate and low density lipoprotein fractions. The results indicate that most, but not all, of the newly synthesized apoprotein B enters plasma in very low density lipoproteins and that the low concentrations of this lipoprotein in squirrel monkey plasma are a consequence of its rapid turnover.     

Plasma metabolism of apolipoprotein A-IV in humans

As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day. The results are consistent with the conclusions that: apoA-IV is present in human plasma in three distinct metabolic pools; apoA-IV associated with the triglyceride-rich lipoproteins is a precursor to the apoA-IV HDL and LFF pools; apoA-IV in LFF is not a free protein and its turnover rate is faster than that of apoA-IV in HDL; since no transfer of apoA-IV from the HDL or the LFF occurs, these pools may represent a terminal pathway for the catabolism of apoA-IV; and the catabolism of apoA-IV in HDL is dissociated from that of apoA-I although both apoproteins may reside on the same lipoprotein particles.     

Acute inhibition of hepatic lipase and increase in plasma lipoproteins after alcohol intake

Chronic alcohol intake is associated with an increase in fasting plasma high density lipoproteins (HDL). To study alcohol's acute effects on plasma lipoproteins, we measured plasma lipoprotein concentrations and activities of postheparin plasma lipases in nine normolipemic males after ingestion of 40 g of ethanol (as whiskey). After alcohol there was no change in lipoprotein lipase activity but hepatic lipase was decreased to 67% of baseline at 6 hr. There were associated increases in HDL phospholipids (12 mg/dl) and cholesterol (10 mg/dl) resulting in prominence of larger, lipid-enriched HDL particles. Changes were most pronounced in the HDL3 and HDL2a subclasses. Very low density lipoprotein (VLDL) phospholipids and cholesterol were also increased by 13 and 9 mg/dl, respectively, with no significant change in triglycerides. Changes in lipoproteins and lipase were largely reversed 10 hr after alcohol intake. The transient increases in VLDL and HDL lipids after alcohol may result in part from acute inhibition of hepatic lipase activity. The results suggest a role of hepatic lipase in the catabolism of phospholipids of VLDL and possibly HDL.     

Synthesis and release of low density lipoproteins by the isolated perfused pig liver.

In previous studies, we have shown that a relatively large amount of low density lipoproteins is released into the perfusate during isolated pig liver perfusion. The present studies were done to determine the source of these lipoproteins. Breakdown of the very low density lipoproteins to low density lipoproteins by the perfusion apparatus or by hepatic catabolism was excluded by adding 125I very low density lipoproteins to the perfusate in the presence and absence of a liver and then measuring the radioactivity in the low density lipoprotein fraction after rate-zonal ultracentrifugation. Release of preformed low density, lipoproteins from the liver was investigated by injecting iodine-labeled low density lipoproteins in vivo several hours prior to perfusion of the liver and then measuring the release of labeled low density lipoproteins into the perfusate. It was shown that intact labeled low density lipoproteins were released by the perfused liver. De novo synthesis of the low density lipoproteins was established by measuring the incorporation of [1-14C]leucine into this lipoprotein fraction. The radioactivity in the low density lipoprotein fraction increased with time and accounted for 20 to 25% of the total radioactivity incorporated into all the lipoprotein fractions. The incorporation of [1-14C]leucine into the low density lipoproteins was confirmed by rate-zonal analysis. We conclude that the low density lipoproteins in the perfusate from pig liver perfusions were derived mainly from a preformed liver pool, but also partly from de novo synthesis by the liver.     

Alcohol and the emergency service patient.

To determine the prevalence of alcohol use in casualty patients breath-alcohol analysis was performed on 702 patients attending the accident and emergency department of a large teaching hospital during the evening. Forty per cent of patients had consumed alcohol before attending and 32% had a blood alcohol concentration exceeding 17.4 mmol/l (80 mg/100 ml). Clinical assessment of intoxication resulted in a false-negative diagnosis in 10% of inebriated patients, indicating that an objective measurement of the blood alcohol concentration by a test, such as breath-alcohol analysis, may be of additional value. These findings confirm that a high proportion of emergency-service patients are affected by alcohol and suggest that alcoholism treatment facilities need to be integrated with accident and emergency services.     

Separation and selective detection of lipoprotein particles of patients with coronary artery disease by frit-inlet asymmetrical flow field-flow fractionation

An analytical method to improve the characterization of lipoprotein fractions is presented. Human plasma samples were treated with Sudan Black B to stain the lipid component in lipoproteins, then the stained lipoproteins were separated by frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF), according to the lipoprotein particle sizes, with the selective detection of eluting lipoprotein fractions, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), at 610 nm. The capability of this technique has been evaluated with plasma samples obtained from patients with coronary artery disease (CAD), and it showed that the retention profile of patients' lipoprotein samples was clearly distinct from those of healthy persons. The potential of this technique comes with the direct injection of a stained lipoprotein sample without a prior procedure such as ultracentrifugation for sample preparation, and the size calculation of lipoprotein particles from the experimental retention time by theory. Since sample relaxation was achieved hydrodynamically in an FI-AFlFFF channel, sample injection and separation processes were continuously made without stopping the separation flow. This study demonstrated the potential of the FI-AFlFFF technique to be utilized as a powerful tool for the determination of the LDL profiles of patients with CAD.     

Effect of serum and serum lipoproteins on testosterone production by adult rat Leydig cells in vitro.

The effect of serum factors other than luteinizing hormone on Leydig cell testosterone secretion was examined using an in vitro bioassay system based on the stimulation of purified adult rat Leydig cells during a 20 h incubation in the presence of a maximal dose of human chorionic gonadotrophin (hCG). Charcoal-extracted serum and testicular interstitial fluid (IF) from normal adult male rats were separated into lipoprotein and lipoprotein-deficient fractions by density ultracentrifugation. Stimulatory bioactivity was found in the lipoprotein fraction of both serum and IF, although the levels of lipoprotein and corresponding bioactivity recovered from IF were significantly lower (25%) than those of serum. There was no difference between the effects of serum lipoproteins on Leydig cell testosterone production stimulated by either hCG or dibutyryl cAMP. In time-course studies, the serum lipoprotein fraction had no effect on hCG-stimulated testosterone production in vitro at 3.0 or 6.0 h, but partially prevented the normal decline in hCG-stimulated testosterone production after 6.0 h. In contrast, unfractionated serum was stimulatory at all time-points. In the absence of hCG, the lipoprotein fraction was stimulatory at both 6.0 and 20 h, although not at 3.0 h. The lipoprotein-deficient protein fraction of serum had no effect on hCG-stimulated testosterone production alone, but significantly enhanced the bioactivity of the lipoprotein fraction, and caused a dose-dependent stimulation of testosterone production in the presence of a constant concentration of serum lipoproteins. Both a stimulatory peak of activity (apparent MW 40-80 kDa), and a large MW (> 100 kDa) inhibitor of testosterone production were identified in serum after fractionation by gel filtration (Sephadex G-100). The data indicate that (i) the stimulatory effect of serum on short-term hCG-stimulated Leydig cell testosterone production in vitro is predominantly due to the serum lipoprotein fraction, possibly by providing additional precursors for testosterone synthesis, (ii) the biological activity of the lipoproteins is influenced by both stimulatory and inhibitory serum proteins in addition to luteinizing hormone, and (iii) that serum lipoproteins may be involved in supporting Leydig cell steroidogenesis in vivo.     

Evaluation of gel chromatography for plasma lipoprotein fractionation

The fractionation of lipoproteins of normal and hyperlipidemic subjects on a column of 2% agarose was compared with ultracentrifugation and paper electrophoresis procedures. The following results were obtained. (a) Plasma lipoproteins were eluted successively from the column in the four overlapping peaks of chylomicrons, very low density lipoproteins, low density lipoproteins, and high density lipoproteins. (b) Very low density lipoproteins and high density lipoproteins (d > 1.063, containing nonlipoprotein proteins) showed continuous progressive changes in lipid composition as these fractions emerged, while low density lipoproteins showed a relatively constant lipid composition. (c) A discontinuous transition of lipid composition was observed when consecutive ultracentrifugal fractions were placed on the column. (d) The "trail" of pre-beta lipoprotein seen on paper electrophoresis was shown to consist of particles whose molecular sizes range between chylomicrons and pre-beta lipoproteins. A reverse relationship was observed between electrophoretic mobilities of "trail" components and their particle size. (e) Gel with an agarose content of 2% seemed to fractionate chylomicrons and very low density lipoproteins more effectively than other lipoprotein classes.     

The Conjugated Plasma Proteins of the American Cockroach : II. Changes during the molting and clotting processes

Evidence has been presented for the possible transport function of conjugated roach plasma proteins during molting. Extensive changes in these proteins are evident during the premolt stage. The remainder of the instar appears to be devoted to a gradual return to the intermolt stage. This recovery process is characterized principally by a transformation of less mobile lipoproteins to lipoproteins of higher electrophoretic mobilities and may be indicative of a lipoprotein-lipase reaction in insects. This transformation also appears to give rise to a second sex-specific lipoprotein. Significant changes in the glycoproteins at the premolt and ecdysial stages may indicate transport of carbohydrate for storage or utilization. A total of six fractions has been shown to occur in roach plasma in the period from one molt to the next. The process of clotting appeared to be concerned primarily with the blood lipoproteins. These protein fractions reacted to form two new lipoproteins, one of which was the relatively insoluble coagulum network. The second major protein fraction appearing as a result of the coagulation process also was a lipoprotein which contained a lower concentration of carbohydrate than the clot. A possible method of assaying or screening new anticoagulants for insect blood is proposed.     

The composition and physicochemical properties of the blood lipoproteins in rats exposed to external gamma irradiation]

Content of lipids, character of chemiluminescence of blood plasma and certain classes of lipoproteins have been studied. Geometrical parameters, nature and quantity of charged groups of lipoprotein particles accessible for titration have been determined 1 and 30 days after a single external gamma irradiation of rats in a dose of 3 Gy. The used irradiation dose exerts an expressed hyperlipidemic effect retained for one month after irradiation. The disturbances in the spectrum of blood lipids and lipoproteins are of hyper-beta and hyper-prebeta lipoproteinemia character. Considerable disturbances of physicochemical properties of different classes of lipoproteins have been detected. They are exhibited in changes of the pattern of free-radical processes, state of the charge of surface ionogenic groups and geometrical parameters of lipoprotein particles. Changes registered by the methods of potentiometric titration and correlation spectroscopy are most expressed in lipoproteins of very low density and those of low density.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

High-carbohydrate diets affect the size and composition of plasma lipoproteins in hamsters (Mesocricetus auratus)

High-carbohydrate diets reduce plasma low-density lipoprotein (LDL)-cholesterol but also provoke the appearance of an atherogenic lipoprotein profile (ALP). Characterized by high plasma triglyceride, small dense LDL, and reduced high-density lipoprotein (HDL) cholesterol, an ALP is associated with insulin resistance. Despite extensive use of the fructose-fed hamster as a model of insulin resistance, little is known about changes that occur in the physical properties of circulating lipoproteins. Therefore, we investigated the metabolic and physical properties of lipoproteins in hamsters fed high-carbohydrate diets of varying complexity (60% carbohydrate as chow, cornstarch, or fructose) for 2 wk. Hamsters fed the high-fructose diet showed significantly increased very- low-density lipoprotein (VLDL)-triglyceride (92.3%), free cholesterol (68.6%), and phospholipid (95%), whereas apolipoprotein B levels remained unchanged. Median diameter of circulating VLDL was larger in fructose-fed hamsters (63 nm) than in cornstarch-fed hamsters. Fructose feeding induced a 42.5% increase LDL-triglyceride concurrent with a 20% reduction in LDL-cholesteryl ester. Compositional changes were associated with reduced LDL diameter. In contrast, fructose feeding caused elevations in all HDL fractions. The physical properties of apolipoprotein-B-containing lipoprotein fractions are similar between fructose-fed hamsters and humans with ALP. However, metabolism of high-density lipoprotein appears to differ in the 2 species.     

Potent inhibitors of glucagon-stimulated adenylate cyclase associated with serum lipoprotein particles

Lipoprotein fractions from some individuals have inhibitory effects on rat liver adenylate cyclase. Precipitation of the lipoprotein fractions with acetone released an inhibitory factor, which was soluble in acetone-H2O (3:1, v/v). The inhibition was greater against glucagon-stimulated activity than against basal activity. Acetone extraction increased the potency of inhibition. All three lipoprotein fractions, i.e., very low, low, and high density lipoproteins, released some inhibitory component after acetone extraction. The inhibitor was concentrated in the lipoprotein fractions, since acetone extraction of plasma did not release an inhibitor. The acetone extract from the very low density lipoprotein was the most inhibitory. This material was further purified and partially characterized. The inhibitor had a molecular mass of about 500. It was inhibitory at micromolar concentrations. The material was sufficiently hydrophobic to migrate in normal-phase thin-layer chromatography (TLC). Nuclear magnetic resonance results indicated that it was not a polar lipid. There were several different inhibitory factors that were separable by TLC. The sequestration of these inhibitors into lipoproteins reduced their effectiveness in inhibiting the action of counter-regulatory hormones, such as glucagon.