Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique µ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.

Extreme Contrast of Postprandial Remnant-Like Particles Formed in Abetalipoproteinemia and Homozygous Familial Hypobetalipoproteinemia.

BACKGROUND: Familial hypobetalipoproteinemia (FHBL) and abetalipoproteinemia (ABL) are rare inherited forms of hypolipidemia. Their differential diagnosis is important for predicting of the prognosis and selecting appropriate therapy. MATERIALS AND METHODS: Genetic analysis was performed in two patients with primary hypocholesterolemia born from consanguineous parents. The oral fat tolerance test (OFTT) was performed in one patient with FHBL (apoB-87.77) and one with ABL as well as in four normal control subjects. After overnight fasting, blood samples were drawn. Serum lipoprotein and remnant-like particle (RLP) fractions were determined by HPLC analysis. RESULTS: Both patients with homozygous FHBL were asymptomatic probably because of preserved levels of fat-soluble vitamins, especially vitamin E. The patients with FHBL were homozygous because of novel apoB-83.52 and apoB-87.77 mutations, and although one of them (apoB-87.77) had fatty liver disease, microscopic findings suggesting nonalcoholic steatohepatitis were absent. Fasting apoB-48 and RLP-triglyceride levels in the patient with homozygous FHBL, which were similar to those in normal control subjects, increased after OFTT both in normal control subjects and the patient with FHBL but not in the patient with ABL, suggesting that the fat load administered was absorbed only in the patient with FHBL. CONCLUSION: Although lipid levels in the patients with homozygous FHBL and ABL were comparable, fasting, postoral fat loading of apoB-48, as well as RLP-triglyceride levels, may help in the differential diagnosis of FHBL and ABL and provide a prompt diagnosis using genetic analysis in the future.

Plasma adiponectin is a more specific marker of fatty liver than a marker of metabolic syndrome in Japanese men.

BACKGROUND: The association of plasma cardiovascular risk markers and metabolic syndrome (MetS) with non-alcoholic fatty liver disease (NAFLD) has not been well defined. METHODS: Japanese men (n = 809) had standard anthropometric measurements done, and had their liver fat quantitated by ultrasound. Three groups were identified: (1) normal controls without significant disease, (2) preliminary-metabolic syndrome (pre-MetS) cases and (3) MetS cases. Plasma adiponectin, high sensitivity-C reactive protein (hs-CRP), HOMA-IR, lipids, lipoproteins and liver enzymes were evaluated among the three groups. RESULTS: The prevalence of fatty liver was 13% in controls, 39% in pre-MetS and 62% in MetS. Plasma adiponectin and high density lipoprotein cholesterol (HDL-C) were significantly decreased, and HOMA-IR, hs-CRP, TG, remnant lipoproteins (RLPs) and small dense-LDL-C (sd LDL-C) were significantly increased in subjects with fatty liver compared to those without fatty liver. Multivariate analyses of serum parameters associated with fatty liver revealed that adiponectin and hs-CRP were more strongly associated with the presence of fatty liver than waist circumference. However, HOMA-IR, HDL-C, TG, RLP-C, RLP-TG and sd LDL-C were more strongly associated with waist circumference than with fatty liver. Factor analysis revealed that adiponectin and HDL-C were linked to liver enzymes, lipoproteins and HOMA-IR associated with fatty liver, but not with waist circumference. CONCLUSIONS: Adiponectin was found to be a more specific diagnostic marker for the presence of fatty liver regardless of MetS status, and was inversely correlated with liver enzyme concentrations. However, RLPs were found to be more specifically associated with the presence of MetS.

Post-prandial remnant lipoprotein metabolism in autosomal recessive hypercholesterolaemia.

BACKGROUND: Phenotype of autosomal recessive hypercholesterolaemia (ARH), a rare lipid disorder, is known to be milder than that of homozygous familial hypercholesterolaemia (FH) with LDL receptor gene mutation. However, few data exist regarding the functional differences in ARH and FH particularly in terms of remnant-like particles' (RLP) metabolism. MATERIALS AND METHODS: Blood sampling was performed up to 6h after OFTT cream loading (50 g/body surface area) with 2-h intervals in a single ARH proband, four heterozygous FH patients with LDL receptor gene mutation and four normal controls. Plasma lipoprotein and RLP fraction were determined by HPLC system. The area under curve (AUC) of each lipoprotein including RLP fractions was evaluated. RESULTS: The AUC of TG, RLP cholesterol (RLP-C) and RLP triglyceride (RLP-TG) levels of heterozygous FH subjects was significantly higher than those of controls (466±71 mg/dL×h vs. 303±111 mg/dL×h, P<0·05; 35±7 mg/dL×h vs. 21±8 mg/dL×h, P<0·05; 124±57 mg/dL×h vs. 51±13 mg/dL×h, P<0·05, respectively). Under these conditions, those values of ARH were close to those of controls (310 mg/dL×h, 22 mg/dL×h, 23 mg/dL×h, respectively). CONCLUSION: These data demonstrate that unlike in FH, RLP clearance is preserved in ARH. The preservation of post-prandial RLP clearance may contribute to the mild phenotype of ARH compared with FH.

The characteristics of remnant lipoproteins in the fasting and postprandial plasma.

BACKGROUND: Remnant-like lipoprotein particles (RLP) have been measured by cholesterol as RLP-C for CHD risk assessment in the fasting plasma. However, RLP-triglyceride (TG) is a better marker of the characteristics of remnant lipoproteins in the postprandial plasma, especially in plasma with TG concentrations <150 mg/dl. METHOD: The RLP-TG and RLP-C concentrations in subjects undergoing a health check-up and in volunteers receiving an oral fat load were determined in the fasting and postprandial plasma. TC, TG, HDL-C, LDL-C, apoB 100, apoB48, RLP apoB-100 and RLP apoB48 were also determined. RESULTS: When fasting TG concentrations were <150 mg/dl, the 95th percentile of RLP-TG was 20mg/dl and the RLP-C 7.5 mg/dl in healthy subjects. The prevalence of RLP-TG and RLP-C above the cut-off values with a TG concentration <150 mg/dl was significantly higher in the metabolic syndrome cases than in the controls. RLP-TG increased significantly in plasma to >20mg/dl after an oral fat load in cases with TG concentrations >80 mg/dl. Further, RLP apoB100, but not RLP apoB48 was highly correlated with the increase of TG in the postprandial plasma. CONCLUSION: RLP-TG and RLP-C were increased significantly above the cut-off values in the postprandial plasma in healthy volunteers from a TG concentration >80 mg/dl. RLP apoB100, but not RLP apoB48, increased significantly when the plasma TG increased after an oral fat load despite the increase of plasma apoB48. The results show that the major lipoproteins which were increased in postprandial plasma were VLDL remnants, not CM remnants.

Metallothionein (MT) 1/2 expression in MT 1/2 and MT 3 knock-out mice and Long-Evans Cinnamon (LEC) rats.

Metallothionein (MT) is known to be involved in various physiological roles and diseases. However, a standard method for MT measurement has not been established until recently. Therefore, we have developed an easy and specific enzyme-linked immunosorbent assays (ELISA) to determine MT-1 and MT-2. In order to evaluate the method we developed, MT-1/2 in liver, kidney and brain was determined in wild type (WT), MT-1/2 knockout (KO) and MT-3 KO mice, with and without Cd treatment. MT 1/2 in urine was determined in genetically disordered LEC rats (an animal model of Wilson disease). MT-1/2 concentrations in the liver, kidney and brain in MT-1/2 KO mice were significantly lower compared to those of WT and MT-3 KO mice. MT-1/2 concentrations in the livers of WT mice significantly increased with Cd administration, but not in MT-1/2 KO mice. Similar results were observed by immunohistochemical staining. To confirm the molecular weight (MW) of MT detected in organs by the ELISA, analysis with a Sephadex G-75 was performed. Two peaks of MT-1/2 (MW small and large) were detected in WT and MT-3 KO mice. The small MT peak was mostly depleted in MT 1/2-KO mice, while a large MT peak remained. A significant increase in MT-1/2 concentration was detected in the urine of LEC rats with age and especially at the hepatitis stage. In conclusion, MT-1/2 ELISA and immunohistochemical staining was highly correlated with MT-1/2 determination in experimental animal specimens and could be a robust analytical tool for physiological and toxicological studies.

Comparative reactivity of remnant-like lipoprotein particles (RLP) and low-density lipoprotein (LDL) to LDL receptor and VLDL receptor: effect of a high-dose statin on VLDL receptor expression.

BACKGROUND: Comparison of the reactivity of remnant-like lipoprotein particles (RLP) and LDL particles to LDL receptor and VLDL receptor has not been investigated. METHODS: LDL receptor- or VLDL receptor-transfected ldlA-7, HepG2 and L6 cells were used. Human LDL and rabbit ß-VLDL were isolated by ultracentrifugation. Human RLP was isolated using an immunoaffinity mixed gel. The effect of statin on lipoprotein receptors was examined. RESULTS: Both LDL receptor and VLDL receptor recognized RLP. In LDL receptor transfectants, RLP, ß-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-ß-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and ß-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than ß-VLDL because of higher apolipoprotein E in RLP. LDL receptor expression was induced in HepG2 by the low concentration of statin while VLDL receptor expression was induced in L6 myoblasts at higher concentration. CONCLUSIONS: RLP are bound to hepatic LDL receptor more efficiently than LDL, which may explain the mechanism by which statins prevent cardiovascular risk by primarily reducing plasma RLP rather than by reducing LDL. Additionally, a high-dose of statins also may reduce plasma RLP through muscular VLDL receptor.

Consumption of fructose and high fructose corn syrup increase postprandial triglycerides, LDL-cholesterol, and apolipoprotein-B in young men and women.

CONTEXT: The American Heart Association Nutrition Committee recommends women and men consume no more than 100 and 150 kcal of added sugar per day, respectively, whereas the Dietary Guidelines for Americans, 2010, suggests a maximal added sugar intake of 25% or less of total energy. OBJECTIVE: To address this discrepancy, we compared the effects of consuming glucose, fructose, or high-fructose corn syrup (HFCS) at 25% of energy requirements (E) on risk factors for cardiovascular disease. PARTICIPANTS, DESIGN AND SETTING, AND INTERVENTION: Forty-eight adults (aged 18-40 yr; body mass index 18-35 kg/m(2)) resided at the Clinical Research Center for 3.5 d of baseline testing while consuming energy-balanced diets containing 55% E complex carbohydrate. For 12 outpatient days, they consumed usual ad libitum diets along with three servings per day of glucose, fructose, or HFCS-sweetened beverages (n = 16/group), which provided 25% E requirements. Subjects then consumed energy-balanced diets containing 25% E sugar-sweetened beverages/30% E complex carbohydrate during 3.5 d of inpatient intervention testing. MAIN OUTCOME MEASURES: Twenty-four-hour triglyceride area under the curve, fasting plasma low-density lipoprotein (LDL), and apolipoprotein B (apoB) concentrations were measured. RESULTS: Twenty-four-hour triglyceride area under the curve was increased compared with baseline during consumption of fructose (+4.7 ± 1.2 mmol/liter × 24 h, P = 0.0032) and HFCS (+1.8 ± 1.4 mmol/liter × 24 h, P = 0.035) but not glucose (-1.9 ± 0.9 mmol/liter × 24 h, P = 0.14). Fasting LDL and apoB concentrations were increased during consumption of fructose (LDL: +0.29 ± 0.082 mmol/liter, P = 0.0023; apoB: +0.093 ± 0.022 g/liter, P = 0.0005) and HFCS (LDL: +0.42 ± 0.11 mmol/liter, P < 0.0001; apoB: +0.12 ± 0.031 g/liter, P < 0.0001) but not glucose (LDL: +0.012 ± 0.071 mmol/liter, P = 0.86; apoB: +0.0097 ± 0.019 g/liter, P = 0.90). CONCLUSIONS: Consumption of HFCS-sweetened beverages for 2 wk at 25% E increased risk factors for cardiovascular disease comparably with fructose and more than glucose in young adults.

Postprandial lipoprotein metabolism: VLDL vs chylomicrons.

Since Zilversmit first proposed postprandial lipemia as the most common risk of cardiovascular disease, chylomicrons (CM) and CM remnants have been thought to be the major lipoproteins which are increased in the postprandial hyperlipidemia. However, it has been shown over the last two decades that the major increase in the postprandial lipoproteins after food intake occurs in the very low density lipoprotein (VLDL) remnants (apoB-100 particles), not CM or CM remnants (apoB-48 particles). This finding was obtained using the following three analytical methods; isolation of remnant-like lipoprotein particles (RLP) with specific antibodies, separation and detection of lipoprotein subclasses by gel permeation HPLC and determination of apoB-48 in fractionated lipoproteins by a specific ELISA. The amount of the apoB-48 particles in the postprandial RLP is significantly less than the apoB-100 particles, and the particle sizes of apoB-48 and apoB-100 in RLP are very similar when analyzed by HPLC. Moreover, CM or CM remnants having a large amount of TG were not found in the postprandial RLP. Therefore, the major portion of the TG which is increased in the postprandial state is composed of VLDL remnants, which have been recognized as a significant risk for cardiovascular disease.

Particle size of apoB-48 carrying lipoproteins in remnant lipoproteins isolated from postprandial plasma.

BACKGROUND: Particle size of apoB-48 carrying lipoproteins in remnant-like lipoprotein particles (RLP) in postprandial plasma has not been well characterized. METHODS: Plasma lipids, lipoproteins and apolipoproteins in 12 healthy subjects were analysed after an oral fat load. RLP isolated by immunoaffinity gel from plasma of a normolipidaemic and a hyperlipidaemic subject in four hours after an oral fat load was fractionated by high-performance liquid chromatography (HPLC) and monitored by total cholesterol (TC), triglycerides (TG), apoB-48 and apoB-100. RESULTS: TC, low-density lipoprotein (LDL)-C and apoB did not change after an oral fat load, while TG, RLP-C, RLP-TG and apoB-48 increased significantly in postprandial plasma. HPLC profiles monitored by TC and TG revealed that major lipoproteins increased in RLP after an oral fat load was VLDL size particles. The percentage of RLP-TG in total TG and the ratio of RLP-TG/RLP-C were significantly increased in four hours after an oral fat load compared with the fasting state (P < 0.01). RLP in four hours after an oral fat load fractionated by HPLC and monitored by TC, TG, apoB-48 and apoB-100 revealed that VLDL size or smaller particles were the major lipoproteins. CONCLUSIONS: ApoB-48 carrying lipoproteins in RLP isolated from a normolipidaemic and a hyperlipidaemic subject after an oral fat load showed a similar particle size with apoB-100 carrying VLDL remnants. Therefore, the most apoB-48 carrying particles found in postprandial RLP can be classified as CM remnants. The majority of remnants in the postprandial state were not CM remnants, but VLDL remnants.

Measurement of serum remnant-like lipoprotein particle-triglyceride (RLP-TG) and RLP-TG/total TG ratio using highly sensitive triglyceride assay reagent.

BACKGROUND: Serum concentration of remnant-like lipoprotein particles (RLP) have been measured by cholesterol as RLP-C for clinical diagnostic purpose. However, the measurement of TG in RLP and the ratio of RLP-TG/total TG has not been well established. METHOD: Highly sensitive triglyceride assay reagent (TG-EX) was used for RLP-TG assay and compared with the previously used TG reagent (Determiner LTGII). Sera in health check-up populations, cardiovascular disease, diabetes and oral fat load cases were used for the evaluation of the new RLP-TG assay. Serum TC, TG, HDL-C, LDL-C and RLP-C concentrations were also determined in above cases. RESULTS: The detection limit of new RLP-TG using TG-EX was 2.0mg/dl. The within-run imprecision (n=10) was CV=3.0% (RLP-TG: 4.1 mg ± 0.7 mg/dl), CV = 1.4% (RLP-TG: 42.0 ± 0.6 mg/dl) and CV=0.5% (RLP-TG: 100.6 ± 0.6 mg/dl). Cut-off value (75 percentile) of RLP-TG determined in the fasting Japanese population was 13.1mg/dl in men and 9.9 mg/dl in women. In patients with metabolic syndrome, cardiovascular disease and diabetes, RLP-TG levels were significantly higher than those in normal control subjects. RLP-TG levels increased significantly after an oral fat load and the ratio of RLP-TG/total TG increased > 3-fold compared to the ratio in the fasting state. Approximately 80% of TG increased after an oral fat load was TG derived from remnant lipoproteins. CONCLUSION: Normal range of plasma RLP-TG in the fasting Japanese population was first determined using a highly sensitive TG assay reagent. RLP-TG was shown to be higher in cases with metabolic syndrome, cardiovascular disease, etc and a better marker than RLP-C for the measurement of postprandial remnant lipoproteins, together with total TG for RLP-TG/total TG ratio.

Remnant lipoproteins induced proliferation of human prostate cancer cell, PC-3 but not LNCaP, via low density lipoprotein receptor.

BACKGROUND: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. METHODS: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. RESULTS: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. CONCLUSIONS: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.

A deficiency of cholesteryl ester transfer protein whose serum remnant-like particle-triglyceride significantly increased, but serum remnant-like particle-cholesterol did not after an oral fat load.

BACKGROUND: We found a unique cholesteryl ester transfer protein (CETP) deficient case with markedly elevated serum triglyceride (TG) as well as high-density lipoprotein cholesterol (HDL-C) levels. Most of the CETP deficiency cases were reported to have normal or reduced serum TG with elevated HDL-C. METHODS: The case subject was a 40-year-old male with a compound heterozygous CETP deficiency. Two heterozygous CETP deficient cases and 10 normal volunteers were also recruited as controls. They underwent an oral fat tolerance test (OFTT) and their blood was taken at fasting and during the OFTT to be used for laboratory tests. RESULTS: The case subject had apolipoprotein E (apo-E) phenotype 4/2 with fatty liver but without any cardiovascular disease. His serum TG, HDL-C, apo-AI and apo-B48 levels were significantly higher, but the low-density lipoprotein cholesterol level was lower than controls. Although post-heparin plasma lipoprotein lipase and hepatic lipase (both mass and activity) were nearly normal, the serum level of angiopoietin-like-protein-3 was extremely elevated. While his serum remnant-like particles-TG (RLP-TG) and total TG levels significantly increased after a fat load, the RLP-cholesterol (RLP-C) level did not increase during OFTT. CONCLUSIONS: The case subject was different from the common CETP deficient cases reported previously. Also, the results indicated that the metabolic pathways of RLP-C and RLP-TG formation in the postprandial state are controlled independently in CETP deficient cases. CETP deficiency itself may not be atherogenic, while one with elevated RLPs may be atherogenic. These cases may have raised the controversy of whether CETP deficiency is atherogenic or not.

Consuming fructose-sweetened, not glucose-sweetened, beverages increases visceral adiposity and lipids and decreases insulin sensitivity in overweight/obese humans.

Studies in animals have documented that, compared with glucose, dietary fructose induces dyslipidemia and insulin resistance. To assess the relative effects of these dietary sugars during sustained consumption in humans, overweight and obese subjects consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Although both groups exhibited similar weight gain during the intervention, visceral adipose volume was significantly increased only in subjects consuming fructose. Fasting plasma triglyceride concentrations increased by approximately 10% during 10 weeks of glucose consumption but not after fructose consumption. In contrast, hepatic de novo lipogenesis (DNL) and the 23-hour postprandial triglyceride AUC were increased specifically during fructose consumption. Similarly, markers of altered lipid metabolism and lipoprotein remodeling, including fasting apoB, LDL, small dense LDL, oxidized LDL, and postprandial concentrations of remnant-like particle-triglyceride and -cholesterol significantly increased during fructose but not glucose consumption. In addition, fasting plasma glucose and insulin levels increased and insulin sensitivity decreased in subjects consuming fructose but not in those consuming glucose. These data suggest that dietary fructose specifically increases DNL, promotes dyslipidemia, decreases insulin sensitivity, and increases visceral adiposity in overweight/obese adults.

The correlation between TG vs remnant lipoproteins in the fasting and postprandial plasma of 23 volunteers.

BACKGROUND: Two recent publications report that non-fasting triglycerides concentrations in plasma are more predictive of cardiovascular events than conventional measurements of fasting triglycerides. While these observations are consistent with the previous studies, direct correlations between remnant lipoprotein triglyceride (RLP-TG) and remnant lipoprotein cholesterol (RLP-C), which are also considered to be risk factors for cardiovascular disease, and fasting and postprandial TG have not been investigated. METHODS: On four different days, both fasting and postprandial blood samples were collected from twenty-three overweight to obese men and women at UC Davis and analyzed for plasma concentrations of TG, RLP-C and RLP-TG. RESULTS: Significantly higher correlations between plasma TG and RLPs were observed in the postprandial state (RLP-C r2 = 0.85; RLP-TG r2 = 0.92) than in the fasting state (RLP-C r2 = 0.61; RLP-TG r2 = 0.73). The differences in the correlations between the fasting and postprandial TG and RLPs were statistically significant (p < 0.001). The increase of RLP-TG (postprandial RLP-TG minus fasting RLP-TG) consisted of approximately 80% of the total increase of TG (postprandial TG minus fasting TG). CONCLUSION: Postprandial TG vs remnant lipoprotein concentrations were significantly more correlated when compared with fasting TG vs RLP concentrations. The increased TG in the postprandial state mainly consisted of TG in remnant lipoproteins. Therefore, the increased sensitivity of non-fasting TG in predicting the risk for cardiovascular events may be directly explained by the increase of remnant lipoproteins in the postprandial state.

Cholesterol efflux from J774 macrophages and Fu5AH hepatoma cells to serum is preserved in CETP-deficient patients.

BACKGROUND: The role of CETP in the development of atherosclerosis is debatable, and few data exist regarding the total impact of CETP inhibition on cholesterol efflux. METHODS: Acceptor capacities of whole serum and HDL subfractions separated by HPLC were compared using 2 different cell systems. Subjects with CETP deficiency (2 homozygous, 1 compound heterozygous, and 5 heterozygous) were analyzed along with 10 normolipidemic controls. The fractional efflux from cholesterol-labeled Fu5AH hepatoma cells was determined to be SR-BI mediated. The efflux difference between control and liver X receptor (LXR) agonist-induced ABCA1-upregulated J774 macrophages was considered as a measure of ABCA1-mediated efflux. RESULTS: For the Fu5AH cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fraction 2 obtained from the homozygous subjects were 38% and 116% higher than the corresponding values for the controls, respectively (p<0.05). For the J774 cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fractions were similar among the CETP-deficient subjects and controls. CONCLUSIONS: Serum from homozygous subjects with CETP-null defects exhibited enhanced acceptor capacity via an SR-BI dependent pathway, which is regulated by the middle HPLC-separated HDL fraction. Further, the cholesterol acceptor capacity of serum obtained from patients having complete and partial CETP deficiency was preserved via an ABCA1-dependent pathway.

Dietary salt restriction increases plasma lipoprotein and inflammatory marker concentrations in hypertensive patients.

BACKGROUND: Dietary salt restriction has been reported to adversely modify the plasma lipoprotein profile in hypertensive and in normotensive subjects. We investigated the effects of the low sodium intake (LSI) on the plasma lipoprotein profile and on inflammation and thrombosis biomarkers during the fasting and postprandial periods. METHODS: Non-obese, non-treated hypertensive adults (n=41) were fed strictly controlled diets. An initial week on a control diet (CD, Na=160 mmol/day) was followed by 3 weeks on LSI (Na=60 mmol/day). At admission and on the last day of each period, the 24-h ambulatory blood pressure was monitored and blood was drawn after an overnight fasting period and after a fat-rich test meal. RESULTS: The dietary adherence was confirmed by 24-h urinary sodium excretion. Fasting triglyceride (TG), chylomicron-cholesterol, hsC-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) concentrations, renin activity, aldosterone, insulin, and homeostasis model assessment insulin resistance (HOMA-IR) values were higher, but non-esterified fatty acids (NEFA) were lower on LSI than on CD. For LSI, areas under the curve (AUC) of TG, chylomicron-cholesterol, apoB and the cholesterol/apoB ratio were increased, whereas AUC-NEFA was lowered. LSI did not modify body weight, hematocrit, fasting plasma cholesterol, glucose, adiponectin, leptin, fibrinogen and factor VII (FVII), and AUC of lipoprotein lipase and of lipoprotein remnants. CONCLUSION: LSI induced alterations in the plasma lipoproteins and in inflammatory markers that are common features of the metabolic syndrome.

Detection of apolipoproteins B-48 and B-100 carrying particles in lipoprotein fractions extracted from human aortic atherosclerotic plaques in sudden cardiac death cases.

BACKGROUND: ApoB-48 is a major apolipoprotein secreted by the small intestine and is the main constitutive apolipoprotein in chylomicrons (CM). In the past, presence of apoB-48 in human aortic atherosclerotic plaques has not been detected. METHODS: A newly developed apoB-48 ELISA together with an HPLC fractionation technique, were applied to investigate the presence of apoB-48 (CM) in aortic atherosclerotic plaques. The atherosclerotic plaques were obtained from aortae of sudden cardiac death cases. Total cholesterol, triglycerides (TG), apoB-100 and apoB-48 were measured in the aortic plaques extracts. RESULTS: HPLC analysis of plaques extracts monitored by cholesterol revealed mainly particle sizes of CM and very low density lipoproteins (VLDL) in the d>1.006 fractions. The plaques extracts were monitored by apoB-48 and apoB-100 ELISA. There were no TG peaks in any lipoprotein fraction extracted from the plaques except as free glycerol. ApoB-100 was detected in VLDL particles and in LDL sizes. In contrast, apoB-48 was detected in particles of CM, VLDL and LDL sizes. Further, in postmortem plasma, apo B-48 was detected in particles sizes of HDL or smaller and the Western blot analysis could not show any 250 kDa molecular weight (MW) protein in the plaque extracts, but smaller and broader MW staining were observed at 20-150 kDa. CONCLUSION: Hitherto there has been lack of an appropriate assay to measure apoB-48 in plaques. Our investigations show that apoB-48 is present in atherosclerotic plaques with denatured or degraded structure. This is the first report describing presence of apoB-48 in human atherosclerotic plaques.

Decreased post-prandial triglyceride response and diminished remnant lipoprotein formation in cholesteryl ester transfer protein (CETP) deficiency.

Plasma cholesteryl ester transfer protein (CETP) mediates CE/TG exchange among various lipoproteins. CETP deficiency results in low LDL and high HDL phenotype including apoE-rich large HDL. Large HDL could provide apoE to chylomicron/VLDL during lipolysis in post-prandial state, accelerating remnant lipoprotein uptake in the liver. To determine the effects of low CETP levels on post-prandial lipoprotein metabolism, lipid levels of plasma remnant-like lipoprotein particles (RLP) fraction were determined in one homozygous and three heterozygous CETP deficiency and controls with apoE3/3 phenotype. After oral fat-load, the area under curve (AUC) of TG levels were remarkably decreased in CETP deficiency as compared to controls (423+/-187 [S.D.] mg/dl x h in three heterozygous CETP deficiency and 926+/-268 [S.D.] in 10 controls, P=0.012). Similarly, the homozygote had a low AUC of TG levels (416 mg/dl x h). Plasma RLP-cholesterol levels were decreased in heterozygotes, but not significantly as compared to controls (P=0.14). HPLC analysis showed that increased RLP-cholesterol level was not due to conventional VLDL-LDL size RLP, but to those in large HDL size in the homozygote. In heterozygotes, bimodal distribution of RLP-cholesterol level was found in lipoprotein sizes of conventional VLDL-LDL and large HDL. Subjects with CETP deficiency appeared to have low levels of TG response and diminished remnant lipoprotein formation after fat-load.

Remnant lipoproteins stimulate proliferation and activate MAPK and Akt signaling pathways via G protein-coupled receptor in PC-3 prostate cancer cells.

BACKGROUND: Hypertriglyceridemia was recently shown to be a risk factor for prostate cancer; however, there are only a few reports about the relationship between prostate cancer and TG (triglycerides) rich lipoproteins. Remnant lipoproteins (RLP) are TG-rich lipoproteins, which are produced by the hydrolysis of very low density lipoproteins and chylomicrons. We examined the direct effect of RLP on the proliferation and signal transduction of prostate cancer cells. METHODS: RLP were isolated from human serum with an immunoaffinity mixed gel containing anti-apoA-1 and anti-apoB-100. We evaluated RLP-induced cell proliferation by using MTS assay. Moreover we examined the direct effect of RLP on the MAPK and Akt signal transductions which are reported to be correlated with prostate cancer by using Western blotting. RESULTS: Incubation in the presence of RLP for 48 h induced the proliferation of prostate cancer PC-3 cells more significantly than prostate cancer LNCaP cells and human prostate stromal cells. In PC-3 cells, RLP also induced the phosphorylation of MEK/ERK via a G protein-coupled receptor-protein kinase C dependent pathway. Moreover, activation of Akt pathway was observed after RLP treatment of PC-3. CONCLUSION: These findings suggested that hypertriglyceridemia, especially remnant hyperlipoproteinemia, might be one of the progressive factors for prostate cancer.