The quantitation of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction, and the effect of increased consumption of n-3 polyunsaturated fatty acids

Objective: To examine the effects of the consumption of fish oils on the gene expression of lipoprotein lipase (LPL, EC 3.1.1.34) in human adipose tissue. In order to measure LPL mRNA in adipose tissue samples obtained by needle biopsy from human volunteers a competitive, reverse transcriptase PCR (RT-PCR) protocol was developed. Design: A randomised controlled, single blind cross over dietary study which compared the effects of a low level n-3 polyunsaturated fatty acids (PUFA) using normal foods enriched with eicosapentaenoic (EPA) and docosahexaenoic (DHA) (test diet), with non-enriched but otherwise identical foods (control). The diets were consumed for a period of 22 d with a wash out period of 5 months between the diets. Setting: Free-living individuals associated with the University of Surrey. Subjects: Six male subjects with a mean (±sd) age of 51.2±3.6 y were recruited. Major Outcome Measures: Pre-and postprandial blood samples were taken for the measurement of triacylglycerol (TAG), postheparin LPL activity and adipose tissue samples for the measurement of LPL mRNA levels. Results: Mean LPL expression values were 4.12´105 molecules of LPL mRNA per ng total RNA on the control diet and 4.60´105 molecules of LPL mRNA per ng total RNA on the n-3 PUFA enriched (test) diet. There was no significant difference between the levels of LPL expression following each diet, consistent with the lack of change in TAG levels in response to increased dietary n-3 PUFA intake. However, the change in LPL expression (Test-Control diet) correlated significantly with the change in fasting TAG levels (P=0.03, R=-0.87 and R2=0.75) and with the total area under the TAG-time response curve (P=0.003, R=-0.96 and R2=0.92) in individuals. Conclusions: These findings, although based on a small number of subjects, suggest that LPL expression may be a determinant of plasma TAG levels. The development of this methodology should allow further elucidation of the effects of dietary manipulation and disease processes on lipid clearance and regulation in human subjects.

The effect of test meal monounsaturated fatty acid: saturated fatty acid ratio on postprandial lipid metabolism

Epidemiological evidence shows that a diet high in monounsaturated fatty acids (MUFA) but low in saturated fatty acids (SFA) is associated with reduced risk of CHD. The hypocholesterolaemic effect of MUFA is known but there has been little research on the effect of test meal MUFA and SFA composition on postprandial lipid metabolism. The present study investigated the effect of meals containing different proportions of MUFA and SFA on postprandial triacylglycerol and non-esterified fatty acid (NEFA) metabolism. Thirty healthy male volunteers consumed three meals containing equal amounts of fat (40 g), but different proportions of MUFA (12, 17 and 24% energy) in random order. Postprandial plasma triacylglycerol, apolipoprotein B-48, cholesterol, HDL-cholesterol, glucose and insulin concentrations and lipoprotein lipase (EC 3.1.1.34) activity were not significantly different following the three meals which varied in their levels of SFA and MUFA. There was a significant difference in the postprandial NEFA response between meals. The incremental area under the curve of postprandial plasma NEFA concentrations was significantly (P = 0.03) lower following the high-MUFA meal. Regression analysis showed that the non-significant difference in fasting NEFA concentrations was the most important factor determining difference between meals, and that the test meal MUFA content had only a minor effect. In conclusion, varying the levels of MUFA and SFA in test meals has little or no effect on postprandial lipid metabolism.

Meal frequency; does it determine postprandial lipaemia?

OBJECTIVE: To determine the effect of altering meal frequency on postprandial lipaemia and associated parameters. DESIGN: A randomized open cross over study to examine the programming effects of altering meal frequency. A standard test meal was given on three occasions following: (i) the normal diet; (ii) a period of two weeks on a nibbling and (iii) a period of two weeks on a gorging diet. SETTING: Free living subjects associated with the University of Surrey. SUBJECTS: Eleven female volunteers (age 22 +/- 0.89 y) were recruited. INTERVENTIONS: The subjects were requested to consume the same foods on either a nibbling diet (12 meals per day) or a gorging diet (three meals per day) for a period of two weeks. The standard test meal containing 80 g fat, 63 g carbohydrate and 20 g protein was administered on the day prior to the dietary intervention and on the day following each period of intervention. MAJOR OUTCOME MEASURES: Fasting and postprandial blood samples were taken for the analysis of plasma triacylglycerol, non-esterified fatty acids, glucose, immunoreactive insulin, glucose-dependent insulinotropic polypeptide levels (GIP) and glucagon-like peptide (GLP-1), fasting total, low density lipoprotein (LDL)- and high density lipoprotein (HDL)-cholesterol concentrations and postheparin lipoprotein lipase (LPL) activity measurements. Plasma paracetamol was measured following administration of a 1.5 g paracetamol load with the meal as an index of gastric emptying. RESULTS: The compliance to the two dietary regimes was high and there were no significant differences between the nutrient intakes on the two intervention diets. There were no significant differences in fasting or postprandial plasma concentrations of triacylglycerol, non-esterified fatty acids, glucose, immunoreactive insulin, GIP and GLP-1 levels, in response to the standard test meal following the nibbling or gorging dietary regimes. There were no significant differences in fasting total or LDL-cholesterol concentrations, or in the 15 min postheparin lipoprotein lipase activity measurements. There was a significant increase in HDL-cholesterol in the subjects following the gorging diet compared to the nibbling diet. DISCUSSION: The results suggest that previous meal frequency for a period of two weeks in young healthy women does not alter the fasting or postprandial lipid or hormonal response to a standard high fat meal. CONCLUSIONS: The findings of this study did not confirm the previous studies which suggested that nibbling is beneficial in reducing the concentrations of lipid and hormones. The rigorous control of diet content and composition in the present study compared with others, suggest reported effects of meal frequency may be due to unintentional alteration in nutrient and energy intake in previous studies.

Effects of dietary fatty acid composition on basal and hormonestimulated hepatic lipogenesis and on circulating lipids in the rat

Thirty male rats were randomly assigned to one of three dietary groups in which the source of dietary fat was either a mixed oil, maize oil or fish oil. Effects of dietary fatty acid composition on in virro rates of [U-'4C]glucose incorporation into hepatic total lipids and into hepatic triacylglycerol were measured under basal, insulin (4 nM)-, gastric inhibitory polypeptide (GIP; 6 mi)- and insulin + GIP (4 nM + 6 n ~ ) - stimulated conditions. Effects of the three diets on postprandial plasma triacylglycerol, cholesterol, insulin and GIP concentrations were also measured. The fish-oil diet decreased rates of basal glucose incorporation into hepatic total lipids (P < 0.05) and hepatic triacylglycerol (P < 0.01) compared with the mixed-oil diet. The presence of insulin + GIP in the incubation medium stimulated glucose incorporation into hepatic total lipids in the maize-oil (P < 0.01) and fish-oil groups (P < OW), as well as into hepatic triacylglycerol in the maize-oil group (P < 0.005). In addition, the fish-oil diet decreased postprandial plasma triacylglycerol levels compared with both other dietary groups (P < 0-05 both cases), and the mixed-oil diet markedly increased postprandial plasma insulin levels compared with the other dietary groups (P c 0.001).

Postprandial lipid and hormone responses to meals of varying fat contents: modulatory role of lipoprotein lipase?

OBJECTIVE: Substrate and hormone responses to meals of differing fat content were evaluated in normal subjects in order to investigate mechanisms underlying the regulation of postprandial lipoprotein concentration. DESIGN: A randomised cross-over study with three different meals on three occasions. SETTING: Free-living subjects associated with Surrey University. SUBJECTS: Ten male volunteers (aged 18-23 years) were recruited. INTERVENTIONS: Three test meals containing 20, 40 or 80 g fat but identical carbohydrate and protein content were randomly allocated to volunteers. MAJOR OUTCOME MEASURES: Pre- and postprandial blood samples were taken for the analysis of plasma triacylglycerol, non-esterified fatty acids, glucose, immunoreactive insulin and glucose-dependent insulinotrophic polypeptide levels and postheparin lipoprotein lipase activity measurements. RESULTS: Peak triacylglycerol concentrations and lipoprotein lipase activity measurements were significantly higher following the 80 g than the 20 g fat meal (P = 0.009 and P = 0.049 respectively). Areas under the glucose-dependent insulinotrophic polypeptide time-response concentration curves were significantly higher following the 80 g compared with the 20 g fat meal (P = 0.04), but no differences in insulin response to the meals were seen. The 30-360 min decrease in the non-esterified fatty acid concentration was less following the 80 g than the 20 g meal (P = 0.001). CONCLUSIONS: The results suggest that glucose-dependent insulinotrophic polypeptide may mediate increased lipoprotein lipase activity in response to fat-containing meals and may play a role in circulating lipoprotein homeostasis. This mechanism may be overloaded with high fat meals with adverse consequences on circulating triacylglycerol and NEFA concentrations.

Postprandial lipoprotein lipase, insulin and gastric inhibitory polypeptide responses to test meals of different fatty acid composition: comparison of saturated, n-6 and n-3 polyunsaturated fatty acids

OBJECTIVE: The present study was carried out to investigate effects of meals, rich in either saturated fatty acids (SFA), or n-6 or n-3 fatty acids, on postprandial plasma lipid and hormone concentrations as well as post-heparin plasma lipoprotein lipase (LPL) activity. DESIGN: The study was a randomized single-blind study comparing responses to three test meals. SETTING: The volunteers attended the Clinical Investigation Unit of the Royal Surrey County Hospital on three separate occasions in order to consume the meals. SUBJECTS: Twelve male volunteers with an average age of 22.5 +/- 1.4 years (mean +/- SD), were selected from the University of Surrey student population; one subject dropped out of the study because he found the test meal unpalatable. INTERVENTIONS: Three meals were given in the early evening and postprandial responses were followed overnight for 11h. The oils used to prepare each of the three test meals were: a mixed oil rich in saturated fatty acids (SFA) which mimicked the fatty acid composition of the current UK diet, corn oil, rich in n-6 fatty acids and a fish oil concentrate (MaxEPA) rich in n-3 fatty acids. The oil under investigation (40 g) was incorporated into the test meals which were otherwise identical [208 g carbohydrates, 35 g protein, 5.65 MJ (1350 kcal) energy]. Postprandial plasma triacylglycerol (TAG), gastric inhibitory polypeptide (GIP), and insulin responses, as well as post-heparin LPL activity (measured at 12 h postprandially only) were investigated. RESULTS: Fatty acids of the n-3 series significantly reduced plasma TAG responses compared to the mixed oil meal (P < 0.05) and increased post-heparin LPL activity 15 min after the injection of heparin (P < 0.01). A biphasic response was observed in TAG, with peak responses occurring at 1 h and between 3-7 h postprandially. GIP and insulin showed similar responses to the three test meals and no significant differences were observed. CONCLUSION: We conclude that fish oils can decrease postprandial plasma TAG levels partly through an increase in post-heparin LPL activity, which however, is not due to increased GIP or insulin concentrations.

Polyunsaturated fatty acids of the n-6 and n-3 series: effects on postprandial lipid and apolipoprotein levels in healthy men

OBJECTIVE: The present study was carried out to determine effects of test meals of different fatty acid compositions on postprandial lipoprotein and apolipoprotein metabolism. DESIGN: The study was a randomized, single blind design. SETTING: The study was carried out in the Clinical Investigation Unit of the Royal Surrey County Hospital. SUBJECTS: Twelve male normal subjects with an average age of 22.4 +/- 1.4 years (mean +/- SD) were selected from the student population of the University of Surrey; one subject dropped out of the study because he found the test meal unpalatable. INTERVENTIONS: The subjects were given three evening test meals on three separate occasions, in which the oils used were either a mixed oil (rich in saturated fatty acids and approximated the fatty acid intake of the current UK diet), corn oil (rich in n-6 fatty acids), or fish oil (rich in n-3 fatty acids) 40 g of the oil under investigation were incorporated into a rice-based test meal. Triacylglycerol-rich lipoproteins-triacylglycerol (TRL-TAG), TRL-cholesterol (TRL-cholesterol), plasma-TAG, plasma cholesterol (T-C), and serum apolipoprotein A-I and B (apo A-I and B) responses were measured. Postprandial responses were followed for 11 h. RESULTS: Postprandial plasma-TAG responses, calculated as incremental areas under the response curves (IAUC) were significantly reduced following the fish oil meal [365.5 +/- 145.4 mmol/l x min (mean +/- SD)[ compared with the mixed oil meal (552.0 +/- 141.7 mmol/l x min) (P < 0.05) and there was a strong trend towards the same direction in the TRL-TAG responses. In all instances, plasma-and TRL-TAG showed a biphasic response with increased concentrations occurring at 1h and between 3 and 7h postprandially. TRL-cholesterol, T-C, and serum apo A-I and B responses to the three meals were similar. CONCLUSIONS: The findings support the view that fish oils decrease postprandial lipaemia and this may be an important aspect of their beneficial effects in reducing risk of coronary heart disease (CHD). Further work is required to determine the mechanisms responsible for this effect.

A novel antiserum specific to apolipoprotein B-48: application in the investigation of postprandial lipidaemia in humans

1. Apolipoprotein B-48, the transport protein for chylomicrons, is identical with apolipoprotein B-100 for the first 48% of its sequence. No antiserum has yet been reported that can recognize apolipoprotein B-48, but not apolipoprotein B-100. 2. In the present study an antiserum was raised to the C-terminal sequence of apolipoprotein B-48, using specific chemical reactions to ensure that the charged carboxyl group of the C-terminal isoleucine residue was free. In a Western blot the antiserum was shown to bind to a protein band having the characteristics of apolipoprotein B-48, but not to apolipoprotein B-100. 3. In the early evening 11 subjects were given a test meal which contained 40 g of mixed oil and retinyl palmitate. Blood samples were collected over 9 h. Chylomicron-enriched fractions were prepared and analysed for triacylglycerol, retinyl palmitate and apolipoprotein B-48, the latter after separation using SDS/PAGE and visualization by chemiluminescence on a Western blot. Both triacylglycerol and apolipoprotein B-48 showed an early peak at 1 h, which was not seen with retinyl palmitate. All three substances gave a broader peak between 5 and 6 h postprandially. Retinyl palmitate concentrations declined rapidly during the late (6-9 h) postprandial period, but apolipoprotein B-48 concentrations remained elevated. 4. This study has shown that an antiserum has been produced which is specific for apolipoprotein B-48. This has enabled measurement of postprandial concentrations of the protein that revealed features of chylomicron metabolism which have not been reported previously.

Pretranslational regulation of the expression of the lipoprotein lipase (EC 3.1, l.34) gene by dietary fatty acids in the rat

Although there have been a number of studies of effects of diet and hormones on lipoprotein lipase (EC 3.1.1.34; LPL) activity and levels of LPL mRNA (Raynolds et al. 1990), there have been no studies which have investigated effects of different dietary fatty acids on LPL gene expression. In the present study male Wistar Albino rats were pair-fed diets containing 50 g fat/kg of different fatty acid composition for 2 weeks. The diets fed were (1) a mixed oil (450 g saturated fatty acids, 420 g monounsaturated fatty acids, 130 g polyunsaturated fatty acids/kg; n 8), (2) maize oil (n 8), or (3) fish oil (n 8). Animals were killed, RNA was extracted from liver and perirenal and epididymal fat pads, and analysed by ‘Northern methodology’. Samples were hybridized to a human cDNA probe for LPL (Gotoda et al. 1989). Two transcripts were identified in epididymai and perirenal adipose tissue which were approximately 3·7 and 1·7 kb in size. The results suggested that (1) fish oil-fed animals had significantly greater production of LPL mRNA in epididymai adipose tissue compared with maize oil-fed animals (P < 0·05), (2) maize oil-fed animals had significantly greater production of LPL mRNA in perirenal fat compared with the other dietary groups (P < 0·05), (3) expression in the liver was not significant. Rats fed on a fish oil diet had significantly reduced plasma triacylglycerol concentrations compared with the mixed-oil group (P < 0·05), but there were no significant differences in plasma cholesterol. The differences in LPL could not be explained directly by the changes in plasma immunoreactive-insulin and glucose-dependent insulinotrophic polypeptide levels in the three groups.

Inulin and oligofructose: effects on lipid metabolism from human studies

Although convincing lipid-lowering effects of the fructo-oligosaccharide, inulin, have been demonstrated in animals, attempts to reproduce similar effects in man have produced conflicting findings. This may be because of the much lower doses which can be used due to the adverse gastrointestinal symptoms exhibited by most subjects consuming in excess of 15 g/d. There are nine studies reported in the literature which have investigated the response of blood lipids (usually total and LDL-cholesterol and triacylglycerol) to inulin or oligofructose supplementation in human volunteers. Three have observed no effects of inulin or oligofructose on blood levels of cholesterol or triacylglycerol, three have shown significant reductions in triacylglycerol, whilst four have shown modest reductions in total and LDL-cholesterol. Studies have been conducted in both normo- and moderately hyperlipidaemic subjects. Differences in study outcomes do not appear to be due to differences in the type or dose of oligosaccharides used nor the duration of the studies. Because animal studies have identified inhibition of hepatic fatty acid synthesis as the major site of action for the triacylglycerol lowering effects of inulin and oligofructose, and because this pathway is relatively inactive in man unless a high carbohydrate diet is fed, variability in response may be a reflection of differences in background diet or the experimental foods used.

A sequential two meal challenge reveals abnormalities in postprandial TAG but not glucose in men with increasing numbers of metabolic syndrome components

Objective To examine the impact of increasing numbers of metabolic syndrome (MetS) components on postprandial lipaemia. Methods Healthy men (n = 112) underwent a sequential meal postprandial investigation, in which blood samples were taken at regular intervals after a test breakfast (0 min) and lunch (330 min). Lipids and glucose were measured in the fasting sample, with triacylglycerol (TAG), non-esterified fatty acids and glucose analysed in the postprandial samples. Results Subjects were grouped according to the number of MetS components regardless of the combinations of components (0/1, 2, 3 and 4/5). As expected, there was a trend for an increase in body mass index, blood pressure, fasting TAG, glucose and insulin, and a decrease in fasting high-density lipoprotein cholesterol with increasing numbers of MetS components (P≤0.0004). A similar trend was observed for the summary measures of the postprandial TAG and glucose responses. For TAG, the area under the curve (AUC) and maximum concentration (maxC) were significantly greater in men with ≥ 3 than < 3 components (P < 0.001), whereas incremental AUC was greater in those with 3 than 0/1 and 2, and 4/5 compared with 2 components (P < 0.04). For glucose, maxC after the test breakfast (0-330 min) and total AUC (0-480 min) were higher in men with ≥ 3 than < 3 components (P≤0.001). Conclusions Our data analysis has revealed a linear trend between increasing numbers of MetS components and magnitude (AUC) of the postprandial TAG and glucose responses. Furthermore, the two meal challenge discriminated a worsening of postprandial lipaemic control in subjects with ≥ 3 MetS components.

Role of lipids in human nutrition

Fat is a major contributor to energy intake in most Western diets, supplying 35–40% of food energy. It is described as being ‘energy-dense’, because a gram of fat (9 kcal/g) yields more than twice as much metabolisable energy as a gram of either carbohydrate or protein (4 kcal/g). Most of the fat we consume in our diet is in the form of triacylglycerol (90-95%), with cholesterol and phospholipids making up the bulk of the remainder. Dietary advice invariably stresses the importance of fat reduction, yet fats have diverse roles in human nutrition. They are important as a source of energy, both for immediate utilisation by the body and in laying down a storage depot (adipose tissue) for later utilisation when food intake is reduced, they act as a vehicle for the ingestion and absorption of fat-soluble vitamins, and they have diverse structural and functional roles in the body. Cholesterol is also an essential component of cell membranes and is the precursor for synthesis of hormones. This chapter describes the structure, digestion, transport and functional properties of dietary fat in the body and explains the basis of associations between fat consumption and chronic disease.

The leptin receptor Gln223Arg polymorphism (rs1137101) mediates the postprandial lipaemic response, but only in males.

Objective: An exaggerated postprandial triacylglycerol (TAG) response is an important determinant of cardiovascular disease risk. With increased recognition of the role of leptin in systemic macronutrient metabolism, we used a candidate gene approach to examine the impact of the common leptin receptor (LEPR) Gln223Arg polymorphism (rs1137101) on postprandial lipaemia. Methods and results: Healthy adults (n ¼ 251) underwent a sequential meal postprandial investigation, in which blood samples were taken at regular intervals after a test breakfast (t ¼ 0) and lunch (t ¼ 330 min). Fasting total- and low-density lipoprotein cholesterol were 9% lower in the ArgArg than GlnArg group (P < 0.04), whereas fasting TAG was 27% lower in the ArgArg than GlnGln group (P < 0.02). The magnitude of the postprandial TAG response was also significantly lower in the ArgArg compared with the GlnArg and GlnGln genotypes, with a 26% lower area under the curve (AUC) and incremental AUC in the ArgArg individuals (P � 0.023). Genotype*gender interactions were evident for fasting and postprandial TAG responses (P < 0.05), with the genotype effect only evident in males. Regression analysis indicated that the LEPR genotype and genotype*gender interactions were independent predictors of the TAG AUC, accounting for 6.3% of the variance. Our main findings were replicated in the independent LIPGENE-Cordoba postprandial cohort of metabolic syndrome subjects (n ¼ 75), with a 52% lower TAG AUC in the ArgArg than GlnGln male subjects (P ¼ 0.018). Conclusion: We report for the first time that the common LEPR Gln223Arg genotype is an important predictor of postprandial TAG in males. The mechanistic basis of these associations remains to be determined.

Dietary fat manipulation has a greater impact on postprandial lipid metabolism than the apolipoprotein E (epsilon) genotype: insights from the SATgenε study

Scope: Our aim was to determine the effects of chronic dietary fat manipulation on postprandial lipaemia according to apolipoprotein (APO)E genotype. Methods and results:Men (mean age 53 (SD 9) years), prospectively recruited for the APOE genotype (n = 12 E3/E3, n = 11 E3/E4), were assigned to a low fat (LF), high fat, high-saturated fat (HSF), and HSF diet with 3.45 g/day docosahexaenoic acid (HSF-DHA), each for an 8-week period in the same order. At the end of each dietary period, a postprandial assessment was performed using a test meal with a macronutrient profile representative of that dietary intervention. A variable postprandial plasma triacylglycerol (TAG) response according to APOE genotype was evident, with a greater sensitivity to the TAG-lowering effects of DHA in APOE4 carriers (p ≤ 0.005). There was a lack of an independent genotype effect on any of the lipid measures. In the groups combined, dietary fat manipulation had a significant impact on lipids in plasma and Svedberg flotation rate (Sf) 60–400 TAG-rich lipoprotein fraction, with lower responses following the HSF-DHA than HSF intervention (p < 0.05). Conclusion: Although a modest impact of APOE genotype was observed on the plasma TAG profile, dietary fat manipulation emerged as a greater modulator of the postprandial lipid response in normolipidaemic men.

Does dairy food intake predict arterial stiffness and blood pressure in men? Evidence from the Caerphilly prospective study

Arterial stiffness is an independent predictor of cardiovascular disease events and mortality, and like blood pressure, may be influenced by dairy food intake. Few studies have investigated the effects of consumption of these foods on prospective measures of arterial stiffness. The present analysis aimed to investigate the prospective relationship between milk, cheese, cream, and butter consumption and aortic pulse wave velocity, augmentation index, systolic and diastolic blood pressure, as well as cross-sectional relationships between these foods and systolic and diastolic blood pressure and metabolic markers using data from the Caerphilly Prospective Study. Included in this cohort were 2512 men, aged 45 to 59 years, who were followed up at 5-year intervals for a mean of 22.8 years (number follow-up 787). Augmentation index was 1.8% lower in subjects in the highest quartiles of dairy product intake compared with the lowest (P trend=0.021), whereas in the highest group of milk consumption systolic blood pressure was 10.4 mm Hg lower (P trend=0.033) than in nonmilk consumers after a 22.8-year follow-up. Cross-sectional analyses indicated that across increasing quartiles of butter intake, insulin (P trend=0.011), triacylglycerol (P trend=0.023), total cholesterol (P trend=0.002), and diastolic blood pressure (P trend=0.027) were higher. Across increasing groups of milk intake and quartiles of dairy product intake, glucose (P trend=0.032) and triglyceride concentrations (P trend=0.031) were lower, respectively. The present results confirm that consumption of milk predicts prospective blood pressure, whereas dairy product consumption, excluding butter, is not detrimental to arterial stiffness and metabolic markers. Further research is needed to better understand the mechanisms that underpin these relationships.