The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

The impact of antioxidant supplements and endurance exercise on genes of the carbohydrate and lipid metabolism in skeletal muscle of mice

To ascertain whether reactive oxygen species (ROS) contribute to training-induced adaptation of skeletal muscle, we administered ROS-scavenging antioxidants (AOX; 140 mg/l of ascorbic acid, 12 mg/l of coenzyme Q10 and 1% N-acetyl-cysteine) via drinking water to 16 C57BL/6 mice. Sixteen other mice received unadulterated tap water (CON). One cohort of both groups (CON(EXE) and AOX(EXE) ) was subjected to treadmill exercise for 4 weeks (16-26 m/min, incline of 5°-10°). The other two cohorts (CON(SED) and AOX(SED) ) remained sedentary. In skeletal muscles of the AOX(EXE) mice, GSSG and the expression levels of SOD-1 and PRDX-6 were significantly lower than those in the CON(EXE) mice after training, suggesting disturbance of ROS levels. The peak power related to the body weight and citrate synthase activity was not significantly influenced in mice receiving AOX. Supplementation with AOX significantly altered the mRNA levels of the exercise-sensitive genes HK-II, GLUT-4 and SREBF-1c and the regulator gene PGC-1alpha but not G6PDH, glycogenin, FABP-3, MCAD and CD36 in skeletal muscle. Although the administration of AOX during endurance exercise alters the expression of particular genes of the ROS metabolism, it does not influence peak power or generally shift the metabolism, but it modulates the expression of specific genes of the carbohydrate and lipid metabolism and PGC-1alpha within murine skeletal muscle.

Attributional styles and stress-related atherogenic plasma lipid reactivity in essential hypertension.

OBJECTIVE Hypertension and an atherogenic lipid profile are known risk factors for coronary heart disease (CHD). Hypertensives show greater changes in atherogenic plasma lipids to acute stress than normotensives. In this study, we investigated whether attribution of failure is associated with lipid stress reactivity in hypertensive compared with normotensive men. METHODS 18 normotensive and 17 hypertensive men (mean±SEM; 45±2.2 years) underwent an acute standardized psychosocial stress task that can be viewed as a situation of experimentally induced failure. We assessed external-stable (ES), external-variable (EV), internal-stable (IS), and internal-variable (IV) attribution of failure and psychological control variables (i.e. extent of depression and neuroticism). Moreover, total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C), and norepinephrine were measured immediately before and several times after stress. RESULTS ES moderated TC- and LDL-C-stress reactivity in hypertensives as compared to normotensives (interaction mean arterial pressure [MAP]-by-ES for TC: F=3.71, p=.015; for LDL-C: F=3.61, p=.016). TC and LDL-C levels were highest in hypertensives with low ES immediately after stress (p≤.039). In contrast, hypertensives with high ES did not differ from normotensives in TC and LDL-C immediately after stress (p's>.28). Controlling for norepinephrine, depression, and neuroticism in addition to age and BMI did not significantly change results. There were no significant associations between lipid baseline levels or aggregated lipid secretion and IS, IV, or EV (p's>.23). CONCLUSION Our data suggest that ES may independently protect from elevated lipid stress reactivity in hypertensive individuals. ES thus might be a protective factor against CHD in hypertension.

Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint(-/-)) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1(-/-) mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1(-/-) mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1(-/-) mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1(-/-) mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.

Prognostic value of PCSK9 levels in patients with acute coronary syndromes.

AIMS Proprotein convertase subtilisin kexin 9 (PCSK9) is an emerging target for the treatment of hypercholesterolaemia, but the clinical utility of PCSK9 levels to guide treatment is unknown. We aimed to prospectively assess the prognostic value of plasma PCSK9 levels in patients with acute coronary syndromes (ACS). METHODS AND RESULTS Plasma PCSK9 levels were measured in 2030 ACS patients undergoing coronary angiography in a Swiss prospective cohort. At 1 year, the association between PCSK9 tertiles and all-cause death was assessed adjusting for the Global Registry of Acute Coronary Events (GRACE) variables, as well as the achievement of LDL cholesterol targets of <1.8 mmol/L. Patients with higher PCSK9 levels at angiography were more likely to have clinical familial hypercholesterolaemia (rate ratio, RR 1.21, 95% confidence interval, CI 1.09-1.53), be treated with lipid-lowering therapy (RR 1.46, 95% CI 1.30-1.63), present with longer time interval of chest pain (RR 1.29, 95% CI 1.09-1.53) and higher C-reactive protein levels (RR 1.22, 95% CI 1.16-1.30). PCSK9 increased 12-24 h after ACS (374 ± 149 vs. 323 ± 134 ng/mL, P < 0.001). At 1 year follow-up, HRs for upper vs. lower PCSK9-level tertiles were 1.13 (95% CI 0.69-1.85) for all-cause death and remained similar after adjustment for the GRACE score. Patients with higher PCSK9 levels were less likely to reach the recommended LDL cholesterol targets (RR 0.81, 95% CI 0.66-0.99). CONCLUSION In ACS patients, high initial PCSK9 plasma levels were associated with inflammation in the acute phase and hypercholesterolaemia, but did not predict mortality at 1 year.

Decreased phosphatidylcholine plasmalogens - A putative novel lipid signature in patients with stable coronary artery disease and acute myocardial infarction.

OBJECTIVE Glycerophospholipids and sphingolipids are structurally heterogeneous due to differences in the O- and N-linked fatty acids and head groups. Sphingolipids also show a heterogeneity in their sphingoid base composition which up to now has been little appreciated. The aim of this study was to investigate the association of certain glycerophospholipid and sphingolipid species with stable coronary artery disease (CAD) and acute myocardial infarction (AMI). METHODS The lipid profile in plasma from patients with stable CAD (n = 18) or AMI (n = 17) was compared to healthy subjects (n = 14). Sixty five glycerophospholipid and sphingolipid species were quantified by LC-MS. The relative distribution of these lipids into lipoprotein fractions was analyzed. RESULTS In the CAD cohort, 45 glycerophospholipid and sphingolipid species were significantly lower compared to healthy controls. In the AMI group, 42 glycerophospholipid and sphingolipid species were reduced. Four PC plasmalogens (PC33:1, PC33:2, PC33:3 and PC35:3) showed the most significant difference. Out of eleven analyzed sphingoid bases, four were lower in the CAD and six in the AMI group. Sphingosine-1-phosphate (S1P) levels were reduced in the AMI group whereas an atypical C16:1 S1P was lower in both groups. Phosphatidylcholine and sphingomyelin species were exclusively present in lipoprotein particles, whereas lysophosphatidylcholines were mainly found in the lipoprotein-free fraction. The observed differences were not explained by the use of statins as confirmed in a second, independent cohort. CONCLUSIONS Reduced levels of four PC plasmalogens (PC33:1, PC33:2, PC33:3 and PC35:3) were identified as a putatively novel lipid signature for CAD and AMI.

Identification of genetic variation influencing apolipoprotein E levels: Follow-up of genome-wide linkage scans in the GENOA study

Apolipoprotein E (ApoE) plays a major role in the metabolism of high density and low density lipoproteins (HDL and LDL). Its common protein isoforms (E2, E3, E4) are risk factors for coronary artery disease (CAD) and explain between 16 to 23% of the inter-individual variation in plasma apoE levels. Linkage analysis has been completed for plasma apoE levels in the GENOA study (Genetic Epidemiology Network of Atherosclerosis). After stratification of the population by lipoprotein levels and body mass index (BMI) to create more homogeneity with regard to biological context for apoE levels, Hispanic families showed significant linkage on chromosome 17q for two strata (LOD=2.93 at 104 cM for a low cholesterol group, LOD=3.04 at 111 cM for a low cholesterol, high HDLC group). Replication of 17q linkage was observed for apoB and apoE levels in the unstratified Hispanic and African-American populations, and for apoE levels in African-American families. Replication of this 17q linkage in different populations and strata provides strong support for the presence of gene(s) in this region with significant roles in the determination of inter-individual variation in plasma apoE levels. Through a positional and functional candidate gene approach, ten genes were identified in the 17q linked region, and 62 polymorphisms in these genes were genotyped in the GENOA families. Association analysis was performed with FBAT, GEE, and variance-component based tests followed by conditional linkage analysis. Association studies with partial coverage of TagSNPs in the gene coding for apolipoprotein H (APOH) were performed, and significant results were found for 2 SNPs (APOH_20951 and APOH_05407) in the Hispanic low cholesterol strata accounting for 3.49% of the inter-individual variation in plasma apoE levels. Among the other candidate genes, we identified a haplotype block in the ACE1 gene that contains two major haplotypes associated with apoE levels as well as total cholesterol, apoB and LDLC levels in the unstratified Hispanic population. Identifying genes responsible for the remaining 60% of inter-individual variation in plasma apoE level, will yield new insights into the understanding of genetic interactions involved in the lipid metabolism, and a more precise understanding of the risk factors leading to CAD. ^

Determine whether markers of lipid metabolism, inflammation and/or liver function are altered in tuberculosis patients with diabetes

The mechanism for higher susceptibility of diabetes patients to TB is unknown. Chronic hyperglycemia has been shown to be associated with altered immunity to Mycobacterium tuberculosis, and may explain the higher risk of TB among diabetes patients. However, it is possible that other conditions that frequently occur in these patients are also contributing to TB susceptibility. Our goal was to determine whether lipid metabolism, liver function and/or chronic inflammation are altered in tuberculosis (TB) patients with diabetes (DM), compared to non-DM.^ Confirmed TB patients who were 20 years or older (n=159) were selected from a database in the south Texas and northeast Mexico area. Differences between serum values for liver function, lipid metabolism and/or chronic inflammation were compared between TB patients with DM to non-DM.^ We found that CRP was the most frequent alteration, with about 80% having high values suggestive of chronic inflammation. The other frequent abnormalities were high triglycerides in about 40% of the patients and low HDL cholesterol in about 60% of the patients. Otherwise, less than 10% of the TB patients had an abnormal finding for any of the other laboratory tests. The abnormalities were not more frequent among the patients with either DM (versus non-DM) or high HbA1c (versus normal).^ A possible explanation for the high levels or CRP may be that everyone in the study had TB, which in itself causes inflammation and may have masked the increased CRP levels that characterize diabetes patients. There was a mild alteration in lipid metabolism in patients with DM, which is unlikely to explain altered immunity to TB. Otherwise, liver function tests were normal in patients with DM. Therefore the processing of anti-TB medications should be no different between the TB patients with and without diabetes. Our findings, however, do not rule out that other study populations have more remarkable metabolic alterations associated with diabetes. Therefore, it would be interesting to conduct a similar study in patients from different ethnic groups (White, African American, or Native American) in order to see if the same pattern is observed.^

Selection of body fat distribution indices in adults and associations with plasma lipid metabolism variables

Body fat distribution is a cardiovascular health risk factor in adults. Body fat distribution can be measured through various methods including anthropometry. It is not clear which anthropometric index is suitable for epidemiologic studies of fat distribution and cardiovascular disease. The purpose of the present study was to select a measure of body fat distribution from among a series of indices (those traditionally used in the literature and others constructed from the analysis) that is most highly correlated with lipid-related variables and is independent of overall fatness. Subjects were Mexican-American men and women (N = 1004) from a study of gallbladder disease in Starr County, Texas. Multivariate associations were sought between lipid profile measures (lipids, lipoproteins, and apolipoproteins) and two sets of anthropometric variables (4 circumferences and 6 skinfolds). This was done to assess the association between lipid-related measures and the two sets of anthropometric variables and guide the construction of indices.^ Two indices emerged from the analysis that seemed to be highly correlated with lipid profile measures independent of obesity. These indices are: 2*arm circumference-thigh skinfold in pre- and post-menopausal women and arm/thigh circumference ratio in men. Next, using the sum of all skinfolds to represent obesity and the selected body fat distribution indices, the following hypotheses were tested: (1) state of obesity and centrally/upper distributed body fat are equally predictive of lipids, lipoproteins and apolipoproteins, and (2) the correlation among the lipid-related measures is not altered by obesity and body fat distribution.^ With respect to the first hypothesis, the present study found that most lipids, lipoproteins and apolipoproteins were significantly associated with both overall fatness and anatomical location of body fat in both sex and menopausal groups. However, within men and post-menopausal women, certain lipid profile measures (triglyceride and HDLT among post-menopausal women and apos C-II, CIII, and E among men) had substantially higher correlation with body fat distribution as compared with overall fatness.^ With respect to the second hypothesis, both obesity and body fat distribution were found to alter the association among plasma lipid variables in men and women. There was a suggestion from the data that the pattern of correlations among men and post-menopausal women are more comparable. Among men correlations involving apo A-I, HDLT, and HDL$\sb2$ seemed greatly influenced by obesity, and A-II by fat distribution; among post-menopausal women correlations involving apos A-I and A-II were highly affected by the location of body fat.^ Thus, these data point out that not only can obesity and fat distribution affect levels of single measures, they also can markedly influence the pattern of relationship among measures. The fact that such changes are seen for both obesity and fat distribution is significant, since the indices employed were chosen because they were independent of one another. ^

(Table 1) Circulating levels of TT3 and TT4, and age, length, girth, and estimated body mass in polar bears (Ursus maritimus) from East Greenland 1999-2001

We investigated the multivariate relationships between adipose tissue residue levels of 48 individual organohalogen contaminants (OHCs) and circulating thyroid hormone (TH) levels in polar bears (Ursus maritimus) from East Greenland (1999-2001, n = 62), using projection to latent structure (PLS) regression for four groupings of polar bears; subadults (SubA), adult females with cubs (AdF_N), adult females without cubs (AdF_S) and adult males (AdM). In the resulting significant PLS models for SubA, AdF_N and AdF_S, some OHCs were especially important in explaining variations in circulating TH levels: polybrominated diphenylether (PBDE)-99, PBDE-100, PBDE-153, polychlorinated biphenyl (PCB)-52, PCB-118, cis-nonachlor, trans-nonachlor, trichlorobenzene (TCB) and pentachlorobenzene (QCB), and both negative and positive relationships with THs were found. In addition, the models revealed that DDTs had a positive influence on total 3,5,3'-triiodothyronine (TT3) in AdF_S, and that a group of 17 higher chlorinated ortho-PCBs had a positive influence on total 3,5,3',5'-tetraiodothyronine (thyroxine, TT4) in AdF_N. TH levels in AdM seemed less influenced by OHCs because of non-significant PLS models. TH levels were also influenced by biological factors such as age, sex, body size, lipid content of adipose tissue and sampling date. When controlling for biological variables, the major relationships from the PLS models for SubA, AdF_N and AdF_S were found significant in partial correlations. The most important OHCs that influenced TH levels in the significant PLS models may potentially act through similar mechanisms on the hypothalamic-pituitary-thyroid (HPT) axis, suggesting that both combined effects by dose and response addition and perhaps synergistic potentiation may be a possibility in these polar bears. Statistical associations are not evidence per se of biological cause-effect relationships. Still, the results of the present study indicate that OHCs may affect circulating TH levels in East Greenland polar bears, adding to the "weight of evidence" suggesting that OHCs might interfere with thyroid homeostasis in polar bears.

(Table 1) Lipid and organochlorine content in blood of common eiders (Somateria mollissima) in Svalbard

Female common eiders (Somateria mollissima) starve during the nesting stage and may lose 30-45% of their initial body mass, mostly through lipid mobilization. In this study, the effects of fasting on the blood concentrations of three lipid-soluble organochlorines (OCs: polychlorinated biphenyl [PCB]-153; 1-dichloro-2,2-bis (p-chlorophenyl) ethylene [p,p'-DDE]; and hexachlorobenzene [HCB]) were examined in eiders breeding in the high Arctic. Blood samples were taken from females (n = 47) at day 5 and day 20 of the incubation period. The mean wet weight concentrations of PCB-153 and p,p'-DDE increased strongly between day 5 and day 20 (3.6 and 8.2-fold, respectively), while HCB increased less (1.7-fold). There was a strong negative association between daily increase in PCB-153 and clutch size, and a weaker relationship for p,p'-DDE, suggesting that maternal transfer to the eggs is a significant pathway of elimination of OCs in eiders. Moreover, poor body condition (body mass controlled for body size) late in the incubation period was associated with strong daily increase of both p,p'-DDE and PCB-153, which may suggest that the release of these compounds increases when lipid reserves become depleted. For HCB, the increase was mainly associated with increase in blood lipid concentrations, and weakly to the amount of burned lipids. The causes for the differences between the compounds are, however, poorly understood. Although the absolute levels of OCs in eiders were relatively low, their rapid build-up during incubation is worrying as it coincides with poor body condition and weakened immune systems.

(Table 1) Lipid content and organohalogen contaminant concentration in plasma of incubating glaucous gulls (Larus hyperboreus) from Bjørnøya, Norwegian Arctic in 2006

The factors influencing prolactin (PRL) variation in birds and in wildlife in general have rarely been investigated with respect to the physiological impacts of exposure to environmental contaminants. We investigated the associations between circulating baseline PRL levels and concentrations of eight persistent organohalogen contaminant (OHC) classes (i.e., major organochlorines and brominated flame retardants, and associated metabolic products) in blood (plasma) of free-ranging glaucous gulls (Larus hyperboreus), a top predator in the Norwegian Arctic, engaged in the process of incubation. We further examined whether plasma OHC concentrations were associated with the variation of PRL in glaucous gulls exposed to a standardized capture/restraint protocol. Plasma OHC concentrations in male glaucous gulls were 2-to 3-fold higher relative to females. Baseline PRL levels tended to be higher in females compared to males, although not significantly (p = 0.20). In both males and females, the 30-min capture/restraint protocol led on average to a 26% decrease in PRL levels, which resulted in a rate of PRL decrease of 0.76 ng/mL/min. The baseline PRL levels and the rate of decrease in PRL levels tended to vary negatively with plasma OHC concentrations in males, but not in females, although several of these associations did not adhere with the criterion of significance (alpha = 0.05). Present results suggest that in highly OHC-exposed male glaucous gulls, the control of PRL release may be affected by the direct or indirect modulating actions of OHCs and/or their metabolically derived products. We conclude that potentially OHC-mediated impact on PRL secretion in glaucous gulls (males) may be a contributing factor to the adverse effects observed on the reproductive behavior, development and population size of glaucous gulls breeding in the Norwegian Arctic.