This study demonstrates that hearts rapidly adapt to caloric excess, developing patterns of substrate utilization that mimic changes observed in longstanding obesity or diabetes (type 1 or type 2).1,2,4–7
They occur in the absence of significant obesity or hyperlipidaemia, but are associated with mildly abnormal glucose tolerance and a two-fold increase in fasting insulin concentrations. As early as 2 weeks of HFD, MVO2
and FA oxidation are increased, whereas rates of glucose oxidation and glycolysis are reduced. PDH activity is not altered, but GLUT4 protein content is reduced via post-transcriptional mechanisms. GLUT4 translocation is also impaired independently of changes in insulin-mediated activation of the upstream regulators of glucose transport Akt/PKB and AS-160. Increased FA utilization occurs prior to the activation of PPAR-α signalling, without any changes in malonyl CoA content and in the absence of mitochondrial uncoupling. Taken together, these data suggest that the initial molecular defect that alters myocardial substrate metabolism early in the course of high-fat feeding is impaired GLUT4-mediated myocardial glucose utilization.
Contraction-mediated GLUT4 translocation in beating hearts might be the major mediator of basal myocardial glucose utilization. Support for this comes from studies in mice with cardiomyocyte deletion of GLUT4 (G4H−/−).29
These animals have increased expression levels of GLUT1, yet after an overnight fast, basal rates of glucose uptake in perfused hearts were negligible.30
Moreover, in mice with cardiomyocyte-restricted KO of insulin receptors (CIRKO), basal rates of glycolysis in perfused hearts were significantly increased, despite >50% reduction in levels of GLUT1 protein, but a two-fold increase in the GLUT4 protein.21
In contrast, in isolated cardiomyocytes, GLUT1 is the major contributor to basal glucose uptake. Thus in CIRKO mice, GLUT1 protein content and basal glucose uptake in cardiomyocytes were proportionately reduced,21
whereas in GLUT4-deficient cardiomyocytes, basal rates of glucose uptake were unchanged.31
Thus, the normal rate of basal glucose uptake in isolated cardiomyocytes in the present study was not unexpected given that expression levels of GLUT1 were unchanged.
PDH flux is an important regulator of glucose oxidation in the heart.32
Prior studies in HFD rats demonstrated reduced activity of the active fraction of PDH (PDHa
) after 28 days but not after 10 days of high-fat feeding. Moreover, PDH kinase activities were increased at 28 days, but not at 10 days.33,34
Other studies in skeletal muscle of humans and rodents have also suggested that the decline in PDH activity with high-fat feeding parallels an increase in PDH kinase activity.32,35,36
In our study, we observed no change in the expression levels of pyruvate dehydrogenase kinase (PDK4) after 2 weeks of high-fat feeding, and consistent with this, we observed no differences in the total or active fraction of PDH, measured at this early time point. We did observe a significant increase in PDK4 activity after 5 weeks of high-fat feeding and would expect that PDHa
(if measured) would be reduced after 5 weeks of high-fat feeding. Taken together, these findings are consistent with the conclusion that reduced GLUT4-mediated glucose uptake may represent the critical mechanism for reduced basal rates of glycolysis and glucose oxidation early in the course of high-fat feeding (2 weeks), but as the duration of high-fat feeding becomes more prolonged, reduced PDH flux will likely contribute to the impairment in glucose oxidation.
High-fat feeding attenuated insulin-mediated glucose uptake in isolated cardiomyocytes and prevented insulin-mediated increases in glycolysis and glucose oxidation in isolated working hearts despite normal insulin signalling to AS-160. We believe this reflects a distal defect in GLUT4 translocation. Evidence for this was obtained by analyzing insulin-mediated redistribution of intracellular GLUT4 vesicles to the sarcolemma and qualitatively by GLUT4 immunohistochemistry. The dissociation of insulin-mediated GLUT4 translocation from Akt/PKB and AS-160 signalling suggests that high-fat feeding initially impairs key steps in the movement of GLUT4 vesicles from their intracellular compartment to the sarcolemma. Studies in palmitate-exposed L6 myotubes and skeletal muscles of HFD mice illustrated that impaired insulin-mediated glucose uptake can occur in the absence of defects in insulin-stimulated phosphorylation of Akt/PKB or AS-160.37
Moreover, increased sarcolemmal cholesterol content, reduced phosphatidylinositol-4,5-bisphosphate (phosphatidylinositol-3,4-bisphosphate) content or disruption of cortical F-actin can impair insulin-mediated GLUT4 translocation in skeletal muscle without reducing insulin-mediated phosphorylation of Akt/PKB.38–40
The molecular mechanisms that are responsible for the transit of GLUT4 from the intracellular compartment to the sarcolemma are complex and incompletely understood, but involve vesicle budding, actin polymerization, and movement of GLUT4 vesicles on microtubules by myosin motors.41
Moreover, there are regulated steps involved in GLUT4 vesicle docking and fusion, which could be perturbed by high-fat feeding in the heart.41
For example, increased expression of the SNARE protein Munc-18, a negative regulator of GLUT4 vesicle docking, was described in skeletal muscle with lipid-induced insulin resistance on the basis of overexpression of lipoprotein lipase.42
Thus, additional studies will be required to elucidate the mechanisms by which short-term high-fat feeding impairs GLUT4 trafficking in the heart.
An intriguing aspect of this study is the difference in tempo of impaired insulin-stimulated glycolysis relative to the ability of insulin to stimulate glucose oxidation. Insulin-stimulated glycolysis was completely absent after 2 weeks of HFD at a time when the ability of insulin to simulate glucose oxidation was relatively preserved, whereas the ability of insulin to stimulate glucose oxidation was abrogated after 5 weeks of HFD. Increased glycolysis following insulin stimulation is due in part to increased GLUT4 translocation, which was clearly impaired as early as 2 weeks of HFD. The oxidative metabolism of glucose while partially dependent on glycolytic flux is also regulated by flux through PDH, which is regulated by PDH phosphatases and kinases whose activities are modulated by allosteric interactions with nucleotides, acetyl CoA, NAD(H), and intracellular [Mg2+
] and [Ca2+
Thus, the regulatory mechanisms for glycolysis might exhibit differential insulin sensitivity relative to mechanisms that regulate glucose oxidation.
Reduced myocardial glucose utilization and increased myocardial FA utilization after 2 weeks of HFD, although reminiscent of changes described in severe diabetes and obesity occurred in the absence of major changes in the serum concentrations of glucose, FFA, or TGs. It is widely believed that an important mediator of altered myocardial substrate metabolism in obesity and diabetes is activation of PPAR-α signalling via increased delivery of FA ligands to the heart.44
The present study suggests that the activation of PPAR-α signalling does not occur early in the course of high-fat feeding at a time when myocardial FA utilization is increased. We propose that the initial increase in FA utilization likely results from reduced basal rates of myocardial glucose utilization, which is secondary to reduced GLUT4 content and translocation, which according to Randle’s hypothesis would be predicted to increase FA oxidation.45
CD36 translocation to the sarcolemma has been described in the hearts of rats after 8 weeks of high-fat feeding.15
We did not determine sarcolemmal CD36 content in the present study, thus this mechanism cannot be ruled out. Perfusion of hearts with FA alone could also clarify the mechanism. If impaired glucose uptake was the sole basis for initial metabolic defects within 2 weeks of high-fat feeding, then FA utilization rates in the absence of glucose in the perfusate would not be expected to be changed.
Convincing evidence for the increased activation of PPAR-α signalling pathways was evident only after 5 weeks of HFD. Thus it is likely that the activation of the PPAR-α pathway may sustain the increase in myocardial FA utilization only when increased dietary lipid intake persists. These results are similar to those of Buchanan et al
., who noted increased myocardial FA utilization and decreased glucose utilization in hearts from obese 4-week-old ob/ob
mice prior to the onset of diabetes, which was not associated with the activation of PPAR-α signalling in young mice. However, PPAR-α signalling increased as animals aged and after hyperglycaemia developed.6
HFD caused an early increase in MVO2
, which commonly accompanies increased FA metabolism. Previous studies from our laboratory suggested that mitochondrial uncoupling may contribute to increased MVO2
in severe obesity.17,18
However, the present study demonstrated that following short-term high-fat feeding, changes in FA oxidation and oxygen consumption occurred in the absence of mitochondrial uncoupling. Thus, increased MVO2
is likely a consequence of altered substrate metabolism. FA is a less efficient substrate than glucose, producing less ATP per oxygen consumed. Thus an increase in FA utilization in HFD hearts would be expected to increase myocardial oxygen consumption.16,46
It is likely that as caloric excess becomes more prolonged, mitochondrial uncoupling could occur as could be sustained by the increased expression of uncoupling proteins (UCP2 and UCP3), which was evident after 5 weeks of HFD. In addition, increased expression of mitochondrial thioesterases at 5 weeks would further reduce myocardial energetic efficiency by promoting futile ATP-wasting FA cycling between the mitochondria and cytosol.47,48
In conclusion, high-fat feeding causes an early reduction in glucose utilization on the basis of reduced GLUT4 content and GLUT4 translocation, which is independent of coordinate reductions in PDH activity or in insulin-mediated Akt/PKB and AS-160 phosphorylation. The reciprocal increase in cardiac FA oxidation (Randle effect) is initially independent of PPAR-α activation, and the increase in MVO2 is not attributable to mitochondrial uncoupling. Thus, cardiac metabolism rapidly adapts to high-fat feeding. These changes precede the development of obesity or diabetes, but recapitulate changes that have classically been associated with longstanding obesity and diabetes.