In this work we showed that chronic consumption of S and F, individually, lead to MetS, as indicated by the presence of IR and the increase in adiposomatic index and in the levels of glucose, insulin, total-cholesterol, TG, and ROS (as assessed by the levels of MDA and -NO2). Moreover, these metabolic alterations were accompanied by enhanced ox-CaMKIIδ and by the development of CH, suggesting that there is a relationship between the metabolic abnormalities produced by these sugars and the remodeling of cardiac structure. Finally, we showed that metabolic alterations as well as the ox-CaMKIIδ and severity of CH are exacerbated when combined S and F (S + F) are consumed.
Experimental studies have reported that high-carbohydrate diets produce metabolic alterations such as IR, glucose intolerance, OS, dyslipidemia, inflammation, increased AP, hepatic steatosis, among others [3, 8]. However, there are no experimental studies that compare the possible metabolic damage between the different sugars consumed from an early age and the cardiac alterations that they can generate. Therefore, in this study we evaluated the effects of long-term consumption of S, F and S + F in recently weaned Wistar rats.
BW in the three groups studied in this investigation did not increase; on the contrary, a significant reduction in weight was observed in the S group. Our results are in agreement with previous studies, which showed that adult male Wistar rats fed with 30% sucrose for 15–18 weeks, starting at a few weeks of age, did not present significant BW gain [3, 20, 21]. Nevertheless, despite the lack of BW gain, our results for the visceral adiposomatic index confirmed that animals on all three diets produced a greater accumulation of adipose tissue compared to C, with the S + F group inducing the greatest accumulation, followed by S, and finally F. These results indicate that the degree of fat accumulation depends on the type of sugar ingested. Our data agree with that reported by Fuente-Martin et al., who found that feeding neonatal Wistar rats with 33% sucrose for two months caused a significant decrease in BW, although there was a significant increase in adipose tissue due to adipocyte hypertrophy .
Although it is unclear why there was no BW gain in response to high-carbohydrate diets, it is likely due to the earlier age at which the animals started on the hypercaloric diets and the lower food intake they had, as seen in Table 1. These results are consistent with our previous reports showing that S-fed adults rats decreased food intake, while keeping constant liquid consumption [20–22]. Moreover, although it has been reported that rodent food provides a sufficient amount of protein and other essential nutrients, the preference of young animals for the intake of sugar-sweetened beverages [21, 22] and the decrease in solid food ingestion, may explain why there is not a BW gain in our animals despite the increase in visceral adipose tissue, as has been suggested by others [21, 22].
Biochemical profile showed that glucose, insulin, cholesterol and TG levels, as well as the HOMA index, increased significantly in S and F groups, which in addition to the increase in VATW and the decrease in QUICKY index, confirms that our animals developed several components of MetS, such as obesity, IR and dyslipidemia. Our data are in agreement with previous reports where the effects of S- and F-sweetened beverages were studied separately [3, 8, 22–24]. In this work, however, we also showed that combined S and F diet exacerbates all these metabolic alterations, indicating that S + F worsen MetS.
IR and sustained hyperglycemia promote increased proliferation of β-pancreatic cells to produce more insulin and compensate for excess blood glucose; however, they also induce the activation of other processes, such as inflammation and overproduction of ROS associated with mitochondrial oxidative stress . According to our results, we confirmed that metabolic damage due to excess consumption of S and F as the main sugars used by the food industry, and especially when consumed together the metabolic damage is exacerbated. On the other hand, the data on hyperglycemia, hyperinsulinemia and IR suggest the development of DM2 in all experimental groups that consumed S, F or S + F. However, further tests are required to confirm the presence of DM2.
The consumption of high-carbohydrate diets produced a significant increase in both MAP and DAP, and only the S + F diet produced an increase in SAP. According to the WHO criteria, and taking into account that normal AP values in rats are similar to those in humans, our data show that hypercaloric diets increase AP. Balderas-Villalobos et al., reported that diet with sucrose (30%) for 18 weeks did not cause a significant change in SAP and DAP, while Baños et al., reported that 3-week-old Wistar rats treated with the same diet for 8 months, resulted in a significant increase in SAP, but did not evaluate DAP [3, 25]. In this sense, the ENIGMA study examined a group of students with an average age of 20 years and with an elevated body mass index, and observed that DAP is the first parameter to rise and then give way to elevation of SAP or else, that only DAP was elevated . This could explain why in our animals the significant increase in DAP was greater in the three diets. Nevertheless, in the combined intake (S + F) the increase in both DAP and SAP (Table 3) may be due to exacerbation of OS (Fig. 1), which can lead to a greater endothelial damage (see below).
In the three groups of experimental animals, the intake of sugar-sweetened beverages significantly increased lipid peroxidation and, therefore, increased ROS generation (Fig. 1). Nonetheless, these effects were more accentuated with the S + F diet, suggesting a greater degree of OS. In the case of serum SOD, levels increased with all three hypercaloric diets, which could suggest that this enzyme is produced in higher amounts to maintain homeostasis between ROS and body antioxidants. However, this was not the case in cardiac tissue, where the sugary diets caused a reduction in SOD levels, probably due to the imbalance between antioxidant enzymes and increased ROS, characteristic of OS. Indeed, it has been reported that young adult Wistar rats (2 months old), placed on a 40% sucrose diet for 6 months, in addition to presenting obesity, IR and dyslipidemias, had a significant increase in hepatic MDA, and a significant decrease in SOD and glutathione, which are characteristic of OS in obesity .
The imbalance between ROS production and the activity of endogenous antioxidant agents in people with MetS has been strongly correlated with the development of different cardiovascular pathologies . Zhang et al., reported that hearts from C57BL/6 mice (8 to 10 weeks old) treated with a 10% F diet for 20 weeks, and neonatal rat ventricular cardiomyocytes cultured with 25 mM F, displayed a significant increase in mitochondrial ROS . Furthermore, echocardiographic and histological evaluation confirmed the presence of CH in these animals, which was accompanied by an increase in molecular markers such as ANP. These results highlighted that F by itself is able to induce an increase in mitochondrial ROS production, which is associated with the development of CH, both in vivo and in vitro . In this sense, our data showed that there is a direct correlation between VATW and the increase in TG and MDA (Fig. 2) and the severity of CH (Fig. 3 and Table 4) generated by the consumption of S and F, thus, the higher the VATW, the higher the TG and OS and the higher CH. Hence, combined consumption of S and F, in addition to causing greatest VATW, generated highest OS and thus the highest CH. On the other hand, it has been reported that presence of DM2, IR, glucose intolerance, inflammation and hepatic steatosis, besides obesity, plays an important role in the development of OS in MetS induced by high carbohydrate diets [5, 28]. Therefore, the higher levels in glucose, insulin and IR generated by S + F diet, could explain why these sugars together caused higher MDA levels.
One of the mechanisms linking increased adipose tissue and AH is endothelial dysfunction, which is associated with decreased endothelial NO in blood vessels. As a result of excessive ROS production, O2•- is highly likely to react with NO leading to an increase in peroxynitrite (OONO•-) . This reaction is responsible for the decrease in NO, which together with the increase in OONO•-, contributes to endothelial dysfunction. Our data showed that hypercaloric diets produced an increase in -NO2 levels, which correlated with an increase in DAP in all groups. Nonetheless, the higher level of -NO2 produced by S + F was accompanied by an increase not only in DAP, but also in SAP. Increase in both DAP and SAP correlates with higher CH when S and F are consumed in combination. A direct relationship in the development of CH by AH-induced pressure overload has been widely reported . It has been described that children with obesity, and with obesity plus AH, had CH, indicating that CH is not only present in adulthood, but also at early ages, and that its development is independently favored by AH and obesity . These observations in children and adolescents support the hypothesis that from an early age obese subjects could develop CH or have a high probability of developing it in adulthood.
Increased expression of ANP has been established as a molecular marker of CH [13, 32]. Furthermore, it is well established that OS-mediated chronic activation of CaMKIIδ plays an important role in the development of CH [6, 13, 17, 18]. Therefore, to determine the presence of CH in MetS and the molecular involvement of ox-CaMKIIδ, we evaluated the effects of hypercaloric diets on expression of ANP and level of ox-CaMKIIδ. All carbohydrate-diets cause CH, as was showing by the increased ANP expression, however, ANP expression was higher in S + F (Fig. 4A). In addition, expression of total CaMKIIδ was not modified by any of the diets (Fig. 4B), which is in agree with a previous report , however, ox-CaMKIIδ was increased by the three experimental groups, but this increase was higher in the case of S + F (Fig. 4C and D). Recently, it was reported that CaMKIIδ plays an important role in the development of obesity-induced CH . Therefore, our results suggest that in MetS, combined S and F consumption exacerbates CH by increasing the level of ox-CaMKIIδ (Fig. 5).
Histopathological analysis confirmed that all three diets, but mainly S + F caused CH as indicated by increased LVWT and RVWT and decreased LVCR and LVLA (Table 4). These effects on cardiac structure decreased LVCR/LVWT index (Table 4), suggesting the development of a concentric-type CH. Our histological studies also showed increased myocyte size and the presence of higher collagen deposition in the hearts of the three experimental groups (Fig. 3B and C). Increased myocyte size is usually accompanied by interstitial growth that allows structural support to CH , while collagen deposition takes place as a compensatory response to the cell loss caused mainly by apoptosis [14, 16]. It has been reported that OS, produced under pathological conditions, plays a role in cardiomyocyte death and replacement of cell loss with collagen fibers . In addition, OS has been implicated in the activation of CaMKIIδ via oxidation of this kinase, which in turn, has been reported to contribute to cell growth and apoptosis in hearts [12–14]. Our data showing that the amount of VAT correlates with the level of OS (Fig. 2) and that S + F causes the higher increase in these parameters and in parallel produces the higher increase in ox-CaMKIIδ and CH, suggest that there is a direct relationship between the degree of MetS and OS with the levels of ox-CaMKIIδ and the worsening of CH (Fig. 5).