Introduction
Diabetic cardiomyopathy (DCM) is a common complication in type 2 diabetes [
1]. Changes in myocardial energy metabolism, due to altered substrate handling, characterise diabetes-related heart disease and DCM [
1]. Studies in rodent models, including rats fed a high-fat diet (HFD) [
2], leptin-deficient and -resistant animals [
3‐
6] and transgenic mice with cardiac-restricted peroxisome proliferator activated receptor
α (PPAR
α) overexpression [
7], indicate that intramyocardial accumulation of triacylglycerol metabolites (lipotoxicity) and myocardial insulin resistance may underlie diabetes-related cardiac dysfunction [
1,
8].
Among the putative causes of myocardial triacylglycerol accumulation are elevations in long-chain fatty acid (LCFA) uptake [
6,
7,
9]. In the heart, approximately 50% of LCFA uptake is mediated by the fatty acid translocase CD36 [
10,
11]. Under physiological conditions, both insulin and contraction stimulate LCFA uptake by inducing the translocation of CD36 from an intracellular pool to the sarcolemma, illustrating that CD36 is also directly involved in the dynamic utilisation of LCFA by cardiomyocytes. This acute translocation of CD36 involves activation of phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (PKB/Akt)- and AMP-kinase (AMPK)-dependent signalling cascades in response to insulin and contraction, respectively [
11].
Interestingly, CD36 deletion rescues the cardiac dysfunction, metabolic abnormalities and myocardial triacylglycerol accumulation observed in transgenic mice with cardiac-restricted PPARα overexpression [
7], suggesting a critical role for CD36 in the development of diabetes-related heart disease [
12]. We hypothesised that long-term alterations in the functional pool of CD36 lead to myocardial accumulation of (toxic) lipid metabolites and insulin resistance and contribute to the development of diabetes-related heart disease. To examine this, we first measured in vivo cardiac dimensions by echocardiography in rats exposed to a high-fat diet (HFD), which impairs myocardial insulin signalling [
2,
13]. Subsequently, isolated cardiomyocytes were used to determine changes in substrate uptake and LCFA metabolism under basal conditions and after incubations with insulin and with oligomycin; the latter compound induces a contraction-like elevation of intracellular AMP levels and concomitant activation of AMPK [
14]. Finally, we assessed whether the observed alterations could be ascribed to CD36 in hearts from rats on the HFD by analysing the effects of the CD36 inhibitor sulfo-
N-succinimidyloleate (SSO) on myocardial LCFA uptake and utilisation in isolated cardiomyocytes, and by studying the expression and subcellular localisation of CD36 in cardiac ventricular tissue.
Discussion
Here we report that HFD feeding induces cardiac contractile dysfunction in rats and that this is associated with a permanent relocation of CD36 to the sarcolemma. The continuous presence of CD36 at the sarcolemmal membrane results in enhanced rates of LCFA uptake and subsequent esterification. We propose that this contributes to a decrease in myocardial insulin action and the development of diabetes-related heart disease. In addition, we show that AMPK-mediated responses are not affected by the composition of the diet.
A key observation in this study is that the alterations in cardiac contractile function in HFD hearts was associated with a continuous presence of CD36 at the sarcolemmal membrane. The present study provides the first morphological evidence for translocation of CD36 to the sarcolemmal membrane. Importantly, the amount of sarcolemmal CD36 closely correlated with enhanced LCFA uptake rates in isolated cardiomyocytes. The observation that SSO inhibited LCFA uptake in HFD cardiomyocytes to the same residual levels as measured in LFD cardiomyocytes provides further pharmacological evidence that the enhanced flux of LCFA in the heart of HFD-fed rats is a direct consequence of the relocalisation of CD36 to the sarcolemmal membrane.
Previously, a redistribution of CD36 to both subsarcolemmal and intramyofibrillar mitochondria has also been observed [
27,
28]. Can the observed CD36 immunoreactivity be ascribed to subsarcolemmal mitochondria rather that the sarcolemma itself? Stimuli inducing translocation of CD36 to the mitochondria are expected not to discriminate between the subsarcolemmal and the intramyofibrillar mitochondria. Hence, if a stimulus or condition, in this case insulin or HFD feeding, were to increase the subsarcolemmal CD36 content, one would expect a similar increase in intramyofibrillar CD36 content. This, however, could not be confirmed in the immunohistochemical experiments. Furthermore, biochemical fractionations performed in previous studies substantiate the idea that a significant fraction of CD36 is present at the sarcolemma of hearts from insulin-resistant rats, and that only a minor portion of CD36 is found within the mitochondrial fractions [
9,
22]. While insulin has been shown to translocate CD36 to the sarcolemma [
9,
22], insulin-mediated CD36 translocation to the mitochondria has never been reported and even seems counterintuitive. Collectively, the available evidence strongly supports the idea that the observed CD36 immunoreactivity can be ascribed to CD36 located at the sarcolemma rather than in subsarcolemmal mitochondria.
The combined data of this and our earlier study on obese Zucker rats [
9] suggest that relocalisation of CD36 is a general phenomenon in insulin-resistant hearts, and raises the question of what mechanism underlies the continuous presence of CD36 at the sarcolemmal membrane in HFD hearts. In the healthy myocardium, CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PI3K/PKB/Akt, respectively [
26]. Activation of AMPK is critical for contraction- and oligomycin-mediated CD36 translocation, whereas PI3K/PKB/Akt is critical for insulin-mediated CD36 trafficking [
10,
14,
26]. Basal phosphorylation of PKB/Akt and its distal target, PRAS40, were elevated in HFD compared with LFD hearts, whereas AMPK activity was not affected by the diet. Furthermore, in cells from HFD-fed rats, the stimulatory effects of insulin on LCFA uptake, CD36 translocation and phosphorylation of PKB/Akt and PRAS40 were abrogated, whereas oligomycin-induced AMPK activation and LCFA uptake was unimpaired between cardiomyocytes from LFD- and HFD-fed rats. Although we cannot exclude a contribution of other kinases or HFD-induced alterations in the as yet undisclosed trafficking machinery regulating the internalisation of CD36 [
11,
26], our observations do not argue against the suggestion that increases in PKB/Akt activity may contribute to the sustained sarcolemmal presence of CD36 in the heart of HFD-fed rats.
Previously, we suggested that increased plasma insulin might contribute to CD36 relocalisation in hearts from obese Zucker rats [
9]. As systemic hyperinsulinaemia was not observed in HFD-fed rats [
2,
13], it remains interesting to examine whether changes in the activity of other (insulin-independent) regulators of PKB/Akt phosphorylation, such as PI3Kγ- and β2-adrenergic receptor signalling pathways, Ca
2+-calmodulin dependent kinase, protein phosphatase 2A and the sympathetic nervous system [
29], can be linked to the observed increase in PKB/Akt phosphorylation in HFD hearts.
The metabolic and biochemical data were paralleled by in vivo cardiac functional changes. Importantly, HFD-induced cardiac contractile dysfunction does not seem to be linked to diet-related elevations in blood pressure. Circadian haemodynamic parameters were monitored using implanted telemetry devices in an experiment performed in a separate group of animals. We found a slight increase in night-time (activity-related) heart rate in HFD rats, but no changes in blood pressure even after 10 weeks of exposure to the diet (Electronic supplementary material Table
1). Others confirmed the absence of relevant hypertension after feeding rats a diet containing 74% fat for 17 weeks [
30]. This underscores the possibility that alterations in metabolism, rather than in haemodynamics, underlie the impairment in cardiac contractile function in HFD rats.
The present study also suggests that HFD-induced alterations in the functional pool of CD36 may contribute to an imbalance in LCFA uptake and in oxidation and esterification rates in HFD vs LFD cells. Whereas LCFA uptake rates were increased 1.4-fold in HFD cells, the LCFA oxidation rates were not increased but rather modestly lower in HFD cells, while the rates of esterification into triacylglycerol and phospholipids were 1.4-fold higher in HFD vs LFD cardiomyocytes. Accordingly, triacylglycerol content was 1.9-fold increased in HFD compared with LFD hearts [
2]. It seems unlikely that changes in malonylCoA levels underlie the reduced rates of LCFA oxidation in HFD hearts, as no diet-induced changes in ACC phosphorylation were found. Rather, degenerative changes in mitochondria, such as matrix dilution, cristolysis and mitochondria-associated lamellar bodies, have been observed in cardiomyocytes from HFD-fed rats [
2] and have been linked to reduced oxidative capacity and lipid accumulation in skeletal muscle from patients with type 2 diabetes. However, further studies are required to examine whether these changes contribute to the reduced basal LCFA oxidation and increased LCFA esterification rates, or whether the extra LCFA taken up by HFD cells are stored as triacylglycerol and phospholipids as a consequence of HFD-induced changes in enzymes promoting LCFA esterification or inhibition of lipolysis.
An important finding of this study is that the HFD-induced CD36 redistribution preceded the onset of cardiac contractile dysfunction. Although we cannot unequivocally link CD36 redistribution to cardiac dysfunction, a recent report provided strong support for this notion as the absence of CD36 was found to prevent myocardial triacylglycerol accumulation in transgenic mice with cardiac PPARα overexpression, both under normal conditions and after HFD feeding [
12]. Similarly, alterations in PKB/Akt activity have been linked to cardiac dysfunction [
31]. Myocardial biopsy samples obtained from patients with advanced heart failure or dilated cardiomyopathy show increased basal phosphorylation of PKB/Akt [
32,
33], and transgenic mouse models with constitutively activated PKB/Akt in the heart develop hypertrophy, decreased cardiac function and impaired recovery from ischaemia–reperfusion injury [
31,
34]. Whereas chronic activation of PKB/Akt increases basal glucose uptake rates in the heart [
35], the effects on myocardial lipid metabolism have not been studied. Interestingly, PKB/Akt signalling has been linked to palmitate-induced beta cell lipotoxicity [
36]. Based on this report and the findings described in this study, it would be of interest to analyse animals expressing chronically active PKB/Akt for translocation of CD36 to the sarcolemma and myocardial lipid accumulation.
It seems plausible that an enhanced CD36-mediated LCFA uptake rate may contribute to the development of cardiac contractile dysfunction. An increased supply of LCFA may influence excitation–contraction coupling as well as other processes linked to cellular Ca
2+ handling [
37,
38]. Furthermore, triacylglycerol accumulation is strongly linked to cardiac insulin resistance and contractile dysfunction [
1,
2,
8]. The dynamic equilibrium between triacylglycerol stores and triacylglycerol metabolites causes accumulation of ceramide and diacylglycerol during prolonged LCFA influx. Both ceramide and diacylglycerol have been implicated in the activation of serine/threonine kinases, such as PKC, Jun N terminal kinase (JNK), and inhibitor of nuclear factor-κB kinase (IKK), which counteract insulin signalling [
39,
40]. Notably, chronic myocardial activation of Akt in transgenic mice has also been linked to feedback inhibition of PI3K activity [
33] and blunted insulin stimulation of glucose uptake [
35]. Importantly, reduced insulin responsiveness reduces the ability of insulin to regulate substrate handling, e.g. metabolic inflexibility [
41]. In this study and previous reports [
2,
13], we show that HFD feeding reduces insulin responsiveness and impairs metabolic flexibility, as illustrated by the blunted effects of insulin on the stimulation of glucose uptake, phosphorylation of PRAS40, LCFA uptake and translocation of CD36 to the sarcolemma.
We conclude that HFD feeding in rats induces cardiac contractile dysfunction, which is preceded by relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The continuous presence of CD36 at the sarcolemma contributes to enhanced rates of fatty acid uptake, resulting in myocardial triacylglycerol accumulation and accompanying insulin resistance. Collectively, these data suggest that alterations in the subcellular localisation of CD36 may contribute to the development of diabetes-related heart disease and that CD36 may be a therapeutic target to prevent cardiac dysfunction and the development of heart failure in diabetes.