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1 From the Oxford Centre for Diabetes, Endocrinology and Metabolism, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom (MDR, BAF, and KNF); the Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, Reading, United Kingdom (KGJ and CMW); and the School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, United Kingdom (LMM).
2 Supported by a grant from the Biotechnology and Biological Sciences Research Council, United Kingdom. 3 Address reprint requests to MD Robertson, Oxford Lipid Metabolism Group, Sheikh Rashid Laboratory, Radcliffe Infirmary, Oxford OX2 6HE, United Kingdom. E-mail: denise.robertson{at}oxlip.ox.ac.uk.
ABSTRACT
Background: n-3 Polyunsaturated fatty acids (PUFAs) have proven benefits for both the development of atherosclerosis and inflammatory conditions. The effects on atherosclerosis may be partly mediated by the observed reduction in fasting and postprandial triacylglycerol concentrations after both acute and chronic n-3 PUFA ingestion.
Objective: The aim of this study was to assess gastric emptying and gastrointestinal hormone release after the consumption of mixed meals rich in n-3 PUFAs or other classes of fatty acids.
Design: Ten healthy women (aged 50–62 y) completed 4 separate study visits in a single-blind, randomized design. On each occasion, subjects consumed 40 g oil rich in either saturated fatty acids, monounsaturated fatty acids, n-6 PUFAs, or n-3 PUFAs as part of a mixed meal. [1-13C]Octanoic acid (100 mg) was added to each oil. Gastric emptying was assessed by a labeled octanoic acid breath test, and concentrations of gastrointestinal hormones and plasma lipids were measured.
Results: Recovery of 13C in breath was enhanced after n-3 PUFA ingestion (P < 0.005). The cholecystokinin response after the n-3 PUFA meal was significantly delayed (P < 0.001), and the glucagon-like peptide 1 response was significantly reduced (P < 0.05).
Conclusion: The inclusion of n-3 PUFAs in a meal alters the gastric emptying rate, potentially as the result of changes in the pattern of cholecystokinin and glucagon-like peptide 1 release.
Key Words: Cholecystokinin • glucagon-like peptide 1 • postprandial responses • breath test • chylomicron • n-3 fatty acids • polyunsaturated fatty acids • gastric emptying • women
INTRODUCTION
Evidence is accumulating that an increased consumption of n-3 polyunsaturated fatty acids (PUFAs) favorably affects atherosclerosis, coronary artery disease, and inflammatory conditions (1). A major factor in atherogenesis is believed to be the triacylglycerol response after a meal (2), as originally proposed by Zilversmit (3). Chronic ingestion of n-3 PUFAs, which are present in large amounts in oily fish, leads to large decreases in both fasting (4) and postprandial triacylglycerol responses (5). This relation has also been shown for the purified long-chain fatty acids eicosapentaenoic acid and docosahexaenoic acid (6). Acute ingestion of specific fatty acids, particularly n-3 PUFAs, also influences postprandial triacylglycerol concentrations (7). The addition of even small amounts (<10 g) of long-chain n-3 PUFAs to a single meal containing fat can significantly reduce the postprandial lipemic response (8). The mechanism of action of acute fatty acid ingestion on postprandial lipemia may include alterations in the rate of gastric emptying, in gut hormones, and in systemic metabolism. These may be mediated via insulin, absorption, chylomicron synthesis and clearance (9), or effects on lipoprotein lipase (8). The present study, however, focuses on the effects of acute n-3 PUFA ingestion on gastric emptying and gut hormone response because this area has not been extensively researched.
Ingestion of triacylglycerol has considerable effects on the digestive tract via the release of an array of gastrointestinal peptides. Of these, glucagon-like peptide 1 (GLP-1) and cholecystokinin can inhibit gastric emptying in a dose-dependent manner (10). Studies in animals (11) and more recently in humans (12,13) have shown the inhibitory effects of n-3 PUFAs on plasma cholecystokinin concentrations, although the effects of this change in cholecystokinin concentrations on gastrointestinal motility remain to be fully resolved. The present study was designed to investigate the acute effects of mixed meals consisting of fatty acids present within different dietary food oils on postprandial lipemia, gastrointestinal hormone release, and gastric emptying by use of the noninvasive [1-13C]octanoic acid breath test (14).
SUBJECTS AND METHODS
Subjects
Ten healthy postmenopausal women [
The experiment was a single-blind, randomized study with 4 separate postprandial study days occurring 1 mo apart. The gastric emptying rate of the dietary fats was determined by using the [13C]octanoic acid breath test (14), with simultaneous measurement of both gastrointestinal hormones and plasma lipids.
Experimental procedure
All studies were performed at 0800, after the subjects had consumed a standardized low-fat evening meal (<10 g fat) and had fasted overnight. In the morning, an indwelling intravenous cannula was inserted into an antecubital vein with the use of a local anesthetic (1% lignocaine). Two fasting blood and end-expired air samples were collected during the 10 min before the meal. At time zero, subjects were given 1 of 4 test meals (Table 1) consisting of cereal, banana, and a warm chocolate drink containing 40 g test oil. The test oils were chosen to provide meals enriched with either saturated fatty acids (SFAs; palm oil), n-6 PUFAs (safflower oil; Anglia Oils Ltd, Hull, United Kingdom), n-3 PUFAs (EPAX 3000TG; Pronova Biocare, Aaslund, Norway), or monounsaturated fatty acids (MUFAs; olive oil; Tesco, Cheshunt, United Kingdom). The EPAX 3000TG fish oil was diluted (1:1) with safflower oil to reduce the proportion of SFAs in the n-3 PUFA test meal and to improve the palatability of the dietary fish oil. One hundred milligrams [1-13C]octanoic acid (99 atom%; Masstrace, Woburn, MA) was added to each test oil. All test meals were fully consumed within 15 min, and the subjects remained in the supine position throughout. Blood and end-expired breath samples were taken at regular intervals for 180 min after the test meal (240 min for triacylglycerol analysis).
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TABLE 1 . Fatty acid composition of the test meals1
Analyses
Plasma measurements
Blood for plasma triacylglycerol analysis was collected in heparin-treated syringes (Sarstedt, Leicester, United Kingdom). A chylomicron-rich (Sf > 400) fraction was prepared from plasma by density-gradient ultracentrifugation (15) with a SW40Ti swing-out rotor (Beckman Instruments, High Wycombe, United Kingdom). The chylomicron fraction was separated by aspiration. Chylomicron triacylglycerol was analyzed enzymatically (Instrumentation Laboratory, Warrington, United Kingdom) with a centrifugal analyzer. Plasma for 3-hydroxybutyrate determination was deproteinized with 7% (wt:vol) perchloric acid, and concentrations were measured enzymatically. The interassay CVs for all metabolite assays were <2%.
Blood for cholecystokinin, GLP-1, gastric insulinotropic polypeptide (GIP), and peptide YY (PYY) analysis was collected into potassium-EDTA coated tubes containing 200 kIU aprotinin/mL whole blood (Bayer plc, Newbury, United Kingdom). Plasma cholecystokinin concentrations were analyzed by using a commercially available radioimmunoassay (RIA; Eurodiagnostica, Boldon, United Kingdom) after ethanol extraction (16). The antibody used was specific for all sulfated forms of the peptide, exhibiting only a 0.5% cross-reactivity with sulfated gastrin 17. Plasma PYY concentrations were analyzed by using a commercially available RIA (Peninsula Laboratories, St Helens, United Kingdom) after solid-phase extraction (Waters, Milwaukee). The detection limit of the assay was 1 pmol/L. GLP-1 was assayed on unextracted plasma according to Elliott et al (17) by a double-antibody disequilibrium RIA method. The antiserum used in this assay exhibited no detectable cross-reactivity with GIP, GLP-2, glucagon, vasoactive intestinal peptide (VIP), secretin, or motilin. It was specific for the C-terminal amidated form of GLP-1, cross-reacting 100% with GLP-1 (the 7–36 amide). The detection limit for this assay was 5 pmol/L. GIP was assayed by RIA as previously described by Morgan et al (18). The cross-reactivity of this antiserum with VIP and glucagon was negligible. The detection limit of this assay was 24 pmol/L. The interassay CVs for all RIAs were <10%.
Vacutainers (Isochem, Wokingham, United Kingdom) of 12-mL breath samples were analyzed for 13C enrichment by isotope ratio mass spectrometry in an ANCA-TG machine (PDZ- Europa Ltd, Crewe, United Kingdom). Isotopic enrichments were expressed as delta relative to a working carbon dioxide standard calibrated against a certified reference standard.
Measurement of gastric emptying
Gastric emptying variables of the fat phase of the meal were calculated by using the [13C]octanoic acid breath test (14). Validation of the model for use in solid-emptying studies is described in detail elsewhere (19–22). This method has been used successfully to measure the gastric emptying rates of fat-rich liquids in both humans (23,24) and animals (22). Labeled octanoic acid, a medium-chain fatty acid, was added to the lipid component of each test meal. When this fatty acid leaves the stomach and enters the duodenum, it is rapidly absorbed and is transported in portal vein plasma without the need for either digestion by lipase or chylomicron formation. The medium-chain fatty acid is preferentially oxidized by the mitochondria of the liver, heart, and kidney (25) rather than being stored as fat (26), and the 13C label appears in expired breath. The time taken for the 13C to appear in breath represents the time taken for the label to leave the stomach and become oxidized to carbon dioxide. The 13C label is known to be diluted within the body glutamine and bicarbonate pools, which would certainly affect the pattern of recovery in breath, although recent studies now suggest that this is constant in an individual consuming a habitual diet (27). Endogenous carbon dioxide production during the study was assumed to be constant (14) at 5 mmol•L CO2-1•m-2 body surface area•min-1 (28). The change in the ratio of 13C to 12C in carbon dioxide was determined as the difference above baseline compared with the international standard for 13C abundance (Pee Dee Belemnite). The results are expressed as both % 13C recovery/min and cumulatively over 180 min.
The cumulative excretion of the 13C label is described by
RESULTS
The apparent palatability of the test meals did not differ as a result of the use of a highly deodorized fish oil. The protocol adopted for this study proved acceptable to the subjects, and all elements of the protocol were completed.
Gastric emptying of [13C]octanoic acid
The mean rate of recovery of 13C in breath after co-ingestion of labeled octanoic acid with the 4 test oils is shown in Figure 1A. In all subjects and after all meals, the recovery pattern of 13C was characterized by a lag period followed by a period of almost constant recovery. The initial rate of label recovery was identical for the 4 test meals, and the time to reach peak recovery was similar. However, the peak recovery rate and the rate of recovery during the postlag period were significantly different for the n-3 PUFA meal (P < 0.005). When the model was fitted to the cumulative 13C recovery data (Figure 1), the fit for all data sets was equally good (SFA meal, mean r2 = 0.995; n-6 PUFA meal, mean r2 = 0.998; n-3 PUFA meal, mean r2 = 0.996; and MUFA meal, mean r2 = 0.996).
FIGURE 1. . Mean (±SEM) rate of recovery of 13CO2 in breath (A) and cumulative recovery of 13C (B) after the co-ingestion of [1-13C]octanoic acid with oils rich in n-3 polyunsaturated fatty acids (), n-6 polyunsaturated fatty acids (), monounsaturated fatty acids (•), or saturated fatty acids (). n = 10. Repeated-measures ANOVA showed a significant meal effect (P = 0.005) and a significant meal x time interaction (P < 0.001).
Summarized in Table 2 are the mean variables for the gastric emptying of the fat phase of the meal. The duration of the lag phase did not differ significantly between the 4 test meals. The half-emptying time was significantly lower after the n-3 PUFA meal than after the other test meals (SFA, P = 0.005; MUFA, P = 0.008; and n-6 PUFA, P = 0.002). The mean gastric emptying rate of the label during the postlag period also differed significantly between the test meals (P = 0.05). The SFA, n-6 PUFA, and MUFA test meals did not differ significantly from one another for any of the gastric emptying variables measured in this study.
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TABLE 2 . Kinetic variables for the gastric emptying of the fat phase of a meal as calculated from the cumulative recovery of 13C in breath after dosing with [1-13C]octanoic acid 1
Plasma hormones
The postprandial cholecystokinin response after the 4 test meals is illustrated in Figure 2. Each response was characterized by a biphasic peak that failed to return to baseline by the end of the study. After the n-6 PUFA, SFA, and MUFA meals, the peak concentration was reached 30 min postprandially. After the n-3 PUFA meal, however, this peak was delayed to 120 min postingestion (P < 0.001). Despite the differences in the pattern of cholecystokinin release, there was no overall difference in the IAUC for cholescystokinin between the test meals (Table 3).
FIGURE 2. . Mean (±SEM) plasma cholecystokinin (CCK) concentrations after meals rich in n-3 polyunsaturated fatty acids (), n-6 polyunsaturated fatty acids (), monounsaturated fatty acids (•), or saturated fatty acids (). n = 10. Repeated-measures ANOVA showed a significant meal effect (P = 0.042) and a significant meal x time interaction (P < 0.001).
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TABLE 3 . Summary data for gastrointestinal hormone release after ingestion of the test meals1
The GLP-1 concentration after all test meals rose quickly to a peak 30 min after the meal. The peak concentration reached was higher for the MUFA meal, although by the end of the study the GLP-1 concentration after the n-3 PUFA meal was significantly lower than those for the other oils (Figure 3). Plasma PYY and GIP data are summarized in Table 3. Postprandial PYY concentrations reached approximately double the fasting concentration by 30 min for all test meals and remained at this level for the duration of the study. Maximal GIP concentrations were achieved 120 min postingestion for all test meals, with declining concentrations by 180 min. No significant differences were detected for either the postprandial PYY or GIP responses between the test meals, although there was a trend for the GIP concentration to be higher after the MUFA meal. The SFA, n-6 PUFA, and MUFA meals did not differ significantly from one another for any of the gastrointestinal hormones measured in this study.
FIGURE 3. . Mean (±SEM) plasma glucagon-like peptide 1 (GLP-1) concentrations after meals rich in n-3 polyunsaturated fatty acids (), n-6 polyunsaturated fatty acids (), monounsaturated fatty acids (•), or saturated fatty acids (). n = 10. Repeated-measures ANOVA showed a significant time effect (P < 0.001) and a significant meal x time interaction (P < 0.05).
Plasma lipids
After n-3 PUFA ingestion, there was an early peak in chylomicron triacylglycerol that was not observed with the other 3 meals (P = 0.014; Figure 4). Plasma 3-hydroxybuytrate concentrations fell after the ingestion of all 4 meals, although no significant differences were noted (data not shown).
FIGURE 4. . Mean (±SEM) chylomicron triacylglycerol (TG) concentrations after meals rich in n-3 polyunsaturated fatty acids (), n-6 polyunsaturated fatty acids (), monounsaturated fatty acids (•), or saturated fatty acids (). n = 10. Repeated-measures ANOVA showed a significant time effect (P < 0.001) and a significant meal x time interaction (P = 0.05).
DISCUSSION
Using the [1-13C]octanoic acid breath test, we showed that the gastric emptying of a fat-rich liquid consumed as part of a mixed meal enriched in n-3 PUFAs was significantly faster than that for meals rich in either SFAs, MUFAs, or n-6 PUFAs. Furthermore, we found that this n-3 PUFA–enriched meal resulted in both a slower release of cholecystokinin and a lower postprandial GLP-1 response, both of which have been implicated in the nutrient-induced slowing of gastric emptying.
Nutrient-induced feedback from the both the small and large intestine is important in the regulation of gastric function and ultimately the gastrointestinal handling of a nutrient load. In the present study, we found that the gastric emptying rate of the SFA, MUFA, and n-6 PUFA components compared well with the gastric emptying rate of a similar load of olive oil administered as part of a mixed meal (31) when measured by using scintigraphy (tlag: 56 min; t50: 206 min).
Cholecystokinin release after fat ingestion is regulated by lipase-dependent fat hydrolysis (32), leading to gallbladder contraction (33), release of bile and pancreatic enzymes into the duodenum, and the inhibition of gastric emptying (34). In this study, we found that the cholecystokinin response to the MUFA, n-6 PUFA, and SFA test meals was biphasic, consisting of an early peak followed by a sustained response. The second phase of cholecystokinin release probably contributes to the maintained slowing of gastric emptying and may be important for the gastric handling of a sequential meal. The most significant observation, however, was the lower initial peak in cholecystokinin after the n-3 PUFA meal. The [13C]octanoic acid data suggest that this early peak is likely of primary importance for the inhibition of gastric emptying.
In a study by Riber et al (12), the intragastric administration by tube of an n-3 PUFA fish oil resulted in a significant reduction in both cholecystokinin release and gall bladder contraction when compared with a MUFA-rich oil, although no effects on gastric emptying were found. The lack of an effect on gastric emptying in that study is surprising because the cholecystokinin response was virtually eliminated, yet may be partially explained by differences in the delivery of the n-3 PUFA oil. We administered the n-3 PUFA oil orally as part of a mixed meal as opposed to as a single oil in the study by Riber et al. There is extensive evidence for the layering of oil on top of the gastric contents (31,35), which becomes more pronounced once the products of fat digestion reach the small intestine (36) as a result of the reduced contractile activity in the gastric antrum (37,38). In the absence of other gastric contents, either solid or aqueous, cholecystokinin would have been unable to initiate the so-called layering of the test oil, and inhibitory effects of the n-3 PUFA on gastric motility may have been masked.
Triacylglycerol digestion is critical for cholecystokinin release and the inhibition of gastric emptying, as was shown recently in a study using orlistat, a lipase inhibitor (39). The slower cholecystokinin response after the n-3 PUFA–enriched meal might have been reflected by a lower plasma chylomicron triacylglycerol concentration, because chylomicron assembly is viewed by some as an integral step in cholecystokinin release (34). This was not observed in our study. After n-3 PUFA meal ingestion, chylomicron triacylglycerols actually peaked more rapidly than after the other 3 test meals. However, chylomicron concentrations in the plasma are not only the result of direct synthesis; differential effects of lipoprotein lipase may also be important for chylomicron clearance. It is not known which aspect of chylomicron assembly is involved in cholecystokinin release and the inhibition of gastric emptying. Chylomicrons in the plasma and lymph show structural similarities to the original triacylglycerol present within the gut lumen (40); thus, it is possible that the delay in cholecystokinin response may be due to the configuration of the n-3 PUFA chylomicrons.
In vitro studies using an STC-1 murine cell line also showed that long-chain fatty acids can trigger cholecystokinin release directly from enteroendocrine cells (41). It is possible that the differences in postprandial cholecystokinin response after the n-3 PUFA meal may have been due to the rate of intraluminal lipolysis (42,43) and release of free fatty acids or the ability of these very-long-chain free fatty acids to stimulate cholecystokinin release. It has long been claimed that fatty acids with >12 carbons are equipotent for the release of cholecystokinin and inhibition of gastric emptying (44). Although more recent studies have confirmed this early observation (34,38), few studies have considered fatty acids with chain lengths of >18.
Chronic n-3 PUFA ingestion has been associated with a lowering of plasma triacylglycerol through changes in mitochondrial fatty acid oxidation, although evidence for the effects of a single n-3 PUFA meal on plasma lipid concentrations is less convincing. In this study, neither the concentrations of 3-hydroxybuytrate (a marker of fatty acid oxidation) nor the plasma concentrations of triacylglycerol differed significantly between the 4 meals (data not shown), despite a more rapid gastric emptying of the n-3 PUFA meal. This finding is in contrast with the results of earlier studies (8).
GLP-1 is released from the endocrine L-cells of the distal gut and has been shown to be not only a physiologic regulator of motor activity in the antro-pyloro-duodenal region (45) but also an inhibitor of gastric lipase secretion (46). In this study, the n-3 PUFA meal induced a significantly lower integrated GLP-1 response than did the MUFA-rich meal (Table 3). This is consistent with the results of other human studies (47,48). In vitro studies using rat fetal intestinal cultures (49) showed that MUFAs with >14 carbons induce a 2–3-fold increase in GLP-1 production. One of the triggers for the early release of GLP-1 in animals is gastrin-releasing peptide via a neural reflex (50). In the small intestine, neuronal gastrin-releasing peptide was found to be colocalized with cholecystokinin on the same enteric neurons (51). In addition, cholecystokinin stimulates the release of bile acids into the gastrointestinal tract, which have also been implicated as a GLP-1 releasing factor during perfusion studies (52). The effects of n-3 PUFAs on both cholecystokinin and GLP-1 release may therefore imply a degree of coordination between the 2 hormone systems. Direct luminal contact of fatty acids with the endocrine cells themselves is also an important potential trigger for GLP-1 (53). GLP-1 and PYY are known to be colocalized in the endocrine L-cells and are often secreted together after luminal stimulation by nutrients (54). In this study, however, the 2 gut peptides were not secreted together. Such differential release has been noted before with short-chain fatty acids (52), potentially because of differences in posttranslational expression of the genes involved.
In summary, our study of physiologic mixed meals rich in different fatty acids showed that n-3 PUFAs are less able to trigger both cholecystokinin and GLP-1, resulting in a more rapid gastric emptying of the fat load. There is a more rapid appearance of chylomicron triacylglycerol in the plasma after an n-3 PUFA meal, although the degree of lipemia (represented by the IAUC) is unaffected.
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