Loss of muscle PDH induces lactic acidosis and adaptive anaplerotic compensation via pyruvate-alanine cycling and glutaminolysis.

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation that links glycolysis-derived pyruvate with the tricarboxylic acid (TCA) cycle. Although skeletal muscle is a significant site for glucose oxidation and is closely linked with metabolic flexibility, the importance of muscle PDH during rest and exercise has yet to be fully elucidated. Here, we demonstrate that mice with muscle-specific deletion of PDH exhibit rapid weight loss and suffer from severe lactic acidosis, ultimately leading to early mortality under low-fat diet provision. Furthermore, loss of muscle PDH induces adaptive anaplerotic compensation by increasing pyruvate-alanine cycling and glutaminolysis. Interestingly, high-fat diet supplementation effectively abolishes early mortality and rescues the overt metabolic phenotype induced by muscle PDH deficiency. Despite increased reliance on fatty acid oxidation during high-fat diet provision, loss of muscle PDH worsens exercise performance and induces lactic acidosis. These observations illustrate the importance of muscle PDH in maintaining metabolic flexibility and preventing the development of metabolic disorders.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury through a sirtuin-3 mediated increase in fatty acid oxidation.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury (IRI) but the mechanisms are unknown. Here, we investigate the role of the mitochondrial NAD+-dependent deacetylase, Sirtuin-3 (SIRT3), which has been shown to influence fatty acid oxidation and cardiac outcomes, as a potential mediator of this effect. Fasting was shown to shift metabolism from glucose towards fatty acid oxidation. This change in metabolic fuel substrate utilisation increased myocardial infarct size in wild-type (WT), but not SIRT3 heterozygous knock-out (KO) mice. Further analysis revealed SIRT3 KO mice were better adapted to starvation through an improved cardiac efficiency, thus protecting them from acute myocardial IRI. Mitochondria from SIRT3 KO mice were hyperacetylated compared to WT mice which may regulate key metabolic processes controlling glucose and fatty acid utilisation in the heart. Fasting and the associated metabolic switch to fatty acid respiration worsens outcomes in WT hearts, whilst hearts from SIRT3 KO mice are better adapted to oxidising fatty acids, thereby protecting them from acute myocardial IRI.

Barth syndrome-related cardiomyopathy is associated with a reduction in myocardial glucose oxidation.

Heart failure presents as the leading cause of infant mortality in individuals with Barth syndrome (BTHS), a rare genetic disorder due to mutations in the tafazzin (TAZ) gene affecting mitochondrial structure and function. Investigations into the perturbed bioenergetics in the BTHS heart remain limited. Hence, our objective was to identify the potential alterations in myocardial energy metabolism and molecular underpinnings that may contribute to the early cardiomyopathy and heart failure development in BTHS. Cardiac function and myocardial energy metabolism were assessed via ultrasound echocardiography and isolated working heart perfusions, respectively, in a mouse model of BTHS [doxycycline-inducible Taz knockdown (TazKD) mice]. In addition, we also performed mRNA/protein expression profiling for key regulators of energy metabolism in hearts from TazKD mice and their wild-type (WT) littermates. TazKD mice developed hypertrophic cardiomyopathy as evidenced by increased left ventricular anterior and posterior wall thickness, as well as increased cardiac myocyte cross-sectional area, though no functional impairments were observed. Glucose oxidation rates were markedly reduced in isolated working hearts from TazKD mice compared with their WT littermates in the presence of insulin, which was associated with decreased pyruvate dehydrogenase activity. Conversely, myocardial fatty acid oxidation rates were elevated in TazKD mice, whereas no differences in glycolytic flux or ketone body oxidation rates were observed. Our findings demonstrate that myocardial glucose oxidation is impaired before the development of overt cardiac dysfunction in TazKD mice, and may thus represent a pharmacological target for mitigating the development of cardiomyopathy in BTHS.NEW & NOTEWORTHY Barth syndrome (BTHS) is a rare genetic disorder due to mutations in tafazzin that is frequently associated with infantile-onset cardiomyopathy and subsequent heart failure. Although previous studies have provided evidence of perturbed myocardial energy metabolism in BTHS, actual measurements of flux are lacking. We now report a complete energy metabolism profile that quantifies flux in isolated working hearts from a murine model of BTHS, demonstrating that BTHS is associated with a reduction in glucose oxidation.

FoxO1 inhibition alleviates type 2 diabetes-related diastolic dysfunction by increasing myocardial pyruvate dehydrogenase activity.

Type 2 diabetes (T2D) increases the risk for diabetic cardiomyopathy and is characterized by diastolic dysfunction. Myocardial forkhead box O1 (FoxO1) activity is enhanced in T2D and upregulates pyruvate dehydrogenase (PDH) kinase 4 expression, which inhibits PDH activity, the rate-limiting enzyme of glucose oxidation. Because low glucose oxidation promotes cardiac inefficiency, we hypothesize that FoxO1 inhibition mitigates diabetic cardiomyopathy by stimulating PDH activity. Tissue Doppler echocardiography demonstrates improved diastolic function, whereas myocardial PDH activity is increased in cardiac-specific FoxO1-deficient mice subjected to experimental T2D. Pharmacological inhibition of FoxO1 with AS1842856 increases glucose oxidation rates in isolated hearts from diabetic C57BL/6J mice while improving diastolic function. However, AS1842856 treatment fails to improve diastolic function in diabetic mice with a cardiac-specific FoxO1 or PDH deficiency. Our work defines a fundamental mechanism by which FoxO1 inhibition improves diastolic dysfunction, suggesting that it may be an approach to alleviate diabetic cardiomyopathy.

Considerations for using isolated cell systems to understand cardiac metabolism and biology.

Changes in myocardial metabolic activity are fundamentally linked to cardiac health and remodeling. Primary cardiomyocytes, induced pluripotent stem cell-derived cardiomyocytes, and transformed cardiomyocyte cell lines are common models used to understand how (patho)physiological conditions or stimuli contribute to changes in cardiac metabolism. These cell models are helpful also for defining metabolic mechanisms of cardiac dysfunction and remodeling. Although technical advances have improved our capacity to measure cardiomyocyte metabolism, there is often heterogeneity in metabolic assay protocols and cell models, which could hinder data interpretation and discernment of the mechanisms of cardiac (patho)physiology. In this review, we discuss considerations for integrating cardiomyocyte cell models with techniques that have become relatively common in the field, such as respirometry and extracellular flux analysis. Furthermore, we provide overviews of metabolic assays that complement XF analyses and that provide information on not only catabolic pathway activity, but biosynthetic pathway activity and redox status as well. Cultivating a more widespread understanding of the advantages and limitations of metabolic measurements in cardiomyocyte cell models will continue to be essential for the development of coherent metabolic mechanisms of cardiac health and pathophysiology.

Insulin directly stimulates mitochondrial glucose oxidation in the heart.

BACKGROUND: Glucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin can increase glucose oxidation indirectly by enhancing glucose uptake and glycolysis, it also directly stimulates mitochondrial glucose oxidation, independent of increasing glucose uptake or glycolysis, through activating mitochondrial pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates PDH is not known. To determine this, we characterized the impacts of modifying mitochondrial insulin signaling kinases, namely protein kinase B (Akt), protein kinase C-delta (PKC-δ) and glycogen synthase kinase-3 beta (GSK-3ß), on the direct insulin stimulation of glucose oxidation. METHODS: We employed an isolated working mouse heart model to measure the effect of insulin on cardiac glycolysis, glucose oxidation and fatty acid oxidation and how that could be affected when mitochondrial Akt, PKC-δ or GSK-3ß is disturbed using pharmacological modulators. We also used differential centrifugation to isolate mitochondrial and cytosol fraction to examine the activity of Akt, PKC-δ and GSK-3ß between these fractions. Data were analyzed using unpaired t-test and two-way ANOVA. RESULTS: Here we show that insulin-stimulated phosphorylation of mitochondrial Akt is a prerequisite for transducing insulin's direct stimulation of glucose oxidation. Inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of glucose uptake or glycolysis. We also show a novel role of mitochondrial PKC-δ in modulating mitochondrial glucose oxidation. Inhibition of mitochondrial PKC-δ mimics insulin stimulation of glucose oxidation and mitochondrial Akt. We also demonstrate that inhibition of mitochondrial GSK3ß phosphorylation does not influence insulin-stimulated glucose oxidation. CONCLUSION: We identify, for the first time, insulin-stimulated mitochondrial Akt as a prerequisite transmitter of the insulin signal that directly stimulates cardiac glucose oxidation. These novel findings suggest that targeting mitochondrial Akt is a potential therapeutic approach to enhance cardiac insulin sensitivity in condition such as heart failure, diabetes and obesity.

Adropin regulates cardiac energy metabolism and improves cardiac function and efficiency.

BACKGROUND: Impaired cardiac insulin signalling and high cardiac fatty acid oxidation rates are characteristics of conditions of insulin resistance and diabetic cardiomyopathies. The potential role of liver-derived peptides such as adropin in mediating these changes in cardiac energy metabolism is unclear, despite the fact that in skeletal muscle adropin can preferentially promote glucose metabolism and improve insulin sensitivity. OBJECTIVES: To determine the influence of adropin on cardiac energy metabolism, insulin signalling and cardiac efficiency. METHODS: C57Bl/6 mice were injected with either vehicle or a secretable form of adropin (450 nmol/kg, i.p.) three times over a 24-h period. The mice were fasted to accentuate the differences between animals in adropin plasma levels before their hearts were isolated and perfused using a working heart system. In addition, direct addition of adropin to the perfusate of ex vivo hearts isolated from non-fasting mice was utilized to investigate the acute effects of the peptide on heart metabolism and ex vivo function. RESULTS: In contrast to the observed fasting-induced predominance of fatty acid oxidation as a source of ATP production in control hearts, insulin inhibition of fatty acid oxidation was preserved by adropin treatment. Adropin-treated mouse hearts also showed a higher cardiac work, which was accompanied by improved cardiac efficiency and enhanced insulin signalling compared to control hearts. Interestingly, acute adropin administration to isolated working hearts also resulted in an inhibition of fatty acid oxidation, accompanied by a robust stimulation of glucose oxidation compared to vehicle-treated hearts. Adropin also increased activation of downstream cardiac insulin signalling. Moreover, both in vivo and ex vivo treatment protocols induced a reduction in the inhibitory phosphorylation of pyruvate dehydrogenase (PDH), the major enzyme of glucose oxidation, and the protein levels of the responsible kinase PDH kinase 4 and the insulin-signalling inhibitory phosphorylation of JNK (p-T183/Y185) and IRS-1 (p-S307), suggesting acute receptor- and/or post-translational modification-mediated mechanisms. CONCLUSIONS: These results demonstrate that adropin has important effects on energy metabolism in the heart and may be a putative candidate for the treatment of cardiac disease associated with impaired insulin sensitivity.

Weight loss enhances cardiac energy metabolism and function in heart failure associated with obesity.

AIMS: Obesity is associated with high rates of cardiac fatty acid oxidation, low rates of glucose oxidation, cardiac hypertrophy and heart failure. Whether weight loss can lessen the severity of heart failure associated with obesity is not known. We therefore determined the effect of weight loss on cardiac energy metabolism and the severity of heart failure in obese mice with heart failure. MATERIALS AND METHODS: Obesity and heart failure were induced by feeding mice a high-fat (HF) diet and subjecting them to transverse aortic constriction (TAC). Obese mice with heart failure were then switched for 8 weeks to either a low-fat (LF) diet (HF TAC LF) or caloric restriction (CR) (40% caloric intake reduction, HF TAC CR) to induce weight loss. RESULTS: Weight loss improved cardiac function (%EF was 38 ± 6% and 36 ± 6% in HF TAC LF and HF TAC CR mice vs 25 ± 3% in HF TAC mice, P < 0.05) and it decreased cardiac hypertrophy post TAC (left ventricle mass was 168 ± 7 and 171 ± 10 mg in HF TAC LF and HF TAC CR mice, respectively, vs 210 ± 8 mg in HF TAC mice, P < 0.05). Weight loss enhanced cardiac insulin signalling, insulin-stimulated glucose oxidation rates (1.5 ± 0.1 and 1.5 ± 0.1 µmol/g dry wt/min in HF TAC LF and HF TAC CR mice, respectively, vs 0.2 ± 0.1 µmol/g dry wt/min in HF TAC mice, P < 0.05) and it decreased pyruvate dehydrogenase phosphorylation. Cardiac fatty acid oxidation rates, AMPKTyr172 /ACCSer79 signalling and the acetylation of ß-oxidation enzymes, were attenuated following weight loss. CONCLUSIONS: Weight loss is an effective intervention to improve cardiac function and energy metabolism in heart failure associated with obesity.

Cardiac-specific deficiency of the mitochondrial calcium uniporter augments fatty acid oxidation and functional reserve.

The mitochondrial calcium uniporter (MCU) relays cytosolic Ca2+ transients to the mitochondria. We examined whether energy metabolism was compromised in hearts from mice with a cardiac-specific deficiency of MCU subjected to an isoproterenol (ISO) challenge. Surprisingly, isolated working hearts from cardiac MCU-deficient mice showed higher cardiac work, both in the presence or absence of ISO. These hearts were not energy-starved, with ISO inducing a similar increase in glucose oxidation rates compared to control hearts, but a greater increase in fatty acid oxidation rates. This correlated with lower levels of the fatty acid oxidation inhibitor malonyl CoA, and to an increased stimulatory acetylation of its degrading enzyme malonyl CoA decarboxylase and of the fatty acid ß-oxidation enzyme ß-hydroxyacyl CoA dehydrogenase. We conclude that impaired mitochondrial Ca2+ uptake does not compromise cardiac energetics due to a compensatory stimulation of fatty acid oxidation that provides a higher energy reserve during acute adrenergic stress.

A novel role of endothelial autophagy as a regulator of myocardial fatty acid oxidation.

BACKGROUND: We sought to determine if endothelial autophagy affects myocardial energy metabolism. METHODS: We used isolated working mouse hearts to compare cardiac function, energy metabolism, and ischemic response of hearts from endothelial cell-specific ATG7 knockout (EC-ATG7-/-) mice to hearts from their wild-type littermates. We also conducted gene analyses on human umbilical vein endothelial cells incubated with scrambled small interfering RNA or small interfering ATG7. RESULTS: In the presence of insulin, working hearts from EC-ATG7-/- mice, relative to those from wild-type littermates, exhibited greater reductions in insulin-associated palmitate oxidation indicating a diminished reliance on fatty acids as a fuel source. Likewise, palmitate oxidation was markedly lower in the hearts of EC-ATG7-/- mice versus wild-type mice during reperfusion of ischemic hearts. Although hearts from EC-ATG7-/- mice revealed significantly lower triacylglycerol content compared with those from wild-type mice, ATG7-silenced human umbilical vein endothelial cells demonstrated appreciably lower fatty acid binding protein 4 and 5 expression relative to those treated with scrambled small interfering RNA. CONCLUSIONS: Disruption of endothelial autophagy reduces cardiac fatty acid storage and dampens reliance on fatty acid oxidation as a cardiac fuel source. The autophagy network represents a novel target for designing new strategies aimed at resetting perturbed myocardial bioenergetics.

Cytosolic carnitine acetyltransferase as a source of cytosolic acetyl-CoA: a possible mechanism for regulation of cardiac energy metabolism.

The role of carnitine acetyltransferase (CrAT) in regulating cardiac energy metabolism is poorly understood. CrAT modulates mitochondrial acetyl-CoA/CoA (coenzyme A) ratios, thus regulating pyruvate dehydrogenase activity and glucose oxidation. Here, we propose that cardiac CrAT also provides cytosolic acetyl-CoA for the production of malonyl-CoA, a potent inhibitor of fatty acid oxidation. We show that in the murine cardiomyocyte cytosol, reverse CrAT activity (RCrAT, producing acetyl-CoA) is higher compared with the liver, which primarily uses ATP-citrate lyase to produce cytosolic acetyl-CoA for lipogenesis. The heart displayed a lower RCrAT Km for CoA compared with the liver. Furthermore, cytosolic RCrAT accounted for 4.6 ± 0.7% of total activity in heart tissue and 12.7 ± 0.2% in H9C2 cells, while highly purified heart cytosolic fractions showed significant CrAT protein levels. To investigate the relationship between CrAT and acetyl-CoA carboxylase (ACC), the cytosolic enzyme catalyzing malonyl-CoA production from acetyl-CoA, we studied ACC2-knockout mouse hearts which showed decreased CrAT protein levels and activity, associated with increased palmitate oxidation and acetyl-CoA/CoA ratio compared with controls. Conversely, feeding mice a high-fat diet for 10 weeks increased cardiac CrAT protein levels and activity, associated with a reduced acetyl-CoA/CoA ratio and glucose oxidation. These data support the presence of a cytosolic CrAT with a low Km for CoA, favoring the formation of cytosolic acetyl-CoA, providing an additional source to the classical ATP-citrate lyase pathway, and that there is an inverse relation between CrAT and the ratio of acetyl-CoA/CoA as evident in conditions affecting the regulation of cardiac energy metabolism.