UCP1-dependent brown adipose activation accelerates cardiac metabolic remodeling and reduces initial hypertrophic and fibrotic responses to pathological stress.

Brown adipose tissue (BAT) is correlated to cardiovascular health in rodents and humans, but the physiological role of BAT in the initial cardiac remodeling at the onset of stress is unknown. Activation of BAT via 48 h cold (16°C) in mice following transverse aortic constriction (TAC) reduced cardiac gene expression for LCFA uptake and oxidation in male mice and accelerated the onset of cardiac metabolic remodeling, with an early isoform shift of carnitine palmitoyltransferase 1 (CPT1) toward increased CPT1a, reduced entry of long chain fatty acid (LCFA) into oxidative metabolism (0.59 ± 0.02 vs. 0.72 ± 0.02 in RT TAC hearts, p < .05) and increased carbohydrate oxidation with altered glucose transporter content. BAT activation with TAC reduced early hypertrophic expression of ß-MHC by 61% versus RT-TAC and reduced pro-fibrotic TGF-ß1 and COL3α1 expression. While cardiac natriuretic peptide expression was yet to increase at only 3 days TAC, Nppa and Nppb expression were elevated in Cold TAC versus RT TAC hearts 2.7- and 2.4-fold, respectively. Eliminating BAT thermogenic activation with UCP1 KO mice eliminated differences between Cold TAC and RT TAC hearts, confirming effects of BAT activation rather than autonomous cardiac responses to cold. Female responses to BAT activation were blunted, with limited UCP1 changes with cold, partly due to already activated BAT in females at RT compared to thermoneutrality. These data reveal a previously unknown physiological mechanism of UCP1-dependent BAT activation in attenuating early cardiac hypertrophic and profibrotic signaling and accelerating remodeled metabolic activity in the heart at the onset of cardiac stress.

Western Diet Causes Heart Failure With Reduced Ejection Fraction and Metabolic Shifts After Diastolic Dysfunction and Novel Cardiac Lipid Derangements.

Western diet (WD) impairs glucose tolerance and cardiac lipid dynamics, preceding heart failure with reduced ejection fraction (HFrEF) in mice. Unlike diabetic db/db mice with high cardiac triglyceride (TG) and rapid TG turnover, WD mice had high TG but slowed turnover, reducing lipolytic PPAR⍺ activation. WD deranged cardiac TG dynamics by imbalancing synthesis and lipolysis, with low cardiac TG lipase (ATGL), low ATGL co-activator, and high ATGL inhibitory peptide. By 24 weeks of WD, hearts shifted from diastolic dysfunction to diastolic dysfunction with HFrEF with decreases in GLUT4 and exogenous glucose oxidation and elevated ß-hydroxybutyrate dehydrogenase 1 without increasing ketone oxidation.

Short-Chain Fatty Acids Outpace Ketone Oxidation in the Failing Heart.

BACKGROUND: The failing heart is energy starved with impaired oxidation of long-chain fatty acids (LCFAs) at the level of reduced CPT1 (carnitine palmitoyltransferase 1) activity at the outer mitochondrial membrane. Recent work shows elevated ketone oxidation in failing hearts as an alternate carbon source for oxidative ATP generation. We hypothesized that another short-chain carbon source, short-chain fatty acids (SCFAs) that bypass carnitine palmitoyltransferase 1, could similarly support energy production in failing hearts. METHODS: Cardiac hypertrophy and dysfunction were induced in rats by transverse-aortic constriction (TAC). Fourteen weeks after TAC or sham operation, isolated hearts were perfused with either the 4 carbon, 13C-labeled ketone (D3-hydroxybutyrate) or the 4 carbon, 13C-labeled SCFA butyrate in the presence of glucose and the LCFA palmitate. Oxidation of ketone and SCFA was compared by in vitro 13C nuclear magnetic resonance spectroscopy, as was the capacity for short-chain carbon sources to compensate for impaired LCFA oxidation in the hypertrophic heart. Adaptive changes in enzyme expression and content for the distinct pathways of ketone and SCFA oxidation were examined in both failing rat and human hearts. RESULTS: TAC produced pathological hypertrophy and increased the fractional contributions of ketone to acetyl coenzyme-A production in the tricarboxylic acid cycle (0.60±0.02 sham ketone versus 0.70±0.02 TAC ketone; P<0.05). However, butyrate oxidation in failing hearts was 15% greater (0.803±0.020 TAC SCFA) than ketone oxidation. SCFA was also more readily oxidized than ketone in sham hearts by 15% (0.693±0.020 sham SCFA). Despite greater SFCA oxidation, TAC did not change short-chain acyl coenzyme-A dehydrogenase content. However, failing hearts of humans and the rat model both contain significant increases in acyl coenzyme-A synthetase medium-chain 3 enzyme gene expression and protein content. The increased oxidation of SCFA and ketones occurred at the expense of LCFA oxidation, with LCFA contributing less to acetyl coenzyme-A production in failing hearts perfused with SCFA (0.190±0.012 TAC SCFA versus 0.3163±0.0360 TAC ketone). CONCLUSIONS: SCFAs are more readily oxidized than ketones in failing hearts, despite both bypassing reduced CPT1 activity and represent an unexplored carbon source for energy production in failing hearts.

Preservation of Acyl Coenzyme A Attenuates Pathological and Metabolic Cardiac Remodeling Through Selective Lipid Trafficking.

BACKGROUND: Metabolic remodeling in heart failure contributes to dysfunctional lipid trafficking and lipotoxicity. Acyl coenzyme A synthetase-1 (ACSL1) facilitates long-chain fatty acid (LCFA) uptake and activation with coenzyme A (CoA), mediating the fate of LCFA. The authors tested whether cardiac ACSL1 overexpression aids LCFA oxidation and reduces lipotoxicity under pathological stress of transverse aortic constriction (TAC). METHODS: Mice with cardiac restricted ACSL1 overexpression (MHC-ACSL1) underwent TAC or sham surgery followed by serial in vivo echocardiography for 14 weeks. At the decompensated stage of hypertrophy, isolated hearts were perfused with 13C LCFA during dynamic-mode 13C nuclear magnetic resonance followed by in vitro nuclear magnetic resonance and mass spectrometry analysis to assess intramyocardial lipid trafficking. In parallel, acyl CoA was measured in tissue obtained from heart failure patients pre- and postleft ventricular device implantation plus matched controls. RESULTS: TAC-induced cardiac hypertrophy and dysfunction was mitigated in MHC-ACSL1 hearts compared with nontransgenic hearts. At 14 weeks, TAC increased heart weight to tibia length by 46% in nontransgenic mice, but only 26% in MHC-ACSL1 mice, whereas ACSL1 mice retained greater ejection fraction (ACSL1 TAC: 65.8±7.5%; nontransgenic TAC: 45.9±7.3) and improvement in diastolic E/E'. Functional improvements were mediated by ACSL1 changes to cardiac LCFA trafficking. ACSL1 accelerated LCFA uptake, preventing C16 acyl CoA loss post-TAC. Long-chain acyl CoA was similarly reduced in human failing myocardium and restored to control levels by mechanical unloading. ACSL1 trafficked LCFA into ceramides without normalizing the reduced triglyceride storage in TAC. ACSL1 prevented de novo synthesis of cardiotoxic C16- and C24-, and C24:1 ceramides and increased potentially cardioprotective C20- and C22-ceramides post-TAC. ACLS1 overexpression activated AMP activated protein kinase at baseline, but during TAC, prevented the reduced LCFA oxidation in hypertrophic hearts and normalized energy state (phosphocreatine:ATP) and consequently, AMP activated protein kinase activation. CONCLUSIONS: This is the first demonstration of reduced acyl CoA in failing hearts of humans and mice, and suggests possible mechanisms for maintaining mitochondrial oxidative energy metabolism by restoring long-chain acyl CoA through ASCL1 activation and mechanical unloading. By mitigating cardiac lipotoxicity, via redirected LCFA trafficking to ceramides, and restoring acyl CoA, ACSL1 delayed progressive cardiac remodeling and failure.

Enhanced Redox State and Efficiency of Glucose Oxidation With miR Based Suppression of Maladaptive NADPH-Dependent Malic Enzyme 1 Expression in Hypertrophied Hearts.

RATIONALE: Metabolic remodeling in hypertrophic hearts includes inefficient glucose oxidation via increased anaplerosis fueled by pyruvate carboxylation. Pyruvate carboxylation to malate through elevated ME1 (malic enzyme 1) consumes NADPH necessary for reduction of glutathione and maintenance of intracellular redox state. OBJECTIVE: To elucidate upregulated ME1 as a potential maladaptive mechanism for inefficient glucose oxidation and compromised redox state in hypertrophied hearts. METHODS AND RESULTS: ME1 expression was selectively inhibited, in vivo, via non-native miR-ME1 (miRNA specific to ME1) in pressure-overloaded rat hearts. Rats subjected to transverse aortic constriction (TAC) or Sham surgery received either miR-ME1 or PBS. Effects of ME1 suppression on anaplerosis and reduced glutathione (GSH) content were studied in isolated hearts supplied 13C-enriched substrate: palmitate, glucose, and lactate. Human myocardium collected from failing and nonfailing hearts during surgery enabled RT-qPCR confirmation of elevated ME1 gene expression in clinical heart failure versus nonfailing human hearts (P<0.04). TAC induced elevated ME1 content, but ME1 was lowered in hearts infused with miR-ME1 versus PBS. Although Sham miR-ME1 hearts showed no further reduction of inherently low anaplerosis in normal heart, miR-ME1 reduced anaplerosis in TAC to baseline: TAC miR-ME1=0.034±0.004; TAC PBS=0.081±0.005 (P<0.01). Countering elevated anaplerosis in TAC shifted pyruvate toward oxidation in the tricarboxylic acid cycle. Importantly, via the link to NADPH consumption by pyruvate carboxylation, ME1 suppression in TAC restored GSH content, reduced lactate production, and ultimately improved contractility. CONCLUSIONS: A maladaptive increase in anaplerosis via ME1 in TAC is associated with reduced GSH content. Suppressing increased ME1 expression in hypertrophied rat hearts, which is also elevated in failing human hearts, reduced pyruvate carboxylation thereby normalizing anaplerosis, restoring GSH content, and reducing lactate accumulation. Reducing ME1 induced favorable metabolic shifts for carbohydrate oxidation, improving intracellular redox state and enhanced cardiac performance in pathological hypertrophy.

Triacylglycerol turnover in the failing heart.

No longer regarded as physiologically inert the endogenous triacylglyceride (TAG) pool within the cardiomyocyte is now recognized to play a dynamic role in metabolic regulation. Beyond static measures of content, the relative rates of interconversion among acyl intermediates are more closely linked to dynamic processes of physiological function in normal and diseased hearts, with the potential for both adaptive and maladaptive contributions. Indeed, multiple inefficiencies in cardiac metabolism have been identified in the decompensated, hypertrophied and failing heart. Among the intracellular responses to physiological, metabolic and pathological stresses, TAG plays a central role in the balance of lipid handling and signaling mechanisms. TAG dynamics are profoundly altered from normal in both diabetic and pathologically stressed hearts. More than just expansion or contraction of the stored lipid pool, the turnover rates of TAG are sensitive to and compete against other enzymatic pathways, anabolic and catabolic, for reactive acyl-CoA units. The rates of TAG synthesis and lipolysis thusly affect multiple components of cardiomyocyte function, including energy metabolism, cell signaling, and enzyme activation, as well as the regulation of gene expression in both normal and diseased states. This review examines the multiple etiologies and metabolic consequences of the failing heart and the central role of lipid storage dynamics in the pathogenic process. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Metabolic efficiency promotes protection from pressure overload in hearts expressing slow skeletal troponin I.

BACKGROUND: The failing heart displays increased glycolytic flux that is not matched by a commensurate increase in glucose oxidation. This mismatch induces increased anaplerotic flux and inefficient glucose metabolism. We previously found adult transgenic mouse hearts expressing the fetal troponin I isoform, (ssTnI) to be protected from ischemia by increased glycolysis. In this study, we investigated the metabolic response of adult mouse hearts expressing ssTnI to chronic pressure overload. METHODS AND RESULTS: At 2 to 3 months of age, ssTnI mice or their nontransgenic littermates underwent aortic constriction (TAC). TAC induced a 25% increase in nontransgenic heart size but only a 7% increase in ssTnI hearts (P<0.05). Nontransgenic TAC developed diastolic dysfunction (65% increase in E/A ratio), whereas the E/A ratio actually decreased in ssTnI TAC. Isolated perfused hearts from nontransgenic TAC mice showed reduced cardiac function and reduced creatine phosphate:ATP (16% reduction), but ssTnI TAC hearts maintained cardiac function and energy charge. Contrasting nontransgenic TAC, ssTnI TAC significantly increased glucose oxidation at the expense of palmitate oxidation, preventing the increase in anaplerosis observed in nontransgenic TAC hearts. Elevated glucose oxidation was mediated by a reduction in pyruvate dehydrogenase kinase 4 expression, enabling pyruvate dehydrogenase to compete against anaplerotic enzymes for pyruvate carboxylation. CONCLUSIONS: Expression of a single fetal myofilament protein into adulthood in the ssTnI-transgenic mouse heart induced downregulation of the gene expression response for pyruvate dehydrogenase kinase to pressure overload. The consequence of elevated pyruvate oxidation in ssTnI during TAC reduced anaplerotic flux, ameliorating inefficiencies in glucose oxidation, with energetic and functional protection against cardiac decompensation.

Dietary fat supply to failing hearts determines dynamic lipid signaling for nuclear receptor activation and oxidation of stored triglyceride.

BACKGROUND: Intramyocardial triglyceride (TG) turnover is reduced in pressure-overloaded, failing hearts, limiting the availability of this rich source of long-chain fatty acids for mitochondrial ß-oxidation and nuclear receptor activation. This study explored 2 major dietary fats, palmitate and oleate, in supporting endogenous TG dynamics and peroxisome proliferator-activated receptor-α activation in sham-operated (SHAM) and hypertrophied (transverse aortic constriction [TAC]) rat hearts. METHODS AND RESULTS: Isolated SHAM and TAC hearts were provided media containing carbohydrate with either (13)C-palmitate or (13)C-oleate for dynamic (13)C nuclear magnetic resonance spectroscopy and end point liquid chromatography/mass spectrometry of TG dynamics. With palmitate, TAC hearts contained 48% less TG versus SHAM (P=0.0003), whereas oleate maintained elevated TG in TAC, similar to SHAM. TG turnover in TAC was greatly reduced with palmitate (TAC, 46.7±12.2 nmol/g dry weight per min; SHAM, 84.3±4.9; P=0.0212), as was ß-oxidation of TG. Oleate elevated TG turnover in both TAC (140.4±11.2) and SHAM (143.9±15.6), restoring TG oxidation in TAC. Peroxisome proliferator-activated receptor-α target gene transcripts were reduced by 70% in TAC with palmitate, whereas oleate induced normal transcript levels. Additionally, mRNA levels for peroxisome proliferator-activated receptor-γ-coactivator-1α and peroxisome proliferator-activated receptor-γ-coactivator-1ß in TAC hearts were maintained by oleate. With these metabolic effects, oleate also supported a 25% improvement in contractility over palmitate with TAC (P=0.0202). CONCLUSIONS: The findings link reduced intracellular lipid storage dynamics to impaired peroxisome proliferator-activated receptor-α signaling and contractility in diseased hearts, consistent with a rate-dependent lipolytic activation of peroxisome proliferator-activated receptor-α. In decompensated hearts, oleate may serve as a beneficial energy substrate versus palmitate by upregulating TG dynamics and nuclear receptor signaling.

Matrix revisited: mechanisms linking energy substrate metabolism to the function of the heart.

Metabolic signaling mechanisms are increasingly recognized to mediate the cellular response to alterations in workload demand, as a consequence of physiological and pathophysiological challenges. Thus, an understanding of the metabolic mechanisms coordinating activity in the cytosol with the energy-providing pathways in the mitochondrial matrix becomes critical for deepening our insights into the pathogenic changes that occur in the stressed cardiomyocyte. Processes that exchange both metabolic intermediates and cations between the cytosol and mitochondria enable transduction of dynamic changes in contractile state to the mitochondrial compartment of the cell. Disruption of such metabolic transduction pathways has severe consequences for the energetic support of contractile function in the heart and is implicated in the pathogenesis of heart failure. Deficiencies in metabolic reserve and impaired metabolic transduction in the cardiomyocyte can result from inherent deficiencies in metabolic phenotype or maladaptive changes in metabolic enzyme expression and regulation in the response to pathogenic stress. This review examines both current and emerging concepts of the functional linkage between the cytosol and the mitochondrial matrix with a specific focus on metabolic reserve and energetic efficiency. These principles of exchange and transport mechanisms across the mitochondrial membrane are reviewed for the failing heart from the perspectives of chronic pressure overload and diabetes mellitus.

Multiphasic triacylglycerol dynamics in the intact heart during acute in vivo overexpression of CD36.

Cardiac triacylglycerol (TAG) stores buffer the intracellular availability of long chain fatty acid (LCFA) that act as nuclear receptor ligands, substrate for lipotoxic derivatives, and high energy-yield fuel. The kinetic characteristics of TAG turnover and homeostatic mechanisms linking uptake and storage dynamics in hearts have until now remained elusive. This work examines TAG pool dynamics in the intact beating heart, under normal conditions and in response to acute gene expression-induced changes in CD36. Dynamic mode (13)C NMR elucidated multiple kinetic processes in (13)C-palmitate incorporation into TAG: an initial, saturable exponential component and a slower linear rate. Although previous work indicates the linear component to reflect TAG turnover, we hypothesized the saturable exponential to reflect transport of LCFA across the sarcolemma. Thus, we overexpressed the LCFA transporter CD36 through cardiac-specific adenoviral infection in vivo. Within 72 h, CD36 expression was increased 40% in intact hearts, accelerating the exponential phase relative to PBS-infused hearts. TAG turnover also increased with elevations in adipose triglyceride lipase (ATGL) and a modest increase in diacylglycerol acyltransferase 1 (DGAT1), without a significant expansion of the intracellular lipid pools. The results demonstrate a dynamic system of reciprocal gene regulation that couples saturable LCFA uptake across the sarcolemma to TAG synthesis/lipolysis rates.

Fatty acid (FFA) transport in cardiomyocytes revealed by imaging unbound FFA is mediated by an FFA pump modulated by the CD36 protein.

Free fatty acid (FFA) transport across the cardiomyocyte plasma membrane is essential to proper cardiac function, but the role of membrane proteins and FFA metabolism in FFA transport remains unclear. Metabolism is thought to maintain intracellular FFA at low levels, providing the driving force for FFA transport, but intracellular FFA levels have not been measured directly. We report the first measurements of the intracellular unbound FFA concentrations (FFA(i)) in cardiomyocytes. The fluorescent indicator of FFA, ADIFAB (acrylodan-labeled rat intestinal fatty acid-binding protein), was microinjected into isolated cardiomyocytes from wild type (WT) and FAT/CD36 null C57B1/6 mice. Quantitative imaging of ADIFAB fluorescence revealed the time courses of FFA influx and efflux. For WT mice, rate constants for efflux (∼0.02 s(-1)) were twice influx, and steady state FFA(i) were more than 3-fold larger than extracellular unbound FFA (FFA(o)). The concentration gradient and the initial rate of FFA influx saturated with increasing FFA(o). Similar characteristics were observed for oleate, palmitate, and arachidonate. FAT/CD36 null cells revealed similar characteristics, except that efflux was 2-3-fold slower than WT cells. Rate constants determined with intracellular ADIFAB were confirmed by measurements of intracellular pH. FFA uptake by suspensions of cardiomyocytes determined by monitoring FFA(o) using extracellular ADIFAB confirmed the influx rate constants determined from FFA(i) measurements and demonstrated that rates of FFA transport and etomoxir-sensitive metabolism are regulated independently. We conclude that FFA influx in cardiac myocytes is mediated by a membrane pump whose transport rate constants may be modulated by FAT/CD36.

Flip-flop is the rate-limiting step for transport of free fatty acids across lipid vesicle membranes.

The mechanism of transport of free fatty acids (FFA) across lipid bilayer membranes remains a subject of debate. The debate is whether the rate-limiting step for transport is flip-flop across the membrane or dissociation into the aqueous phase. Recently, a new method for assessing dissociation was described in which fluorescein phosphatidylethanolamine (FPE) introduced into the outer leaflet of lipid vesicles was used to monitor FFA dissociation. Transport of FFA into vesicles containing both FPE in the outer leaflet and pyranine trapped in the inside aqueous phase revealed identical rate constants for quenching of FPE and pyranine fluorescence. Because no difference was observed in the time for FFA binding to the outer surface and flip-flop across the bilayer, it was concluded that dissociation was slower than flip-flop. Here, we used FPE and BSA to assess dissociation of oleate from lipid vesicles. In separate pyranine- or ADIFAB-containing vesicles, we assessed flip-flop. We found that the FPE and BSA transfer methods yielded equivalent rate constants for dissociation, which were 3-10-fold faster than that of flip-flop. We found that in vesicles containing both FPE and pyranine, pyranine fluorescence cannot be separated from FPE fluorescence. The identical rate constants for FPE and pyranine observed with vesicles containing both fluorophores reflected the dominance (20-fold) of FPE fluorescence at pyranine excitation and emission wavelengths. Because the dissociation rate constants are 3-10 times faster than the rate constants for flip-flop, flip-flop must be the rate-limiting step for the transport of FFA across lipid vesicles.

What are the biochemical mechanisms responsible for enhanced fatty acid utilization by perfused hearts from type 2 diabetic db/db mice?

INTRODUCTION: It is generally accepted that diabetic hearts have an altered metabolic phenotype, with enhanced fatty acid (FA) utilization. The over-utilization of FA by diabetic hearts can have deleterious functional consequences, contributing to a distinct diabetic cardiomyopathy. The objective of this review will be to examine which biochemical mechanisms are responsible for enhanced FA utilization by diabetic hearts. METHODOLOGY AND RESULTS: Studies were performed with db/db mice, a monogenic model of type 2 diabetes with extreme obesity and hyperglycemia. Perfused db/db hearts exhibit enhanced FA oxidation and esterification. Hypothesis 1: Cardiac FA uptake is enhanced in db/db hearts. The plasma membrane content of two FA transporters, fatty acid translocase/CD36 (FAT/CD36) and plasma membrane fatty acid binding protein (FABPpm), was increased in db/db hearts, consistent with hypothesis 1. Hypothesis 2: Cardiac FA oxidation is enhanced in db/db hearts due to mitochondrial alterations. However, the activity of carnitine palmitoyl transferase-1 (CPT-1) and sensitivity to inhibition by malonyl CoA was unchanged in mitochondria from db/db hearts. Furthermore, total malonyl CoA content was increased, not decreased as predicted for elevated FA oxidation. Finally, the content of uncoupling protein-3 was unchanged in db/db heart mitochondria. CONCLUSION: Increased plasma membrane content of FA transporters (FAT/CD36 and FABPpm) will increase FA uptake into db/db cardiomyocytes and thus increase FA utilization. On the other hand, mitochondrial mechanisms do not contribute to elevated rates of FA oxidation in db/db hearts.

Fatty acid metabolism is enhanced in type 2 diabetic hearts.

The metabolic phenotype of hearts has been investigated using rodent models of type 2 diabetes which exhibit obesity and insulin resistance: db/db and ob/ob mice, and Zucker fatty and ZDF rats. In general, cardiac fatty acid (FA) utilization is enhanced in type 2 diabetic hearts, with increased rates of FA oxidation (db/db, ob/ob and ZDF models) and increased FA esterification into cellular triacylglycerols (db/db hearts). Hearts from db/db and ob/ob mice and ZDF rat hearts all have elevated levels of myocardial triacylglycerols, consistent with enhanced FA utilization. A number of mechanisms may be responsible for enhanced FA utilization in type 2 diabetic hearts: (i) increased FA uptake into cardiac myocytes and into mitochondria; (ii) altered mitochondrial function, with up-regulation of uncoupling proteins; and (iii) stimulation of peroxisome proliferator-activated receptor-alpha. Enhanced cardiac FA utilization in rodent type 2 diabetic models is associated with reduced cardiac contractile function, perhaps as a consequence of lipotoxicity and/or reduced cardiac efficiency. Similar results have been obtained with human type 2 diabetic hearts, suggesting that pharmacological interventions that can reduce cardiac FA utilization may have beneficial effects on contractile function.

Metabolic effects of insulin on cardiomyocytes from control and diabetic db/db mouse hearts.

Diabetic db/db mice exhibit profound insulin resistance in vivo, but the specific degree of cardiac insensitivity to insulin has not been assessed. Therefore, the effect of insulin on cardiomyocytes from db/db hearts was assessed by measuring two metabolic responses (deoxyglucose uptake and fatty acid oxidation) and the phosphorylation of two enzymes in the insulin-signaling cascade [Akt and AMP-activated protein kinase (AMPK)]. Maximal insulin-stimulated deoxyglucose transport was reduced to 58 and 40% of control in cardiomyocytes from db/db mice at two ages (6 and 12 wk). Insulin-stimulated deoxyglucose uptake was also reduced in myocytes from transgenic db/db mice overexpressing the insulin-sensitive glucose transporter (db/db-hGLUT4). Treatment of db/db mice for 1 wk with an insulin-sensitizing peroxisome proliferator-activated receptor-gamma agonist (COOH) completely normalized insulin-stimulated deoxyglucose uptake. Insulin had no direct effect on palmitate oxidation by either control or db/db cardiomyocytes, but the combination of insulin and glucose reduced palmitate oxidation, likely an indirect effect secondary to increased glucose uptake. Insulin had no effect on AMPK phosphorylation from either control or db/db cardiomyocytes. Insulin increased the phosphorylation of Akt in all cardiomyocyte preparations (control, db/db, COOH-treated db/db) to the same extent. Thus insulin has selective metabolic actions in mouse cardiomyocytes; deoxyglucose uptake and Akt phosphorylation are increased, but fatty acid oxidation and AMPK phosphorylation are unchanged. Insulin resistance in db/db cardiomyocytes is manifested by reduced insulin-stimulated deoxyglucose uptake.

Treatment of type 2 diabetic db/db mice with a novel PPARgamma agonist improves cardiac metabolism but not contractile function.

Hearts from insulin-resistant type 2 diabetic db/db mice exhibit features of a diabetic cardiomyopathy with altered metabolism of exogenous substrates and reduced contractile performance. Therefore, the effect of chronic oral administration of 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH), a novel ligand for peroxisome proliferator-activated receptor-gamma that produces insulin sensitization, to db/db mice (30 mg/kg for 6 wk) on cardiac function was assessed. COOH treatment reduced blood glucose from 27 mM in untreated db/db mice to a normal level of 10 mM. Insulin-stimulated glucose uptake was enhanced in cardiomyocytes from COOH-treated db/db hearts. Working perfused hearts from COOH-treated db/db mice demonstrated metabolic changes with enhanced glucose oxidation and decreased palmitate oxidation. However, COOH treatment did not improve contractile performance assessed with ex vivo perfused hearts and in vivo by echocardiography. The reduced outward K+ currents in diabetic cardiomyocytes were still attenuated after COOH. Metabolic changes in COOH-treated db/db hearts are most likely indirect, secondary to changes in supply of exogenous substrates in vivo and insulin sensitization.

Chylomicron and palmitate metabolism by perfused hearts from diabetic mice.

Hydrolysis of triacylglycerols (TG) in circulating chylomicrons by endothelium-bound lipoprotein lipase (LPL) provides a source of fatty acids (FA) for cardiac metabolism. The effect of diabetes on the metabolism of chylomicrons by perfused mouse hearts was investigated with db/db (type 2) and streptozotocin (STZ)-treated (type 1) diabetic mice. Endothelium-bound heparin-releasable LPL activity was unchanged in both type 1 and type 2 diabetic hearts. The metabolism of LPL-derived FA was examined by perfusing hearts with chylomicrons containing radiolabeled TG and by measuring (3)H(2)O accumulation in the perfusate (oxidation) and incorporation of radioactivity into tissue TG (esterification). Rates of LPL-derived FA oxidation and esterification were increased 2.3-fold and 1.7-fold in db/db hearts. Similarly, LPL-derived FA oxidation and esterification were increased 3.4-fold and 2.5-fold, respectively, in perfused hearts from STZ-treated mice. The oxidation and esterification of [(3)H]palmitate complexed to albumin were also increased in type 1 and type 2 diabetic hearts. Therefore, diabetes may not influence the supply of LPL-derived FA, but total FA utilization (oxidation and esterification) was enhanced.