The ketogenic diet does not improve cardiac function and blunts glucose oxidation in ischaemic heart failure.

AIMS: Cardiac energy metabolism is perturbed in ischaemic heart failure and is characterized by a shift from mitochondrial oxidative metabolism to glycolysis. Notably, the failing heart relies more on ketones for energy than a healthy heart, an adaptive mechanism that improves the energy-starved status of the failing heart. However, whether this can be implemented therapeutically remains unknown. Therefore, our aim was to determine if increasing ketone delivery to the heart via a ketogenic diet can improve the outcomes of heart failure. METHODS AND RESULTS: C57BL/6J male mice underwent either a sham surgery or permanent left anterior descending coronary artery ligation surgery to induce heart failure. After 2 weeks, mice were then treated with either a control diet or a ketogenic diet for 3 weeks. Transthoracic echocardiography was then carried out to assess in vivo cardiac function and structure. Finally, isolated working hearts from these mice were perfused with appropriately 3H or 14C labelled glucose (5 mM), palmitate (0.8 mM), and ß-hydroxybutyrate (ß-OHB) (0.6 mM) to assess mitochondrial oxidative metabolism and glycolysis. Mice with heart failure exhibited a 56% drop in ejection fraction, which was not improved with a ketogenic diet feeding. Interestingly, mice fed a ketogenic diet had marked decreases in cardiac glucose oxidation rates. Despite increasing blood ketone levels, cardiac ketone oxidation rates did not increase, probably due to a decreased expression of key ketone oxidation enzymes. Furthermore, in mice on the ketogenic diet, no increase in overall cardiac energy production was observed, and instead, there was a shift to an increased reliance on fatty acid oxidation as a source of cardiac energy production. This resulted in a decrease in cardiac efficiency in heart failure mice fed a ketogenic diet. CONCLUSION: We conclude that the ketogenic diet does not improve heart function in failing hearts, due to ketogenic diet-induced excessive fatty acid oxidation in the ischaemic heart and a decrease in insulin-stimulated glucose oxidation.

Ketones provide an extra source of fuel for the failing heart without impairing glucose oxidation.

BACKGROUND: Cardiac glucose oxidation is decreased in heart failure with reduced ejection fraction (HFrEF), contributing to a decrease in myocardial ATP production. In contrast, circulating ketones and cardiac ketone oxidation are increased in HFrEF. Since ketones compete with glucose as a fuel source, we aimed to determine whether increasing ketone concentration both chronically with the SGLT2 inhibitor, dapagliflozin, or acutely in the perfusate has detrimental effects on cardiac glucose oxidation in HFrEF, and what effect this has on cardiac ATP production. METHODS: 8-week-old male C57BL6/N mice underwent sham or transverse aortic constriction (TAC) surgery to induce HFrEF over 3 weeks, after which TAC mice were randomized to treatment with either vehicle or the SGLT2 inhibitor, dapagliflozin (DAPA), for 4 weeks (raises blood ketones). Cardiac function was assessed by echocardiography. Cardiac energy metabolism was measured in isolated working hearts perfused with 5 mM glucose, 0.8 mM palmitate, and either 0.2 mM or 0.6 mM ß-hydroxybutyrate (ßOHB). RESULTS: TAC hearts had significantly decreased %EF compared to sham hearts, with no effect of DAPA. Glucose oxidation was significantly decreased in TAC hearts compared to sham hearts and did not decrease further in TAC hearts treated with high ßOHB or in TAC DAPA hearts, despite ßOHB oxidation rates increasing in both TAC vehicle and TAC DAPA hearts at high ßOHB concentrations. Rather, increasing ßOHB supply to the heart selectively decreased fatty acid oxidation rates. DAPA significantly increased ATP production at both ßOHB concentrations by increasing the contribution of glucose oxidation to ATP production. CONCLUSION: Therefore, increasing ketone concentration increases energy supply and ATP production in HFrEF without further impairing glucose oxidation.

Deletion of BCATm increases insulin-stimulated glucose oxidation in the heart.

BACKGROUNDS: Branched chain amino acid (BCAA) oxidation is impaired in cardiac insulin resistance, leading to the accumulation of BCAAs and the first products of BCAA oxidation, the branched chain ketoacids. However, it is not clear whether it is the BCAAs, BCKAs or both that are mediating cardiac insulin resistance. To determine this, we produced mice with a cardiac-specific deletion of BCAA aminotransferase (BCATm-/-), the first enzyme in the BCAA oxidation pathway that is responsible for converting BCAAs to BCKAs. METHODS: Eight-week-old BCATm cardiac specific knockout (BCATm-/-) male mice and their α-MHC (myosin heavy chain) - Cre expressing wild type littermates (WT-Cre+/+) received tamoxifen (50 mg/kg i.p. 6 times over 8 days). At 16-weeks of age, cardiac energy metabolism was assessed in isolated working hearts. RESULTS: BCATm-/- mice have decreased cardiac BCAA oxidation rates, increased cardiac BCAAs and a reduction in cardiac BCKAs. Hearts from BCATm-/- mice showed an increase in insulin stimulation of glucose oxidation and an increase in p-AKT. To determine the impact of reversing these events, we perfused isolated working mice hearts with high levels of BCKAs, which completely abolished insulin-stimulated glucose oxidation rates, an effect associated with decreased p-AKT and inactivation of pyruvate dehydrogenase (PDH), the rate-limiting enzyme in glucose oxidation. CONCLUSION: This implicates the BCKAs, and not BCAAs, as the actual mediators of cardiac insulin resistance and suggests that lowering cardiac BCKAs can be used as a therapeutic strategy to improve insulin sensitivity in the heart.

Ketones can become the major fuel source for the heart but do not increase cardiac efficiency.

AIMS: Ketones have been proposed to be a 'thrifty' fuel for the heart and increasing cardiac ketone oxidation can be cardioprotective. However, it is unclear how much ketone oxidation can contribute to energy production in the heart, nor whether increasing ketone oxidation increases cardiac efficiency. Therefore, our goal was to determine to what extent high levels of the ketone body, ß-hydroxybutyrate (ßOHB), contributes to cardiac energy production, and whether this influences cardiac efficiency. METHODS AND RESULTS: Isolated working mice hearts were aerobically perfused with palmitate (0.8 mM or 1.2 mM), glucose (5 mM) and increasing concentrations of ßOHB (0, 0.6, 2.0 mM). Subsequently, oxidation of these substrates, cardiac function, and cardiac efficiency were assessed. Increasing ßOHB concentrations increased myocardial ketone oxidation rates without affecting glucose or fatty acid oxidation rates where normal physiological levels of glucose (5 mM) and fatty acid (0.8 mM) are present. Notably, ketones became the major fuel source for the heart at 2.0 mM ßOHB (at both low or high fatty acid concentrations), with the elevated ketone oxidation rates markedly increasing tricarboxylic acid (TCA) cycle activity, producing a large amount of reducing equivalents and finally, increasing myocardial oxygen consumption. However, the marked increase in ketone oxidation at high concentrations of ßOHB was not accompanied by an increase in cardiac work, suggesting that a mismatch between excess reduced equivalents production from ketone oxidation and cardiac adenosine triphosphate production. Consequently, cardiac efficiency decreased when the heart was exposed to higher ketone levels. CONCLUSIONS: We demonstrate that while ketones can become the major fuel source for the heart, they do not increase cardiac efficiency, which also underscores the importance of recognizing ketones as a major fuel source for the heart in times of starvation, consumption of a ketogenic diet or poorly controlled diabetes.

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.

Impaired branched chain amino acid oxidation contributes to cardiac insulin resistance in heart failure.

BACKGROUND: Branched chain amino acids (BCAA) can impair insulin signaling, and cardiac insulin resistance can occur in the failing heart. We, therefore, determined if cardiac BCAA accumulation occurs in patients with dilated cardiomyopathy (DCM), due to an impaired catabolism of BCAA, and if stimulating cardiac BCAA oxidation can improve cardiac function in mice with heart failure. METHOD: For human cohorts of DCM and control, both male and female patients of ages between 22 and 66 years were recruited with informed consent from University of Alberta hospital. Left ventricular biopsies were obtained at the time of transplantation. Control biopsies were obtained from non-transplanted donor hearts without heart disease history. To determine if stimulating BCAA catabolism could lessen the severity of heart failure, C57BL/6J mice subjected to a transverse aortic constriction (TAC) were treated between 1 to 4-week post-surgery with either vehicle or a stimulator of BCAA oxidation (BT2, 40 mg/kg/day). RESULT: Echocardiographic data showed a reduction in ejection fraction (54.3 ± 2.3 to 22.3 ± 2.2%) and an enhanced formation of cardiac fibrosis in DCM patients when compared to the control patients. Cardiac BCAA levels were dramatically elevated in left ventricular samples of patients with DCM. Hearts from DCM patients showed a blunted insulin signalling pathway, as indicated by an increase in P-IRS1ser636/639 and its upstream modulator P-p70S6K, but a decrease in its downstream modulators P-AKT ser473 and in P-GSK3ß ser9. Cardiac BCAA oxidation in isolated working hearts was significantly enhanced by BT2, compared to vehicle, following either acute or chronic treatment. Treatment of TAC mice with BT2 significantly improved cardiac function in both sham and TAC mice (63.0 ± 1.8 and 56.9 ± 3.8% ejection fraction respectively). Furthermore, P-BCKDH and BCKDK expression was significantly decreased in the BT2 treated groups. CONCLUSION: We conclude that impaired cardiac BCAA catabolism and insulin signaling occur in human heart failure, while enhancing BCAA oxidation can improve cardiac function in the failing mouse heart.

Increased ketone body oxidation provides additional energy for the failing heart without improving cardiac efficiency.

AIMS: The failing heart is energy-starved and inefficient due to perturbations in energy metabolism. Although ketone oxidation has been shown recently to increase in the failing heart, it remains unknown whether this improves cardiac energy production or efficiency. We therefore assessed cardiac metabolism in failing hearts and determined whether increasing ketone oxidation improves cardiac energy production and efficiency. METHODS AND RESULTS: C57BL/6J mice underwent sham or transverse aortic constriction (TAC) surgery to induce pressure overload hypertrophy over 4-weeks. Isolated working hearts from these mice were perfused with radiolabelled ß-hydroxybutyrate (ßOHB), glucose, or palmitate to assess cardiac metabolism. Ejection fraction decreased by 45% in TAC mice. Failing hearts had decreased glucose oxidation while palmitate oxidation remained unchanged, resulting in a 35% decrease in energy production. Increasing ßOHB levels from 0.2 to 0.6 mM increased ketone oxidation rates from 251 ± 24 to 834 ± 116 nmol·g dry wt-1 · min-1 in TAC hearts, rates which were significantly increased compared to sham hearts and occurred without decreasing glycolysis, glucose, or palmitate oxidation rates. Therefore, the contribution of ketones to energy production in TAC hearts increased to 18% and total energy production increased by 23%. Interestingly, glucose oxidation, in parallel with total ATP production, was also significantly upregulated in hearts upon increasing ßOHB levels. However, while overall energy production increased, cardiac efficiency was not improved. CONCLUSIONS: Increasing ketone oxidation rates in failing hearts increases overall energy production without compromising glucose or fatty acid metabolism, albeit without increasing cardiac efficiency.

Empagliflozin Increases Cardiac Energy Production in Diabetes: Novel Translational Insights Into the Heart Failure Benefits of SGLT2 Inhibitors.

SGLT2 inhibitors have profound benefits on reducing heart failure and cardiovascular mortality in individuals with type 2 diabetes, although the mechanism(s) of this benefit remain poorly understood. Because changes in cardiac bioenergetics play a critical role in the pathophysiology of heart failure, the authors evaluated cardiac energy production and substrate use in diabetic mice treated with the SGTL2 inhibitor, empagliflozin. Empagliflozin treatment of diabetic db/db mice prevented the development of cardiac failure. Glycolysis, and the oxidation of glucose, fatty acids and ketones were measured in the isolated working heart perfused with 5 mmol/l glucose, 0.8 mmol/l palmitate, 0.5 mmol/l ß-hydroxybutyrate (ßOHB), and 500 µU/ml insulin. In vehicle-treated db/db mice, cardiac glucose oxidation rates were decreased by 61%, compared with control mice, but only by 43% in empagliflozin-treated diabetic mice. Interestingly, cardiac ketone oxidation rates in db/db mice decreased to 45% of the rates seen in control mice, whereas a similar decrease (43%) was seen in empagliflozin-treated db/db mice. Overall cardiac adenosine triphosphate (ATP) production rates decreased by 36% in db/db vehicle-treated hearts compared with control mice, with fatty acid oxidation providing 42%, glucose oxidation 26%, ketone oxidation 10%, and glycolysis 22% of ATP production in db/db mouse hearts. In empagliflozin-treated db/db mice, cardiac ATP production rates increased by 31% compared with db/db vehicle-treated mice, primarily due to a 61% increase in the contribution of glucose oxidation to energy production. Cardiac efficiency (cardiac work/O2 consumed) decreased by 28% in db/db vehicle-treated hearts, compared with control hearts, and empagliflozin did not increase cardiac efficiency per se. Because ketone oxidation was impaired in db/db mouse hearts, the authors determined whether this contributed to the decrease in cardiac efficiency seen in the db/db mouse hearts. Addition of 600 µmol/l ßOHB to db/db mouse hearts perfused with 5 mmol/l glucose, 0.8 mmol/l palmitate, and 100 µU/ml insulin increased ketone oxidation rates, but did not decrease either glucose oxidation or fatty acid oxidation rates. The presence of ketones did not increase cardiac efficiency, but did increase ATP production rates, due to the additional contribution of ketone oxidation to energy production. The authors conclude that empagliflozin treatment is associated with an increase in ATP production, resulting in an enhanced energy status of the heart.

FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4, the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4/PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4/PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4/PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates.NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report here, for the first time, that FoxO1 inhibition increases glucose oxidation in the isolated working mouse heart.