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Rationale The nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown. Objective To examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart. Methods and results We developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1iSA) through deletion of Tsc2. Prior to hypertrophy, rates of glucose uptake and oxidation, as well as protein and enzymatic activity of glucose 6-phosphate isomerase (GPI) were decreased, while intracellular levels of glucose 6-phosphate (G6P) were increased. Subsequently, hypertrophy developed. Transcript levels of the fetal gene program and pathways of exercise-induced hypertrophy increased, while hypertrophy did not progress to heart failure. We therefore examined the hearts of wild-type mice subjected to voluntary physical activity and observed early changes in GPI, followed by hypertrophy. Rapamycin prevented these changes in both models. Conclusion Activation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

Diabetic patients with acute myocardial infarction are more likely to die than nondiabetic patients. In the present study we examined the effect of insulin resistance on myocardial ischemia tolerance. Hearts of rats, rendered insulin resistant by high-sucrose feeding, were subjected to ischemia/reperfusion ex vivo. Cardiac power of control hearts from chow-fed rats recovered to 93%, while insulin-resistant hearts recovered only to 80% (P

Insulin resistance is a prominent feature in heart failure, while hyperglycemia impairs cardiac contraction. We propose that decreased insulin-mediated glucose uptake by the heart preserves cardiac function in response to metabolic and hemodynamic stress. To test this hypothesis, we fed rats a high-sucrose diet (HSD). Energy substrate metabolism and cardiac work were determined ex vivo in a sequential protocol simulating metabolic and hemodynamic stress. Compared to chow-fed, control rats, HSD impaired myocardial insulin responsiveness and induced profound metabolic changes in the heart, characterized by reduced rates of glucose uptake (7.91±0.30 vs. 10.73±0.67 μmol/min/g dry weight; P

When stressed, the heart remodels metabolically and structurally. Metabolically, increases its reliance on carbohydrates for energy provision. Structurally, it hypertrophies. A common pathway for both of these changes is the activation of the mechanistic Target Of Rapamycin Complex 1 (mTORC1).

Objective Insulin regulates both glucose uptake and postnatal cardiac growth. The anabolic effects of insulin are mediated by the mammalian target of rapamycin (mTOR), an evolutionarily conserved kinase which is also a convergence point between nutrient sensing and cell growth. We postulated that mTOR signalling in the heart requires the metabolism of glucose. Methods We interrogated the insulin-mediated mTOR signalling pathway in response to different metabolic interventions regulating substrate metabolism in the isolated working rat heart and in isolated cardiomyocytes. Results Although insulin enhanced Akt activity, phosphorylation of mTOR and its downstream targets (p70S6K and 4EBP1) required the addition of glucose. Glucose-dependent p70S6K phosphorylation was independent of the hexosamine biosynthetic pathway, the AMP kinase pathway, and the pentose phosphate pathway. However, inhibition of glycolysis downstream of hexokinase markedly enhanced p70S6K phosphorylation. Furthermore, 2-deoxyglucose activated p70S6K suggesting that phosphorylation of glucose is required for carbohydrate-mediated mTOR signalling in the heart. Lastly, we also found enhanced p70S6K phosphorylation in the hearts of diabetic rats. Conclusion Phosphorylation of glucose is necessary for insulin-dependent mTOR activity in the heart, suggesting a link between intermediary metabolism and cardiac growth.

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 μU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of13C from glutamine to glutamate was not detected ( n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.

The heart, like other organs, possesses an internal circadian clock. These clocks provide the selective advantage of anticipation, enabling the organ to prepare for a given stimulus, thereby optimizing the appropriate response. The heart in diabetes is associated with alterations in morphology, gene expression, metabolism and contractile performance. The present study investigated whether diabetes also alters the circadian clock in the heart. Insulin-dependent diabetes mellitus was induced in rats by treatment with streptozotocin (STZ; 65 mg/kg). STZ increased humoral (glucose and non-esterified fatty acids) and heart gene expression (myosin heavy chain beta, pyruvate dehydrogenase kinase 4 and uncoupling protein 3) markers of diabetes. The circadian patterns of gene expression of seven components of the mammalian clock (bmal1, clock, cry1, cry2, per1, per2 and per3), as well as three clock output genes (dbp, hlf and tef), were compared in hearts isolated from control and STZ-induced diabetic rats. All components of the clock investigated possessed circadian rhythms of gene expression. In the hearts isolated from STZ-induced diabetic rats, the phases of these circadian rhythms were altered (approximately 3 h early) compared to those observed for control hearts. The clock in the heart has therefore lost normal synchronization with its environment during diabetes. Whether this loss of synchronization plays a role in the development of contractile dysfunction of the heart in diabetes remains to be determined.

Diurnal variation of cardiac function in vivo has been attributed primarily to changes in factors such as sympathetic activity. No study has investigated previously the intrinsic properties of the heart throughout the day. We therefore investigated diurnal variations in metabolic flux and contractile function of the isolated working rat heart and how this related to circadian expression of metabolic genes. Contractile performance, carbohydrate oxidation, and oxygen consumption were greatest in the middle of the night, with little variation in fatty acid oxidation. The expression of all metabolic genes investigated (including regulators of carbohydrate utilization, fatty acid oxidation, and mitochondrial function) showed diurnal variation, with a general peak in the night. In contrast, pressure overload–induced cardiac hypertrophy completely abolished this diurnal variation of metabolic gene expression. Thus, over the course of the day, the normal heart anticipates, responds, and adapts to physiological alterations within its environment, a trait that is lost by the hypertrophied heart. We speculate that loss of plasticity of the hypertrophied heart may play a role in the subsequent development of contractile dysfunction.

Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, an important modulator of fatty acid oxidation. We hypothesized that increased fatty acid availability would increase the expression and activity of heart and skeletal muscle MCD, thereby promoting fatty acid utilization. The results show that high-fat feeding, fasting, and streptozotocin-induced diabetes all significantly increased the plasma concentration of nonesterified fatty acids, with a concomitant increase in both rat heart and skeletal muscle MCD mRNA. Upon refeeding of fasted animals, MCD expression returned to basal levels. Fatty acids are known to activate peroxisome proliferator-activated receptor-α (PPARα). Specific PPARα stimulation, through Wy-14643 treatment, significantly increased the expression of MCD in heart and skeletal muscle. Troglitazone, a specific PPARγ agonist, decreased MCD expression. The sensitivity of MCD induction by fatty acids and Wy-14643 was soleus > extensor digitorum longus > heart. High plasma fatty acids consistently increased MCD activity only in solei, whereas MCD activity in the heart actually decreased with high-fat feeding. Pressure overload-induced cardiac hypertrophy, in which PPARα expression is decreased (and fatty acid oxidation is decreased), resulted in decreased MCD mRNA and activity, an effect that was dependent on fatty acids. The results suggest that fatty acids induce the expression of MCD in rat heart and skeletal muscle. Additional posttranscriptional mechanisms regulating MCD activity appear to exist.

The regulatory neuropeptide calcitonin-gene related peptide (CGRP) has been shown to evoke a hypertrophic response in isolated cardiomyocytes in vitro, an effect which was attributed to PKC activation. Activation of PKC has previously been implicated in the development of cardiac hypertrophy. We therefore investigated the role of CGRP in pressure overload-induced hypertrophy in vivo, which has not previously been reported. Constriction of the ascending aorta of rats resulted in an increase in the heart weight to body weight ratio, increased myocyte diameter, re-expression of the fetal genes ANF, MHCbeta and skeletal alpha-actin, and decreased expression of the adult genes GLUT4 and SERCA2a. Treatment of neonatal rat pups (1-2 days old) with capsaicin (50 mg/kg), resulted in the permanent de-afferentation of small-diameter unmyelinated CGRP-containing sensory C-fibres. Such treatment caused a 68% decrease in the CGRP-like immunoreactivity of hearts isolated from 10 week old rats (p < 0.001). Contrary to expectations, aortic constriction of capsaicin treated rats had no effect on the development of hypertrophy at the trophic, morphometric or gene expression levels. The results suggest that the development of pressure overload-induced hypertrophy in vivo does not require the regulatory neuropeptide CGRP.

Myocardial glucose use is regulated by competing substrates and hormonal influences. However, the interactions of these effectors on the metabolism of exogenous glucose and glucose derived from endogenous glycogen are not completely understood. In order to determine changes in exogenous glucose uptake, glucose oxidation, and glycogen enrichment, hearts were perfused with glucose (5 mM) either alone, or glucose plus insulin (40 microU/ml), glucose plus acetoacetate (5 mM), or glucose plus insulin and acetoacetate, using a three tracer (3H, 14C, and 13C) technique. Insulin-stimulated glucose uptake and lactate production in the absence of acetoacetate, while acetoacetate inhibited the uptake of glucose and the oxidation of both exogenous glucose and endogenous carbohydrate. Depending on the metabolic conditions, the contribution of glycogen to carbohydrate metabolism varied from 20-60%. The addition of acetoacetate or insulin increased the incorporation of exogenous glucose into glycogen twofold, and the combination of the two had additive effects on the incorporation of glucose into glycogen. In contrast, the glycogen content was similar for the three groups. The increased incorporation of glucose in glycogen without a significant change in the glycogen content in hearts perfused with glucose, acetoacetate, and insulin suggests increased glycogen turnover. We conclude that insulin and acetoacetate regulate the incorporation of glucose into glycogen as well as the relative contributions of exogenous glucose and endogenous carbohydrate to myocardial energy metabolism by different mechanisms.

We tested the hypothesis that low-flow ischemia increases glucose uptake and reduces insulin responsiveness. Working hearts from fasted rats were perfused with buffer containing glucose alone or glucose plus a second substrate (lactate, octanoate, or β-hydroxybutyrate). Rates of glucose uptake were measured by3H2O production from [2-3H]glucose. After 15 min of perfusion at a physiological workload, hearts were subjected to low-flow ischemia for 45 min, after which they were returned to control conditions for another 30 min. Insulin (1 mU/ml) was added before, during, or after the ischemic period. Cardiac power decreased by 70% with ischemia and returned to preischemic values on reperfusion in all groups. Low-flow ischemia increased lactate production, but the rate of glucose uptake during ischemia increased only when a second substrate was present. Hearts remained insulin responsive under all conditions. Insulin doubled glucose uptake when added under control conditions, during low-flow ischemia, and at the onset of the postischemic period. Insulin also increased net glycogen synthesis in postischemic hearts perfused with glucose and a second substrate. Thus insulin stimulates glucose uptake in normal and ischemic hearts of fasted rats, whereas ischemia stimulates glucose uptake only in the presence of a cosubstrate. The results are consistent with two separate intracellular signaling pathways for hexose transport, one that is sensitive to the metabolic requirements of the heart and another that is sensitive to insulin.

Background Changes in energy substrate metabolism are first responders to hemodynamic stress in the heart. We have previously shown that hexose‐6‐phosphate levels regulate mammalian target of rapamycin ( mTOR ) activation in response to insulin. We now tested the hypothesis that inotropic stimulation and increased afterload also regulate mTOR activation via glucose 6‐phosphate (G6P) accumulation. Methods and Results We subjected the working rat heart ex vivo to a high workload in the presence of different energy‐providing substrates including glucose, glucose analogues, and noncarbohydrate substrates. We observed an association between G6P accumulation, mTOR activation, endoplasmic reticulum ( ER ) stress, and impaired contractile function, all of which were prevented by pretreating animals with rapamycin ( mTOR inhibition) or metformin ( AMPK activation). The histone deacetylase inhibitor 4‐phenylbutyrate, which relieves ER stress, also improved contractile function. In contrast, adding the glucose analogue 2‐deoxy‐ d ‐glucose, which is phosphorylated but not further metabolized, to the perfusate resulted in mTOR activation and contractile dysfunction. Next we tested our hypothesis in vivo by transverse aortic constriction in mice. Using a micro‐ PET system, we observed enhanced glucose tracer analog uptake and contractile dysfunction preceding dilatation of the left ventricle. In contrast, in hearts overexpressing SERCA2a, ER stress was reduced and contractile function was preserved with hypertrophy. Finally, we examined failing human hearts and found that mechanical unloading decreased G6P levels and ER stress markers. Conclusions We propose that glucose metabolic changes precede and regulate functional (and possibly also structural) remodeling of the heart. We implicate a critical role for G6P in load‐induced mTOR activation and ER stress.

We tested the hypothesis that improved ischemia tolerance in an isolated working rat heart preparation can be achieved by interventions other than ischemic preconditioning. Hearts were perfused at near-physiological workload with bicarbonate buffer containing glucose (10 mM). A preischemic period of 25 min was followed by 15 min of global ischemia and 30 min of reperfusion under preischemic conditions. Hearts came from either fed or fasted animals (groups 1 and 2). In group 3 lactate (10 mM) and insulin (10 mU/ml) were added to the perfusate of fasted animals. In group 4 hearts from fed animals were perfused with glucose (10 mM) and were ischemically preconditioned by one cycle of ischemia between 10 and 15 min of the preischemic perfusion. Cardiac power and glucose uptake were measured continuously to assess functional and metabolic recovery. In addition, we measured the time to return of aortic flow. Glucose metabolites and the ratio of latent of free citrate synthase activity (citrate synthase ratio, a marker for the structural integrity of mitochondria) were determined at selected time points. Groups 2, 3, and 4 recovered significantly faster than group 1, whereas recovery of power showed an improvement in groups 3 and 4 only. In addition, there was an early increase in glucose uptake during reperfusion in these two groups, suggesting an early need for glucose substrate. Glycogen levels decreased with ischemia in all groups and returned to preischemic levels in groups 2, 3, and 4. The citrate synthase ratio was low in the control group and preserved in the groups showing improved functional recovery. We conclude that metabolic interventions may be as effective as ischemic preconditioning in protecting the heart from ischemic injury.

The acute adaptation of myocardial glucose metabolism in response to low-flow ischemia and reperfusion was investigated in isolated working rat hearts perfused with bicarbonate saline containing glucose (10 mM) and insulin (40 microU/ml). Reversible low-flow ischemia was induced by reducing coronary perfusion pressure from 100 to 35 cmH2O. Tritiated glucose was used to assess rates of glucose transport and phosphorylation, flux from glucose to pyruvate, and oxidation of exogenous glucose. Rates of glycogen synthesis and glycolysis were also assessed. With ischemia, cardiac power decreased by more than two-thirds. Rates of glucose uptake and flux from glucose to pyruvate remained unchanged, while glucose oxidation declined by 61%. Rates of lactate release more than doubled, and fractional enrichment of glycogen remained the same. During reperfusion, glucose oxidation returned to the preischemic values. When isoproterenol was added during ischemia, glucose uptake increased, glycogen decreased, and lactate release increased. No effect was seen with pacing. We conclude that during low-flow ischemia and with glucose as the only exogenous substrate, net glucose uptake remains unchanged. There is a reversible redirection between glycolysis and glucose oxidation, while glycogen synthesis continues during ischemia and is enhanced with reperfusion.

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC2) is a key regulator of mitochondrial fatty acid (FA) uptake via carnitine palmitoyltransferase 1 (CPT1). To test the hypothesis that oxidative metabolism is upregulated in hearts from animals lacking ACC2 (employing a transgenic Acc2-mutant mouse), we assessed cardiac function in vivo and determined rates of myocardial substrate oxidation ex vivo. When examined by echocardiography, there was no difference in systolic function, but left ventricular mass of the Acc2-mutant (MUT) mouse was significantly reduced ( approximately 25%) compared with wild-types (WT). Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts. Exogenous oxidation rates of oleate were increased approximately 22%, and, unexpectedly, exogenous glucose oxidation rates were also increased in MUT hearts. Using a hyperinsulinemic-euglycemic clamp, we found that glucose uptake in MUT hearts was increased by approximately 83%. Myocardial triglyceride levels were significantly reduced in MUT vs. WT while glycogen content was the same. In parallel, transcript levels of PPARalpha and its target genes, pyruvate dehydrogenase kinase-4 (PDK-4), malonyl-CoA decarboxylase (MCD), and mCPT1, were downregulated in MUT mice. In summary, we report that 1) Acc2-mutant hearts exhibit a marked preference for the oxidation of both glucose and FAs coupled with greater utilization of endogenous fuel substrates (triglycerides), 2) attenuated mTOR signaling may result in reduced heart sizes observed in Acc2-mutant mice, and 3) Acc2-mutant hearts displayed normal functional parameters despite a significant decrease in size.

Background During pressure overload-induced hypertrophy, unloading-induced atrophy, and diabetes mellitus, the heart induces ‘fetal’ genes (e.g. myosin heavy chain β; mhcβ). Hypothesis We propose that altered glucose homeostasis within the cardiomyocyte acts as a central mechanism for the regulation of gene expression in response to environmental stresses. The evidence is as follows. Methods and Results Forced glucose uptake both ex vivo and in vivo results in mhc isoform switching. Restricting dietary glucose prevents mhc isoform switching in hearts of both GLUT1-Tg mice and rats subjected to pressure overload-induced hypertrophy. Thus, glucose availability correlates with mhc isoform switching under all conditions investigated. A potential mechanism by which glucose affects gene expression is through O-linked glycosylation of specific transcription factors. Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the flux generating step in UDP-N-acetylglucosamine biosynthesis, the rate determining metabolite in protein glycosylation. Ascending aortic constriction increased intracellular levels of UDP-N-acetylglucosamine, and the expression of gfat2, but not gfat1, in the rat heart. Conclusions Collectively, the results strongly suggest glucose-regulated gene expression in the heart, and the involvement of glucose metabolites in isoform switching of sarcomeric proteins characteristic for the fetal gene program.

It is suggested that insulin resistance and metabolic maladaptation of the heart are causes of contractile dysfunction. We tested the hypothesis whether systemic PPARgamma activation, by changing the metabolic profile in a model of insulin resistance and type 2 diabetes (the ZDF rat) in vivo, improves contractile function of the heart in vitro. Male Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats, at 53-56 days of age, were treated with either GI-262570 (a nonthiazolidinedione PPARgamma agonist; A) or vehicle (V) for 1 wk. Agonist treatment resulted in correction of hyperglycemia and dyslipidemia, as well as in reduced hyperinsulinemia. The accumulation of triacylglycerols in the myocardium, characteristic of the ZDF rat, disappeared with treatment. Cardiac power and rates of glucose oxidation in the isolated working heart were significantly reduced in ZDF-V rats, but both parameters increased to nondiabetic levels with agonist treatment. In ZDF-V hearts, transcript levels of PPARalpha-regulated genes and of myosin heavy chain-beta were upregulated, whereas GLUT4 was downregulated compared with ZL. Agonist treatment of ZDF rats reduced PPARalpha-regulated genes and increased transcripts of GLUT4 and GLUT1. In conclusion, by changing the metabolic profile, reducing myocardial lipid accumulation, and promoting the downregulation of PPARalpha-regulated genes, PPARgamma activation leads to an increased capacity of the myocardium to oxidize glucose and to a tighter coupling of oxidative metabolism and contraction in the setting of insulin resistance and type 2 diabetes.

We investigated whether decreased responsiveness of the heart to physiological increases in fatty acid availability results in lipid accumulation and lipotoxic heart disease. Lean and obese Zucker rats were either fed ad libitum or fasted overnight. Fasting increased plasma nonesterified fatty acid levels in both lean and obese rats, although levels were greatest in obese rats regardless of nutritional status. Despite increased fatty acid availability, the mRNA transcript levels of peroxisome proliferator-activated receptor (PPAR)-α-regulated genes were similar in fed lean and fed obese rat hearts. Fasting increased expression of all PPAR-α -regulated genes in lean Zucker rat hearts, whereas, in obese Zucker rat hearts, muscle carnitine palmitoyltransferase and medium-chain acyl-CoA dehydrogenase were unaltered with fasting. Rates of oleate oxidation were similar for hearts from fed rats. However, fasting increased rates of oleate oxidation only in hearts from lean rats. Dramatic lipid deposition occurred within cardiomyocytes of obese, but not lean, Zucker rats upon fasting. Cardiac output was significantly depressed in hearts isolated from obese rats compared with lean rats, regardless of nutritional status. Fasting increased cardiac output in hearts of lean rats only. Thus, the heart’s inability to increase fatty acid oxidation in proportion to increased fatty acid availability is associated with lipid accumulation and contractile dysfunction of the obese Zucker rat.

Preliminary evidence has suggested that hexokinase in rat heart changes its kinetic properties in response to insulin through translocation to the outer mitochondrial membrane. We reexamined this hypothesis in light of tracer kinetic evidence to the contrary. Our methods were as follows. Working rat hearts were perfused with Krebs-Henseleit buffer containing glucose (5 mmol/l) and sodium oleate (0.4 mmol/l). Dynamic glucose uptake was measured with [2-3H]glucose and with 2-deoxy-2-[18F]fluoroglucose (2-[18F]DG). Hexokinase activity was determined in the cytosolic and mitochondrial fractions. Our results are as follows. Uptake of glucose and uptake of 2-[18F]DG were parallel. Insulin (1 mU/ml) increased glucose uptake threefold but had no effect on 2-[18F]DG uptake. The tracer-to-tracee ratio decreased significantly. The Michaelis-Menten constant of hexokinase for 2-deoxyglucose was up to 10 times higher than for glucose. There was no difference in maximal reaction velocity. Two-thirds of hexokinase was bound to mitochondria. Insulin neither caused translocation nor changed Michaelis-Menten constant or maximal reaction velocity. In conclusion, the insulin-induced changes in the tracer-to-tracee ratio are due to a shift of the rate-limiting step for glucose uptake from transport to phosphorylation by hexokinase. Insulin does not affect the intracellular distribution or the kinetics of hexokinase.

We tested the hypothesis that glycogen levels at the beginning of ischemia affect lactate production during ischemia and postischemic contractile function.

We recently observed that glycogen loading of the heart in vivo improves ischemia tolerance to the same extent as ischemic preconditioning (IP). We have now tested the hypothesis that glycogen loading is beneficial in a protocol where IP per se is not effective. We perfused hearts from fed animals with bicarbonate buffer containing glucose (10 mM). Five min IP with 5 min preconditioning-reperfusion (IP5/5) or 10 min normoxic perfusion (Control, C) were preceded by a 15 min stabilization period and followed by 15 min global. no flow ischemia witl 30 min of reperfusion. Glycogen loaded hearts came from fasted animals, were perfused with buffer containing glucose (10 mM). lactate (10 mM) and insulin (10 mU/ml) and were subjected to the same protocol as IP5/5. Cardiac power and heart rate were assessed continuously. Postischemic recovery was assessed by postischemic power as % of preischemic power. Hearts were freeze-clamped for metabolites. Results Glycogen levels in vivo were higher in the glycogen loaded hearts than in C (140 ± 4.5 vs. 70 ± 6.6 μ mol/g dry). IP5/5 did not improve postischemic recovery compared to C (68.5 ± 3.7% vs. 71.4 ± 3.5% of preischemic power, n = 9 each). However, glycogen loading of this group resulted in a full return of cardiac power post ischemia (101 ± 6.1%, n = 8). Levels of glucose6-phosphate (G-6-P), pyruvate, and lactate were not different in IP5/5 and C and returned to preischemic levels with reperfusion. Glycogen loaded hearts maintained elevated G-6-P and lactate levels with reperfusion. Conclusions Glycogen loading improves ischemia tolerance where IP fails to do so. Thus, glycogen or glycogen breakdown may playa crucial role in myocardial protection.

Abstract 3779 Poster Board III-715 Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect, i.e. the inhibition of anaerobic fermentation by oxygen. We have recently demonstrated in leukemia cells that mitochondrial uncoupling, i.e. the abrogation of ATP synthesis in response to mitochondrial membrane potential (MMP), promotes the Warburg effect, contributes to chemo-resistance and represents a metabolic shift to fatty acid oxidation (FAO). Exposure of leukemic cells to marrow-derived mesenchymal stromal cells (MSC) promotes accumulation of lactate and reduces MMP. Stroma/leukemia co-cultures protect leukemia cells from chemotherapy-induced apoptosis. We found that he decrease in MMP was mediated by mitochondrial uncoupling accompanied by increased expression of mitochondrial uncoupling protein (UCP2) (Cancer Res. 68:5198,2008). We therefore proposed that the Warburg effect may be the result of preferential oxidation of fatty acids in cancer cell mitochondria (Cancer Res.69:2163,2009). Here we demonstrate that leukemia cells uncouple FAO from ATP synthesis, and that pharmacological inhibition of FAO with etomoxir or ranolazine inhibits proliferation and sensitizes leukemia cells – cultured alone or on bone marrow stromal cells – to apoptosis induction by the BH3 mimetic ABT-737 and the MDM-2 antagonist Nutlin 3a. Results suggest that leukemia cells rely, at least in part, on de novo fatty acid synthesis (FAS) to support FAO. Furthermore, treatment with the FAS inhibitor orlistat sensitized leukemia cells to apoptosis induction by ABT-737. Mechanistically, mitochondria derived from etomoxir treated leukemia cells were sensitized to release of cytochrome C and apoptosis-inducing-factor (AIF) upon treatment with ABT-737. Etomoxir (EX) facilitated the formation of Bak oligomers after treatment with ABT-737 suggesting that FAO regulates the activity of Bak-dependent mitochondrial permeability transition. Lastly, we present evidence that EX, in combination with liposomal ABT-737 or cytosine arabinoside (AraC), provides significant therapeutic benefit in a murine model of human leukemia (luciferase/GFP marked MOLT13 cells) as evidenced by reduced in vivo growth kinetics (BLI) and prolonged median survival (ABT-737 vs. EX+ABT-737, p= Disclosures: No relevant conflicts of interest to declare.

More than half a century ago, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect, i.e. the inhibition of anaerobic fermentation by oxygen. However, permanent and transmissible defects in the respiratory capacity of cancer cells that could broadly support Warburg’s hypothesis have not been identified. Notably, we have recently demonstrated that mitochondrial uncoupling – the abrogation of ATP synthesis in response to mitochondrial membrane potential – can promote the Warburg effect in leukemia cells, and may contribute to chemoresistance, via in part, the expression of the highly conserved thermogenic protein UCP2. Here we demonstrate that mitochondrial uncoupling in leukemia cells is supported by the oxidation of fatty acids, and provide evidence that etomoxir (EX) or ranolazine (RAN), pharmacological inhibitors of fatty acid oxidation utilized for the treatment of heart failure, sensitize leukemia cell lines and primary samples to apoptosis induced by the BH3 mimetic ABT-737 and the MDM-2 inhibitor Nutlin 3a. EX and RAN, but not 2-deoxyglucose (2DG), markedly inhibited oxygen consumption in leukemia cell lines and primary samples. In contrast, 2DG, but not EX or RAN, potently depleted ATP levels, suggesting that the oxidation of fatty acids is uncoupled from ATP synthesis – and conversely, the synthesis of ATP primarily depends on the non-oxidative, glycolytic metabolism of glucose. It is noteworthy that albeit EX and RAN inhibited the growth of p53-wild type and -mutant leukemia cells, neither agent induced marked apoptosis. Nonetheless, a pronounced induction of the proapoptotic BH3-only proteins Noxa and Bim was observed regardless of p53 status, suggesting a potential mechanism by which these agents enhance apoptosis by ABT-737. In addition, EX and RAN abrogated the chemoprotective effects of bone marrow-derived stromal feeder layers, and EX provided a survival advantage in combination with ABT-737 in a murine model of leukemia suggesting that inhibition of mitochondrial fatty acid oxidation represents a novel therapeutic strategy for the treatment of leukemia. Intriguingly, C13-NMR analysis, H3–oleate oxidation, and oxymetry experiments revealed that leukemia cells do not oxidize exogenous fatty acids, but rather depend on glucose and glutamine-supported de novo synthesis of fatty acids to maintain mitochondrial function. Accordingly, depletion of glutamine, inhibition of fatty acid synthesis, or reduced pentose phosphate shunt-derived NADPH significantly decreased oxygen consumption and potentiated ABT-737 induced apoptosis. The above results support the hypothesis that glutamine and glucose-dependent anaplerotic reactions sustain fatty acid metabolism and survival of leukemia cells. Our results suggest that the dependence of cancer cells on glycolysis for energy generation indicates a metabolic shift to the ATP-uncoupled oxidation of non-glucose substrates, and most importantly, support the clinical investigation of fatty acid oxidation and synthesis inhibitors as a therapeutic strategy in hematological malignancies.