*3.4. E*ff*ect of Betaine Supplementation on Aerobic Capacity*

All participants completed EEE, which involved running at an intensity of 80% VO2max for 30 min on a treadmill, followed by a 0.2 m/s increase in speed every min until exhaustion. Table 2 indicates no significant differences between the trials regarding the time to exhaustion, peak oxygen consumption, HRmax, or AHR (*p* > 0.05). The result shows that two weeks of betaine supplementation had no effects on the indicators of aerobic capacity (time to exhaustion, VO2max, HRmax, and AHR).

**Figure 5.** Changes of (**A**) TBARS and (**B**) PC in the exhaustive exercise after 2 weeks of betaine supplementation. Pre, before exercise; Post-0, immediately after exercise; Post-3, 3 h after exercise.

**Table 2.** Changes in aerobic capacity after 2 weeks of betaine supplementation.


The data are presented as mean ± SD (*n* = 10). TTE, time to exhaustion; HRmax, maximal heart rate; AHR, average heart rate; VO2peak, peak oxygen intake.

#### **4. Discussion**

This study examined the effects of 2 weeks of betaine supplementation on lymphocyte apoptosis, MTP decline, oxidative stress (TBARS and PC), and aerobic capacity (time to exhaustion, VO2max, HRmax, and AHR) after EEE. The findings revealed that twice per day supplementation of 1.25 g of betaine mixed in 300 mL of sports beverage for 2 weeks attenuated lymphocyte apoptosis immediately after EEE. However, a 14-day betaine supplementation had no effects on MTP decline, oxidative stress, or aerobic capacity. In addition, the results revealed that the concentration of betaine in participants' plasma increased significantly with 2 weeks of betaine supplementation. This result was consistent with a previous study that indicated 2 weeks of betaine supplementation (1.25 g of betaine mixed in 300 mL of sports beverage twice per day) can effectively enhance the concentration of betaine in plasma [24]. Moreover, no significant differences were noted in the concentration of choline in plasma between trials (Figure 3B). Therefore, the reason for the elevated betaine concentration in plasma with betaine supplementation was due to of the supplementation rather than choline metabolism. Therefore, the beneficial effects of attenuation of lymphocyte apoptosis after EEE can be attributed to the elevation in plasma betaine concentration. This study also revealed that 2 weeks of betaine supplementation could not reduce the production of TBARS and PC after EEE. The reactive oxygen species production is considered essential for exercise adaptations to occur [35], our result indicated the effectiveness of the betaine supplementation to reduce lymphocyte apoptosis is not via the redox processes.

The MTP is negative inside and positive outside under the normal status; however, when apoptosis is initiated through the depolarization of MTP, which opens the permeability transition pore of mitochondria, the increased membrane permeability of mitochondria results in MTP decline [36]. Therefore, mitochondria are believed to play a pivotal role in the intrinsic apoptosis pathway [37]. In addition, the extrinsic pathway is triggered by pro-inflammatory marker like tumor necrosis factor-α or by death receptors and ligands (Fas/ FasL complex) [38]. Therefore, apoptosis is induced by two distinct cell death pathways, either the intrinsic or extrinsic pathway [39,40]. This study showed that

betaine supplementation could not reduce the production of TBARS and PC after EEE (Figure 4A,B), potentially because betaine supplementation is unable to effectively inhibit MTP decline after EEE. Craig [12] indicated that active coupled sodium and chloride ions and passive sodium ion independent transport systems promote the cellular absorption of betaine. Based on this finding, most studies regarding betaine supplementation have used betaine mixed in a sports beverage, with the sports beverage employed as the placebo [23,24,41–43]. Notably, sodium and chloride ions are essential electrolytes present in the blood and extracellular fluids. Moreover, sodium plays a crucial role in muscle contractions, and its depletion may affect the whole body [44]. Therefore, sodium and chloride ions, the essential ingredients of a sports drink, may have had an ergogenic effect on MTP, causing the consistent changes in MTP decline in both betaine and placebo trials. Hence, further studies should consider only betaine intake to evaluate its effects on MTP decline after EEE. In addition, this study revealed that 2 weeks of betaine supplementation can effectively attenuate lymphocyte apoptosis after EEE (Figure 3A). This result is consistent with in vitro studies, which have indicated that betaine can effectively decrease lymphocytes apoptosis in cell cultures [45,46]. Moreover, this result is consistent with a previous study in our laboratory that determined a single dose of betaine supplementation can effectively attenuate lymphocyte apoptosis immediately after EEE [30]. However, the present study revealed that 2 weeks of betaine supplementation can significantly attenuate lymphocyte apoptosis not only immediately after EEE but also up to 3 h after EEE. Therefore, the present study demonstrated that the ergogenic effect of 2 weeks of betaine supplementation is better than that of acute betaine supplementation. Lymphocyte apoptosis represents an extrinsic apoptosis pathway, which is initiated when the death ligands, such as tumor necrosis factor-α, bind to the death receptor [47]. Nevertheless, future studies might evaluate the inflammatory response after betaine supplementation to clarify the mechanism of lymphocyte apoptosis attenuation.

Animal studies have indicated that betaine can decrease oxidative stress [48,49]. Therefore, the present study hypothesized that betaine supplementation effectively attenuates oxidative stress caused by exhaustive exercise and enhances aerobic capacity. The present study did not observe an attenuation in lipid and protein peroxidation with 2 weeks of betaine supplementation. This result differs from that of previous studies involving cultured cells and animal tissue, which indicated that betaine can attenuate oxidative stress [48–53]. Halliwell [54] reviewed numerous studies with antioxidant paradox and concluded that the levels of oxidative damage measured in laboratory animals seem more responsive to being decreased by dietary antioxidants than they are in humans. This may one of the reasons with the inconsistent results. Moreover, the validity of measuring TBARS via colorimetry to be the indicator of lipid peroxidation is questionable [55]. This may the limitation of this study. Furthermore, some studies reported positive effects of betaine supplementation related to oxidative stress were conducted over an experimental period lasting more than 3 weeks [52,53,56]. More human studies with longer supplementation periods should be conducted to clarify the effects of betaine supplementation on oxidative stress.

The present study revealed that 1.25 g of betaine mixed in 300 mL of sports beverage twice per day for 2 weeks had no effects on the time to exhaustion, peak oxygen consumption, HRmax, or AHR (Table 2). This finding is consistent with that of the previous study in our laboratory, which determined that acute betaine supplementation cannot enhance aerobic capacity [30]. Mitochondrial density can affect the consumption of oxygen during oxidative phosphorylation [57]. The fact that MTP decline is not suppressed by betaine supplementation may be one of the reasons for the lack of aerobic capacity enhancement. Furthermore, one study indicated that betaine supplementation effectively increases the force and power in participants who had a minimum of 3 months of prior resistance training [24]. And betaine supplementation had no effects on muscular strength in untrained participants [41]. The participants in the present study were healthy untrained men, which may explain why 2 weeks of betaine supplementation did not enhance aerobic capacity. Study of Schwab et al. [58] indicated that consuming 6 g per day of betaine for at least 4 weeks elevates plasma betaine concentration. However, other studies have revealed that consuming 6 g of betaine per day for 6 weeks and 12 weeks

can increase risks to human health by elevating concentrations of low-density lipoprotein cholesterol and triglycerides as a consequence of increasing the concentration of homocysteine in blood [58,59]. Future studies should attempt to evaluate how various periods and dosages of betaine supplementation affect blood lipids to understand the effects of betaine supplementation on exercise.
