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Background and Aims: Adipose tissue (AT) fatty acid (FA) composition is considered to be the gold standard long-term biomarker of dietary fatty acid intake. Typically this measurement is made directly from samples collected via large-needle-biopsy or incision. However, with growing interest in the role of AT in relation to health, ideally the fatty acid composition would be analysed along with other measurements, such as gene expression or histology, on a single AT sample. Here we assess alternative ways of obtaining AT for measuring FA composition, in some cases in conjunction with other measurements., Methods and Results: The FA composition of tissue obtained via different methods was compared to that of tissue collected via large-needle or surgical biopsy. Fatty acid composition was not significantly different in AT collected by small-needle mini-biopsy (n = 10), from an RNA 'lipid layer' (obtained during RNA extraction, 2 sites, n = 6 for each), or from cryosectioned tissue prepared for histology (n = 10). We also assessed the usefulness of the composition of plasma NEFA as a surrogate marker of subcutaneous AT (n = 58-80). Most FAs in plasma NEFA correlated strongly with those in AT (P < 0.05)., Conclusion: It is feasible to measure the FA composition of AT on very small amounts of tissue. Additionally, it is possible to measure FA composition on the lipid rich 'by-product' of AT samples undergoing RNA extraction for gene expression. Samples sectioned for histology are also suitable. This provides further opportunities for multidisciplinary collaborations that may lead to a better application of dietary biomarkers., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: A high dietary protein intake has been shown to blunt the deposition of intrahepatic lipids in high-fat- and high-carbohydrate-fed rodents and humans., Objective: The aim of this study was to evaluate the effect of essential amino acid supplementation on the increase in hepatic fat content induced by a high-fructose diet in healthy subjects., Design: Nine healthy male volunteers were studied on 3 occasions in a randomized, crossover design after 6 d of dietary intervention. Dietary conditions consisted of a weight-maintenance balanced diet (control) or the same balanced diet supplemented with 3 g fructose · kg(-1) · d(-1) and 6.77 g of a mixture of 5 essential amino acids 3 times/d (leucine, isoleucine, valine, lysine, and threonine) (HFrAA) or with 3 g fructose · kg(-1) · d(-1) and a maltodextrin placebo 3 times/d (HFr); there was a washout period of 4 to 10 wk between each condition. For each condition, the intrahepatocellular lipid (IHCL) concentration, VLDL-triglyceride concentration, and VLDL-[(13)C]palmitate production were measured after oral loading with [(13)C]fructose., Results: HFr increased the IHCL content (1.27 ± 0.31 compared with 2.74 ± 0.55 vol %; P < 0.05) and VLDL-triglyceride (0.55 ± 0.06 compared with 1.40 ± 0.15 mmol/L; P < 0.05). HFr also enhanced VLDL-[(13)C]palmitate production. HFrAA significantly decreased IHCL compared with HFr (to 2.30 ± 0.43 vol%; P < 0.05) but did not change VLDL-triglyceride concentrations or VLDL-[(13)C]palmitate production., Conclusions: Supplementation with essential amino acids blunts the fructose-induced increase in IHCL but not hypertriglyceridemia. This is not because of inhibition of VLDL-[(13)C]palmitate production. This trial was registered at www.clinicaltrials.gov as NCT01119989.

Aim: This study investigated the impact of fatty acid (FA) composition on the echo time behavior of triglyceride resonances in a clinical setting. The feasibility of (1)H NMR spectroscopy to detect these resonances was also evaluated in human adipose tissue in vivo., Method: Ten edible oils chosen to cover a wide spectrum of FA compositions were used as phantom material. The detailed FA composition and intrinsic proton spectra of the oils were characterized by gas chromatography and high-resolution (1)H NMR spectroscopy (11.7T), respectively. The detailed echo time behavior of the oils were subsequently measured by (1)H NMR spectroscopy in a clinical scanner (1.5T) using PRESS. The effect of temperature was investigated in five oils., Results: The olefinic (5.3 ppm) and diallylic (2.8 ppm) resonances exhibited distinct J-modulation patterns independent of oil FA composition. The methylene resonance (1.3 ppm) displayed an exponential decay, with the apparent T(2) showing a weak positive correlation with oil unsaturation (R=0.628, P=0.052), probably a result of changes in viscosity. For the methyl resonance (0.9 ppm), oils high in omega-3 FA displayed a markedly different J-modulation pattern compared to non-omega-3 oils. The characteristic J-modulation of the omega-3 methyl group could be attributed to the phase behavior of the omega-3 methyl triplet signal (all triplet lines in-phase at TE of 135 ms), a result of the omega-3 methyl end forming a first order spin system. The omega-3 methyl outer triplet line at 1.08ppm of the TE=140 ms spectrum was found to be useful for determining the omega-3 content of the oils (R=0.999, standard error of estimate (SE) 0.80). The olefinic and diallylic proton resonance (measured at TE=50 ms) areas correlated with the olefinic (R=0.993, SE 0.33) and diallylic (R=0.997, SE 0.19) proton contents calculated from the GC data. Information derived from long echo time spectra (TE=200) demonstrated good correlations to GC data and showed no change with increasing temperature (and T(2)). In (1)H NMR spectra (1.5T) of adipose tissue in five healthy subjects, the analytically important olefinic and diallylic resonances were clearly resolved with a coefficient of variation of 1.6% and 8.4%, respectively, for repeated measurements. The characteristic phase behavior of the omega-3 methyl outer triplet line at 1.08 ppm could also be detected at very long echo times (470 and 540 ms)., Conclusion: Fatty acid composition has an impact on the echo time behavior of triglyceride resonances. Long TE spectra can resolve omega-3 FA in adipose tissue in vivo. These findings will benefit long TE studies of tissue lipids.

Background: Vagal stimulation in response to nutrients is reported to elicit an array of digestive and endocrine responses, including an alteration in postprandial lipid metabolism., Objective: The objective of this study was to assess whether neural stimulation could alter hormone and substrate metabolism during the late postprandial phase, with implications for body fat mobilization., Design: Vagal stimulation was achieved by using the modified sham feeding (MSF) technique, in which nutrients are chewed and tasted but not swallowed. Ten healthy subjects were studied on 3 separate occasions, 4 wk apart. Five hours after a high-fat breakfast (56 g fat), the subjects were given 1 of 3 test meals allocated in random order: water, a lunch containing a modest amount of fat (38 g), or MSF (38 g fat). Blood was collected for 3 h poststimulus for hormone and metabolite analyses., Results: Plasma insulin and pancreatic polypeptide concentrations peaked at 250% and 209% of baseline concentrations within 15 min of MSF. The plasma glucose concentration increased significantly (P = 0.038) in parallel with the changes observed in the plasma insulin concentration. The nonesterified fatty acid concentration was significantly suppressed (P: = 0.006); maximum suppression occurred at a mean time of 114 min after MSF. This fall in nonesterified fatty acid was accompanied by a fall in the plasma glucagon concentration from 122 to 85 pmol/L (P = 0.018) at a mean time of 113 min after MSF., Conclusions: Effects on substrate metabolism after MSF in the postprandial state differ from those usually reported in the postabsorptive state. The effects of MSF were prolonged beyond the period of the cephalic response and these may be relevant for longer-term metabolic regulation.

The objective of this study was to test the hypothesis that increased fatty acid trapping by subcutaneous adipose tissue might contribute to the development and/or maintenance of obesity. To do so, venoarterial (V-A) gradients across subcutaneous adipose tissue for triglycerides, glycerol, nonesterified fatty acid (NEFA), and acylation-stimulating protein (ASP) were determined in eight lean females [body mass index (BMI), 22.2 +/- 0.6] and eight obese females (BMI, 34.4 +/- 3.4). Plasma insulin was also measured at intervals throughout this period. Fasting plasma triglyceride was significantly higher in the obese group and postprandial triglyceride was also significantly delayed. In contrast, both triglyceride clearance and fatty acid uptake by subcutaneous adipose tissue were significantly greater in the obese group compared with the lean group. Fasting insulin did not differ between the groups, but postprandial insulin values were significantly higher in the obese group. The pattern of ASP release from subcutaneous adipose tissue also appeared to differ in that it was significantly greater in the early postprandial period (0;-90 min) in the obese group versus the lean group and this correlated with greater triglyceride clearance during this period. Moreover, there were strong, positive correlations between BMI and the V-A gradient for fasting ASP, the 0- to 90-min area under the curve (AUC) for ASP V-A gradient fasting insulin, and the 0- to 90-min AUC for fatty acid incorporation into adipose tissue. Taken together, these data demonstrate that fatty acid trapping by adipose tissue can be increased even when overall plasma triglyceride clearance is delayed. The postprandial pattern of insulin, in particular, was altered in the obese, although it is certainly possible that differences in ASP release or response could also contribute to increased fatty acid trapping in the obese. The data, therefore, suggest that increased fatty acid trapping by adipose tissue may be a feature of some forms of obesity.

Background: The fatty acid composition of adipose tissue triacylglycerol reflects, but is not identical to, the fatty acid composition of the habitual diet., Objective: We investigated whether the fatty acid composition of adipose tissue is explained by differences between fatty acids in early storage in adipose tissue after a meal., Design: Nine healthy men ate a meal containing several fatty acids. Blood samples were taken for 6 h after the meal from an arterialized hand vein and a vein draining the anterior abdominal subcutaneous adipose tissue., Results: Net storage of fatty acids in adipose tissue occurred between 1 and 4 h after the meal. In relation to the amount fed, storage of fatty acids differed (P < 0. 01) between classes (n-3 polyunsaturated < saturated < n-6 polyunsaturated < monounsaturated); oleic acid was stored in the greatest amounts. These differences agreed closely with published data, except for n-3 polyunsaturated fatty acids. The only individual metabolic step at which significant differences between fatty acids was shown was incorporation of fatty acids into chylomicron triacylglycerol. Differences between fatty acids in rate of extraction from chylomicron triacylglycerol and net uptake into adipose tissue in the postprandial period were significant (P < 0. 01), but not when expressed in relation to proportions in chylomicron triacylglycerol., Conclusions: The characteristic fatty acid pattern of adipose tissue may predominantly reflect the early metabolic handling of different fatty acids. Adipose tissue uptake of n-3 polyunsaturated fatty acids is slow in relation to that of other fatty acids.

Early events in the metabolic processing of dietary triacylglycerol may have an important impact on subsequent development of risk factors for coronary heart disease. We have used structured triacylglycerols containing predominantly stearic or oleic acids at the sn -2 position to probe aspects of the processing of dietary fatty acids presented to adipose tissue in chylomicron-triacylglycerol. Studies were conducted on 14 healthy women who were given meals containing 85 g carbohydrate and 60 g of either of the two structured triacylglycerols in random order. Systemic concentrations and arterio-venous differences across adipose tissue for plasma triacylglycerol and non-esterified fatty acids were measured, together with analysis of the fatty acid composition of the relevant fractions. The stereo-specific structure of the ingested triacylglycerol was largely preserved in chylomicron-triacylglycerol. Systemic concentrations of total and individual non-esterified fatty acids were not significantly different after ingestion of the two fats, nor were their rates of release across adipose tissue. The composition of non-esterified fatty acids released from adipose tissue changed after the meal to reflect more closely the composition of the triacylglycerol ingested, but again no significant differences were observed between the two test meals. There was no detectable release of monoacylglycerol from adipose tissue after either test meal. We conclude that the environment for lipoprotein lipase action in adipose tissue in vivo is likely to be highly organized, such that there is no release of monoacylglycerol, nor preferential uptake or release of fatty acids from chylomicron-triacylglycerol according to the nature or the position within triacylglycerol of the fatty acid.

The objective of this study was to determine whether Acylation Stimulating Protein (ASP) is generated in vivo by human adipose tissue during the postprandial period. After a fat meal, samples from 12 subjects were obtained (up to 6 h) from an arterialized hand vein and an anterior abdominal wall vein that drains adipose tissue. Veno-arterial (V-A) gradients across the subcutaneous adipose tissue bed were calculated. The data demonstrate that ASP is produced in vivo (positive V-A gradient) With maximal production at 3-5 h postprandially. The plasma triacylglycerol (TAG) clearance was evidenced by a negative V-A gradient. It increased substantially after 3 h and remained prominent until the final time point. There was, therefore, a close temporal coordination between ASP generation and TAG clearance. In contrast, plasma insulin and non-esterified fatty acid (NEFA) had an early (1-2 h) postprandial change. Fatty acid incorporation into adipose tissue (FIAT) was calculated from V-A glycerol and non-esterified fatty acid (NEFA) differences postprandially. FIAT was negative during the first hour, implying net fat mobilization. FIAT then became increasingly positive, implying net fat deposition, and overall followed the same time course as ASP and TAG clearance. There was a direct positive correlation between total ASP production and total FIAT (r = 0.566, P < 0.05). These data demonstrate that ASP is generated in vivo by human adipocytes and that this process is accentuated postprandially, supporting the concept that ASP plays an important role in clearance of TAG from plasma and fatty acid storage in adipose tissue.

The objective of this study was to investigate the mobilization of individual fatty acids from human subcutaneous adipose tissue in vivo. Concentrations of individual non-esterified fatty acids were measured in arterialized plasma and in the venous drainage from subcutaneous abdominal adipose tissue in eight normal subjects, after an overnight fast, and for the subsequent 6 h. Whilst the veno-arterial concentration difference for non-esterified fatty acids increased over this period, the relative proportions of different fatty acids remained constant. There was a close relationship between veno-arterial difference and arterialized concentration for the different fatty acids. The arterialized concentration of stearic acid consistently lay above the regression line drawn for unsaturated fatty acids (P = 0.001), probably reflecting lower clearance of stearic acid. The release of individual fatty acids was compared with their prevalence in adipose tissue triacylglycerol in biopsies taken from six subjects. Relative release decreased with increasing chain length, and for a given chain length increased with increasing unsaturation, in agreement with animal studies (P < 0.001 for differences in relative mobilization according to these two factors). The results suggest that the systemic plasma concentration of individual non-esterified fatty acids is determined by their release from adipose tissue, but that the relationship between release and systemic concentration for stearic acid is different from that for other fatty acids. The results confirm, in humans, differences in relative mobilization found previously in animal studies.

Previous studies have noted the presence of an early postprandial peak in plasma triacylglycerol concentration, particularly when successive meals have been consumed. We tested the hypothesis that fat from a previous meal contributes to this early postprandial lipemia. We investigated the effect of consuming a lunch containing 61 g fat 5 h after a breakfast containing 54 g fat. The predominant fatty acids in the first meal, expressed as % by wt of total fatty acids, were 18:2 (linoleic acid), 68%, and 18:1 (oleic acid), 19%. The main fatty acids in the second meal were 18:1 (75%) and 18:2 (8%). After lunch, the early peak (at 50-60 min) in chylomicron triacylglycerol was found to contain a large proportion of 18:2, the main constituent of the first meal, whereas at later time points the chylomicron triacylglycerol fatty acid profile more closely resembled that of the second meal. Control studies in three subjects showed the complete absence of the early peaks in plasma and chylomicron triacylglycerol concentrations when either the lunch was omitted or the first meal was low in fat. The plasma nonesterified fatty acid profile also showed a corresponding peak in 18:2 at 50-60 min, which may represent the release into the plasma of fatty acids arising from the hydrolysis of chylomicron triacylglycerol by adipose tissue lipoprotein lipase.

We tested the hypothesis that when fat is ingested in combination with carbohydrate, direct release of fatty acids into the plasma may occur. Eight normal subjects ingested two meals: high-fat (80 g fat, 80 g carbohydrate, and 18 g protein) and low-fat (< 1 g fat, otherwise closely matched). Over the subsequent 6 h, net fat oxidation was greater after the high-fat meal (20.7 vs 10.6 g, P < 0.01). Plasma nonesterified fatty acid (NEFA) concentrations were markedly suppressed after the low-fat meal, but relatively maintained after the high-fat meal (P < 0.01). The profile of plasma NEFAs changed after the high-fat meal, consistent with entry of meal-derived fatty acids into the NEFA pool. We suggest that after ingestion of combinations of carbohydrate and fat, the action of lipoprotein lipase on chylomicron-triacylglycerol leads to direct release of fatty acids into the plasma and increased fat oxidation.

A sensitive method has been developed to measure specific mono-, di- and triacylglycerol concentrations in human plasma, using thin-layer chromatography and enzymatic assay. The levels of partial acylglycerols in human plasma from fasting subjects were lower than previous reports had suggested and amounted to less than 3% of the total acylglycerols. After heparin injection the plasma monoacylglycerol concentration increased markedly (P < 0.01) while the triacylglycerol concentration decreased significantly (P < 0.001). The plasma diacylglycerol concentration did not change significantly although it increased as a percentage of the total (P < 0.05); after heparin partial acylglycerols accounted for more than 10% of the total. After a high-fat meal the plasma concentrations of di- and triacylglycerol increased approximately two-fold (P < 0.005) but no significant change was observed for mono-acylglycerol. The percentage contribution of partial acylglycerols was unchanged (2.6% fasting, 2.4% postprandially).