Effects of n-3 fatty acids and exercise on oxidative stress parameters in type 2 diabetic: a randomized clinical trial

The main finding of this study was that 8 weeks of n-3 PUFA supplementation reduces TRAP concentration after a high intensity exercise, with no other statistically significant changes in other oxidative stress parameters or inflammation. Additionally, as previously demonstrated, n-3 PUFA supplementation reduced triglycerides levels. Our hypothesis was partially rejected, due to the fact that our primary outcomes (TBARS and F2-isoprostanes) were not attenuated by n-3 PUFA supplementation.

As previously mentioned, physical exercise is considered a powerful tool to prevent and to treat diabetes and the associated comorbidities. Nowadays, due to the lack of time, many people are using high intensity exercise sessions (with a lower volume), as an alternative, to improve their health, metabolic function and body composition. However, high intensity exercise can induce a transient inflammatory state and increase oxidative stress [10, 11] that may promote, in diabetic population, an undesired effect. The increase in VO2 (load dependent) caused by exercise can lead to increased blood and tissue levels of reactive oxygen species [30]. Laaksonen et al. [31] and Atalay et al. [32] showed increased reactive oxygen species production after 40 min of sub-maximal exercise (intensity equivalent to 60% of VO2peak) in type 1 diabetic subjects. In our study, exercise performed with intensity equivalent to 70–80% of VO2peak was not able to increase the production of lipoperoxides as measured by TBARS and F2-Isoprostanes (primary outcomes). If exercise intensity was to be an issue, our data are corroborated by the findings of Davison et al. [33], that did not observe differences in the concentrations of malondialdehyde even after strenuous exercise. In addition, the lack of changes in TBARS and F2-isoprostanes may be attributed, at least in part, by the shorter period of supplementation of the present work in comparison with others.

We hypothesized that including n-3 PUFA to our subjects would induce benefits when they exercise at high intensity, however, with except of changes in TRAP, our intervention fail to change levels of lipoperoxidation, SOD and UA. Accinni and colleagues [34] also found an increase in TRAP in dyslipidemic volunteers supplemented with n-3 PUFA for 4 months, however, with a significant reduction in the levels of TBARS (P = 0,002). After treating healthy volunteers with 3.6 g/day of n-3 PUFA, Nalsen et al. [16] did not observe changes in the values of TRAP; however, they found a significant (P = 0.015) reduction in lipoperoxidation as measured by F2-isoprostanes concentration. The difference in results among studies may be related to different exercise protocols, duration of the tests, and different methods employed when measuring the oxidative stress markers.

Regarding SOD and UA, our secondary outcomes, there were no significant changes after the exercise, similarly to the results described by Laaksonen et al. [31] and Atalay et al. [32]. If our subjects were more obese or dyslipidemic, the results could be different (49). There is still very limited information on the effects of exercise in acute oxidative stress in subjects with type 2 diabetes. The trend for increased activity of SOD after supplementation with n-3 PUFA was not reported in the work of Kesavulu et al. [4]. However, Smaoui et al. [35] showed a positive correlation between SOD activity and concentrations of n-3 PUFA in erythrocytes. Erdogan et al. [36] also found increased activity of SOD in rats after 0.4 / kg body weight fish oil supplementation for 30 days. This increase in SOD and TRAP could be explained by the formation of cyclopentenone prostaglandins. Supplementation with n-3 PUFA, particularly with EPA and DHA, has been documented to trigger a momentary lipoperoxidation process, thus leading to the formation of prostaglandins, particularly cyclopentenone prostaglandins. These are responsible for the dissociation of Keap1/Nrf2 (NF-E2-related factor). Nrf2 is the main transcription factor for the activation of the expression of antioxidant enzymes (e.g. SOD, catalase, glutathione reductase) as well as phase 2 enzymes (e.g. glutathione S-transferase, NADPH oxidase) and thus also increases TRAP [37]. The production of lipoperoxide is not considered enough to cause damage to the body, but important to stimulate the defense systems.

Subjects who develop T2DM have high levels of inflammatory markers such as hs-CRP when compared to healthy subjects [37, 38]. Supplementation with n-3 PUFA has been suggested to minimize the inflammatory processes [39]. However, our study failed to detect any significant change in the plasma levels of hs-CRP after supplementation with n-3 PUFA. Our findings corroborate the data published by Madsen et al. [40] who worked with healthy subjects supplemented with 2 or 6.6 g / day of n-3 PUFA, for a period of 12 weeks. Both groups showed no change in the values of hs-CRP. Mori et al. [15] and Fatemeh Azizi-Soleiman et al. [41] also found no change in plasma levels of CRP after supplementation with EPA or DHA, for 6 weeks and 8 weeks, respectively, in type 2 diabetic subjects. Other studies also have shown that either EPA or DHA reduce in vivo oxidant stress without changing markers of inflammation, in treated, hypertensive, type 2 diabetic subjects [15]. Finally, despite the lack of changes of hs-CRP, we cannot exclude the potential anti-inflammatory effects of our supplementation, since we did not look at other inflammatory markers such as TNF-α, IL-8, eHSP70 etc. This is a limitation of our study.

Plasma glucose and HbA1c did not change significantly after the period of supplementation. Some studies reported increased levels of glucose, HbA1c and low insulin activity, in type 2 diabetes, after the consumption of large amounts of fish oil (around 10 g/day) [42]. However, when of n-3 PUFA is given in smaller quantities (1 to 4 g/day), no changes in glycemic parameters are observed [43]. In our study, a significant reduction (17%) in the values of plasma triglycerides was observed after the period of supplementation with n-3 PUFA. These results can be explained by the reduction of fractions of VLDL – thus minimizing the hepatic production of triglycerides – and a possible reduction of free fatty acids [44]. As in other studies, there were no significant changes in other lipid parameters [43, 4547]. This can be partially explained by the fact that the diabetic subjects in the present study were not dyslipidemic, and were relatively lean (overweight on average). This differs from many studies in the literature. In a meta-analysis covering studies between 1966 and 2006, Hartweg et al. [48], showed that n-3 PUFA supplementation is, indeed, efficient to reduce triglycerides and VLDL levels, without changing other lipid sub fractions. In contrast, Abe et al. [49], after 6 weeks of supplementation with 4 g/day n-3 PUFA, found an 11% reduction in total cholesterol and a 14% increase in HDL. These changes are not a consensus in the available literature. In addition, a 4-h infusion of intralipid (n-3 FA) failed to affect insulin sensitivity, insulin secretion, or markers of oxidative stress in subjects with T2DM [50]. It could explain the lack of changes in the glycemic profile.

To the best of our knowledge, this is the first study to examine the interactions between n-3 PUFA supplementation and high-intensity exercise on oxidative stress and inflammation in subjects with T2DM. However, we recognize that the study has several limitations. First, the sample size was small, although the effect size on the variables with statistical significance was high. So, at this point, should be interpreted with caution before being extrapolated to the general population. Second, we have only studied acute effect of high intensity exercise; therefore, it is not possible to state that the effects would be similar after other intensities of exercise. Finally, we did not analyze the supplement offered to the participants, so we cannot be 100% sure if the product provides the amount of n-3 PUFA. We believe in the suitability of the company, but we suggest that future studies test the product before the beginning of the trial. Despite some limitations, the strength of this study is that we analyzed a large number of variables related to oxidative stress, including two measurements of lipoperoxidation (TBARS and F2-siprostanes) and measurements of enzymatic (SOD) and non-enzymatic antioxidants (uric acid and TRAP). Thus, this study contributes to narrow the gap in the literature on the effects of antioxidants nutrients in oxidative stress parameters after acute exercise. Further clarification is needed regarding the clinical relevance of the n-3 PUFA supplementation for T2DM in different intensities of exercise.