The high-sucrose diet rat as a model of prediabetes
Many reports have demonstrated that consumption of high-sucrose diet induces impaired glucose tolerance and hyperinsulinemia, and decreases insulin sensitivity [27–29]. However, data from models of prediabetes, namely the HSu-treated rat, are scarce [15, 16]. Our study demonstrates no differences on glycemia in the fasting state, but a significant increase in the fed (postprandial) state in HSu-treated rats, together with impaired glucose tolerance, hyperinsulinemia and insulin resistance. This is line with previous studies having demonstrated that sucrose consumption produces insulin resistance in rats [19, 20], which is characterized by fasting normoglycemia [30, 31]. Thus, HSu-evoked impaired glucose tolerance as well as hyperinsulinemia might be triggered by peripheral insulin resistance. In fact, T2DM is recognized as a progressive disorder, which is associated with diminishing pancreatic function over time. However, at an early stage, as it may occur after 9 weeks of high sucrose intake, the presence of insulin resistance must be compensated by hyperinsulinemia to maintain normoglycemia. This is the case in individuals who develop diabetes, a progressive loss of the insulin secretory capacity of β-cells can appear much before the clinical diagnosis . Furthermore, TRB3 (a member of mammalian tribbles homologs protein family), has been recently considered to be a mediator of insulin resistance in DCM , explaining our interest in its evaluation in this model. However, our result showed no differences in TRB3 mRNA expression between the groups, suggesting that it might not be impaired in the sucrose-induced prediabetic state.
The present study demonstrated that sucrose intake induced no significant changes on body weight and total cholesterol levels, suggesting that insulin resistance produced in this model is, at least initially, independent of the overt obesity, which is consistent with a previous study . However, elevated serum TGs contents were observed in the HSu-treated rats consistent with other reports [28, 34]. This may be a result of de novo lipogenesis, the enhanced rate of hepatic TGs synthesis and the decrease in peripheral clearance . In addition, no significant differences in blood pressure and heart rate were observed between HSu-treated rats and the control animals, indicating that 9 weeks of 35% sucrose treatment was unable to cause a significant impact on these parameters. The data is in agreement with a previous work, which failed to show hypertension in high-sucrose diet fed rats .
The mechanisms underlying the development of diabetic cardiomyopathy have been widely discussed and seem to involve complex and multifactorial influences. Indeed, as in diabetes, hyperglycemia, hyperinsulinemia and hyperlipidemia might be the initial triggers of significant cellular and molecular changes ultimately leading to cardiac functional and structural impairment, key features of the systolic and diastolic dysfunction associated with diabetic cardiomyopathy. Oxidative stress, endothelial dysfunction, inflammation and apoptosis, are important triggers of cardiac fibrosis and hypertrophy, which are viewed as major structural changes associated with cardiac dysfunction inherent to DCM [15–18]. The existence of earlier cardiac changes in prediabetes has been suggested, but the human studies, and even the animal models, are usually limited because of the coexistence of other perturbations. Our data is consistent with a model of prediabetes/insulin resistance, characterized by fasting normoglycemia, impaired glucose tolerance, hyperinsulinemia, insulin resistance, together with hypertriglyceridemia, important features to regarding their direct impact on cardiac tissue, independently of other confounding factors, such as hypertension and obesity.
Early markers of cardiac hypertrophy and/or fibrosis: focus on BNP overexpression
Cardiomyocyte hypertrophy, as well as subsequent apoptosis and myocardial fibrosis, are structural hallmarks of DCM [36, 37]. Previous studies have shown that cardiomyocyte hypertrophy in diabetes is regulated at the transcriptional level [38, 39]. Thus, it is important to examine molecular biomarkers of the putative cardiac alterations. In this study, we evaluated cardiac mRNA levels of TGF-β1, TSP-1, pro-collagen III, CTGF and BNP, which have been associated with cardiac hypertrophy and/or fibrosis. Indeed, TGF-β1 plays a role in cell growth, differentiation, apoptosis, inflammatory processes and gene expression  and has consistent implications in DCM due to pro-fibrotic properties, including the expression of many matrix proteins, such as collagens. In the hearts of diabetic patients, collagen remodeling occurs in the perimysium and perivascular region, mainly as a result of an increased accumulation of type III collagen , which has been related to left-ventricular diastolic and systolic dysfunction in DCM [42, 43]. Additionally, TSP-1 is an adhesive glycoprotein that influences cellular phenotype and the structure of the extracellular matrix . It has been reported that high glucose levels stimulates TGF-β1 bioactivation by fibroblasts through up-regulation of TSP-1 . CTGF is another potent pro-fibrotic protein that plays important roles in tissue and organ fibrosis and it is increasingly being implicated in structural and functional abnormalities in the diabetic heart [46, 47]. In addition, BNP, a peptide hormone released from the cardiac ventricles in response to pressure and volume overload, is among the most relevant molecular markers of cardiac hypertrophy . In our study, no significant differences were found in cardiac gene expression of TGF-β1, pro-collagen III, TSP-1 and CTGF between the groups, despite a trend to increased values in the HSu animals for all mediators. However, BNP gene expression was significantly higher in animals under sucrose treatment, which is in agreement with the LV hypertrophy, viewed by the relative increase in LV weight. Thus, collectively, this data indicate that a high sucrose diet can promote cardiac trophism in a prediabetic state, with BNP the earlier biomarker of those alterations, in agreement with a recent study suggesting BNP as a useful marker for screening preclinical ventricular diastolic dysfunction in patients prone to develop cardiovascular complications, such as uncontrolled diabetics . Cardiac hypertrophy and fibrosis are usually associated with apoptosis . In this study, no significant changes in Bax and Bcl-2 cardiac gene expression were found. Whether these are or not related with the anti-apoptotic effect of insulin in the absence of hyperglycemia, deserves further evaluation .
Markers of endothelial dysfunction and angiogenesis
Endothelial dysfunction is one of the mechanisms involved in the pathogenesis of DCM. Inducible NOS is an enzyme with recognized implications in ROS production, particularly under inflammatory conditions, and it is also involved in insulin resistance . Activation of iNOS contributes to the pathogenesis of CVD, probably because of excessive release of NO and generation of ROS. Ropelle et al. (2013) recently demonstrated that iNOS overexpression induced by aging is associated with increased S-nitrosation of major proteins involved in insulin signalling, thus reducing insulin sensitivity in skeletal muscle, which is prevented in aged iNOS-null mice . In addition, iNOS deficiency protects from high-fat diet-induced insulin resistance in the obese Zucker rat model . In our model, no significant changes in cardiac iNOS gene levels were found, despite a trend for higher values in the insulin resistant animals. Lee et al. (2009) suggest that upregulation of iNOS may be a protective mechanism against excessive contraction, abnormal signalling resulting from oxidative stress and enhanced inflammation in the diabetic vasculature . Whether this hypothetic protection is present or not in our model, needs additional investigation. Endothelial dysfunction was also assessed by the expression of adhesion molecules, such as ICAM-1 and VCAM-1, which have been associated with the development of CVD in diabetic patients [56, 57]. In our model, however, no differences in cardiac ICAM-1 and VCAM-1 gene expression were found, suggesting they could be later markers of these changes. Another mediator of vascular changes found in diabetic patients is VEGF, promoting angiogenesis and increasing vascular permeability [58, 59]. We observed a trend for increased levels in cardiac tissue VEGF in HSu-treated rats, which was accompanied by increased serum protein levels, suggesting its direct involvement.
Markers of inflammation and redox status
Elevated inflammatory cytokines have been found in circulation and in the diabetic hearts of T2DM patients, contributing to heart failure [60–62]. Cardiac overexpression of TNF-α has been associated with cardiac hypertrophy and fibrosis, as well with left ventricular dysfunction [63, 64] and IL-6 has been also described as an inducer of myocardial damage by promoting LV dysfunction and cardiac hypertrophy under acute myocardial infarction . In our study, however, no significant changes were found in either the serum or cardiac levels for IL-6 and cardiac TNF-α, suggesting that inflammation is not the key factor of the early changes related to cardiomyopathy.
In diabetes, oxidative stress has been recognized as an important player in the mechanism underlying DCM. However, in a state of insulin resistance without hyperglycemia, as it occurs in the early stages of diabetes (prediabetes), the contribution of ROS and consequent oxidative stress to the heart remains to be elucidated. In our model, the absence of markers of increased inflammation was accompanied by slight changes in the redox status measured. We found a trend for increased MDA levels (a marker of lipid peroxidation) in both the serum and heart tissue of HSu-treated rats. However, a significantly increased in serum TAS (a measure of total antioxidant capacity) was observed in the HSu rats, accompanied by an overexpression of the cardiac SOD gene, as well as a reduced expression of RAGE. These might collectively be viewed as compensatory actions against the putative increase in ROS. Furthermore, our model presented hyperinsulinemia, which was still able to normalize blood glucose levels, despite the impaired glucose tolerance. Thus, the putative generation of ROS, as suggested by the small increase in serum and heart lipid peroxidation, seems to be compensated by additional generation of antioxidants, observed by the significantly increased serum TAS and cardiac gene expression of SOD. Furthermore, the observed under-expression of RAGE in cardiac tissue might be also viewed as a compensatory mechanism, protecting the cardiac tissue. Indeed, we cannot exclude the possibility that longer exposure times of sucrose may lead to reduction of these antioxidant defence mechanisms and, consequently, to toxic effects induced by the oxidative stress aggravating cardiac function.
Hyperglycemia has been indicated as one of the major players in DCM, due to activation of several mechanisms, including promotion of oxidative stress. In our model, the modifications present at an early stage (hyperinsulinemia, insulin resistance, fasting normoglycemia) before over diabetes (defined as prediabetes), might be eventually linked with the impact of impaired glucose tolerance (fed, or postprandial, hyperglycemia). Additional studies are needed to better understand the underlying molecular and cellular mechanisms responsible for the effects of HSu in prediabetes, as well as to elucidate whether hyperglycemia is, or not, the major driving force for the overt cardiac changes underlying cardiomyopathy.