Olive oil consumption was claimed as the basic food supply responsible for the low incidence of cardiovascular diseases in some societies 1. Recent research has suggested that olive oil minor constituents might have more effects on health than once believed 12345.

On this concern, a group of olive oil phenolic compounds, such as oleuropein and cafeic acid have attracted considerable attention because of their anti-diabetic 67, anti-atherosclerotic 8 and anti-inflammatory 9 properties. Olive oil minor components have been also associated with the antioxidant activity of olive oil 910, but little information is available on cardiac oxidative stress and energy metabolism in obesity conditions.

Cardiac muscle utilizes a variety of substrates to produce energy, and the heart can shift from one substrate to another depending on food intake and pathophysiological state 11. There is a growing awareness that during energy metabolism, the mitochondrial respiratory chain represents a major intracellular source of reactive oxygen species (ROS). Alterations in food constituents or substrate for energy generation, as found in hypercaloric diet 12 may result in higher ROS, thus inducing oxidative stress, an imbalance between oxidants and antioxidants systems in favor of the former 1314. Recent research in our laboratory demonstrated that enhanced energy intake reduced the mitochondrial membrane fluidity, increasing ROS generation 1215.

Furthermore, it has been shown that blocking mitochondrial oxidative phosphorylation system increases lipid accumulation in adipocytes, and that in obesity there is decreased oxygen consumption, indicating impairment in mitochondrial activity 16. Therefore, weight gain can be viewed not only as the consequence of an initial positive energy balance, but also as the mechanism by which energy balance is eventually stored. Whether obesity-related cardiac effects are due to an increase in caloric intake, a decrease in metabolic rate, or a combination of both factors is still unclear.

An excess of energy intake and/or a decrease in energy expenditure may reflect important metabolic dysfunction, for instance exacerbated fat storage and/or a deficit in oxidative metabolism 17. However, it was not studied whether olive oil intake can alter calorimetric parameters and can improve energy metabolism in cardiac tissue of obese rats, which certainly would bring new insights on obesity-related cardiac metabolic changes control.

Thus, the aim of this study was to investigate the effects of olive oil and its minor phenolic constituents, oleuropein and cafeic acid, on calorimetric parameters, myocardial oxidative stress and energy metabolism in cardiac tissue of control and obese rats. Animal models are useful for increasing the understanding of the effects of diet and the development of obesity related abnormalities, particularly on cardiac tissue 1518. Therefore, we used a rat model to study the effects of olive oil and its phenolic compounds on an unresolved issue in the field of obesity and health that is the control of obesity-related cardiac metabolic alterations.

Dietary olive oil supplementation 20, and more recently olive oil phenols 21 have been recommended as important therapeutic interventions in preventive medicine. However, a question remains to be addressed: what are the effects of olive oil and its minor phenolic compounds on obesity-induced cardiac metabolic changes?

To the best of our knowledge this is the first study that evaluated the relative potency of olive oil and its phenolic constituents, oleuropein and cafeic acid, on some markers of metabolic pathways in cardiac tissue of obese rats, as well their relationships with calorimetric parameters and oxidative stress. The present study brought new insights into the long term and low-dose intake of olive oil and its compounds on cardiac energy metabolism.

To demonstrate the efficiency of obesity induction 1934 despite the same food intake, Ob rats showed higher final body weight, surface area and BMI than C (Table 1). Prospective studies 35 demonstrated that a relatively low RMR and a high RQ 36 are predictors of body weight gain. In fact, as previously observed 34 obesity was characterized in Ob rats from reduced RMR, oxygen consumption, VO₂/final body weight and VO₂/surface area, corroborating with lower fat oxidation (Table 1). There was also higher energy balance indicating that energy intake exceeded energy expenditure for vital functions (RMR), thus providing greater amount of energy to be stored as fat.

The detected fasting carbohydrate oxidation in Ob group was associated with liver glycogen storage due hypercaloric diet intake in these animals. Since the influence of the meal had essentially ended in the post absorptive state, the fasting RQ value gives an indication of the glycogen storage 27.

As previously found 2021, in both dietary conditions there were no significant alterations in morphometric parameters by olive oil and its phenolic compounds. There were no significant changes in heart weight, heart weight/body weight, as well as in cardiac protein, indicating adequate protein supply in both dietary conditions.

The lower RMR in C-Oleuropein rats, as well the higher RMR in C-Olive and C-Cafeic rats were not enough to significantly change the energy available for fat storage (Table 2). Note that in C-Olive and C-Cafeic rats there was a better fat utilization and this was evidenced by the lower RQ 34 in these animals. The detected carbohydrate oxidation in C-Oleuropein rats was associated with the highest RQ, RQ/body weight and RQ/surface area found in these animals (Table 2). Therefore, the lower fat oxidation in C-Oleuropein animals was not surprising, since the change in fuel selection is controlled by carbohydrate intake, and when carbohydrate oxidation rises in response to intake, there is a profound counterregulatory suppression of fat oxidation, because triacylglycerol lipase enzyme from the adipose tissue is inhibited by the insulin secretion 11. Oleuropein enhanced glucose uptake in tissues, increasing insulin response 6 and the glycogen storage 36.

It was widely accepted that oxygen consumed for carbon dioxide liberation, during substrate oxidation depends upon the oxygen amount in dietary nutrient. Because fat oxidation requires more oxygen, there was a significant reduction in the RQ, RQ/body weight and RQ/surface area in Ob-Olive, Ob-Oleuropein and Ob-Cafeic rats (Table 3).

Several mechanisms may be associated with increased fat oxidation in obese rats receiving olive oil and its phenolic components. It is well known that absorption and delivery of dietary compounds by intestinal cells are part of a complex process, which is influenced by the physiological state of enterocytes. A persistent situation of redox imbalance, due to hypercaloric diet intake has been associated with gastrointestinal alterations 37. Under these conditions, it might be expected an imbalance in the absorption of dietary compounds and the metabolic utilization for energy generation 38. Olive oil and its compounds modulating cellular signal 39 and the activity of peptidases 340 would allow the adequate balance between uptake and metabolism of dietary compounds.

Judging from our experimental results, it was evident that the beneficial effects of olive oil and its minor constituents enhancing fat oxidation were reflected in cardiac tissue of obese rats. Note that there was no antioxidant activity of olive oil and its phenolic compounds in cardiac tissue of animals fed with hypercaloric diet.

The enhanced fat oxidation was demonstrated in cardiac tissue by higher OHADH and CS activities in Ob-Olive rats (Figure 1 and 2). OHADH is a key enzyme for fatty acid oxidation, and CS is the key enzyme for the control of the flux of metabolites through tricarboxylic acid cycle. A clear link between triacylglycerol accumulation and the cardiomyopathy was established in experimental models in which the rate of fatty acid uptake by the heart was increased, or the capacity for fatty acid oxidation was reduced in the mitochondria 41. Myocardial OHADH activity was also significantly enhanced by oleuropein and cafeic acid in obese animals, despite the maintenance of CS activity in these animals. It has been shown that hypercaloric diet induces adverse effects on cardiac function through changes in fatty acid metabolism, by inappropriate activation and expression of PPARα (peroxisome-proliferator-activated receptor α) as a result of glucotoxicity. The decrease in the transcription of PPARα may be a regulatory event for the reduced use of fatty acids 42. Thus, cafeic acid and oleuropein could allow adequate PPARα activation and fatty acid utilization, increasing the OHADH activity under obesity conditions, reducing triacylglycerol accumulation in cardiac muscle. Optimizing cardiac energy metabolism in obese conditions may be one approach to prevent and treat cardiac dysfunction 1113.

Curiously, no significant changes were found in myocardial metabolic enzymes of C-Olive rats, whereas, CS was significantly reduced in C-Oleuropein rats, despite no changes in OHADH (Figure 1 and 2). This indicated enhanced glycolytic pathway relative to aerobic metabolism, or delayed flux of metabolites through tricarboxylic acid cycle in cardiac tissue. Glycolysis preferentially serves energy channeling to sarcolemmal membranes, were glucose transport into cells occurs, by providing this readily available substrate for glycolytic enzymes bound to sarcolemmal molecular complexes 13. Glycolytic pathway thus represents low capacity, but high specificity modules of the integrated metabolic network of a cardiac myocite 11. On the other hand, the reduced OHADH, and the maintenance of CS activity, clearly indicated that C-Cafeic rats had depressed fatty acid degradation, relative to aerobic metabolism in cardiac tissue, as compared to C, C-Olive and C-Oleuropein rats. Therefore, in standard fed conditions, dietary supplementation with olive oil phenolic compounds induced changes in the substrate used for energy generation in cardiac tissue. Further studies may be considered to clear this open question, and to show the importance of these changes on cardiac function.

Anyhow, the beneficial effect of olive oil in standard-fed condition was evidenced from the reduced myocardial LH in these animals (Table 4). Lipids accumulation within the myocardium induces cardiac lipotoxicity 1213. Oxidative stress and lipotoxicity with increased LH may affect myocardial function, in a fashion that mimics reperfusion injury including persistent cellular loss of K⁺, depletion of energy phosphates and decreased metabolic function 43. Note that despite the effects of cafeic acid reducing myocardial SOD, no significant changes were found in LH and TAS concentrations as compared with C rats.

Although both oleuropein and olive oil have reduced LH, the antioxidant responses in both groups occurred by different ways. The antioxidant defense system includes SOD that catalyzes the destruction of superoxide radical (O₂^_) by dismutation and hydrogen peroxide formation, catalase and GSH-peroxidase that catalyzes the conversion of hydrogen peroxide to water. The total antioxidant substances (TAS) include non-enzymatic, lipophilic and aqueous antioxidants 11. Enhanced TAS was associated with direct antioxidant activity of olive oil and oleuropein, because of the free radical scavenger ability 44. This property depends on the number of hydroxyl radicals in the phenolic molecule 21. Oleuropein and hydroxytyrosol, free radical scavengers, have antioxidant activity as found in other antioxidants, such as vitamin E and butylated hydroxytoluene (BHT) 20. On the other hand, it has been demonstrated that olive oil may interfere in iron absorption 24. Considering the importance of iron in the oxidative stress induction, the reduced iron uptake and diminished serum concentration of iron may be alternative mechanism for reduced myocardial LH in C-Olive animals.

In conclusion, the present study brought new insights of olive oil effects on obesity related cardiac metabolic changes, demonstrating for the first time that olive oil enhanced fat oxidation and regulated myocardial metabolic enzymes, optimizing cardiac energy metabolism in obesity conditions. Olive oil and its minor phenolic compounds, oleuropein and cafeic acid had myocardial antioxidant activity in standard-fed conditions.

The authors declare that they have no competing interests.

GMXE took part in planning the study design, sample analysis, analyzed the data and involved in drafting and writing of manuscript. FRFS contributed to the study design, sample analysis and performed the statistical analysis. KKHRR and GSA involved in drafting and sample analysis. ELBN took part in planning and supervising the study design, contributed to the analysis, writing of manuscript, edited the paper, revising it critically for important intellectual content and provided the final version. All authors read and approved the final manuscript.