We report in this study that while DIO itself per se
does not elevate intra-myocardial ceramide levels, inhibition of SPT I to prevent de novo
synthesis of ceramide results in a marked reduction in overall intra-myocardial ceramide levels, and improves the DIO-associated reduction in myocardial glycolysis rates. Such findings yield provocative insights into the role of obesity on cardiac lipotoxicity and function. It has been proposed that during states of obesity, the accumulation of lipid metabolites in the heart such as TAG, long chain acyl CoA, DAG, and ceramide results in contractile dysfunction and contributes to the development of cardiomyopathy 
. Ceramide, in particular, is believed to play a major role in the development of cardiac lipotoxicity, as its accumulation has been shown in a number of in vitro
and in vivo
models to cause apoptosis of cardiac myocytes, which itself would contribute to the contractile dysfunction associated with cardiac lipotoxicity 
In contrast, our findings suggest that DIO does not result in ceramide accumulation in the heart, suggesting that perhaps the ceramide-induced apoptosis, which contributes to cardiac dysfunction, is the result of extreme situations induced by transgene overexpression 
. Nonetheless, the ceramide/sphingolipid pool is under dynamic regulation, being influenced by both its rates of synthesis and degradation 
. It is possible that enzymes involved in ceramide degradation were also upregulated by DIO, thereby masking any increase in its synthesis. Studies in humans support this possibility, as the mRNA expression of ceramide metabolizing/degrading enzymes such as ceramidase and sphingosine kinase is increased in right atrial appendages of obese humans with and without a history of type 2 diabetes, explaining why intra-myocardial ceramide content was similar to lean humans despite an elevation in SPT I mRNA expression 
. Furthermore, our measurement of overall intra-myocardial ceramide content does not separate ceramide originating from de
synthesis via SPT I versus that originating from phospholipid hydrolysis.
Our findings differ slightly from a recent study by Park TS et al.
, who demonstrated that cardiac-specific overexpression of glycosylphosphatidylinositol anchored human lipoprotein lipase in mice is associated with cardiac dysfunction and elevated levels of intra-myocardial ceramide 
. However, treatment of these animals with myriocin prevented intra-myocardial ceramide accumulation, restored glucose oxidation, and restored cardiac function. Although we also observed improved glucose metabolism in our DIO mice treated with myriocin, we did not observe any form of cardiac dysfunction. It may be possible that our high fat diet model did not result in cardiac dysfunction because intra-myocardial ceramides did not accumulate. A recent study in middle-aged mice (40–44 weeks) has shown that ceramides do accumulate in the heart following a 12-week high fat feeding regimen 
. However, systolic cardiac function was not altered, suggesting that an elevation of ceramide content in the heart may not negatively impact cardiac function. Nonetheless, future studies should be aimed at investigating the time points required for DIO in mice to induce cardiac dysfunction, and whether manipulation of SPT 1 and ceramide content in the heart can improve this deficit.
In our model, DIO did not result in a rise in intra-myocardial TAG content. While such findings may seem unexpected, we have previously shown that in C57BL/6 mice, intra-myocardial TAG content initially increases following 3 weeks of DIO, but reverts to normal levels seen in lean mice at 10 weeks of DIO, due to a decrease in DGAT activity, contributing to the DIO-associated elevation in DAG content 
. This is consistent with the observations of Somoza et al.
, as they demonstrated an increase in intra-myocardial TAG content at 4 weeks of DIO in C57BL/6J mice, followed by a reversion to levels seen in lean mice after 8 weeks of DIO 
. Contrary to our previous observations, DIO also did not increase intra-myocardial long chain acyl CoA levels 
. It is important to note that this observation is likely due to the high palmitate concentration used in the perfusate (1.2 mM), which would arbitrarily increase the palmitoyl CoA levels in perfused hearts from low fat fed animals. As such, palmitoyl CoA levels accounted for at least 70% of the total long chain acyl CoA content measured in all groups, explaining why no increase in long chain acyl CoA was observed in hearts following DIO in this study. High levels of palmitoyl CoA may also account for a lack of increase in intra-myocardial ceramide content, as palmitoyl CoA is the substrate for SPT I-mediated ceramide biosynthesis, and increased palmitoyl CoA levels in hearts from lean mice may also arbitrarily increase their ceramide content, masking any difference between lean and obese mice. However, our previous studies looking at ceramide content in non-perfused hearts from DIO mice also do not demonstrate an increase in ceramide content 
. Another factor that may contribute to the lack of observed increase in intra-myocardial ceramide content involves the young age of the mice in our study, as a recent study by Sung et al.
demonstrated no change in ceramide content following DIO in young mice, but a significant increase if middle-aged mice were subjected to DIO 
. Interestingly, we did observe an increase in intra-myocardial DAG content following DIO, which was prevented in mice treated with myriocin. This finding is of particular significance, as we have previously reported that intra-myocardial DAG accumulation is more strongly associated with the development of myocardial insulin resistance and impaired glucose utilization versus the other lipid metabolites 
. Whether the effect of myriocin on intra-myocardial DAG content alone is responsible for improving myocardial glycolysis rates in DIO mice has not been conclusively demonstrated in this study, as myriocin treatment also resulted in a marked reduction in intra-myocardial ceramide levels. However, in db/db
mice myriocin treatment reduced ceramide content without altering DAG levels, and had no effect on insulin signaling, supporting the premise that DAG is the more important lipid metabolite in the heart affecting cardiac insulin sensitivity and subsequent glucose utilization. Nevertheless, further characterization on the individual contributions of both ceramide and DAG towards myocardial glucose utilization is required.
The improvement in myocardial glycolysis rates in DIO mice treated with myriocin suggests an increase in myocardial glucose uptake. However, because our protocol for measuring glycolysis did not factor in the 3
H radiolabel incorporating into glycogen, we cannot conclude for certain that glucose uptake is increased in hearts from DIO mice treated with myriocin. Despite this limitation, insulin stimulated phosphorylation of Akt, a key molecule involved in the insulin signaling cascade that regulates glucose uptake 
, at serine 473 (indicative of increased Akt activity) is increased in DIO mice treated with myriocin. It is also important to note that while myriocin treated DIO mice exhibited an improvement in myocardial glycolytic rates, they were not normalized to normal values observed in lean mice, illustrating that lipotoxic metabolites such as ceramide and DAG are not the sole contributors to obesity-induced impairments in myocardial glucose utilization. Indeed, inflammation and oxidative stress may also impair myocardial glucose utilization 
, and it is possible that while SPT I inhibition improves myocardial lipid status, it has no impact on these other mechanisms that may be activated in response to DIO.
Our previous findings have also demonstrated that DIO impairs myocardial glucose oxidation rates 
, but due to the high variability in glucose oxidation rates in hearts from low fat fed animals, we did not report a decrease in this study, though there was a very strong trend to a reduction in hearts from control treated DIO mice. Further support for this observation in control treated DIO mice is evident with the 44% reduction in myocardial PDH activity, the rate-limiting enzyme for myocardial glucose oxidation. Interestingly, PDH activity was not reduced in hearts from myriocin treated DIO mice, once more suggesting that SPT I inhibition improves obesity-induced impairments on myocardial glucose utilization.
Although myriocin treatment improved myocardial glucose utilization in DIO mice, we observed a slight increase in H+
production in hearts from myriocin treated DIO mice, however this did not reach statistical significance. In the aerobic setting where we observed no difference in cardiac function between low fat fed and DIO mice, as such, the consequences of this H+
load are likely minimal. Nonetheless, as even minor changes in intracellular pH in the heart can significantly affect the recovery of cardiac function during acute metabolic stresses such as ischemia and reperfusion, the functional consequences of this slight increase in H+
production need to be further characterized 
. Interestingly, in the presence of elevated fatty acid concentrations we have actually shown that treatment of the mouse heart with insulin decreases the recovery of cardiac function during ischemia/reperfusion, due to insulin increasing H+
production as a result of increasing glycolytic rates to a greater extent than glucose oxidation rates 
. Since myriocin treatment can reverse obesity-induced insulin resistance and enhance energy metabolism in mice 
, while also improving beta cell function in obese rats 
, our future and ongoing studies are addressing the potential role of SPT 1 in ischemic heart disease.
In summary, we demonstrate that DIO impairs myocardial glucose utilization, which can be improved via SPT I inhibition. The effect of SPT I inhibition was associated with a marked reduction in intra-myocardial ceramide levels and prevented the DIO-associated increase in intra-myocardial DAG content, both of which likely contribute to the improved myocardial glucose utilization. Although SPT I inhibition may represent a potential novel target for the treatment of myocardial insulin resistance, because glucose utilization was not restored to basal levels observed in lean mice, further characterization of obesity-induced myocardial insulin resistance and its associated mechanisms is required.