The present study was aimed at determining the effects of a 3-month HF diet rich in SFAs and MUFAs on the cardiac function and at investigating the underlying mechanism at the level of coronary circulation. Despite its high percentage in fat content, the high-fat diet chosen for this study did not induce severe obesity in the animals but increased significantly their adiposity, which was accompanied or not by body weight gain. The increased adiposity of the animals was related to changes observed in the in vivo and ex vivo cardiac function as well as in the ex vivo coronary reactivity. However, the body weight change and thus the adipose tissue distribution seemed to affect the mechanism through which the changes at the coronary level occurred.
Feeding rats for 3 months with a HF diet containing large amounts of SFAs and MUFAs was expected to generally increase the body weight of the animals. During the experiments two distinct groups were observed in the HF-fed animals with one of them being characterized by no body weight change compared to the control group (HF-) and the other showing an increased body weight (HF+). These two groups displayed a similar increase in abdominal adiposity and were thus clearly different from the C group. The HF + group was composed of obese animals, since their body weight and abdominal adiposity were increased. However, the body weight was moderately increased compared to the C group. These animals were thus considered as moderately obese. The HF- rats were not distinctly obese, since they had an increased abdominal adiposity but no augmentation of body weight. As the case with high body weight and abdominal adiposity can occur in humans, the situation with low body weight and high abdominal adiposity also exists and refers to abnormal and hazardous body composition with high risk of chronic pathological events. Indeed, this situation is interesting to study since it has been recently recognized in the literature the existence of different subtypes of obesity such as metabolically healthy but obese (elevated body fat but normal metabolic profiles) and metabolically obese, normal weight individuals that may be or not at increased cardiovascular risk. Furthermore, a new syndrome has been described lately in humans, the normal weight obesity syndrome, which is defined as a normal body mass index associated with increased body fat. Thus, the study of these two HF subgroups allowed the study of two different subtypes of obesity that correspond to different situations of obesity occurring in humans.
The fact that the fat content of the HF diet chosen was not consisted entirely by saturated fat or that the protein content was doubled in our HF diet might explain the state of obesity that we found in our rats. It could also explain any differences concerning basic characteristics of the animals from previous studies, such as body weight and glucose levels[11, 29]. Futhermore, Buettner et al. have shown that PUFA- or medium-chain fatty acids-rich diets did not induce insulin resistance in rats after a 12 week period. Indeed, in the HF diet chosen for this study one can find not only SFA but also a percentage of PUFAs that could explain the metabolic results of the rats. This diet affected only the triglycerides and cholesterol levels of the rats indicating the beginning of a dyslipidemia. Furthermore, it provoked the development of glucose intolerance in the HF+ rats, which is consistent with previous studies. Thus, the high-fat diet altered the metabolic profile of the HF-fed rats according to the percentage of the adiposity in their bodies with the HF- rats having a less obese profile compared to the HF+ rats.
The reason why the HF- animals did not gain body weight is not known, since we did not evaluate the food intake and the energy expenditure in our study. For that reason, we analyzed a previous study in which we determined the food intake. In that last study, the C diet was given to five rats and the HF diet to ten rats for a period of 50 days. As in the present study, the HF diet-fed animals were divided into two groups of equal sample size (n = 5) with the lightest and heaviest animals. The animal weight at the beginning of the experiment was similar in the three groups (321 ± 3, 318 ± 6 and 316 ± 3 g for the C, HF- and HF + groups, not significant). At the end of the fifty day-diet, the animal weight was significantly higher in the HF + group (445 ± 8 g) compared to the two other groups (411 ± 5 and 406 ± 8 g for the C and HF- groups, respectively). This perfectly fits with the results of the present study indicating that the HF-fed rats can either take weight compared to the control group or not. Indeed, the weight gain of the animals during the 3 month-feeding period was higher in the HF+ group compared to the two other ones. Interestingly, the weight gain paralleled that of the cumulative dietary intake in the HF groups, suggesting that the difference in weight gain was due to a difference in food intake. The palatability of the HF diet could thus be responsible for the observed differences. It could be high enough for the subgroup of rats becoming obese and insufficient for the other ones. However, the reduction of the n-6 to n-3 PUFA ratio of cardiac phospholipids observed in the HF- group could also limit the food intake. In the present study, despite the different body weight gains, the abdominal fat mass was of similar magnitude in the two subgroups of HF diet-fed rats, suggesting that the excessive caloric intake observed in the HF+ group was used to build up other tissues in the body. Another explanation for the lack of body weight gain in the HF- group could be an insufficient intake of proteins and a low lean mass build-up[32–35]. In the present study, the protein content of the HF diet was planned to be twice as high as that of the C diet in order to compensate for the lower food intake due to the dietary lipid enrichment. This could be not enough for certain animals in order to build up a sufficient amount of muscle proteins. Moreover, a reduced respiratory chain complex 3 activity which paralleled the decreased n-6 to n-3 PUFA ratio of membrane phospholipids was observed in the hearts of the HF- animals. This could lead to a lower rate of ATP production. If the energy available for biochemical synthesis was also reduced in the skeletal muscle, this could lead to decreased protein build-up and muscle mass formation. The phenomenon would not occur in the HF+ group, since the respiratory chain complex 3 activity and n-6 to n-3 PUFA ratio of membrane phospholipids were as high as in the C group. However, the formation of these two HF subgroups reveals that a HF diet can have differential effects on the body and blood composition of the individual.
The HF diet chosen for this study triggered an increase in the in vivo contractile function of the animals especially that of the HF- rats, whereas the HF+ rats had an intermediate profile between control and HF- rats. However, these results were not found in the ex vivo situation. Indeed, the ex vivo cardiac mechanical function was reduced by the HF diet, following the elevated adiposity of the animals irrespective of their body weight. That observation has already been presented in the literature after a HF diet period[9, 31], after weight gain through post-natal overfeeding in the mouse and in the rat as well as in the ZDF rat. This depressed ex vivo cardiac mechanical activity observed in this study could be related to changes in the cardiac metabolism related to the whole body glucose intolerance, the increased degree of saturation of the cardiac membranes as shown by the increase in the SFAs at the detriment of MUFAs and the pro-inflammatory environment as indicated by the low ratio EPA/AA that predisposes to a balance of eicosanoids favoring platelet aggregation and inflammatory signaling[37, 38]. However, an increased cardiac output is expected to occur with nascent low- and moderate-severity obesity. Indeed, our in vivo cardiac function measurements suggest an augmented inotropism after the 3-month HF diet intake as already shown by measurements of the in vivo ejection fraction after post-natal overfeeding. That parameter is firstly increased at the age of 3 months before being reduced from the age of 5 months. Thus, nascent obesity may lead to an increased cardiac output resulting from an increased cardiac mechanical function.
The further study of these two subgroups revealed the same profile of the ex vivo cardiac and coronary function after the HF diet but the results were related to different mechanisms at the level of coronary vessels depending on the body composition of the animals. These results indicate that the high-fat diet has an important effect on the adiposity of the individual, but not necessarily on the body weight, and that these changes in the adiposity are related to changes occurring at the level of cardiovascular function.
We then evaluated the ex vivo coronary reactivity of the animals according to Langendorff mode. We evaluated the global cardiac reactivity through estimation of changes in the aortic pressure, which in our model of Langendorff perfusion at fixed flow reflected mainly the pressure of the coronary micro vessels. The conductance vessels may also contribute to the aortic pressure, but no spasm and no atheroma plaque was expected to occur in our experimental conditions. This study reports for the first time that a 3 month HF diet triggered an increase in EDV of the coronary microvasculature. HF diet- or post-natal overfeeding-induced obesity has been associated with either a reduced[14–16, 18] or a maintained[7, 10, 12] EDV of the coronary vessels. It has also been reported that glucose intolerance due to high-fat feeding does not alter myocardial perfusion during hyperemia. However, Jerebolvszki et al. reported a HF diet-induced increase in the sensitivity of pressurized coronary arterioles to NO, suggesting that the coronary reactivity can be increased in certain circumstances. This increase in the EDV of the HF-fed animals could augment the coronary reserve explaining the results of the in vivo situation.
The HF diet-induced inotropic effect that encountered in vivo in our experiments fits perfectly with the increased coronary reserve reported ex vivo. This mechanism could also explain results from previous studies reporting maintenance of myocardial perfusion or preserved contractile function after high-fat feeding[31, 39, 40]. Hence, early obesity triggers an in vivo increase in contractile function which is supported by an augmentation of the coronary reserve. The discrepancies between the ex vivo and in vivo situations observed in our study could be due to an increased left end diastolic volume reported to occur in overfed rats, which would stimulate the cardiac contractile function through the Frank Starling’s law in the in vivo situation.
The augmented EDV observed in our study paralleled the increase in abdominal fat mass, but was not related to an augmentation of body weight. It is possible that the increased fat mass at the abdominal level or at the pericardiac level if it also occurred acted on the coronary vessels through a change in adipocytokine release. Systemic leptin is increased with augmented adiposity while adiponectin is reduced. The resulting in vivo adiposity-related changes in coronary function could be retained ex vivo and contribute to the adaptation of myocardial function in nascent obesity. Indeed, it has been shown that obesity necessitates higher cardiac mechanical activity[43–45] due to augmented whole body energy expenditure. As already indicated, in our model of cardiac perfusion, we measured mainly the reactivity of the coronary microvasculature which determines myocardial perfusion. The increased coronary EDV observed in our study could reflect an augmented in vivo coronary perfusion due to an obesity-related increase in cardiac output.
Previous studies suggest that obesity may reduce NO levels mostly through increased oxidative stress and that when NO bioavailability is reduced a compensatory mechanism takes place in order to maintain a normal coronary function. Adaptation of coronary vessels is particularly important, as in the coronary circulation oxygen extraction is near maximal and any mismatch between blood supply and metabolic demand would deteriorate myocardial contractile function. Furthermore, the increase in body mass, either muscular or adipose, requires higher cardiac output and expanded intravascular volume to meet the elevated metabolic requirements. Thus, the vascular alterations observed in our study could help the coronary microvasculature to adjust the organ perfusion during physiological processes such as exercise. Otherwise the heart would not be able to respond to increased metabolic demands and lead eventually to ischemic incidents.
In order to evaluate the contribution of the main vasodilator pathways in the observed EDV, inhibitors that block NO production and COX were used during the perfusion protocol. The main results were the following: i) L-NAME reduced EDV in the HF- group, indicating the implication of NOS pathway in the enhanced ACh response in the HF group; ii) indomethacin decreased EDV in the HF+ group, implying an altered balance between COX-derived vasodilators and vasoconstrictors in the HF group. The HF diet seems to reduce the availability of vasoconstrictor mediators and maintain or even enhance that of vasodilators contributing eventually to the enhanced EDV. The analysis of the fatty acid content of the cardiac phospholipids also revealed that the arachidonic acid (AA, C20:4n-6) was increased in the HF- rat hearts which could lead eventually to an increase in the COX-vasoactive agents; iii) association of L-NAME and indomethacin decreased the EDV in both HF- and HF+ groups. Thus, both NOS and COX pathways seem to be implicated in the HF-induced ACh response.
The study of the two HF subgroups (HF-, HF+) during the last set of experiments helped to elucidate the involvement of the NOS and COX pathways in the increased ACh-response of the HF rats. The augmented ACh- response of the HF- rats was due to an increase in the activity of endothelial cells, as shown by the ECVA diagram while that of the HF+ rats was due to an increased sensitivity of the smooth muscle cells to NO, as shown by the response to SNP injections. The mechanism explaining the increased EDV observed in the HF+ rats has already been described in the literature and was explained by an increased sensitivity to NO of the SMC guanylate cyclase with consequent augmented cyclic guanosine monophosphate (cGMP) production and SMC relaxation. This fits well our results since L-NAME did not affect the EDV of the HF+ rats, indicating that NOS pathway was not affected, but since SMCs are more sensitive to NO which leads finally to increased EDV. Furthermore, the implication of COX-derived vasodilators seem to participate in the increased EDV of the HF+ rats as shown by the results of the indomethacin experiments. However, more original was the mechanism explaining the increased EDV observed in the HF- group. Indeed, these rats with normal weight obesity displayed an improved EDV which was strictly due to an increased ECVA, which was probably due to increased NOS signaling as shown by the results of the L-NAME experiments. Since the content of AA of myocardial phospholipids was increased in that group and not in the HF+ group, we also suspected the involvement of COX products in order to explain the increased ECVA. Indeed, indomethacin decreased the EDV of the HF-rats but not significantly, indicating that the NOS pathway remains the prominent pathway for the vasodilatation together with the activity of the endothelial cells. This relationship between NOS/COX pathways and endothelial/smooth muscle cells in the EDV seems to be reversed in the HF+ group, with the COX pathway having the most important role and the smooth muscle cells becoming more sensitive. Thus, the adipose tissue distribution seems to affect the mechanism through which the increased ACh response occurs in the HF-fed rats.