Mitochondrial hyperfusion induces metabolic remodeling in lung endothelial cells by modifying the activities of electron transport chain complexes I and III.

OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease with vascular remodeling as a critical structural alteration. We have previously shown that metabolic reprogramming is an early initiating mechanism in animal models of PH. This metabolic dysregulation has been linked to remodeling the mitochondrial network to favor fission. However, whether the mitochondrial fission/fusion balance underlies the metabolic reprogramming found early in PH development is unknown. METHODS: Utilizing a rat early model of PH, in conjunction with cultured pulmonary endothelial cells (PECs), we utilized metabolic flux assays, Seahorse Bioassays, measurements of electron transport chain (ETC) complex activity, fluorescent microscopy, and molecular approaches to investigate the link between the disruption of mitochondrial dynamics and the early metabolic changes that occur in PH. RESULTS: We observed increased fusion mediators, including Mfn1, Mfn2, and Opa1, and unchanged fission mediators, including Drp1 and Fis1, in a two-week monocrotaline-induced PH animal model (early-stage PH). We were able to establish a connection between increases in fusion mediator Mfn1 and metabolic reprogramming. Using an adenoviral expression system to enhance Mfn1 levels in pulmonary endothelial cells and utilizing 13C-glucose labeled substrate, we found increased production of 13C lactate and decreased TCA cycle metabolites, revealing a Warburg phenotype. The use of a 13C5-glutamine substrate showed evidence that hyperfusion also induces oxidative carboxylation. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels secondary to the disruption of cellular bioenergetics and higher levels of mitochondrial reactive oxygen species (mt-ROS). The elevation in mt-ROS correlated with attenuated ETC complexes I and III activities. Utilizing a mitochondrial-targeted antioxidant to suppress mt-ROS, limited HIF-1α protein levels, which reduced cellular glycolysis and reestablished mitochondrial membrane potential. CONCLUSIONS: Our data connects mitochondrial fusion-mediated mt-ROS to the Warburg phenotype in early-stage PH development.

Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing 13C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

Mitochondrial pyruvate carriers are required for myocardial stress adaptation.

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1-/-) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1-/- hearts. Although concurrent short-term KDs did not rescue cMPC1-/- hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function.

Comparison of the Effects of High-Fat Diet on Energy Flux in Mice Using Two Multiplexed Metabolic Phenotyping Systems.

OBJECTIVE: Multiplexed metabolic phenotyping systems are available from multiple commercial vendors, and each system includes unique design features. Although expert opinion supports strengths and weaknesses of each design, empirical data from carefully controlled studies to test the biological impact of design differences are lacking. METHODS: Wild-type C57BL/6J mice of both sexes underwent phenotyping in OxyMax (Columbus Instruments International) and Promethion (Sable Systems International) systems located within the same room of a newly constructed animal research facility in a crossover design study. Phenotypes were examined under chow (2920×)-fed conditions and again after 4 weeks of 60% high-fat diet (D12492) feeding. RESULTS: Food intake, physical activity, and respiratory gas exchange data significantly diverged between systems, depending upon sex of animals and diet supplied. Estimates of energy expenditure based on gas exchange in both systems accounted for a fraction of consumed calories that was greater in males than females. CONCLUSIONS: Design differences quantitatively impact the assessment of metabolic end points and thus the qualitative interpretation of various interventions. Importantly, current multiplexed systems remain blind to multiple additional end points, including digestive efficiency and selected forms of energy flux (nitrogenous, anaerobic, etc.), that account for a physiologically and/or pathophysiologically significant fraction of total energy flux.

Deletion of GLUT1 in mouse lens epithelium leads to cataract formation.

The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1loxP/loxP mice to generate Slc2a1loxP/loxP;GFAP-Cre+/0 (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.

Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induce Post-Translational Modifications of AKAP121, DRP1, and OPA1 That Promote Mitochondrial Fission.

RATIONALE: Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. OBJECTIVE: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. METHODS AND RESULTS: Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. CONCLUSIONS: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy.

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging.

Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity.

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart.

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Antioxidant treatment normalizes mitochondrial energetics and myocardial insulin sensitivity independently of changes in systemic metabolic homeostasis in a mouse model of the metabolic syndrome.

Cardiac dysfunction in obesity is associated with mitochondrial dysfunction, oxidative stress and altered insulin sensitivity. Whether oxidative stress directly contributes to myocardial insulin resistance remains to be determined. This study tested the hypothesis that ROS scavenging will improve mitochondrial function and insulin sensitivity in the hearts of rodent models with varying degrees of insulin resistance and hyperglycemia. The catalytic antioxidant MnTBAP was administered to the uncoupling protein-diphtheria toxin A (UCP-DTA) mouse model of insulin resistance (IR) and obesity, at early and late time points in the evolution of IR, and to db/db mice with severe obesity and type-two diabetes. Mitochondrial function was measured in saponin-permeabilized cardiac fibers. Aconitase activity and hydrogen peroxide emission were measured in isolated mitochondria. Insulin-stimulated glucose oxidation, glycolysis and fatty acid oxidation rates were measured in isolated working hearts, and 2-deoxyglucose uptake was measured in isolated cardiomyocytes. Four weeks of MnTBAP attenuated glucose intolerance in 13-week-old UCP-DTA mice but was without effect in 24-week-old UCP-DTA mice and in db/db mice. Despite the absence of improvement in the systemic metabolic milieu, MnTBAP reversed cardiac mitochondrial oxidative stress and improved mitochondrial bioenergetics by increasing ATP generation and reducing mitochondrial uncoupling in all models. MnTBAP also improved myocardial insulin mediated glucose metabolism in 13 and 24-week-old UCP-DTA mice. Pharmacological ROS scavenging improves myocardial energy metabolism and insulin responsiveness in obesity and type 2 diabetes via direct effects that might be independent of changes in systemic metabolism.

GLUT1 reductions exacerbate Alzheimer's disease vasculo-neuronal dysfunction and degeneration.

The glucose transporter GLUT1 at the blood-brain barrier (BBB) mediates glucose transport into the brain. Alzheimer's disease is characterized by early reductions in glucose transport associated with diminished GLUT1 expression at the BBB. Whether GLUT1 reduction influences disease pathogenesis remains, however, elusive. Here we show that GLUT1 deficiency in mice overexpressing amyloid ß-peptide (Aß) precursor protein leads to early cerebral microvascular degeneration, blood flow reductions and dysregulation and BBB breakdown, and to accelerated amyloid ß-peptide (Aß) pathology, reduced Aß clearance, diminished neuronal activity, behavioral deficits, and progressive neuronal loss and neurodegeneration that develop after initial cerebrovascular degenerative changes. We also show that GLUT1 deficiency in endothelium, but not in astrocytes, initiates the vascular phenotype as shown by BBB breakdown. Thus, reduced BBB GLUT1 expression worsens Alzheimer's disease cerebrovascular degeneration, neuropathology and cognitive function, suggesting that GLUT1 may represent a therapeutic target for Alzheimer's disease vasculo-neuronal dysfunction and degeneration.

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity.

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCßII. These findings reveal a novel mechanism linking lipotoxicity with a PKCß-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs.

GLUT1 deficiency in cardiomyocytes does not accelerate the transition from compensated hypertrophy to heart failure.

The aim of this study was to determine whether endogenous GLUT1 induction and the increased glucose utilization that accompanies pressure overload hypertrophy (POH) are required to maintain cardiac function during hemodynamic stress, and to test the hypothesis that lack of GLUT1 will accelerate the transition to heart failure. To determine the contribution of endogenous GLUT1 to the cardiac adaptation to POH, male mice with cardiomyocyte-restricted deletion of the GLUT1 gene (G1KO) and their littermate controls (Cont) were subjected to transverse aortic constriction (TAC). GLUT1 deficiency reduced glycolysis and glucose oxidation by 50%, which was associated with a reciprocal increase in fatty acid oxidation (FAO) relative to controls. Four weeks after TAC, glycolysis increased and FAO decreased by 50% in controls, but were unchanged in G1KO hearts relative to shams. G1KO and controls exhibited equivalent degrees of cardiac hypertrophy, fibrosis, and capillary density loss after TAC. Following TAC, in vivo left ventricular developed pressure was decreased in G1KO hearts relative to controls, but+dP/dt was equivalently reduced in Cont and G1KO mice. Mitochondrial function was equivalently impaired following TAC in both Cont and G1KO hearts. GLUT1 deficiency in cardiomyocytes alters myocardial substrate utilization, but does not substantially exacerbate pressure-overload induced contractile dysfunction or accelerate the progression to heart failure.

Inducible overexpression of GLUT1 prevents mitochondrial dysfunction and attenuates structural remodeling in pressure overload but does not prevent left ventricular dysfunction.

BACKGROUND: Increased glucose transporter 1 (GLUT1) expression and glucose utilization that accompany pressure overload-induced hypertrophy (POH) are believed to be cardioprotective. Moreover, it has been shown that lifelong transgenic overexpression of GLUT1 in the heart prevents cardiac dysfunction after aortic constriction. The relevance of this model to clinical practice is unclear because of the life-long duration of increased glucose metabolism. Therefore, we sought to determine if a short-term increase in GLUT1-mediated myocardial glucose uptake would still confer cardioprotection if overexpression occurred at the onset of POH. METHODS AND RESULTS: Mice with cardiomyocyte-specific inducible overexpression of a hemagglutinin (HA)-tagged GLUT1 transgene (G1HA) and their controls (Cont) were subjected to transverse aortic constriction (TAC) 2 days after transgene induction with doxycycline (DOX). Analysis was performed 4 weeks after TAC. Mitochondrial function, adenosine triphosphate (ATP) synthesis, and mRNA expression of oxidative phosphorylation (OXPHOS) genes were reduced in Cont mice, but were maintained in concert with increased glucose utilization in G1HA following TAC. Despite attenuated adverse remodeling in G1HA relative to control TAC mice, cardiac hypertrophy was exacerbated in these mice, and positive dP/dt (in vivo) and cardiac power (ex vivo) were equivalently decreased in Cont and G1HA TAC mice compared to shams, consistent with left ventricular dysfunction. O-GlcNAcylation of Ca2+ cycling proteins was increased in G1HA TAC hearts. CONCLUSIONS: Short-term cardiac specific induction of GLUT1 at the onset of POH preserves mitochondrial function and attenuates pathological remodeling, but exacerbates the hypertrophic phenotype and is insufficient to prevent POH-induced cardiac contractile dysfunction, possibly due to impaired calcium cycling.

Regulation of fatty acid metabolism by mTOR in adult murine hearts occurs independently of changes in PGC-1α.

Mechanistic target of rapamycin (mTOR) is essential for cardiac development, growth, and function, but the role of mTOR in the regulation of cardiac metabolism and mitochondrial respiration is not well established. This study sought to determine cardiac metabolism and mitochondrial bioenergetics in mice with inducible deletion of mTOR in the adult heart. Doxycycline-inducible and cardiac-specific mTOR-deficient mice were generated by crossing cardiac-specific doxycycline-inducible tetO-Cre mice with mice harboring mTOR floxed alleles. Deletion of mTOR reduced mTORC1 and mTORC2 signaling after in vivo insulin stimulation. Maximum and minimum dP/dt measured by cardiac catheterization in vivo under anesthesia and cardiac output, cardiac power, and aortic pressure in ex vivo working hearts were unchanged, suggesting preserved cardiac function 4 wk after doxycycline treatment. However, myocardial palmitate oxidation was impaired, whereas glucose oxidation was increased. Consistent with reduced palmitate oxidation, expression of fatty acid metabolism genes fatty acid-binding protein 3, medium-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein)-α and -ß was reduced, and carnitine palmitoyl transferase-1 and -2 enzymatic activity was decreased. Mitochondrial palmitoyl carnitine respiration was diminished. However, mRNA for peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and -1ß, protein levels of PGC-1α, and electron transport chain subunits, mitochondrial DNA, and morphology were unchanged. Also, pyruvate-supported and FCCP-stimulated respirations were unchanged, suggesting that mTOR deletion induces a specific defect in fatty acid utilization. In conclusion, mTOR regulates mitochondrial fatty acid utilization but not glucose utilization in the heart via mechanisms that are independent of changes in PGC expression.

Solid phase synthesis of mitochondrial triphenylphosphonium-vitamin E metabolite using a lysine linker for reversal of oxidative stress.

Mitochondrial targeting of antioxidants has been an area of interest due to the mitochondria's role in producing and metabolizing reactive oxygen species. Antioxidants, especially vitamin E (α-tocopherol), have been conjugated to lipophilic cations to increase their mitochondrial targeting. Synthetic vitamin E analogues have also been produced as an alternative to α-tocopherol. In this paper, we investigated the mitochondrial targeting of a vitamin E metabolite, 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (α-CEHC), which is similar in structure to vitamin E analogues. We report a fast and efficient method to conjugate the water-soluble metabolite, α-CEHC, to triphenylphosphonium cation via a lysine linker using solid phase synthesis. The efficacy of the final product (MitoCEHC) to lower oxidative stress was tested in bovine aortic endothelial cells. In addition the ability of MitoCEHC to target the mitochondria was examined in type 2 diabetes db/db mice. The results showed mitochondrial accumulation in vivo and oxidative stress decrease in vitro.

Genetic loss of insulin receptors worsens cardiac efficiency in diabetes.

AIMS: To determine the contribution of insulin signaling versus systemic metabolism to metabolic and mitochondrial alterations in type 1 diabetic hearts and test the hypothesis that antecedent mitochondrial dysfunction contributes to impaired cardiac efficiency (CE) in diabetes. METHODS AND RESULTS: Control mice (WT) and mice with cardiomyocyte-restricted deletion of insulin receptors (CIRKO) were rendered diabetic with streptozotocin (WT-STZ and CIRKO-STZ, respectively), non-diabetic controls received vehicle (citrate buffer). Cardiac function was determined by echocardiography; myocardial metabolism, oxygen consumption (MVO(2)) and CE were determined in isolated perfused hearts; mitochondrial function was determined in permeabilized cardiac fibers and mitochondrial proteomics by liquid chromatography mass spectrometry. Pyruvate supported respiration and ATP synthesis were equivalently reduced by diabetes and genotype, with synergistic impairment in ATP synthesis in CIRKO-STZ. In contrast, fatty acid delivery and utilization was increased by diabetes irrespective of genotype, but not in non-diabetic CIRKO. Diabetes and genotype synergistically increased MVO(2) in CIRKO-STZ, leading to reduced CE. Irrespective of diabetes, genotype impaired ATP/O ratios in mitochondria exposed to palmitoyl carnitine, consistent with mitochondrial uncoupling. Proteomics revealed reduced content of fatty acid oxidation proteins in CIRKO mitochondria, which were induced by diabetes, whereas tricarboxylic acid cycle and oxidative phosphorylation proteins were reduced both in CIRKO mitochondria and by diabetes. CONCLUSIONS: Deficient insulin signaling and diabetes mediate distinct effects on cardiac mitochondria. Antecedent loss of insulin signaling markedly impairs CE when diabetes is induced, via mechanisms that may be secondary to mitochondrial uncoupling and increased FA utilization.

Oxidative phosphorylation flexibility in the liver of mice resistant to high-fat diet-induced hepatic steatosis.

OBJECTIVE: To identify metabolic pathways that may underlie susceptibility or resistance to high-fat diet-induced hepatic steatosis. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of the livers of A/J and C57Bl/6 mice, which are, respectively, resistant and susceptible to high-fat diet-induced hepatosteatosis and obesity. Mice from both strains were fed a normal chow or a high-fat diet for 2, 10, and 30 days, and transcriptomic data were analyzed by time-dependent gene set enrichment analysis. Biochemical analysis of mitochondrial respiration was performed to confirm the transcriptomic analysis. RESULTS: Time-dependent gene set enrichment analysis revealed a rapid, transient, and coordinate upregulation of 13 oxidative phosphorylation genes after initiation of high-fat diet feeding in the A/J, but not in the C57Bl/6, mouse livers. Biochemical analysis using liver mitochondria from both strains of mice confirmed a rapid increase by high-fat diet feeding of the respiration rate in A/J but not C57Bl/6 mice. Importantly, ATP production was the same in both types of mitochondria, indicating increased uncoupling of the A/J mitochondria. CONCLUSIONS: Together with previous data showing increased expression of mitochondrial ß-oxidation genes in C57Bl/6 but not A/J mouse livers, our present study suggests that an important aspect of the adaptation of livers to high-fat diet feeding is to increase the activity of the oxidative phosphorylation chain and its uncoupling to dissipate the excess of incoming metabolic energy and to reduce the production of reactive oxygen species. The flexibility in oxidative phosphorylation activity may thus participate in the protection of A/J mouse livers against the initial damages induced by high-fat diet feeding that may lead to hepatosteatosis.

Central leptin signaling is required to normalize myocardial fatty acid oxidation rates in caloric-restricted ob/ob mice.

OBJECTIVE: ob/ob and db/db mice manifest myocardial hypertrophy, insulin resistance, altered substrate utilization, mitochondrial dysfunction, and lipid accumulation. This study was designed to determine the contribution of central and peripheral leptin signaling to myocardial metabolism and function in ob/ob and db/db mice in the absence of diabetes and morbid obesity. RESEARCH DESIGN AND METHODS: Male ob/ob mice (aged 4 weeks) were caloric restricted by pairfeeding to a leptin-treated ob/ob group. In addition to determining glucose tolerance and circulating lipid concentrations, myocardial substrate metabolism and mitochondrial function were determined in saponin-permeabilized cardiac fibers. Second, experiments were performed to determine whether leptin treatment by intraperitoneal injection or intracerebroventricular infusion could normalize myocardial palmitate oxidation in caloric-restricted ob/ob mouse hearts. RESULTS: Despite normalizing body weight and glucose tolerance, fat mass and circulating lipid levels remained increased in caloric-restricted ob/ob animals. Palmitate oxidation remained elevated in caloric-restricted ob/ob hearts and was normalized by intraperitoneal or intracerebroventricular leptin. Intraperitoneal and intracerebroventricular treatment also normalized circulating free fatty acid levels, myocardial fatty acid oxidation gene expression, and myocardial insulin sensitivity. CONCLUSIONS: These data suggest that impaired hypothalamic leptin signaling is sufficient to increase myocardial fatty acid oxidation by increasing delivery of free fatty acid substrates and peroxisome proliferator-activated receptor-α ligands to the heart.