INTRODUCTION
Oxamate is a well-known inhibitor of lactate dehydrogenase (LDH) and its chemical structure is analogous to that of pyruvate. By competing with pyruvate, oxamate inhibits LDHA rather than LDHB, resulting in a blockade of pyruvate-to-lactate conversion, thereby disrupting glycolysis [1,2]. Owing to its inhibitory characteristics, oxamate has been commonly used in cancer research to develop therapeutic agents [3-5] because the overflow of glycolysis is essential for the survival of most cancer cells [6]. In addition to these metabolic properties, oxamate has been suggested to have very low toxicity even after long-term (~12 weeks) and high-dose (~ 750 mg/kg) treatments [3,7]. Therefore, oxamate is a promising candidate for the development of anticancer strategies.
As mentioned previously, oxamate disrupts glycolysis by acting as a competitive inhibitor of pyruvate. However, this also implies that oxamate may affect various metabolic pathways centered on pyruvate, including gluconeogenesis and mitochondrial translocation, in addition to glycolysis. Indeed, in an early study on the effects of oxamate on energy metabolic pathways, oxamate hindered gluconeogenesis in isolated rat hepatocytes [8]. A recent study has revealed that oxamate can inhibit various energy metabolic enzymes, including LDH, pyruvate kinase, and enolase [9]. These findings suggest that oxamate is not selective for glycolysis. In other words, the broader inhibitory effects on metabolism have not yet been identified.
Although this suggestion requires an examination of the effects of oxamate on whole-body energy metabolism, no in vivo experiments have been conducted to date. Metabolic regulation in response to external stimuli in vivo is inherently far more sophisticated than that in vitro because a given metabolic statement is ultimately determined by intricate communication among multiple organs. To develop oxamate as a chemotherapeutic drug, it is essential to understand how oxamate alters its systemic metabolism. Energy metabolism is a dependent variable influenced by physiological demands; however, it has also been recognized as an important independent variable that determines cell survival and fate [10-13], which are closely related to pathophysiology [14,15].
Therefore, we aimed to investigate the effects of oxamate on whole-body energy metabolism in mice using a metabolic chamber. Specifically, we examined whether oxamate injection altered the whole-body energy metabolism at rest. Subsequently, we evaluated the effects of oxamate on the energy metabolism during exercise. As exercise requires substantial production of adenosine triphosphate (ATP), the turnover flux of energy metabolism increases significantly during exercise. Thus, we considered that this heightened metabolic demand would provide a clearer context to assess the effects of oxamate on whole-body energy metabolism. Given the central role of glycolysis in energy production and the effects of oxamate, we hypothesized that oxamate suppresses whole-body energy expenditure, both at rest and during exercise, by inhibiting glycolytic flux. This inhibition is expected to reduce the substrate availability for oxidative phosphorylation, thereby decreasing the overall energy expenditure. To the best of our knowledge, this is the first study to investigate the in vivo effects of oxamate on whole-body energy metabolism in mice, thereby providing novel insights into its systemic metabolic effects.
METHODS
Animals
Ten-week-old male ICR mice (Orient Bio Inc., Seongnam, Republic of Korea) were acclimatized to the laboratory environment for at least 1 week. All mice were housed in standard transparent plastic cages under a controlled temperature of 25-26°C with 40-50% humidity and a 12-h light/dark cycle (lights on: 07:00-19:00). A standard chow diet (Orient Bio, Inc., Seongnam, Republic of Korea) and water were provided ad libitum. The animal study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Konkuk University (KU23160). All methods were performed in accordance with relevant guidelines and regulations. This study was performed in compliance with the ARRIVE guidelines: https://arriveguidelines.org/. Furthermore, efforts were made to minimize discomfort and stressful situations.
Oxamate solution
Sodium oxamate (sc-215880A; Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in phosphate-buffered saline (PBS, pH 7.4). To ensure the inhibitory action of oxamate in vivo, 750 mg/kg body weight of oxamate (concentration: approximately 0.56 mol/L) was intraperitoneally injected based on previous studies [3,7].
Experimental design
This study was designed as an acute experiment investigated the effects of oxamates on whole-body energy metabolism. The mice were randomly assigned to one of two groups (n = 8 per group): the control group, which received an injection of vehicle solution (vehicle), and the experimental group, which received an injection of oxamate (oxamate). To comprehensively evaluate the effects of oxamate, whole-body energy metabolism was measured at rest, immediately after injection. After a 1-week washout period to eliminate residual effects, the effect of oxamate on whole-body energy metabolism during exercise was assessed.
Measurement of whole-body energy metabolism at rest
Mice were injected with either oxamate or vehicle solution and immediately placed in isolated metabolic chambers to measure whole-body energy metabolism at rest. To avoid metabolic interference from nutrition, metabolism was measured for 3 h during the active phase without food intake. The open-circuit method was used for the metabolic chambers. The average flow rate in each chamber was set to 1 L/min. An acrylic tube was connected to each chamber to manipulate the air volume. Respiratory gas analysis (O2 uptake and CO2 production) was conducted using a mass spectrometer (ARCO-2000; ARCO System, Chiba, Japan) with a switching system (ARCO-2000-GS-8, ARCO System, Chiba, Japan) that allowed the spectrometer to sample the gas from each chamber every 15 s. Respiratory exchange ratio (RER), fat oxidation, carbohydrate oxidation, and energy expenditure were calculated from respiratory gas measurements.
Measurement of whole-body energy metabolism during exercise
The mice were injected with either oxamate or vehicle solution and immediately placed in individually isolated metabolic treadmill chambers to measure whole-body energy metabolism during exercise. Metabolism during exercise was measured for 30 min. Treadmill exercise protocols are shown in Table 1. Given that oxamate is widely known to inhibit LDHA, the treadmill velocity was increased to 25 m/min, which corresponds to the lactate threshold [16,17]. We considered the lactate threshold as the most appropriate point at which oxamate-induced metabolic differences could be clearly observed. In addition, the protocol featured a gentle increase in velocity to prevent oxamate-injected mice, whose energy metabolism was likely to be disrupted, from being unable to run due to a sudden rise in exercise intensity. The mice were not fasted prior to the measurements. To motivate the mice to run, a mild electrical stimulation was provided on a grid at the rear of the treadmill. Electrical stimulation was set at a constant current of 0.4-0.6 mA, which has previously been validated as an appropriate electrical level to avoid major distress [18-20]. The characteristics of the chamber and its associated system were identical to those mentioned above.
Blood analysis
Resting blood glucose and lactate concentrations were measured using a glucose analyzer (Accu-Chek Performa, Roche, Switzerland) and lactate analyzer (LT-1730, Lactate Pro 2, ARKRAY, Kyoto, Japan), respectively. For analysis, tail-tip blood was used, and measurements were taken at 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, and 180 min after the injection of either the oxamate or vehicle solution.
Post-exercise blood lactate concentration was measured immediately after the 30-min exercise bout, as presented in Table 1. Tailtip blood was used for the analysis.
Statistical analysis
All data were analyzed using the IBM SPSS Statistics 25 software. Graphs were constructed using GraphPad Prism software (version 10.2). All data were checked for normality of distribution using the Shapiro-Wilk test and were found to exhibit normality. Comparisons between the two groups over time were performed using two-way mixed analysis of variance (ANOVA), and post-hoc pairwise comparisons were performed to compare the two groups at each time point using Bonferroni correction to adjust for multiple comparisons. Comparisons between two groups were performed using independent Student’s t-test. Data are presented as the mean ± standard error of the mean (S.E.M.). Differences were considered statistically significant at p < 0.05.
RESULTS
Oxamate suppressed whole-body energy metabolism at rest
We assessed the effects of acute oxamate injection on whole-body energy metabolism at rest using metabolic chambers (Figure 1a). Oxygen uptake and carbon dioxide production were significantly suppressed immediately after oxamate injection (p < 0.001 and p < 0.001, respectively) and remained low for up to 2 h (at 2 h: p = 0.037 and p = 0.039, respectively) (Figure 1b-c). The cumulative 3-h measurement of oxygen uptake and carbon dioxide production were also significantly lower (p = 0.002 and p = 0.002, respectively) in the oxamate-injected mice than in the vehicle group (Figure 1h-i). However, the RER was not affected by oxamate injection (p = 0.136) (Figure 1d, 1j). These results suggest that both fat and carbohydrate oxidation were significantly reduced by oxamate, and we founded a more pronounced decrease in fat oxidation (p = 0.003 and p = 0.049, respectively) (Figure 1e-f, 1k-l). Consequently, energy expenditure was significantly suppressed following oxamate injection (p = 0.002) (Figure 1g, 1m).
Oxamate reduced blood concentration of glucose, but elevated that of lactate at rest
As whole-body energy metabolism was significantly altered following oxamate injection, we assessed blood glucose and lactate concentrations to capture the changes in systemic metabolites. Consistent with the resting metabolic data, the blood glucose concentration was significantly reduced following oxamate injection (at 10 min: p = 0.029), and this reduction was sustained for 3 h (at 180 min: p = 0.023) (Figure 2a). The area under the curve (AUC) analysis further confirmed the significance of this reduction (p = 0.002) (Figure 2b). Notably, blood lactate concentration was significantly elevated following oxamate injection (at 10 min: p = 0.019), which was also confirmed by AUC analysis (p = 0.005) (Figure 2c-d).
Oxamate disturbed energy metabolism during exercise
Next, using treadmill-equipped metabolic chambers, we examined how oxamate injection affects whole-body energy metabolism during exercise (Figure 3a), as sustaining exercise requires a substantial increase in metabolic flux, including glycolysis, gluconeogenesis, and fat oxidation [21].
During the warm-up session (4-8 m/min), oxygen uptake and carbon dioxide production were significantly lower in the oxamate-injected mice than in the vehicle group (p = 0.002 and p = 0.002, respectively) (Figure 3b-c), similar to the resting metabolic data. However, as the exercise session (10-25 m/min) began, this difference disappeared, and by the end of the exercise, a sharp increase in both oxygen uptake and carbon dioxide production was observed in the oxamate-injected mice (at 30 min: p = 0.029 and p < 0.001, respectively) (Figure 3b-c). In the cumulative data, total oxygen uptake was significantly reduced by the oxamate injection (p = 0.015), whereas total carbon dioxide production remained unchanged (p = 0.140) (Figure 3h-i). These results suggest that the RER of oxamate-injected mice was elevated throughout exercise (Figure 3d), and the average RER during exercise was significantly increased by oxamate injection (p = 0.02) (Figure 3j). Nonetheless, total carbohydrate oxidation did not differ significantly between the two groups (p = 0.257) (Figure 3l), although a sharp increase was observed in oxamate-injected mice towards the end of exercise (at 30 min: p = 0.001) (Figure 3f). Fat oxidation during exercise was substantially reduced by oxamate injection (p = 0.009) (Figure 3e, 3k). Consequently, the energy expenditure of the oxamate-injected mice was significantly lower than that of the vehicle group (p = 0.024) (Figure 3g, 3m).
Oxamate elevated the post-exercise blood lactate concentration
Blood lactate concentration was measured immediately after the exercise. Although all mice ran for the same duration at the same speed, the post-exercise blood lactate concentration in oxamate-injected mice was significantly higher than that in the vehicle group (p = 0.005) (Figure 4a).
DISCUSSION
Our study demonstrated that oxamate suppressed whole-body energy metabolism at rest. Both fat and carbohydrate oxidation were reduced following oxamate injection, leading to a significant decrease in resting energy expenditure, whereas RER remained unchanged. Meanwhile, blood glucose concentration significantly decreased following oxamate injection. Unexpectedly, blood lactate concentration was significantly elevated, a finding that has not been previously reported. From the metabolic results during exercise, oxamate reduced total energy expenditure, similar to its effect on resting metabolism, whereas an increase in RER was observed only during exercise. Additionally, the post-exercise blood lactate concentration in oxamate-injected mice was significantly higher than that in vehicle-injected mice.
In the present study, we found that oxamate elevated the resting blood lactate levels in vivo. Mechanistically, oxamate is known to inhibit LDHA, leading to reduced lactate production. Most previous in vitro studies have reported that oxamate treatment decreases lactate production [5,22,23] by suppressing glycolytic capacity [4,24]. Nevertheless, our in vivo study showed the opposite results. We speculate that this discrepancy may be attributed to changes in the interorgan lactate shuttle, which occurs in vivo but not in vitro [25]. In other words, the net blood lactate level in vivo, as opposed to that in vitro, is determined by the balance between lactate production and oxidation, which is regulated by multiple organs, including the liver, skeletal muscle, heart, kidneys, lungs, and brain [25]. The putative mechanism by which oxamate increases blood lactate levels is as follows. If oxamate inhibits gluconeogenesis to a greater extent than glycolysis, blood lactate concentration would increase. In other words, if oxamate reduces lactate uptake from the blood into tissues that favor gluconeogenesis more than it reduces lactate secretion into the blood from tissues that favor glycolysis, the net blood lactate level would increase. As mentioned above, an early ex vivo biochemical study demonstrated that oxamate disrupts gluconeogenesis [8]. However, no studies have directly compared the inhibitory effects of oxamate on different metabolic pathways, including gluconeogenesis and glycolysis. Therefore, further research is needed to validate the proposed mechanism, including studies examining whether oxamate alters the expression of lactate transporters, such as monocarboxylate transporters 1, 2, and 4.
Our findings support this hypothesis. The reduction in blood glucose concentration in the oxamate-injected mice provided supportive evidence for this mechanism (Figure 2a-b). The inhibition of gluconeogenesis inevitably leads to a reduction in glucose production [26,27], and fluctuations in whole-body energy expenditure align with those in blood glucose levels [28]. Consistent with this logical sequence, our study showed that oxamate injection led to reduced blood glucose levels, whole-body carbohydrate oxidation, and energy expenditure (Figure 1-2). Additionally, the substantial reduction in resting fat oxidation following oxamate injection (Figure 1e, 1k) could be attributed to the lactate-induced inhibition of lipolysis [29-31].
Another noteworthy finding was that the suppressive effect of oxamate on resting energy metabolism was sustained for up to 2 h and then gradually diminished (Figure 1b-g). This result suggests that oxamate may be oxidized and metabolized within that timeframe in vivo. However, to date, no study has reported the pharmacokinetics of oxamate in vivo, specifically, how long it takes for oxamate to be metabolized or cleared. Therefore, our findings on the resting energy metabolism may provide preliminary pharmacokinetic insights that could be valuable for the development of oxamates as anticancer agents.
The whole-body energy metabolism results during exercise (Figure 3-4) exhibited a pattern similar to that of resting metabolism. Oxamate injection suppressed fat oxidation and energy expenditure during exercise. Carbohydrate oxidation showed an increasing tendency, although the change was not statistically significant. Consequently, the RER during exercise significantly increased in oxamate-injected mice (Figure 3j), in contrast to the unchanged resting RER (Figure 1j). Exercise requires a substantial increase in the turnover flux of energy metabolism, including gluconeogenesis, glycolysis, beta-oxidation, and oxidative phosphorylation [21]. Blocking a single energy metabolism pathway can result in an inability to sustain exercise [32]. Similarly, we observed that the oxamate-injected mice had difficulty running (please refer to this video. Link: https://docs.google.com/presentation/d/1x3fGJ6kQqEsBqOCzJRPtQIZHpH1J3TX-6CXVXwPqAlGI/edit?usp=sharing) approximately 5 min before the end of the exercise. This phenomenon was also reflected in the metabolic data during exercise. The RER of oxamate-injected mice increased sharply at the end of exercise (Figure 3d), and the average post-exercise blood lactate level in oxamate-injected mice was approximately 5mM, compared to approximately 3 mM in vehicle-injected mice (Figure 4a). These results indicated that oxamate injection suppressed energy metabolism both at rest and during exercise.
The metabolic changes induced by oxamate during exercise can be attributed to its inhibitory effects on gluconeogenesis. The proposed mechanism is as follows: As mentioned above, oxamate injection suppresses energy expenditure by inhibiting gluconeogenesis, leading to elevated blood lactate levels [8], which in turn inhibit lipolysis [31,33]. The oxamate-induced suppression of energy metabolism conflicts with the physiological demand for increased energy production during exercise. Therefore, oxamate-injected mice may rely more on carbohydrate oxidation during running. Furthermore, the accumulation of blood lactate is likely to inhibit lipolysis in exercising muscles via the autocrine lactate loop [34], which accelerates the physiological demand for carbohydrate oxidation during running.
Thus far, we have confirmed the significant inhibitory effects of oxamate on whole-body energy metabolism in vivo and have discussed the potential mechanisms underlying these effects. However, several factors should be considered when interpreting these findings. Most previous studies on oxamates have been conducted using cancer cells, which exhibit highly distinctive metabolic characteristics compared to normal tissues [35,36]. To our knowledge, our study is the first to investigate the effects of oxamates on whole-body energy metabolism in vivo. Considering that different exercise intensities and durations distinctly modulate gluconeogenesis and glycolysis, future studies should explore whether the effects of oxamate on energy metabolism vary under different exercise conditions. In addition, we did not perform molecular analyses, resulting in a lack of direct evidence to support the proposed mechanisms. To address this, future studies using metabolomics are needed to elucidate the oxamate-induced alterations in metabolites in vivo.
Despite these limitations, our findings clearly demonstrated that oxamate suppressed whole-body energy metabolism in vivo. These results suggest that when developing oxamate as an anticancer therapeutic drug, it is crucial to investigate whether systemic alterations in energy metabolism induced by oxamate could lead to unexpected secondary effects. Therefore, a comprehensive understanding of the effects of oxamate on energy metabolism is essential for safe and effective therapeutic applications.
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