Lipid metabolism in women

Differences in whole-body lipid metabolism between men and women are indicated by lower-body fat accumulation in women but more marked accumulation of fat in the intra-abdominal visceral fat depots of men. Circulating blood lipid concentrations also show gender-related differences. These differences are most marked in premenopausal women, in whom total cholesterol, LDL-cholesterol and triacylglycerol concentrations are lower and HDL-cholesterol concentration is higher than in men. Tendency to accumulate body fat in intra-abdominal fat stores is linked to increased risk of CVD, metabolic syndrome, diabetes and other insulin-resistant states. Differential regional regulation of adipose tissue lipolysis and lipogenesis must underlie gender-related differences in the tendency to accumulate fat in specific fat depots. However, empirical data to support current hypotheses remain limited at the present time because of the demanding and specialist nature of the methods used to study adipose tissue metabolism in human subjects. In vitro and in vivo data show greater lipolytic sensitivity of abdominal subcutaneous fat and lesser lipolytic sensitivity of femoral and gluteal subcutaneous fat in women than in men. These differences appear to be due to fewer inhibitory alpha adrenergic receptors in abdominal regions and greater a adrenergic receptors in gluteal and femoral regions in women than in men. There do not appear to be major gender-related differences in rates of fatty acid uptake (lipogenesis) in different subcutaneous adipose tissue regions. In visceral fat rates of both lipolysis and lipogenesis appear to be greater in men than in women; higher rates of lipolysis may be due to fewer alpha adrenergic receptors in this fat depot in men. Fatty acid uptake into this depot in the postprandial period is approximately 7-fold higher in men than in women. Triacylglycerol concentrations appear to be a stronger cardiovascular risk factor in women than in men, with particular implications for cardiovascular risk in diabetic women. The increased triacylglycerol concentrations observed in women taking hormone-replacement therapy (HRT) may explain the paradoxical findings of increased rates of CVD in women taking HRT that have been reported from recent primary and secondary prevention trials of HRT.

Phosphoinositide (PI) 3-kinase assays

The regulation of phosphoinositide (PI) 3-kinase activities has been linked to many normal and disease-related processes, including cell survival, cell growth and proliferation, cell differentiation, cell motility, and intracellular vesicle trafficking. However, as the family of enzymes has now grown to include eight true members, in three functional classes, plus several related protein kinases that are also inhibited by the widely used PI 3-kinase selective inhibitors, wortmannin and LY294002, extended methodologies are required to identify which type of kinase is involved in a particular cellular process, or protein complex, under study. A robust in vitro PI 3-kinase assay, suitable for use with immunoprecipitates, or purified proteins, is described here together with a series of modifications of substrate and assay conditions that will aid researchers in the identification of the particular class and isoform of PI 3-kinase that is involved in a signaling process under investigation.

S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E-3-ligase MIR

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E3-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin-ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.

Long-term monounsaturated fatty acid diets reduce platelet aggregation in healthy young subjects

The aim of the present study was to compare the response of a range of atherogenic and thrombogenic risk markers to two dietary levels of saturated fatty acid (SFA) substitution with monounsaturated fatty acids (MUFA) in students living in a university hall of residence. Although the benefits of such diets have been reported for plasma lipoproteins in high-risk groups, more needs to be known about effects of more modest SFA-MUFA substitutions over the long term and in young healthy adults. In a parallel design over 16 weeks, fifty-one healthy young subjects were randomised to one of two diets: (1) a moderate-MUFA diet in which 16 g dietary SFA/100 g total fatty acids were substituted with MUFA (n 25); (2) a high-MUFA diet in which 33 g dietary SFA/100 g total fatty acids were substituted with MUFA (n 26). All subjects followed an 8-week run-in diet (reference diet), with a fatty acid composition close to the UK average values. There were no differences in plasma lipid responses between the two diets over 16 weeks of the study with similar reductions in total cholesterol (P<0.001) and LDL-cholesterol (P<0.01) in both groups; a small but significant reduction in HDL-cholesterol was also observed in both groups (P<0.01). Platelet responses to ADP (P<0.01) and arachidonic acid (P<0.05) differed with time on the two diets; at 16 weeks, platelet aggregatory response to ADP was significantly lower on the high-MUFA than the moderate-MUFA (P<0.01) diet; ADP responses were also significantly lower within this group at 8 (P< 0.05) and 16 (P< 0.01) weeks compared with baseline. There were no differences in fasting factor VII activity (factors VIII and VIIag), fibrinogen concentration or tissue-type plasminogen activator activity between the diets. There were no differences in postprandial factor VIII responses to a standard meal (area under the curve) between the diets after 16 weeks, but postprandial factor VIII response was lower than on the high-MUFA diet compared with baseline (P<0.01). In conclusion, a high-MUFA diet sustains potentially beneficial effects on platelet aggregation and postprandial activation of factor VII. Moderate or high substitution of MUFA for SFA achieves similar reductions in fasting blood lipids in young healthy subjects.

ACTH reduces the rise in ApoB-48 levels after fat intake

It has been repeatedly demonstrated that ACTH administration lowers plasma lipid concentrations in man. The present study was designed to test the hypothesis, based on observations of decreased apolipoprotein B (ApoB) synthesis and secretion in vitro, that ACTH administration inhibits the postprandial output of ApoB in man. Therefore, we studied the response to a fat-rich meal supplemented with Vitamin A in eight healthy volunteers, who underwent this test without premedication, after 4 days administration of ACTH, and after 4 days administration of a glucocorticoid (betamethasone). As expected, fasting plasma levels of low-density lipoproteins (LDL)-cholesterot (-25%) and ApoB (-17%) decreased after ACTH, but not after betamethasone administration. Also, the elevation of plasma ApoB-48 in response to fat intake (to twice the basal levels) was markedly reduced after ACTH administration. However, the postprandial rise in plasma triglycerides and retinyl palmitate was unimpaired, suggesting that ACTH administration induced the secretion of fewer but larger chylomicrons. The effect of betamethasone on the postprandial response was similar but less pronounced. This study confirms earlier reports on the lipid-lowering effects of ACTH and supports our theory, based on in vitro studies, that the lipid-lowering effects of ACTH administration in man involves an inhibition of ApoB production. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Meal ingestion provokes entry of lipoproteins containing fat from the previous meal: possible metabolic implications

Background: Prolonged and exaggerated postprandial plasma triacylglycerol (TAG) concentrations are considered as an independent risk factor for coronary artery disease. Western populations eat many meals at regular intervals, and can be in a postprandial state for at least 17h of a 24h period. After consuming 2 meals an early plasma TAG peak has been observed after the second meal, the origin of which is unclear. Aim of the study: To test the hypothesis that the early TAG peak observed following sequential meals was of intestinal origin and represented fat derived from the previous meal. Methods: Postprandial plasma lipaemic responses of 17 healthy postmenopausal women were studied by giving a test breakfast followed by a lunch. Watermiscible retinyl palmitate (RP) was added to the breakfast, but not the lunch test meal. Plasma TAG, retinyl esters (RE) and apo B-48 were determined for a 10h period following breakfast. Results: In response to the test meals, RE, apo B-48 and TAG showed multiple peaks. Despite omission of RP from the lunch, RE showed an early peak response after ingestion of lunch in 15 of 17 subjects. The peak response after lunch of all three markers appeared significantly earlier compared with their respective peak responses after the breakfast (P < 0.0001). The area of RE response after lunch was significantly correlated with the RE lipaemic response to the breakfast (r = 0.67; P < 0.004) and to the fasting TAG concentration (r = 0.48; P < 0.05). Conclusions: Since the lunch did not contain RP, the distinctive second influx of RE after lunch was believed to have originated from the breakfast. This, together with the fact that all three markers showed an earlier response to the lunch than the breakfast, supports the view that ingestion of a second meal provokes entry of fat from the previous meal, from an as yet unidentified site (gut, enterocytes, lymph). The results indicate that the degree of TAG "storage" from previous meals might be a function of TAG tolerance and provide a possible site of regulation of the entry of fat into the systemic circulation.

Chylomicron particle size and number, factor VII activation and dietary monounsaturated fatty acids

This study evaluated the effects of substituting dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) on postprandial chylomicron (triacylglycerol (TAG), apolipoprotein B-48 (apo B-48) and retinyl ester (RE)), chylomicron particle size and factor VII (FVII) response when subjects were given a standard meal. In a controlled sequential design, 51 healthy young subjects followed an SFA-rich diet (Reference diet) for 8 weeks after which half of the subjects followed a moderate MUFA diet (n = 25) and half followed a high MUFA diet (n = 26) for 16 weeks. Fasting lipoprotein and lipid measurements were evaluated at baseline and at 8-week intervals during the Reference and MUFA diets. In 25 of the subjects (n = 12 moderate MUFA, n = 13 high MUFA), postprandial responses to a standard test meal containing RE and 13 C-tripalmitin were investigated at the end of the Reference and the MUFA diet periods. Although there were no differences in the postprandial lipid markers (TAG, RE, C-13-TAG) on the two diets, the postprandial apo B-48 response (incremental area under the curve (IAUC) was reduced by 21% on the moderate MUFA diet (NS) and by 54% on the high MUFA diet (P < 0.01). The postprandial peak concentrations of apo B-48 were reduced by 33% on the moderate MUFA diet (P < 0.01) and 48% on the high MUFA diet (P < 0.001). Fasting values for factor VII activity (FVIIc), activated factor VII (FVIIa) or factor VII antigen (FVIIag) did not differ significantly when subjects were transferred from Reference to MUFA diets. However, the postprandial increases in coagulation FVII activity (FVIIc) were 18% lower and of activated FVII (FVIIa) were 17% lower on the moderate MUFA diet (NS). Postprandial increases in FVIIc and FVIIa were 50% (P < 0.05) and 29% (P < 0.07) lower on the high MUFA diet and the area under the postprandial FVIIc response curve (AUC) was also lower on the high MUFA diet (P < 0.05). Significantly higher ratios of RE:apo B-48 (P < 0.001) and 13 C-palmitic acid:apo B-48 (P < 0.01) during both MUFA diets suggest that the CMs formed carry larger amounts of dietary lipids per particle, reflecting an adaptation to form larger lipid droplets in the enterocyte when increased amounts of dietary MUFAs are fed. Smaller numbers of larger chylomicrons may explain attenuated activation of factor VII during the postprandial state when the background diet is rich in MUFA. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Mobilisation of enterocyte fat stores by oral glucose in humans

Background and aims: When a high fat oral load is followed several hours later by further ingestion of nutrients, there is an early postprandial peak in plasma triacylglycerol (TG). The aim of this study was to investigate the location and release of lipid from within the gastrointestinal tract. Methods: Ten healthy patients undergoing oesopho-gastro-duodenoscopy (OGD) were recruited. At t=0, all patients consumed a 50 g fat emulsion and at t=5 hours they consumed either water or a 38 g glucose solution. OGD was performed at t=6 hours and jejunal biopsy samples were evaluated for fat storage. A subgroup of five subjects then underwent a parallel metabolic study in which postprandial lipid and hormone measurements were taken during an identical two meal protocol. Results: Following oral fat at t=0, samples from patients that had subsequently ingested glucose exhibited significantly less staining for lipid within the mucosa and submucosa of the jejunum than was evident in patients that had consumed only water (p=0.028). There was also less lipid storage within the cytoplasm of enterocytes (p=0.005) following oral glucose. During the metabolic study, oral glucose consumed five hours after oral fat resulted in a postprandial peak in plasma TG, chylomicron-TG, and apolipoprotein B48 concentration compared with oral water. Conclusion: After a fat load, fat is retained within the jejunal tissue and released into plasma following glucose ingestion, resulting in a peak in chylomicron-TG which has been implicated in the pathogenesis of atherosclerosis.

Nutrition Society Silver Medal Lecture Nutrigenetics and personalised nutrition: how far have we progressed and are we likely to get there?

Nutrigenetics and personalised nutrition are components of the concept that in the future genotyping will be used as a means of defining dietary recommendations to suit the individual. Over the last two decades there has been an explosion of research in this area, with often conflicting findings reported in the literature. Reviews of the literature in the area of apoE genotype and cardiovascular health, apoA5 genotype and postprandial lipaemia and perilipin and adiposity are used to demonstrate the complexities of genotype-phenotype associations and the aetiology of apparent between-study inconsistencies in the significance and size of effects. Furthermore, genetic research currently often takes a very reductionist approach, examining the interactions between individual genotypes and individual disease biomarkers and how they are modified by isolated dietary components or foods. Each individual possesses potentially hundreds of 'at-risk' gene variants and consumes a highly-complex diet. In order for nutrigenetics to become a useful public health tool, there is a great need to use mathematical and bioinformatic tools to develop strategies to examine the combined impact of multiple gene variants on a range of health outcomes and establish how these associations can be modified using combined dietary strategies.

Olive oil and haemostasis

Olive oil is a key component of the traditional Mediterranean diet; a diet that may explain the low rate of cardiovascular disease (CVD) in Southern European. (Extra virgin) Olive oil is a good source of monounsaturated fatty acids (MUFA) and phenolic compounds, both of which have been investigated for their effects on plasma lipids and lipoproteins, measures of oxidation and factors related to thrombosis. This issue aims to summarise the current understanding of the effects of such dietary components on the haemostatic system and subsequent risk of CVD. To date, evidence suggests that diets rich in MUFA and thus in olive oil attenuate the thrombotic response via a reduction in platelet aggregation and in postprandial FVII levels. Thrombosis is a key event in causing heart attacks and strokes, which if modulated by diet could pose a cost-effective way of reducing CVD incidence in populations that adhere to MUFA/olive oil-rich diets long-term.

Greater enrichment of triacylglycerol-rich lipoproteins with apolipoproteins E and C-III after meals rich in saturated fatty acids than after meals rich in unsaturated fatty acids

Background: Although there is considerable interest in the postprandial events involved in the absorption of dietary fats and the subsequent metabolism of diet-derived triacylglycerol-rich lipoproteins, little is known about the effects of meal fatty acids on the composition of these particles. Objective: We examined the effect of meal fatty acids on the lipid and apolipoprotein contents of triacylglycerol-rich lipoproteins. Design: Ten normolipidemic men received in random order a mixed meal containing 50 L, of a mixture of palm oil and cocoa butter [rich in saturated fatty acids (SFAs)], safflower oil [n-6 polyunsaturated fatty acids (PUFAs)]. or olive oil [monounsaturated fatty acids (MUFAs)] on 3 occasions. Fasting and postprandial apolipoproteins B-48. B-100, E. C-II, and C-III and lipids (triacylglycerol and cholesterol) were measured in plasma fractions with Svedberg flotation rates (S-f) >400 S-f 60-400, and S-f 20 - 60. Results: Calculation of the composition of the triacylglycerol-rich lipoproteins (expressed per mole of apolipoprotein B) showed notable differences in the lipid and apolipoprotein contents of the SFA-enriched particles in the S-f > 400 and S-f 60-400 fractions. After the SFA meal, triacylglycerol-rich lipoproteins in these fractions showed significantly greater amounts of triacylglycerol and of apolipoproteins C-II (Sf 60-400 fraction only), C-III, and E than were found after the MUFA meal (P < 0.02) and more cholesterol, apolipoprotein C-III (Sf > 400 fraction only), and apolipoprotein E than after the PUFA meal (P < 0.02). Conclusions: Differences in the composition of S-f > 400 and S-f 60-400 triacylglycerol-rich lipoproteins formed after saturated compared with unsaturated fatty acid-rich meals may explain differences in the metabolic handling of dietary fats.

Saturated fat-induced changes in S-f 60-400 particle composition reduces uptake of LDL by HepG2 cells

The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of I-125-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of I-125-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (S-f) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of I-125-labeled LDL compared with PUFA- and MUFA-rich particles (P = 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of I-125-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.-Jackson, K. G., V. Maitin, D. S. Leake, P. Yaqoob, and C. M. Williams. Saturated fat-induced changes in Sf 60 400 particle composition reduces uptake of LDL by HepG2 cells.

Impact of saturated, polyunsaturated and monounsaturated fatty acid-rich micelles on lipoprotein synthesis and secretion in Caco-2 cells

Meal fatty acids have been shown to modulate the size and composition of triacylglycerol (TAG)-rich lipoproteins influencing the magnitude and duration of the postprandial plasma TAG response. As a result there is considerable interest in the origin of these meal fatty-acid induced differences in particle composition. Caco-2 cells were incubated over 4 days with fatty acid mixtures resembling the composition of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA)-rich meals fed in a previous postprandial study to determine their impact on lipoprotein synthesis and secretion. The MUFA- and PUFA-rich mixtures supported greater intracellular TAG, but not cholesterol accumulation compared with the SFA-rich mixture (P < 0.001). The MUFA-rich mixture promoted significantly greater TAG and cholesterol secretion than the other mixtures and significantly more apolipoprotein B-100 secretion than the PUFA-rich mixture (P < 0.05). Electron microscopy revealed the SFA-rich mixture had led to unfavourable effects on cellular morphology, compared with the unsaturated fatty acid-rich mixtures. Our findings suggest the MUFA-rich mixture, may support the formation of a greater number of TAG-rich lipoproteins, which is consistent with indirect observations from our human study. Our electron micrographs are suggestive that some endocytotic uptake of MUFA-rich taurocholate micelles may promote greater lipoprotein synthesis and secretion in Caco-2 cells.

Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite

Protein, generally agreed to be the most satiating macronutrient, may differ in its effects on appetite depending on the protein source and variation in digestion and absorption. We investigated the effects of two milk protein types, casein and whey, on food intake and subjective ratings of hunger and fullness, and on postprandial metabolite and gastrointestinal hormone responses. Two studies were undertaken. The first study showed that energy intake from a buffet meal ad libitum was significantly less 90 min after a 1700 kJ liquid preload containing 48 g whey, compared with an equivalent casein preload (P<0.05). In the second study, the same whey preload led to a 28 % increase in postprandial plasma amino acid concentrations over 3 h compared with casein (incremental area under the curve (iAUC), P<0.05). Plasma cholecystokinin (CCK) was increased by 60 % (iAUC, P<0.005), glucagon-like peptide (GLP)-1 by 65 % (iAUC, P<0.05) and glucose-dependent insulinotropic polypeptide by 36 % (iAUC, P<0.01) following the whey preload compared with the casein. Gastric emptying was influenced by protein type as evidenced by differing plasma paracetamol profiles with the two preloads. Greater subjective satiety followed the whey test meal (P<0.05). These results implicate post-absorptive increases in plasma amino acids together with both CCK and GLP-1 as potential mediators of the increased satiety response to whey and emphasise the importance of considering the impact of protein type on the appetite response to a mixed meal.