An attempt to reverse cardiac lipotoxicity by aerobic interval training in a high-fat diet- and streptozotocin-induced type 2 diabetes rat model

Diabetic cardiomyopathy (DCM) is primarily caused by diabetes and is independent of coronary artery disease and hypertension, leading to cardiac diastolic dysfunction during the initial stage and systolic dysfunction at later stages [1]. The main pathological characteristics of DCM are suppressed glucose metabolism, elevated fatty acid metabolism and lipid accumulation within myocardial cells [2, 3]. Myocardial triacylglycerol is significantly higher in diabetes patients but is mobilized rapidly into intermediates, such as free fatty acid (FFA) and diacylglycerol (DAG) [4]. Lipid overload results in an accumulation of toxic intermediates that lead to cardiac lipotoxicity, further activating protein kinase C signalling, producing reactive oxygen species (ROS) and inducing apoptosis [5].

Because no significant clinical symptoms are observed in the early stage of DCM, it is hard to identify until patients present with heart failure. At that time, DCM is difficult to control effectively, so acting at in the early stage of DCM is crucial. Pharmacological treatment may reverse DCM in the early stage; however, pharmacological intervention will cause substantial economic pressure on the family and society [6]. Apart from pharmacological treatment, exercise intervention may also work [7]. Physical activity and exercise have been found to lower the risk of cardiovascular disease and metabolic syndrome, and regular physical activity is associated with improved longevity and reduced burden of diabetes-related complications. Therefore, exercise intervention is attracting the attention of medical and sports scientists for treating diabetes mellitus patients with heart failure [8]. Traditional exercise interventions are aimed at low-to-moderate-intensity exercise (55–70% VO2max), such as jogging and cycling [9]; however, the intensity is not high enough to stimulate adaption of the cardiopulmonary system. Aerobic interval training (AIT) consists of 1–4 min of high-intensity exercise (≥ 70% maximal aerobic capacity) with active low intensity exercise, which has been proved to improve fasting and postprandial blood glucose levels, improve VO2max, and speed up rehabilitation progress [10]. AIT is beneficial to DCM, and previous studies pointed out that it may reduce sarcoplasmic reticulum (SR) Ca2+ leak [11] and improve cardiac function [12], but few studies have directly explored the effect of AIT on cardiac steatosis.

This study aimed to investigate the effects of an 8-week AIT intervention on reducing cardiac steatosis and to further explore the underlying mechanisms involved in the reversal of DCM progression after AIT intervention.

Numerous investigations have demonstrated that diabetes mellitus is positively correlated with cardiac dysfunction and heart failure [19]. The American Heart Association and American College of Cardiology [20] illustrated that diabetes mellitus, a grade A level heart failure risk factor, may directly damage cardiomyocytes by lipid toxicity, potentially contributing to the development of heart failure in diabetes patients even in the absence of coronary artery disease and hypertension, which is called DCM [21]. In the present study, the DCM model with increased FBG levels, insulin resistance, and impaired cardiac diastolic and systolic functions was successfully established. In addition, the HE staining results showed injured cardiac tissues, which further indicated that the DCM rats suffered structural and functional damage. This finding is consistent with previous studies and is suitable for exploring the pathologic mechanisms underlying DCM [22]. AIT intervention may significantly reverse these phenomena.

Cardiac lipotoxicity is characteristic of an accumulation of lipid droplets within cardiomyocytes [23]. Cardiomyocytes in healthy hearts have few droplets, while excessive lipids are stored in diabetic hearts [24]. In cardiomyocytes, lipids were stored in the form of TG but are rapidly mobilized into FFA. TG is non-toxic, and its intermediates, DAG and ceramides, of the non-oxidizing pathway are toxic [25]. This result is consistent with our study, in which histology and cardiac tissue results indicated that DCM hearts accumulate lipid drops inside cardiomyocytes, accompanied by high levels of FFA and DAG. AIT intervention attenuated the levels of cardiac FFA and DAG, therefore reducing cardiac lipid deposition and steatosis.

Cardiac lipotoxicity could be due to increased myocardial FFA uptake. FFA can be taken up by cardiomyocytes in two major ways: 20% of FFA is absorbed by passive diffusion, and 80% of FFA by protein-mediated transport [26]. From the serum lipid profile measurements, we can conclude that DCM rats have higher TG and LDL-c levels, which increase the source of cardiac lipids. Long-chain fatty acids (LCFAs) can be taken up through the LCFA translocase protein CD36 [27]. In the present study, we found that cardiac CD36 expression was elevated in the DCM group. CD36 takes up LCFAs into cardiomyocytes, and inner-membrane LCFA can activate PPARα, which may result in transcriptional upregulation of the enzymes involved in FA transport (FAT/CD36) and FA oxidation (CPT-1), which in turn aggravate cardiac lipid disturbance [28]. In the present study, increased FFA levels and PPARα expression were observed in DCM rats in parallel with the upregulation of CD36. Exercise has beneficial effects on regulating cardiac lipid disturbance. Notably, Chen’s study, which subjected diabetic rats to an 8-week swimming intervention, found that heart PPARα and CD36 mRNA levels were decreased and lipid toxicity was relieved by the intervention [29]. This result is consistent with our study, which showed that an 8-week AIT intervention significantly reduced PPARα and CD36 expression which restricted the source of cardiac steatosis.

AMPK, a heterotrimeric enzyme activated by an increase in the AMP/ATP ratio within cardiomyocytes, is a metabolic regulator that is involved in glucose and lipid metabolism [30]. Recently, AMPK has become a target for treating diabetes, and exercise may significantly activate AMPK signalling. Furthermore, aerobic exercise may reverse insulin resistance by activating AMPK signalling in skeletal muscle [31]. AMPK may directly phosphorylate and activate the nuclear receptor farnesoid X receptor (FXR) in the liver to promote metabolic homeostasis [32]. In addition, AMPK may modulate cardiac FFA metabolism by regulating CPT-1 through phosphorylation of acetyl CoA carboxylase (ACC) and release of the malonyl-CoA-mediated inhibition of CPT-1 [33]. Can AMPK modulate the uptake of FFA? Hay et al. proposed that the FOXO-AMPK-mTOR signalling pathway plays a key role in signal transduction in mammalian cells [34]. Metformin can inhibit the growth of oestrogen-dependent endometrial cancer cells by activating the AMPK–FOXO1 signalling pathway [35]. Moreover, AMPK–FOXO1 signalling may also regulate proteolysis in rat cardiomyocytes [36]. The FOXO transcription factor family includes FOXO1, FOXO3, FOXO4 and FOXO6. These transcription factors participate in stress response, protein hydrolysis, apoptosis, etc. [37]. Forkhead box protein O1 (FOXO1) has been reported to participate in abnormal myocardial metabolism in vivo and in vitro [18]. Interestingly, FOXO1 is overexpressed in the diabetic heart, and the FOXO1-iNOS-CD36 axis was reported to be involved in enhancing fatty acid flux into cardiomyocytes [38]. FOXO1-knockout mice also showed inhibition of CD36 gene expression [39]. Furthermore, fenofibrate reduces lipid accumulation in myotubes by modulating the PPARα/AMPK/FOXO1/ATGL pathway [40]. This finding is consistent with our results in the present study showing that improved AMPK phosphorylation after AIT intervention significantly reduced cardiac FOXO1 expression, in parallel with decreased CD36 expression and reduced PPARα expression, eventually reducing cardiac steatosis.

Low cardiorespiratory fitness is a risk factor for DCM patients, and exercise training is the most effective and costless way to increase cardiorespiratory fitness [41]. Traditional exercise focused on low-to-moderate-intensity exercise, while more vigorous exercise may be associated with cardiovascular risk [9]. Vered’s study pointed out that acute exercise may induce ventricular dysfunction in young men with asymptomatic diabetic cardiomyopathy [42]. Thus, the safety of exercise for DCM patients is crucial. BNP is a hormone that is secreted as a reaction to ventricular distention and stretching [43]. Increased serum BNP concentrations indicate cardiac hypertrophy and diastolic dysfunction. In our study, DCM rats showed increased serum and cardiac BNP levels. However, the effect of exercise on BNP levels is controversial. Our results illustrated that AIT training also reduced BNP levels, which contrasts with Hsfstad’s research, which pointed out that both high-intensity and moderate-intensity training normalized left ventricular (LV) diastolic and systolic function but did not change BNP levels in obese C57BL/6J mice [44].

In conclusion, this study is the first to demonstrate that AIT intervention may reverse diabetic cardiomyopathy at the onset against myocardial steatosis, at least in part, by activation of AMPK–FOXO1 signalling.