Myocardial Glycolysis Research Articles

Abstract Tu087: Knockout of TIGAR Rescues Cardiac Dysfunction in SIRT3 Knockout Mice

Introduction: Heart failure (HF) is the leading cause of death in the United States, despite advances in treatment, it maintains the highest disease mortality rate in the country. Previously it has been established that Sirtuin 3 (SIRT3), a mitochondrial NAD+- dependent deacetylase, and the Tp53-induced glycolysis and apoptosis regulator (TIGAR) are associated with the development of HF. SIRT3 plays a key role in the maintenance of cardiac function. Decreases in SIRT3 are associated with human aging and diabetes, whereas knockout results in cardiac dysfunction in mice. We have shown: (1) overexpression of SIRT3 downregulates TIGAR and attenuates diabetic cardiomyopathy; (2) knockout of TIGAR enhances myocardial glycolysis and reduces pathological remodeling while maintaining cardiac function in pressure-overload-induced heart failure. To date, the direct interactions between TIGAR and SIRT3 in the development of HF remain unexplored. Aim: By crossing TIGAR knockout mice (TIGARKO) with SIRT3 knockout (SIRT3KO) mice, this study tested if genetic targeting of TIGAR could rescue cardiac dysfunction in SIRT3KO mice. Methods and Results: Echocardiography was performed on SIRT3KO, TIGARKO, TIGARKO/SIRT3KO, and SIRT3 Control mice. Analysis of cardiac function showed that SIRT3KO mice had significant decreases in coronary flow reserve (CFR), ejection fraction (EF) SIRT3KO (41.52 ± 4.50) vs SIRT3 Control (60.91 ± 4.91, p<0.001) and fractional shortening (FS) SIRT3KO (20.11 ± 2.48) vs SIRT3 Control (32.07 ± 3.37, p<0.001). The diastolic E’/A’ ratio was decreased; IVRT and MPI were significantly increased. Knockout of TIGAR in mice maintained systolic function by increasing EF (62.25 ± 9.84) vs (41.52 ± 4.50, p<0.001), FS (33.33 ± 7.12 vs 20.11 ± 2.48, p<0.05), and CFR, as well as improved diastolic function by decreasing IVRT, MPI, and increasing E’/A’ ratio in SIRT3KO mice. SIRT3KO and TIGARKO mice had a significant increase in weight/tibia length ratio (HW/TL). Interestingly, the knockout of TIGAR significantly reduced LV mass and HW/TL in SIRT3KO mice. Conclusions: The knockout of TIGAR can rescue the HF phenotype of SIRT3KO mice, but the underlying mechanisms require further investigation to develop potential treatments for HF.

Abstract Tu103: SIRT2 Inhibition Decreases Glycolysis and Attenuates Hypertrophic Response in H9c2 Cardiomyocytes

Introduction: Myocardial glycolysis increases in hypertrophic and failing hearts. Hyperacetylation also occurs in the failing heart, and many glycolytic enzymes are known to be subject to acetylation. However, it is generally considered that acetylation has inhibitory effects on glycolysis. As a result, it is not clear whether acetylation changes directly contribute to glycolysis changes in cardiac hypertrophy. We therefore determined whether changes in the acetylation of glycolytic enzymes and the activity of the cytosolic deacetylase SIRT2 regulate cardiac glycolysis. Methods: Glycolysis rates were directly measured in rat heart-derived H9c2 cardiomyocytes perfused with 5 mM [5- 3 H] glucose, 0.8 mM palmitate, and 4% bovine serum albumin. Before these metabolic measurements, H9c2 cells were treated with either a SIRT2 inhibitor (10 µM AGK2) or a vehicle for 24 hours. In separate experiments, SIRT2 was also knocked down in H9c2 cells using siRNA, followed by glycolysis rate determinations. The impact of SIRT2 inhibition or SIRT2 knockdown on the acetylation status of glycolytic enzyme was also assessed. Furthermore, the effects of SIRT2 inhibition on hypertrophic signalling were assessed by treating H9c2 cells with phenylephrine. Results: SIRT2 inhibition markedly decreased glycolysis rates in H9c2 cells compared to vehicle-treated cells (524±108 vs 2631±372 nmol . g dry wt -1. min -1 , p<0.05). Similarly, SIRT2 knockdown resulted in a significant reduction in glycolysis rates compared to scrambled siRNA-treated H9c2 cells (745±31 vs 1659±168 nmol . g dry wt -1. min -1 , p<0.05). The decrease in SIRT2 was accompanied by an increase in the acetylation status of the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GPDH). Moreover, a trend towards increased phosphofructokinase (PFK) acetylation was also observed in SIRT2 knockdown H9c2 cells compared to scrambled siRNA-treated cells. Lastly, AGK2 treatment also attenuated phenylephrine-mediated hypertrophic responses in H9c2 cells. Conclusions: Increased acetylation of glycolytic enzymes is associated with a decrease in glycolysis, and SIRT2 inhibition or deletion in H9c2 cells significantly decreases glycolysis rates and attenuates hypertrophy. SIRT2 may therefore contribute to the increased glycolysis seen in hypertrophy and heart failure.

Muscone ameliorates myocardial ischemia‒reperfusion injury by promoting myocardial glycolysis

ObjectiveThe incidence of acute myocardial infarction (AMI) is increasing yearly. With the use of thrombolysis or percutaneous coronary intervention (PCI), the mortality rate of acute myocardial infarction has been significantly reduced. However, reperfusion can cause additional myocardial injury. There is still a lack of effective drugs to treat I/R injury, and it is urgent to find new therapeutic drugs. MethodsIn this study, network pharmacology was used to predict potential targets and biological processes involved in Muscone-mediated treatment of acute myocardial infarction. To model ischemia‒reperfusion injury, a hypoxia-reoxygenation model and in vivo ischemia‒reperfusion injury C57BL/6 mice model was constructed. Mice were treated with Muscone i.p. for 4 weeks. We detected the cardiac function on day 28.The expression levels of the apoptotic proteins Caspase-3 and Bax and the anti-apoptotic protein Bcl-2 were detected by immunoblotting after Muscone treatment of AC16 cells and in vivo. Additionally, the gene expression levels of the PUMA and p53 were analyzed by qRT‒PCR. Molecular docking was used to evaluate the binding energy between Muscone and NLRP3-related proteins. Immunoblotting and qRT‒PCR were used to assess the expression levels of NLRP3 signaling pathway-related proteins (NLRP3, ASC, and Caspase-1) and the NLRP3 gene, respectively. Moreover, the extracellular acidification rate of AC16 cells was measured using the Seahorse system to evaluate glycolysis levels after Muscone treatment. The expression of the key glycolytic enzyme PKM2 was analyzed by immunoblotting and qRT‒PCR. Finally, ChIP‒qPCR was performed to determine the levels of histone modifications (H3K4me3, H3K27me3, and H2AK119Ub) in the PKM2 promoter region. ResultsGO functional enrichment analysis revealed that muscone was involved in regulating the biological processes (BP) of AMI, which mainly included negative regulation of the apoptosis signaling pathway, the response to lipopolysaccharide, and blood pressure regulation. The cellular components (CC) involved in muscone-mediated regulation of AMI mainly included lipid rafts, membrane microdomains, and membrane regions. The molecular functions (MF) involved in muscone-mediated regulation of AMI mainly included oxidoreductase activity, nuclear receptor activity, and transcription factor activity. In vitro results indicated that muscone treatment could inhibit the expression levels of Bax and Caspase-3 in AC16 cells after ischemia‒reperfusion while increasing the expression level of the antiapoptotic protein Bcl-2. Muscone significantly suppressed the transcription levels of p53 and PUMA in AC16 cells. Molecular docking suggested that muscone could bind well with the Cryo-EM structure of NEK7(PDB ID:6NPY). Further investigation of inflammatory pathways revealed that muscone could inhibit the expression level of NLRP3 in AC16 cells and reduce the expression levels of Caspase-1 and Caspase recruitment domain. Fluorescent quantitative PCR experiments showed that muscone significantly inhibited the transcription of NLRP3. Moreover, we found that muscone could enhance the glycolytic efficiency of AC16 cells, which may be related to the increased protein expression of PKM2 in AC16 cells. Fluorescent quantitative PCR showed that muscone could increase the transcription level of PKM2. Chromatin immunoprecipitation assays showed that muscone treatment increased the expression level of H3K4me3 in the PKM2 promoter region and inhibited the levels of H3K27me3 and H2AK119Ub in the PKM2 promoter region. ConclusionMuscone promoted myocardial glycolysis and inhibited NLRP3 pathway activation to improve myocardial ischemia‒reperfusion injury.

Hypoxia-Inducible Factor 1-Alpha and Glucose Metabolism during Cardiac Remodeling Progression from Hypertrophy to Heart Failure.

In pathological cardiac hypertrophy, the heart is more dependent on glucose than fatty acids. This shift in energy metabolism occurs due to several factors, including the oxygen deficit, which activates hypoxia-inducible factor-1α (HIF-1α), a critical molecule related to glucose metabolism. However, there are gaps regarding the behavior of key proteins in the glycolytic pathway and HIF-1α during the transition from hypertrophy to heart failure (HF). This study assesses the hypothesis that there is an early change and enhancement of HIF-1α and the glycolytic pathway, as well as an association between them during cardiac remodeling. Sham and aortic stenosis Wistar rats were analyzed at 2, 6, and 18 weeks and in HF (n = 10-18). Cardiac structure and function were investigated by echocardiogram. Myocardial glycolysis, the aerobic and anaerobic pathways and glycogen were analyzed by enzymatic assay, Western blot, and enzyme-linked immunosorbent assay (ELISA). The following were observed: increased left ventricular hypertrophy; early diastolic function change and severe systolic and diastolic dysfunction in HF; increased HIF-1α in the 2nd week and in HF; precocious alteration and intensification of glycolysis with a shift to anaerobic metabolism from the 6th week onwards; association between HIF-1α, glycolysis, and the anaerobic pathway. Our hypothesis was confirmed as there was an early change and intensification in glucose metabolism, alteration in HIF-1α, and an association between data during the progression from hypertrophy to heart failure.

Abstract 11989: Cardiac Muscle-Specific Disruption of the Exocyst Trafficking Complex Results in Heart Failure

Increased myocardial glycolysis is protective against ischemic injury. Glucose uptake via glucose transporter GLUT4 is the rate-limiting step of glycolysis in cardiomyocytes. GLUT4 delivery to the cardiomyocyte membrane can be triggered by insulin or by increased ATP-demand (i.e. during ischemia) via the AMP-activated protein kinase (AMPK). Previous studies of adipocytes and our recent study in skeletal muscle demonstrated that the exocyst trafficking complex is critical for GLUT4 exocytosis in response to insulin. But it is not known if the insulin-insulin receptor-exocyst-GLUT4 signaling axis is conserved in the cardiomyocytes, or if the exocyst also regulates cardiac glucose uptake via AMPK-induced GLUT4 translocation. Investigating glucose transporter trafficking in cardiomyocytes will be key to better understanding the mechanism of cardiac glucose metabolism under normal and disease conditions. We hypothesized that the exocyst is necessary for myocardial glucose uptake and metabolism and its activity protects the heart from ischemic injury. We have generated tamoxifen-inducible cardiac muscle-specific knockout mice of the central exocyst subunit Exoc5 (Exoc5-CMKO) to evaluate the role of the exocyst in cardiac fuel metabolism in vivo . Unexpectedly we found that cardiac muscle-specific Exoc5 knockout shortened the lifespan of Exoc5-CMKO mice (median survival after tamoxifen treatment was 60.5 days). Echocardiography showed cardiac dysfunction in Exoc5-CMKO mice, with reduced ejection fraction (Ctrl: 0.77; CMKO: 0.43; p<0.0001) and fractional shortening (Ctrl: 45.57; CMKO:21.62; p<0.0001), and increased systolic and diastolic LV diameters (LVID s (mm): Ctrl: 1.81; CMKO: 3.32; LVID d (mm): Ctrl: 3.47; CMKO:4.2; p<0.0001) compared to controls. Histological analysis revealed increased fibrosis in Exoc5-CMKO hearts, while isolated knockout primary cardiomyocytes showed decreased contractility and altered calcium handling. Our findings suggest that the exocyst is necessary for proper cardiac muscle function. Future studies will investigate how impaired exocyst-mediated membrane trafficking leads to heart failure in our Exoc5-CMKO mice.

Glucose 6-Phosphate Accumulates via Phosphoglucose Isomerase Inhibition in Heart Muscle.

Metabolic and structural remodeling is a hallmark of heart failure. This remodeling involves activation of the mTOR (mammalian target of rapamycin) signaling pathway, but little is known on how intermediary metabolites are integrated as metabolic signals. We investigated the metabolic control of cardiac glycolysis and explored the potential of glucose 6-phosphate (G6P) to regulate glycolytic flux and mTOR activation. We developed a kinetic model of cardiomyocyte carbohydrate metabolism, CardioGlyco, to study the metabolic control of myocardial glycolysis and G6P levels. Metabolic control analysis revealed that G6P concentration is dependent on phosphoglucose isomerase (PGI) activity. Next, we integrated ex vivo tracer studies with mathematical simulations to test how changes in glucose supply and glycolytic flux affect mTOR activation. Nutrient deprivation promoted a tight coupling between glucose uptake and oxidation, G6P reduction, and increased protein-protein interaction between hexokinase II and mTOR. We validated the in silico modeling in cultured adult mouse ventricular cardiomyocytes by modulating PGI activity using erythrose 4-phosphate. Inhibition of glycolytic flux at the level of PGI caused G6P accumulation, which correlated with increased mTOR activation. Using click chemistry, we labeled newly synthesized proteins and confirmed that inhibition of PGI increases protein synthesis. The reduction of PGI activity directly affects myocyte growth by regulating mTOR activation.

Cardiac metabolic modulation upon low-carbohydrate low-protein ketogenic diet in diabetic rats studied in vivo using hyperpolarized 13 C pyruvate, butyrate and acetoacetate probes.

To investigate the effects of long-term low-carbohydrate low-protein ketogenic diet (KD) on cardiac metabolism and diabetic cardiomyopathy status in lean diabetic Goto-Kakizaki (GK) rats. Diabetic GK rats were fed with KD for 62 weeks. Cardiac function and metabolism were assessed using magnetic resonance imaging and 13 C magnetic resonance spectroscopy (13 C-MRS), at rest and under dobutamine stress. 13 C-MRS was performed following injection of hyperpolarized [3-13 C]acetoacetate, [1-13 C]butyrate or [1-13 C]pyruvate to assess ketone body, short-chain fatty acid or glucose utilization, respectively. Protein expression and cardiomyocyte structure were determined via Western blotting and histology, respectively. KD lowered blood glucose, triglyceride and insulin levels while increasing blood ketone body levels. In KD-fed diabetic rats, myocardial ketone body and glucose oxidation were lower than in chow-fed diabetic rats, while myocardial glycolysis and short-chain fatty acid oxidation were unaltered. Dobutamine stress revealed an increased cardiac preload and reduced cardiac compliance in KD-fed diabetic rats. Dobutamine-induced stimulation of myocardial glycolysis was more enhanced in KD-fed diabetic rats than in chow-fed diabetic rats, which was potentially facilitated via an upregulation in basal expression of proteins involved in glucose transport and glycolysis in the hearts of KD-fed rats. The metabolic profile induced by KD was accompanied by cardiac hypertrophy, a trend for increased myocardial lipid and collagen content, and an increased marker of oxidative stress. KD seems to exacerbate diabetic cardiomyopathy in GK rats, which may be associated with maladaptive cardiac metabolic modulation and lipotoxicity.

P1589Metabolomic impact of glucagon-like peptide-1 on diabetic myocardium - glucagon-like peptide-1 accelerates myocardial glycolysis and utilizes more lactate as myocardial substrate

P1589Metabolomic impact of glucagon-like peptide-1 on diabetic myocardium - glucagon-like peptide-1 accelerates myocardial glycolysis and utilizes more lactate as myocardial substrate

Effect of Gegen Qinlian Decoction on Cardiac Gene Expression in Diabetic Mice.

The aim of this research is to investigate the therapeutic effect of GGQL decoction on cardiac dysfunction and elucidate the pharmacological mechanisms. db/db mice were divided into DB group or GGQL group, and WT mice were used as control. All mice were accessed by echocardiography. And the total RNA of LV tissue samples was sequenced, then differential expression genes were analyzed. The RNA-seq results were validated by the results of RT-qPCR of 4 genes identified as differentially expressed. The content of pyruvate and ceramide in myocardial tissue was also measured. The results showed that GGQL decoction could significantly improve the diastolic dysfunction, increase the content of pyruvate, and had the trend to reduce the ceramide content. The results of RNA-seq showed that 2958 genes were differentially expressed when comparing the DB group with the WT group. Among them, compared with the DB group, 26 genes were differentially regulated in the GGQL group. The expression results of 4 genes were consistent with the RNA-seq results. Our study reveals that GGQL decoction has a therapeutic effect on diastolic dysfunction of the left ventricular and the effect may be related to its role in promoting myocardial glycolysis and decreasing the content of ceramide.

Cardiac Insulin-Resistance and Decreased Mitochondrial Energy Production Precede the Development of Systolic Heart Failure After Pressure-Overload Hypertrophy

Cardiac hypertrophy is accompanied by significant alterations in energy metabolism. Whether these changes in energy metabolism precede and contribute to the development of heart failure in the hypertrophied heart is not clear. Mice were subjected to cardiac hypertrophy secondary to pressure-overload as a result of an abdominal aortic constriction (AAC). The rates of energy substrate metabolism were assessed in isolated working hearts obtained 1, 2, and 3 weeks after AAC. Mice subjected to AAC demonstrated a progressive development of cardiac hypertrophy. In vivo assessment of cardiac function (via echocardiography) demonstrated diastolic dysfunction by 2 weeks (20% increase in E/E'), and systolic dysfunction by 3 weeks (16% decrease in % ejection fraction). Marked cardiac insulin-resistance by 2 weeks post-AAC was evidenced by a significant decrease in insulin-stimulated rates of glycolysis and glucose oxidation, and plasma membrane translocation of glucose transporter 4. Overall ATP production rates were decreased at 2 and 3 weeks post-AAC (by 37% and 47%, respectively) because of a reduction in mitochondrial oxidation of glucose, lactate, and fatty acids that was not accompanied by an increase in myocardial glycolysis rates. Reduced mitochondrial complex V activity was evident at 3 weeks post-AAC, concomitant with a reduction in the ratio of phosphocreatine to ATP. The development of cardiac insulin-resistance and decreased mitochondrial oxidative metabolism are early metabolic changes in the development of cardiac hypertrophy, which create an energy deficit that may contribute to the progression from hypertrophy to heart failure.

Inhibition of Serine Palmitoyl Transferase I Reduces Cardiac Ceramide Levels and Increases Glycolysis Rates following Diet-Induced Insulin Resistance

ObjectiveDiet-induced obesity (DIO) leads to an accumulation of intra-myocardial lipid metabolites implicated in causing cardiac insulin resistance and contractile dysfunction. One such metabolite is ceramide, and our aim was to determine the effects of inhibiting de novo ceramide synthesis on cardiac function and insulin stimulated glucose utilization in mice subjected to DIO.Materials and MethodsC57BL/6 mice were fed a low fat diet or subjected to DIO for 12 weeks, and then treated for 4 weeks with either vehicle control or the serine palmitoyl transferase I (SPT I) inhibitor, myriocin. In vivo cardiac function was assessed via ultrasound echocardiography, while glucose metabolism was assessed in isolated working hearts.ResultsDIO was not associated with an accumulation of intra-myocardial ceramide, but rather, an accumulation of intra-myocardial DAG (2.63±0.41 vs. 4.80±0.97 nmol/g dry weight). Nonetheless, treatment of DIO mice with myriocin decreased intra-myocardial ceramide levels (50.3±7.7 vs. 26.9±2.7 nmol/g dry weight) and prevented the DIO-associated increase in intra-myocardial DAG levels. Interestingly, although DIO impaired myocardial glycolysis rates (7789±1267 vs. 2671±326 nmol/min/g dry weight), hearts from myriocin treated DIO mice exhibited an increase in glycolysis rates.ConclusionsOur data reveal that although intra-myocardial ceramide does not accumulate following DIO, inhibition of de novo ceramide synthesis nonetheless reduces intra-myocardial ceramide levels and prevents the accumulation of intra-myocardial DAG. These effects improved the DIO-associated impairment of cardiac glycolysis rates, suggesting that SPT I inhibition increases cardiac glucose utilization.

Adenosine-mediated inhibition of 5′-AMP-activated protein kinase and p38 mitogen-activated protein kinase during reperfusion enhances recovery of left ventricular mechanical function

Attenuation of excessive rates of myocardial glycolysis limits proton production and Ca(2+) overload during reperfusion and improves recovery of post-ischemic left ventricular (LV) function. In order to elucidate mechanisms underlying glycolytic inhibition by adenosine (ADO), this study tested the hypothesis that the beneficial effects of ADO are due to Ser/Thr protein phosphatase (PP)-mediated inhibition of 5'-AMP-activated protein kinase (AMPK) and phosphofructokinase-2 (PFK-2). In isolated perfused working rat hearts subjected to global ischemia (GI) and reperfusion, ADO (500μmol/l), added 5min prior to the onset of GI and present throughout reperfusion, inhibits glycolysis and proton production during reperfusion and improves post-ischemic LV work. These metabolic effects of ADO are also evident during aerobic perfusion. Assays of glycolytic intermediates show that ADO-induced glycolytic inhibition occurs at the step catalyzed by PFK-1, an effect mediated by reduced activation of PFK-2 by AMPK. The PP1 and PP2A inhibitors, cantharidin (5μmol/l) or okadaic acid (0.1μmol/l), added 10min prior to ADO prevent ADO-induced inhibition of glycolysis and AMPK, as well as ADO-induced cardioprotection. ADO also inhibits p38 MAPK phosphorylation during reperfusion in a cantharidin-sensitive manner, and pharmacological inhibition of p38 MAPK (by SB202190, 10μmol/l) during reperfusion also reduces glycolysis and is cardioprotective. These results indicate that attenuation of glycolysis during reperfusion and cardioprotection can be achieved by inhibition of the stress kinases, AMPK and p38 MAPK.

Long-term effects of intrauterine growth restriction on cardiac metabolism and susceptibility to ischaemia/reperfusion

Adult offspring who are born intrauterine growth restricted (IUGR) are at risk of developing cardiovascular diseases during adulthood. Additionally, several cardiac diseases are associated with changes in myocardial energy metabolism. However, the potential long-term effects of being born IUGR on cardiac energetics are unknown. The aim of this study was to assess the long-term effect of IUGR on cardiac performance and energy metabolism under aerobic conditions and after ischaemia/reperfusion (IR) injury. To induce IUGR, pregnant Sprague-Dawley rats were randomly assigned to hypoxic (11.5% O(2)) or control (21% O(2)) environments from day 15 to 21 of pregnancy. Cardiac susceptibility to IR was evaluated in male and female offspring at 4 (young-adult) or 12 (ageing) months of age using isolated working hearts. Cardiac production of energy was evaluated using radiolabelled substrates. Both male and female IUGR offspring exhibited an increased susceptibility to IR injury compared with controls (P< 0.05) as well as an increased post-ischaemic production of protons (P< 0.001) secondary to a mismatch between myocardial glycolysis and glucose oxidation rates. Moreover, offspring born IUGR exhibited an increased myocardial production of acetyl-CoA during reperfusion. The mismatch between energy production and cardiac performance indicates that in IUGR offspring, cardiac efficiency during reperfusion was decreased relative to controls. Our results suggest that hypoxia-induced IUGR has long-term effects on cardiac susceptibility to IR injury that are independent of sex and age. Moreover, we identified a mismatch in glucose metabolism, leading to proton accumulation in the post-ischaemic myocardium of offspring born IUGR as a potential mechanism involved.

Cardiac-Specific Overexpression of HIF-1α Prevents Deterioration of Glycolytic Pathway and Cardiac Remodeling in Streptozotocin-Induced Diabetic Mice

Defective glycolysis and angiogenesis in the heart of diabetic patients and in experimental diabetic animal models have been reported. The aim of this study was to determine whether overexpression of hypoxia-inducible factor (HIF)-1alpha protects from myocardial injury in diabetic mice by increasing myocardial glycolysis and angiogenesis. Cardiac-specific HIF-1alpha-overexpressing transgenic and age-matched wild-type control mice were treated with streptozotocin to induce diabetes. Changes in glucose transporters, glycolytic enzymes, angiogenic factors and cardiac morphology were examined in the hearts by real-time RT-PCR, Western blotting, enzymatic assay, and histological assays. HIF-1alpha overexpression elevated hexokinase II (HK-II) protein level and total HK activity in nondiabetic heart and prevented the decreases in HK-II mRNA, protein, and total HK activity in diabetic heart. In addition, the reduction of glucose transporter I, but not glucose transporter 4, was restored in HIF transgenic mouse heart along with a recovery of myocardium ATP production. HIF-1alpha overexpression also normalized diabetes-reduced vascular endothelial growth factor concentration along with a sustained myocardial capillary density and an inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. Therefore, elevation of HIF-1alpha provides a cardiac protection from diabetic-induced impairment in glucose metabolism and angiogenesis via up-regulation of HIF-1 target genes.

PET detection of the impact of dobutamine on myocardial glucose metabolism in women with type 1 diabetes mellitus

Our objective was to determine, in the hearts of women with type 1 diabetes mellitus (T1DM), whether the fate of extracted glucose is altered and, if so, what the impact of dobutamine is on myocardial substrate metabolism. In experimental models of T1DM, myocardial glycolysis and glucose oxidation are reduced with the impairment becoming more pronounced with dobutamine. Whether similar changes occur in humans with T1DM is unclear. Myocardial perfusion, oxygen consumption, and glucose and fatty acid metabolism were measured with positron emission tomography in 19 women, 7 normal volunteers (NVs) and 12 with T1DM. The NVs and 6 T1DM (DM1) patients were studied under baseline metabolic conditions and 6 T1DM patients were studied during hyperinsulinemic-euglycemic clamp (DM1-C), both at rest and during dobutamine. At rest, myocardial glucose uptake, glycolysis, glycogen storage, and oxidation were reduced by similar levels in DM1 patients compared with NVs ( P < .05). During dobutamine, although myocardial glucose uptake was not different from DM1 patients at rest, fractional glycolysis was lower compared with NVs or DM1-C patients and reflected a lower glucose oxidation rate ( P < .001). Measurements of myocardial glucose metabolism at rest and during dobutamine were comparable between NVs and DM1-C patients. During dobutamine, myocardial fatty acid uptake and oxidation increased in all 3 groups. In women with T1DM, (1) myocardial glucose metabolism is impaired downstream from initial uptake, (2) these abnormalities become more pronounced with dobutamine and are paralleled by an increase in myocardial fatty acid metabolism, and (3) insulin restores glucose metabolism to levels observed in normal control subjects.

PET detection of the impact of dobutamine on myocardial glucose metabolism in women with type 1 diabetes mellitus

Our objective was to determine, in the hearts of women with type 1 diabetes mellitus (T1DM), whether the fate of extracted glucose is altered and, if so, what the impact of dobutamine is on myocardial substrate metabolism. In experimental models of T1DM, myocardial glycolysis and glucose oxidation are reduced with the impairment becoming more pronounced with dobutamine. Whether similar changes occur in humans with T1DM is unclear. Myocardial perfusion, oxygen consumption, and glucose and fatty acid metabolism were measured with positron emission tomography in 19 women, 7 normal volunteers (NVs) and 12 with T1DM. The NVs and 6 T1DM (DM1) patients were studied under baseline metabolic conditions and 6 T1DM patients were studied during hyperinsulinemic-euglycemic clamp (DM1-C), both at rest and during dobutamine. At rest, myocardial glucose uptake, glycolysis, glycogen storage, and oxidation were reduced by similar levels in DM1 patients compared with NVs (P < .05). During dobutamine, although myocardial glucose uptake was not different from DM1 patients at rest, fractional glycolysis was lower compared with NVs or DM1-C patients and reflected a lower glucose oxidation rate (P < .001). Measurements of myocardial glucose metabolism at rest and during dobutamine were comparable between NVs and DM1-C patients. During dobutamine, myocardial fatty acid uptake and oxidation increased in all 3 groups. In women with T1DM, (1) myocardial glucose metabolism is impaired downstream from initial uptake, (2) these abnormalities become more pronounced with dobutamine and are paralleled by an increase in myocardial fatty acid metabolism, and (3) insulin restores glucose metabolism to levels observed in normal control subjects.

Contribution of glycogen to aerobic myocardial glucose utilization.

We determined glycogen turnover and the contribution of glycogen as a source of glucose for aerobic myocardial glycolysis and glucose oxidation in parallel series of isolated, working rat hearts subjected to pulse-chase perfusions. Myocardial glycogen of isolated, working rat hearts was radiolabeled, after 30 minutes of substrate-free glycogen depletion, by perfusion for 60 minutes with buffer designed to stimulate resynthesis of glycogen (1.2 mmol/L palmitate, 11 mmol/L [U-14C]- or [5-3H]glucose, 0.5 mmol/L lactate, and 100 microU/mL insulin). Rates of glucose oxidation (14 CO2 production) and glycolysis (3H20 production) were then measured by perfusing the hearts for 40 minutes with buffer designed to simulate physiological conditions (0.4 mmol/L palmitate, 0.5 mmol/L lactate, 11 mmol/L [5-3H]- or [U-14C]- glucose, 100 microU/mL insulin) containing radiolabeled glucose different from that used during resynthesis. During the chase perfusion, rates of glycolysis and glucose oxidation from exogenous glucose were significantly greater than those from endogenous glycogen. Nevertheless, glycogen contributed significantly to myocardial energy production (41% of the overall ATP produced from glucose), and a significantly greater fraction of the glucose from glycogen that passed through glycolysis was oxidized (>50%) compared with the fraction oxidized from exogenous glucose (<20%, P<.05). Myocardial glycogen was simultaneously synthesized and degraded (ie, glycogen turnover) during the chase perfusion, despite net glycogenolysis. Furthermore, enrichment of labeled glucose in glycogen at the end of the chase perfusion, when corrected for newly synthesized glycogen, did not differ from that at the end of the labeling period. Thus, glycogen contributes significantly to aerobic myocardial glucose use under these experimental conditions, and the glucose derived from glycogen is oxidized preferentially compared with exogenous glucose. Additionally, substantial myocardial glycogen turnover occurs, and the manner in which glycogen is degraded does not fit the ordered hypothesis of "last glucose on, first glucose off."

Acute insulin withdrawal from diabetic BB rats decreases myocardial glycolysis during low-flow ischemia

We have previously demonstrated that withdrawal of insulin treatment from BB diabetic rats for a 24-hour period will increase the failure rate of hearts subjected to low-flow ischemia. The purpose of this study was to determine if this increased severity of ischemia was related to a decrease in glycolytic rates during ischemia. Two groups of insulin-dependent diabetic BB Wistar rats were used; in one group, insulin treatment was withheld from rats 24 hours prior to study (uncontrolled), while in the second group, the daily insulin injection was not withheld (insulin-treated). Isolated working hearts obtained from these animals were perfused with 30 mmol/L ( 2- 3H U- 14C )-glucose and 1.2 mmol/L palmitate, at an 11.5 mm Hg left atrial preload and 80 mm Hg aortic afterload. Hearts were subjected to a 15-minute aerobic perfusion followed by 60 minutes of low-flow ischemia (coronary flow, 0.5 mL/min). Under aerobic conditions, steady-state glucose oxidation rates (measured as 14CO 2 production) were decreased in the uncontrolled group compared with the insulin-treated group (85.3 ± 21.5 v 406.2 ± 120.1 nmol/min/g dry weight, respectively; P < .05). Steady-state glycolytic rates (measured as 3H 2O production) were also decreased in the uncontrolled group compared with the insulin-treated group (1.73 ± 0.30 v 5.57 ± 1.26 μmol/min/g dry weight, respectively; P < .05). During low-flow ischemia, glucose-oxidation rates markedly decreased in both groups (23.9 ± 8.7 and 38.3 ± 25.2 nmol/min/g dry weight in the uncontrolled and insulin-treated diabetic rats, respectively). Glycolytic rates during ischemia decreased slightly, but remained significantly depressed in the uncontrolled group compared with the insulin-treated group (1.28 ± 0.28 v 2.15 ± 0.25 μmol/min/g dry weight, respectively; P < .05). During reperfusion, glucose oxidation recovered to preischemic values in hearts from uncontrolled diabetic rats, and remained depressed compared with the insulin-treated diabetic rats (84.7 ± 9.2 and 366.9 ± 81.3 nmol/min/g dry weight, respectively, P < 0.05). Glycolytic rates also returned to preischemic values and remained depressed in these hearts (2.27 ± 0.42 and 4.71 ± 0.91 μmol/min/g dry weight, respectively, P < .05). Combined with our previous studies, these data suggest that poor control of insulin-dependent diabetic BB rats increases the sensitivity of the heart to ischemia, possibly by decreasing the ability of hearts to maintain high glycolytic rates during ischemia.

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.

Crossover plot study of glycolytic intermediates in the ischemic canine heart.

Effects of regional ischemia on myocardial glycolysis were studied by measuring the levels of glycolytic intermediates in the endo- and epicardial layers of the left ventricle in dogs anesthetized with pentobarbital. Regional ischemia was induced by ligating a small branch of the left anterior descending coronary artery. The myocardial tissue samples were removed before and 1.5, 3, 7, or 30 min after coronary artery ligation. Based on a crossover plot study of the glycolytic intermediates, it is suggested that the activity of glycogen phosphorylase was accelerated, while that of phosphofructokinase was inhibited in ischemic myocardium samples removed 1.5, 3, 7, and 30 min after ligation. When the frozen myocardium was allowed to stand at room temperature for 10 min, the crossover plot study revealed acceleration of phosphofructokinase activity. The metabolic response to regional ischemia of the endocardial layers was more marked than that of the epicardial. During ischemia the levels of adenine nucleotides did not change significantly, but those of citrate and hydrogen ions increased significantly. It appears that inhibition of myocardial phosphofructokinase activity during ischemia is partly due to an increase in the levels of citrate and hydrogen ions in the ischemic tissue.