Lipids in Health and Disease BioMed Central

Background: Docosahexaenoic acid (22:6n-3, DHA) and n-6 docosapentaenoic acid (22:5n-6, DPAn-6) are highly unsaturated fatty acids (HUFA, ≥ 20 carbons, ≥ 3 double bonds) that differ by a single carbon-carbon double bond at the Δ19 position. Membrane 22:6n-3 may support skeletal muscle function through optimal ion pump activity of sarcoplasmic reticulum and electron transport in the mitochondria. Typically n-3 fatty acid deficient feeding trials utilize linoleic acid (18:2n-6, LA) as a comparison group, possibly introducing a lower level of HUFA in addition to n3 fatty acid deficiency. The use of 22:5n-6 as a dietary control is ideal for determining specific requirements for 22:6n-3 in various physiological processes. The incorporation of dietary 22:5n-6 into rat skeletal muscles has not been demonstrated previously. A one generation, artificial rearing model was utilized to supply 22:6n-3 and/or 22:5n-6 to rats from d2 after birth to adulthood. An n-3 fatty acid deficient, artificial milk with 18:2n-6 was supplemented with 22:6n-3 and/or 22:5n-6 resulting in four artificially reared (AR) dietary groups; AR-LA, AR-DHA, AR-DPAn-6, ARDHA+DPAn-6. A dam reared group (DAM) was included as an additional control. Animals were sacrificed at 15 wks and soleus, white gastrocnemius and red gastrocnemius muscles were collected for fatty acid analyses. Results: In all muscles of the DAM group, the concentration of 22:5n-6 was significantly lower than 22:6n-3 concentrations. While 22:5n-6 was elevated in the AR-LA group and the AR-DPAn-6 group, 20:4n-6 tended to be higher in the AR-LA muscles and not in the AR-DPAn-6 muscles. The AR-DHA+DPAn-6 had a slight, but non-significant increase in 22:5n-6 content. In the red gastrocnemius of the AR-DPAn-6 group, 22:5n-6 levels (8.1 ± 2.8 wt. %) did not reciprocally replace the 22:6n-3 levels observed in AR-DHA reared rats (12.2 ± 2.3 wt. %) suggesting a specific preference/requirement for 22:6n-3 in red gastrocnemius. Conclusion: Dietary 22:5n-6 is incorporated into skeletal muscles and appears to largely compete with 22:6n-3 for incorporation into lipids. In contrast, 18:2n-6 feeding tends to result in elevations of 20:4n-6 and restrained increases of 22:5n-6. As such, 22:5n-6 dietary comparison groups may be useful in elucidating specific requirements for 22:6n-3 to support optimal health and disease prevention. Published: 25 April 2007 Lipids in Health and Disease 2007, 6:13 doi:10.1186/1476-511X-6-13 Received: 26 March 2007 Accepted: 25 April 2007 This article is available from: http://www.lipidworld.com/content/6/1/13 © 2007 Stark et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Background
Many studies have reported beneficial effects of exercise in reducing risk factors for a range of diseases including cardiovascular disease (CVD). Until recently the majority of studies have examined the effects of a chronic exercise intervention on fasting CVD risk factors. Although the results from such studies have been variable the most consistent findings are that an exercise intervention leads to an increase in HDL cholesterol and a reduction in fasting triacylglycerol [1][2][3]. Numerous studies have observed that performing a single session of exercise leads to a reduction in both fasting and postprandial triacylglycerol concentration following consumption of a high fat load on the day subsequent to the exercise session [4,5].
We have reported that obese, insulin resistant individuals exhibit elevated fasting and postprandial chylomicron particle number [6] and there is increasing evidence that chylomicron remnants are able to penetrate, and hence contribute cholesterol to the arterial wall [7]. It is possible that the mechanisms responsible for the reduction in fasting and postprandial triacylglycerol concentrations following exercise also lead to a reduction in chylomicron concentration. The exercise-induced reduction in postprandial lipaemia may be due, at least in part, to an increased lipolysis or clearance of chylomicron-associated triacylglycerol. As a consequence of this improved chylomicron metabolism we may also predict a decrease in chylomicron particle number following exercise. In support of this the total lipoprotein concentration in a chylomicron rich lipoprotein fraction has been reported to be reduced following exercise [8]. However the interpretation of such measurements may be confounded by the presence of hepatically derived lipoproteins in the chylomicron rich fraction and/or the existence of chylomicron particles in other, more dense, lipoprotein fractions. By specifically measuring apolipoprotein (apo) B48 concentration in serum we are able to determine the specific effect of exercise on chylomicron particle number.
We have aimed to investigate whether a single session of exercise of the duration and intensity previously reported to reduce fasting and postprandial triacylglycerol concentrations also leads to a reduction in chylomicron particle number and hence may further reduce CVD risk. In order to allow comparison to the previous studies we have also used young healthy subjects who are not overweight or obese.

Dietary Standardisation
The dietary standardisation for three days prior to each postprandial test day was well adhered to. Subjects replicated their diet for the subsequent dietary standardisation with no difference in macronutrient composition and only minor variations in the reported time of consumption of various meals (Table 2).

Exercise Session
During the exercise session subjects exercised at an average RPE level of 13.3 ± 0.3 which was within the requested level of 12-14 and corresponds to the descriptor "somewhat hard". The average energy expenditure during the total 90 minutes of exercise was 2426 ± 230 kJ. Average heart rate during the exercise session was 123.3 ± 4.7 beats/min which corresponds to an average HR max of 65 ± 2.2%. Blood lactate concentration during the exercise session was on average 4.2 ± 0.6 mM.

Fasting and Postprandial Lipid Measures
The fasting serum apo B48 concentration was not affected by prior exercise (Figure 1, Table 3). Following consumption of the test meal no change in either the total or incremental areas under the postprandial apo B48 curves was observed ( Figure 1, Table 3).
The fasting triacylglycerol concentration was reduced by 16% following the exercise session (P < 0.05; Figure 1, Table 3). Following consumption of the test meal there was a trend towards a reduction in the total area under the postprandial triacylglycerol curves following exercise (23%, P = 0.053, Figure 1, Table 3). The incremental area under the triacylglycerol curve (area under the curve corrected for baseline concentration) was not affected by prior exercise.
Following the exercise session there was no difference in fasting total-, LDL-or HDL-cholesterol concentrations compared to control measurements (Table 3). Postprandial cholesterol concentrations were also not affected by prior exercise (data not shown).
Measures of insulin sensitivity were not affected by prior exercise with no change in fasting insulin and glucose concentrations or HOMA score after exercise (Table 3). Following the mixed meal insulin concentration increased rapidly before returning to baseline at 4 h. However both the total and incremental areas under the postprandial insulin curves were not affected by prior exercise ( Figure 2, Table 3).
The fasting serum NEFA concentration was not different following exercise compared to control. Following consumption of the mixed moderate fat meal, serum NEFA concentrations were initially suppressed at 1-2 h before increasing and remaining elevated at 8 h postprandial ( Figure 2). The total area under the postprandial NEFA curve was not affected by prior exercise (Table 3). Incremental area under the curve was not calculated as a measure of postprandial NEFA as this curve displayed a suppression below baseline ( Figure 2).

Discussion
In agreement with a range of previous studies we report that a single session of moderate intensity exercise decreases fasting triacylglycerol concentration on the subsequent day. There was a trend towards a significant decrease in postprandial triacylglycerol concentration fol-lowing the moderate fat mixed meal; however when postprandial triacylglycerol concentrations were corrected for their corresponding fasting concentration, no decrease was observed. Despite the improvement in triacylglycerol concentration there was no reduction in fasting or postprandial chylomicron particle number. Insulin sensitivity, measured by HOMA score, and NEFA levels in either the fasting or postprandial states were also not altered in this group of subjects.
The extent of the reduction in fasting triacylglycerol concentration observed in the present study was 16% which is comparable to that reported in other studies [9-12]. In the fasting state the majority of circulating triacylglycerol resides associated with VLDL particles, with chylomicron associated triacylglycerol making only a small contribution [13]. Therefore the observed reduction in fasting triacylglycerol concentration is likely due to a reduction in the circulating concentration of VLDL associated triacylglycerol, a notion strengthened by the observation that fasting chylomicron particle number remained unchanged following exercise.
The majority of studies reporting a significant reduction in postprandial triacylglycerol concentration in response to prior exercise have utilised a relatively high (≥ 1 g fat/kg body weight) oral fat load on the postprandial test day [9-11,14,15]. However the physiological relevance of such a fat load (representing >60% fat by energy intake) to a typical western diet is questionable. With this in mind in the present study we have chosen an oral fat load containing a moderate fat content (0.44 g/kg body weight; 45% total energy) being more typical of a meal that may be consumed as part of a western diet. Our analysis of the postprandial triacylglycerol concentrations following exercise revealed the total area under the postprandial triacylglycerol curve was reduced (with a trend towards significance). However when corrected for fasting triacylglycerol concentration, as measured by incremental area under the postprandial triacylglycerol curve no change was observed following exercise. Hence the reduction in total area under the postprandial triacylglycerol curve results primarily from the decrease in fasting triacylglycerol concentration. A similar observation was made by Kolifa et al [16] who also utilised a fat load of moderate fat content (0.66 g/kg body weight; 35% total energy). In contrast, studies where a higher fat load (≥ 1 g/kg body weight) was given, have reported changes in both the total and incremental areas under the postprandial triacylglycerol curves following exercise [9-11,14,15].
An improved action of lipoprotein lipase (LPL) has been proposed as a mechanism whereby triacylglycerol concentrations are reduced following exercise. Indeed LPL activity is higher in endurance trained athletes compared to sedentary controls [17,18]. However improvements in LPL activity following a single session of moderate intensity exercise are lower in magnitude [19] and may only contribute to part of the observed reductions in postprandial lipaemia [20]. In light of the present findings we can speculate about the involvement of an increased LPL activity on circulating triacylglycerol concentration. If we assume that LPL activity is limiting the hydrolysis of VLDL particles, and that following exercise an increased LPL Shown are means and standard errors (in brackets) for each parameter. Abbreviations: BMI, Body mass index.   Table 3. lomicron associated triacylglycerol concentration, particularly as there is evidence that chylomicrons are hydrolysed preferentially over VLDL [21,22]. However our findings suggest that this is not the case as an improved hydrolysis of chylomicron-associated triacylglycerol would likely result in a reduction in postprandial chylomicron particle number following exercise (assuming chylomicron remnant uptake is not limiting). In other studies the effect of prior exercise on chylomicron metabolism has produced variable results. It has been reported that prior exercise is associated with a reduction in chylomicron concentration in a chylomicron rich (Sf>400) lipoprotein fraction postprandially either by measuring triacylglycerol concentration [15] or particle number (apo B48) [8]. However in another study, prior exercise did not significantly reduce triacylglycerol concentration in this chylomicron rich fraction [11]. A reduction in the concentration of triacylglycerol in VLDL fractions has been consistently reported and represents the majority of the observed reduction in postprandial triacylglycerol concentration [8,11,15]. In the present study we have measured chylomicron particle number directly in serum, hence allowing us to determine the total concentration of chylomicron particles in all lipoprotein fractions. Our observation that prior exercise does not reduce fasting or postprandial concentration of apo B48 is consistent with the above findings and suggests that the majority of the triacylglycerol lowering effect of exercise involves reductions in VLDL-associated triacylglycerol. Chylomicron

Conclusion
In conclusion, we found that the concentration of proatherogenic chylomicra and postprandial chylomicron response was not influenced by a single session of moderate intensity exercise. This finding contrasts with the sig-  Table 3.
nificantly lower concentration of fasting plasma triglyceride observed following a single session of exercise. We found no evidence that moderate intensity exercise significantly reduced postprandial triacylglycerolemia following a physiologically relevant dose of ingested fat. Collectively, our results and those of others show that triacylglycerol is a poor surrogate marker of chylomicron homeostasis which may be important when considering cardiovascular risk factors.

Subjects
Eight healthy male and female subjects, not overweight or obese, aged 29.8 ± 2.0 years were recruited (n = 5 male, and 3 female). Exclusion criteria included smoking within two years prior, liver or endocrine dysfunction, malabsorption syndrome, anaemia, hypothyroidism, and the use of lipid lowering or hypertensive agents. Diabetes was excluded based on fasting serum glucose being less than 7 mM. Subjects with total serum cholesterol greater than 6 mM or LDL-cholesterol greater than 4 mM were excluded to avoid the potential confounder of genetic hyperlipidemia. We also screened to exclude insulin resistant (assessed by homeostasis model assessment (HOMA) see below; HOMA score >2) [28], overweight or obese (BMI >25 kg/m 2 ; waist circumference >100 cm). Informed consent was obtained from all subjects. All procedures were approved by the Human Ethics Committee, Curtin University and conformed to the Helsinki Declaration.

Design
The study was a randomised crossover design where subjects were required to attend two postprandial testing days. In the afternoon of the day prior to one of the postprandial testing days the subjects performed 90 minutes of moderate intensity exercise. On the day preceding the control postprandial test day the subjects did not perform any exercise. For the female subjects there was an interval of four weeks between each postprandial test day to minimise the confounding effects of menstrual status on lipid metabolism [29].

Dietary Standardisation
There is evidence that a subject's prior diet may influence the ensuing fasting and postprandial lipid measurements [30,31]; it is therefore important to standardise their dietary intake. In this study we provided the subjects with a standardised meal based on the Australian Guide to Healthy Eating [32], allowing for variations in subject's energy needs by providing a range of "snack" options. This diet was provided for the three days prior to the postprandial test days. All the food to be consumed for each of the three days was pre-packaged and given to the subjects in order to increase dietary compliance. During the first dietary standardisation period the subjects completed a dietary checklist and the meal composition and timing was replicated exactly on the second occasion. Furthermore on the evening prior to the postprandial testing a standard low-fat evening meal was consumed at 7:30 pm (<10 g fat). No other food was consumed following the evening meal.

Exercise Session
At 3 pm on the day prior to one of the postprandial test days, subjects performed a 90 minute moderate intensity exercise session. The exercise consisted of an initial five minutes of treadmill "familiarisation" followed by three 30 minute treadmill sessions with five minutes of rest between each session. Borg Scale ratings of perceived exertion (RPE) were collected every 5 minutes, and based on these results the treadmill incline and speed were adjusted to keep the subject within the ratings 12-14 (which corresponds to "somewhat hard but still feels okay to continue") [33]. In addition heart rate was measured every five minutes (by short range telemetry) using Polar heart rate monitors however subjects were blinded to their heart rate so as not to influence their RPE. Energy expenditure was calculated by formula according to American College of Sports Medicine [34].
Capillary blood samples were taken at 25 minutes during each of the three exercise sessions for blood lactate determination. Briefly 10 to 25 μL of capillary blood was collected via fingerprick for blood lactate measurements. Measurements were conducted employing a dry-chemistry methodology according to the manufacturer's instructions using an Accusport lactate meter (type 1488767, Boehringer Mannheim) and BM-Lactate Test strips.

Postprandial lipoprotein assessment
Subjects fasted for 12 hours prior to the start of their postprandial lipoprotein assessment day. In the morning upon arrival at the clinical rooms the subject's body weight, height, waist, and hip circumference were measured following standardised procedures and using a single trained observer.
Fasting blood samples were collected prior to the subjects consuming the moderate fat mixed meal. The moderate fat mixed meal consisted of a breakfast cereal (Uncle Toby's Sports Plus™); cream, skim milk, and skim milk powder. The amount of each component given to the subjects was based on their body weight. The macronutrient composition (expressed per kg body weight) was 0.44 g fat, 0.94 g carbohydrate, 0.27 g protein. After the mixed meal, venous blood samples were collected at the following time points: 1 h, 2 h, 4 h, 6 h, and 8 h. Blood samples were collected in vacuum tubes containing clotting activators for isolation of serum. After clotting blood was centrifuged at approximately 2,000 g for 10 min at 4°C.