ABSTRACT
BACKGROUND: Elevated myocardial intracellular sodium ([Na+]i) was shown to decrease mitochondrial calcium ([Ca2+]MITO) via mitochondrial sodium/calcium exchanger (NCXMITO), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na+]i in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na+]i. METHODS: Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by 31P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCXMITO inhibitor. Myocardial [Na+]i was measured by 23Na NMR spectroscopy. RESULTS: HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na+]i was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na+]i to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts. CONCLUSIONS: Elevated myocardial [Na+]i contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na+]i and/or NCXMITO may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na+]i.
Subject(s)
Diabetes Mellitus , Diabetic Cardiomyopathies , Sodium-Glucose Transporter 2 Inhibitors , Mice , Male , Animals , Diabetic Cardiomyopathies/drug therapy , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Sodium , Calcium , Deoxycytidine Monophosphate , Myocardial Contraction , Mice, Inbred C57BL , Myocardium , Adenosine TriphosphateABSTRACT
Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using 31 P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG~ATP ), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG~ATP and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/biosynthesis , Butyrates/pharmacology , Cardiovascular Diseases/metabolism , Metabolic Diseases/metabolism , Mitochondria, Heart/metabolism , Myocardial Contraction/drug effects , Animals , Cardiovascular Diseases/diagnostic imaging , Cardiovascular Diseases/physiopathology , Energy Metabolism/drug effects , Hemodynamics/drug effects , Hydrolysis , Magnetic Resonance Spectroscopy , Male , Metabolic Diseases/diagnostic imaging , Metabolic Diseases/physiopathology , Mice, Inbred C57BL , Mitochondria, Heart/drug effects , ThermodynamicsABSTRACT
Aims: Metabolic syndrome is associated with metabolic heart disease (MHD) that is characterized by left ventricular (LV) hypertrophy, interstitial fibrosis, contractile dysfunction, and mitochondrial dysfunction. Overexpression of catalase in mitochondria (transgenic expression of catalase targeted to the mitochondria [mCAT]) prevents the structural and functional features of MHD caused by a high-fat, high-sucrose (HFHS) diet for ≥4 months. However, it is unclear whether the effect of mCAT is due to prevention of reactive oxygen species (ROS)-mediated cardiac remodeling, a direct effect on mitochondrial function, or both. To address this question, we measured myocardial function and energetics in mice, with or without mCAT, after 1 month of HFHS, before the development of cardiac structural remodeling. Results: HFHS diet for 1 month had no effect on body weight, heart weight, LV structure, myocyte size, or interstitial fibrosis. Isolated cardiac mitochondria from HFHS-fed mice produced 2.2- to 3.8-fold more H2O2, and 16%-29% less adenosine triphosphate (ATP). In isolated beating hearts from HFHS-fed mice, [phosphocreatine (PCr)] and the free energy available for ATP hydrolysis (ΔGâ¼ATP) were decreased, and they failed to increase with work demands. Overexpression of mCAT normalized ROS and ATP production in isolated mitochondria, and it corrected myocardial [PCr] and ΔGâ¼ATP in the beating heart. Innovation: This is the first demonstration that in MHD, mitochondrial ROS mediate energetic dysfunction that is sufficient to impair contractile function. Conclusion: ROS produced and acting in the mitochondria impair myocardial energetics, leading to slowed relaxation and decreased contractile reserve. These effects precede structural remodeling and are corrected by mCAT, indicating that ROS-mediated energetic impairment, per se, is sufficient to cause contractile dysfunction in MHD.
Subject(s)
Energy Metabolism , Heart Diseases/metabolism , Metabolic Diseases/metabolism , Mitochondria, Heart/metabolism , Reactive Oxygen Species/metabolism , Adenosine Triphosphate/metabolism , Animals , Biomarkers , Disease Susceptibility , Echocardiography , Fibrosis , Heart Diseases/diagnostic imaging , Heart Diseases/etiology , Heart Diseases/pathology , Hydrogen Peroxide/metabolism , Immunohistochemistry , Metabolic Diseases/etiology , Metabolic Diseases/pathology , Mice , Myocardial Contraction , Myocardium/metabolism , Myocardium/pathologyABSTRACT
BACKGROUND AND PURPOSE: Tai Chi is a mind-body exercise that has been shown to improve both mental and physical health. As a result, recent literature suggests the use of Tai Chi to treat both physical and psychological disorders. However, the underlying physiological changes have not been characterized. The aim of this pilot study is to assess the changes in brain metabolites and muscle energetics after Tai Chi training in an aging population using a combined brain-muscle magnetic resonance spectroscopy (MRS) examination. METHODS: Six healthy older adults were prospectively recruited and enrolled into a 12-week Tai Chi program. A brain 1 H MRS and a muscle 31 P MRS were scanned before and after the training, and postprocessed to measure N-acetylaspartate to creatine (NAA/Cr) ratios and phosphocreatine (PCr) recovery time. Wilcoxon-signed rank tests were utilized to assess the differences between pre- and post-Tai Chi training. RESULTS: A significant within-subject increase in both the NAA/Cr ratios (P = .046) and the PCr recovery time (P = .046) was observed between the baseline and the posttraining scans. The median percentage changes were 5.38% and 16.51% for NAA/Cr and PCr recovery time, respectively. CONCLUSIONS: Our pilot study demonstrates significant increase of NAA/Cr ratios in posterior cingulate gyrus and significantly improved PCr recovery time in leg muscles in older adults following short-term Tai Chi training, and thus provides insight into the beneficial mechanisms.
Subject(s)
Brain/diagnostic imaging , Muscle, Skeletal/diagnostic imaging , Tai Ji , Aged , Aspartic Acid/analogs & derivatives , Aspartic Acid/metabolism , Brain/metabolism , Creatine/metabolism , Female , Humans , Magnetic Resonance Spectroscopy , Male , Middle Aged , Muscle, Skeletal/metabolism , Pilot ProjectsABSTRACT
Metabolic syndrome is a cluster of obesity-related metabolic abnormalities that lead to metabolic heart disease (MHD) with left ventricular pump dysfunction. Although MHD is thought to be associated with myocardial energetic deficiency, two key questions have not been answered. First, it is not known whether there is a sufficient energy deficit to contribute to pump dysfunction. Second, the basis for the energy deficit is not clear. To address these questions, mice were fed a high fat, high sucrose (HFHS) 'Western' diet to recapitulate the MHD phenotype. In isolated beating hearts, we used 31P NMR spectroscopy with magnetization transfer to determine a) the concentrations of high energy phosphates ([ATP], [ADP], [PCr]), b) the free energy of ATP hydrolysis (∆G~ATP), c) the rate of ATP production and d) flux through the creatine kinase (CK) reaction. At the lowest workload, the diastolic pressure-volume relationship was shifted upward in HFHS hearts, indicative of diastolic dysfunction, whereas systolic function was preserved. At this workload, the rate of ATP synthesis was decreased in HFHS hearts, and was associated with decreases in both [PCr] and ∆G~ATP. Higher work demands unmasked the inability of HFHS hearts to increase systolic function and led to a further decrease in ∆G~ATP to a level that is not sufficient to maintain normal function of sarcoplasmic Ca2+-ATPase (SERCA). While [ATP] was preserved at all work demands in HFHS hearts, the progressive increase in [ADP] led to a decrease in ∆G~ATP with increased work demands. Surprisingly, CK flux, CK activity and total creatine were normal in HFHS hearts. These findings differ from dilated cardiomyopathy, in which the energetic deficiency is associated with decreases in CK flux, CK activity and total creatine. Thus, in HFHS-fed mice with MHD there is a distinct metabolic phenotype of the heart characterized by a decrease in ATP production that leads to a functionally-important energetic deficiency and an elevation of [ADP], with preservation of CK flux.
Subject(s)
Adenosine Triphosphate/metabolism , Heart Diseases/metabolism , Heart Diseases/physiopathology , Myocardial Contraction , Animals , Body Weight , Creatine Kinase/metabolism , Diastole , Diet, High-Fat , Dietary Sucrose , Energy Metabolism , Hydrolysis , Magnetic Resonance Spectroscopy , Male , Mice, Inbred C57BL , Organ Size , PerfusionABSTRACT
This study aims to test the hypothesis that thiazolidinedione rosiglitazone (RSG), a selective peroxisome proliferator-activated receptor γ (PPARγ) agonist, causes cardiotoxicity independently of PPARγ. Energy metabolism and mitochondrial function were measured in perfused hearts isolated from C57BL/6, cardiomyocyte-specific PPARγ-deficient mice, and their littermates. Cardiac function and mitochondrial oxidative stress were measured in both in vitro and in vivo settings. Treatment of isolated hearts with RSG at the supratherapeutic concentrations of 10 and 30 µM caused myocardial energy deficiency as evidenced by the decreases in [PCr], [ATP], ATP/ADP ratio, energy charge with a concomitant cardiac dysfunction as indicated by the decreases in left ventricular systolic pressure, rates of tension development and relaxation, and by an increase in end-diastolic pressure. When incubated with tissue homogenate or isolated mitochondria at these same concentrations, RSG caused mitochondrial dysfunction as evidenced by the decreases in respiration rate, substrate oxidation rates, and activities of complexes I and IV. RSG also increased complexes I- and III-dependent O2â» production, decreased glutathione content, inhibited superoxide dismutase, and increased the levels of malondialdehyde, protein carbonyl, and 8-hydroxy-2-deoxyguanosine in mitochondria, consistent with oxidative stress. N-acetyl-L-cysteine (NAC) 20 mM prevented RSG-induced above toxicity at those in vitro settings. Cardiomyocyte-specific PPARγ deletion and PPARγ antagonist GW9662 did not prevent the observed cardiotoxicity. Intravenous injection of 10 mg/kg RSG also caused cardiac dysfunction and oxidative stress, 600 mg/kg NAC antagonized these adverse effects. In conclusion, this study demonstrates that RSG at supratherapeutic concentrations causes cardiotoxicity via a PPARγ-independent mechanism involving oxidative stress-induced mitochondrial dysfunction in mouse hearts.
Subject(s)
Cardiotoxins/toxicity , Heart/drug effects , Mitochondria, Heart/drug effects , Oxidative Stress/drug effects , PPAR gamma/metabolism , Thiazolidinediones/toxicity , Anilides/pharmacology , Animals , Dose-Response Relationship, Drug , Energy Metabolism/drug effects , Heart Function Tests , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/metabolism , PPAR gamma/agonists , PPAR gamma/antagonists & inhibitors , PPAR gamma/genetics , Perfusion , RosiglitazoneABSTRACT
The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial ß-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death. However, the contribution of different tissues to development of these phenotypes has not been studied. We generated cardiac-specific VLCAD-deficient (cVLCAD(-/-)) mice by Cre-mediated ablation of the VLCAD in cardiomyocytes. By 6 mo of age, cVLCAD(-/-) mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Surprisingly, selective VLCAD gene ablation in cardiomyocytes was sufficient to evoke severe cold intolerance in mice who rapidly developed severe hypothermia, bradycardia, and markedly depressed cardiac function in response to fasting and cold exposure (+5°C). We conclude that cardiac-specific VLCAD deficiency is sufficient to induce cold intolerance and cardiomyopathy and is associated with reduced ATP production. These results provide strong evidence that fatty acid oxidation in myocardium is essential for maintaining normal cardiac function under these stress conditions.
Subject(s)
Acyl-CoA Dehydrogenase, Long-Chain/deficiency , Cardiomyopathy, Dilated/enzymology , Hypothermia/enzymology , Adenosine Triphosphate/metabolism , Animals , Cardiomyopathy, Dilated/etiology , Cardiomyopathy, Dilated/metabolism , Cold Temperature , Congenital Bone Marrow Failure Syndromes , Disease Models, Animal , Hypothermia/etiology , Hypothermia/metabolism , Lipid Metabolism, Inborn Errors , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondrial Diseases , Muscular Diseases , Oxidation-Reduction , Stress, PhysiologicalABSTRACT
Various biochemical and genomic mechanisms are considered to be a hallmark of metabolic remodeling in the stressed heart, including the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in ß-oxidation of fatty acids. Collectively, our results demonstrate similar increases in the expression and activities of key ATP-generating enzymes in LQT1 and LQT2 rabbit hearts, suggesting an increased demand, and in turn, increased energy supply across the entire metabolic pathway by virtue of the upregulation of enzymes involved in energy generation.
Subject(s)
Energy Metabolism , Enzymes/metabolism , Long QT Syndrome/pathology , Myocardium/chemistry , Proteomics , Romano-Ward Syndrome/pathology , Animals , Animals, Genetically Modified , ERG1 Potassium Channel , Energy Metabolism/genetics , Energy Metabolism/physiology , Enzyme Activation/physiology , Enzymes/analysis , Enzymes/genetics , Ether-A-Go-Go Potassium Channels/genetics , Gene Expression Regulation, Enzymologic/physiology , Heart , KCNQ1 Potassium Channel/genetics , Long QT Syndrome/genetics , Long QT Syndrome/metabolism , Male , Myocardium/metabolism , Myocardium/pathology , Proteome/analysis , Proteomics/methods , Rabbits , Romano-Ward Syndrome/genetics , Romano-Ward Syndrome/metabolismABSTRACT
Chemical Exchange Saturation Transfer (CEST) contrast utilizes selective pre-saturation of a small pool of exchanging protons and subsequent detection of the decrease in bulk water signal. The CEST contrast is negative and requires detection of small signal change in the presence of a strong background signal. Here we develop a Positive CEST (pCEST) detection scheme utilizing the analogous nature of the CEST and off-resonance T(1)(ρ) experiments and exploring increased apparent relaxation rates in the presence of the selective pre-saturation. pCEST leads to the positive contrast, i.e., increased signal intensity as the result of the presence of the agent and RF pre-saturation. Simultaneously substantial background suppression is achieved. The contrast can be switched "ON" and "OFF", similar to the original CEST.
Subject(s)
Magnetic Resonance Imaging/methods , Agar , Algorithms , Electromagnetic Fields , Electron Spin Resonance Spectroscopy , Image Processing, Computer-Assisted , Indicators and Reagents , Phantoms, ImagingABSTRACT
Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal (1)H(2)O NMR relaxation time constant (T(1)) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τ(i)), where τ(i)(-1) is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O(2)/5%CO(2) (O(2)) or 95%N(2)/5%CO(2) (N(2)). ATP was high during O(2), and τ(i)(-1) was 3.1 s(-1) at 25°C. After changing to N(2), ATP decreased and τ(i)(-1) was 1.8 s(-1). The principal active yeast ion transport protein is the plasma membrane H(+)-ATPase. Studies using the H(+)-ATPase inhibitor ebselen or a yeast genetic strain with reduced H(+)-ATPase found reduced τ(i)(-1), notwithstanding high ATP. Steady-state water exchange correlates with H(+)-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity.
Subject(s)
Cell Membrane/metabolism , Magnetic Resonance Spectroscopy/methods , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Water/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Biological Transport, Active , Extracellular Space/metabolism , Intracellular Space/metabolism , Kinetics , Models, Biological , Oxygen/metabolism , Time FactorsABSTRACT
Pharmacological activation of peroxisome proliferator-activated receptor δ/ß (PPARδ/ß) improves glucose handling and insulin sensitivity. The target tissues of drug actions remain unclear. We demonstrate here that adenovirus-mediated liver-restricted PPARδ activation reduces fasting glucose levels in chow- and high fat-fed mice. This effect is accompanied by hepatic glycogen and lipid deposition as well as up-regulation of glucose utilization and de novo lipogenesis pathways. Promoter analyses indicate that PPARδ regulates hepatic metabolic programs through both direct and indirect transcriptional mechanisms partly mediated by its co-activator, PPARγ co-activator-1ß. Assessment of the lipid composition reveals that PPARδ increases the production of monounsaturated fatty acids, which are PPAR activators, and reduces that of saturated FAs. Despite the increased lipid accumulation, adeno-PPARδ-infected livers exhibit less damage and show a reduction in JNK stress signaling, suggesting that PPARδ-regulated lipogenic program may protect against lipotoxicity. The altered substrate utilization by PPARδ also results in a secondary effect on AMP-activated protein kinase activation, which likely contributes to the glucose-lowering activity. Collectively, our data suggest that PPARδ controls hepatic energy substrate homeostasis by coordinated regulation of glucose and fatty acid metabolism, which provide a molecular basis for developing PPARδ agonists to manage hyperglycemia and insulin resistance.
Subject(s)
Energy Metabolism/physiology , Hyperglycemia/metabolism , Insulin Resistance/physiology , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Adenylate Kinase/metabolism , Animals , Blood Glucose/metabolism , Fatty Acids, Monounsaturated/metabolism , Gene Expression Regulation/physiology , Homeostasis/physiology , Hyperglycemia/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Receptors, Cytoplasmic and Nuclear/genetics , Transcription, Genetic/physiologyABSTRACT
This study tested the ability of MR relaxography (MRR) to discriminate intra- (Nai+) and extracellular (Nae+)23Na+ signals using their longitudinal relaxation time constant (T1) values. Na+-loaded yeast cell (Saccharomyces cerevisiae) suspensions were investigated. Two types of compartmental 23Na+T1 differences were examined: a selective Nae+T1 decrease induced by an extracellular relaxation reagent (RRe), GdDOTP5-; and, an intrinsic T1 difference. Parallel studies using the established method of 23Na MRS with an extracellular shift reagent (SRe), TmDOTP5-, were used to validate the MRR measurements. With 12.8 mM RRe, the 23Nae+T1 was 2.4 ms and the 23Nai+T1 was 9.5 ms (9.4 T, 24 degrees C). The Na+ amounts and spontaneous efflux rate constants were found to be identical within experimental error whether measured by MRR/RRe or by MRS/SRe. Without RRe, the Na+-loaded yeast cell suspension 23Na MR signal exhibited two T1 values, 9.1 (+/-0.3) ms and 32.7 (+/-2.3) ms, assigned to 23Nai+ and 23Nae+, respectively. The Nai+ content measured was lower, 0.88 (+/-0.06); while Nae+ was higher, 1.43 (+/-0.12) compared with MRS/SRe measures on the same samples. However, the measured efflux rate constant was identical. T1 MRR potentially may be used for Nai+ determination in vivo and Na+ flux measurements; with RRe for animal studies and without RRe for humans.
Subject(s)
Saccharomyces cerevisiae/chemistry , Sodium/analysis , Indicators and Reagents , Kinetics , Magnetic Resonance Spectroscopy , Saccharomyces cerevisiae/metabolism , Sodium/metabolism , Sodium Isotopes/analysisABSTRACT
PTP1B(-/-) mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptin's metabolic effects. We find that alpha1 and alpha2 AMPK activity are elevated and acetyl-coenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B(-/-) mice. The effects of PTP1B deficiency on alpha2, but not alpha1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron- but not muscle-specific PTP1B(-/-) mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B(-/-) mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Hypothalamus/enzymology , Isoenzymes/metabolism , Neurons/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , AMP-Activated Protein Kinases/genetics , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Animals , Body Weight , Enzyme Activation , Isoenzymes/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Signal Transduction/physiology , Tissue DistributionABSTRACT
In this report, the On resonance PARamagnetic CHemical Exchange Effects (OPARACHEE) method was implemented in vivo using WALTZ-16* as a preparation pulse with a standard spin echo sequence to detect the accumulation and clearance of the TmDOTA-4AmC(-) in mouse kidney. The performance of the technique in vivo is described in terms of the magnitude of the contrast effect versus the bolus agent concentration and signal-to-noise ratio (SNR) levels. The lowest injected concentration of TmDOTA-4AmC(-), 200 microL of a 2-mM stock solution (corresponds to approximately 0.2 mM agent in plasma), reduced the total water signal in the kidney papilla by 45% 3 min after the a bolus injection. The results show that the OPARACHEE methodology employing low-amplitude RF trains can detect paramagnetic exchanging agents in vivo.
Subject(s)
Kidney/anatomy & histology , Magnetic Resonance Imaging , Animals , Female , Male , Mice , Organometallic CompoundsABSTRACT
Angiotensin (Ang) II is a potent mediator of vascular inflammation. A central mechanism by which Ang II promotes inflammation is through the generation of reactive oxygen species (ROS). In the current study, we investigated the role of the transcription factor Ets-1 in regulating Ang II-induced ROS generation. ROS generation was measured in the thoracic aorta of Ets-1(-/-) mice compared with littermate controls after continuous infusion of Ang II. H2O2 and superoxide anion (O2(-)) production were significantly blunted in the Ets-1(-/-) mice. Inhibition of Ets-1 expression by small interfering RNA in primary human aortic smooth muscle cells also potently inhibited ROS production and the induction of the NAD(P)H oxidase subunit p47(phox) in response to Ang II. To evaluate the therapeutic potential of inhibiting Ets-1 in wild-type mice, dominant negative Ets-1 membrane-permeable peptides were administered systemically. Ang II-induced ROS production and medial hypertrophy in the thoracic aorta were markedly diminished as a result of blocking Ets-1. In summary, Ets-1 functions as a critical downstream transcriptional mediator of Ang II ROS generation by regulating the expression of NAD(P)H oxidase subunits such as p47(phox).
Subject(s)
Angiotensin II/metabolism , NADPH Oxidases/genetics , Proto-Oncogene Protein c-ets-1/metabolism , Reactive Oxygen Species/metabolism , Vasoconstrictor Agents/metabolism , Angiotensin II/pharmacology , Animals , Aorta, Thoracic/cytology , Cells, Cultured , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Hydrogen Peroxide/metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Muscle, Smooth, Vascular/cytology , Mutagenesis, Site-Directed , NADPH Oxidases/metabolism , Oxidative Stress/drug effects , Oxidative Stress/physiology , Promoter Regions, Genetic/physiology , Proto-Oncogene Protein c-ets-1/genetics , Rats , Transcriptional Activation/drug effects , Transcriptional Activation/physiology , Vasoconstrictor Agents/pharmacologyABSTRACT
AMP-activated protein kinase (AMPK) responds to impaired cellular energy status by stimulating substrate metabolism for ATP generation. Mutation of the gamma2 regulatory subunit of AMPK in humans renders the kinase insensitive to energy status and causes glycogen storage cardiomyopathy via unknown mechanisms. Using transgenic mice expressing one of the mutant gamma2 subunits (N488I) in the heart, we found that aberrant high activity of AMPK in the absence of energy deficit caused extensive remodeling of the substrate metabolism pathways to accommodate increases in both glucose uptake and fatty acid oxidation in the hearts of gamma2 mutant mice via distinct, yet synergistic mechanisms resulting in selective fuel storage as glycogen. Increased glucose entry in the gamma2 mutant mouse hearts was directed through the remodeled metabolic network toward glycogen synthesis and, at a substantially higher glycogen level, recycled through the glycogen pool to enter glycolysis. Thus, the metabolic consequences of chronic activation of AMPK in the absence of energy deficiency is distinct from those previously reported during stress conditions. These findings are of particular importance in considering AMPK as a target for the treatment of metabolic diseases.
Subject(s)
Energy Metabolism/genetics , Glycogen Storage Disease/metabolism , Glycogen/metabolism , Multienzyme Complexes/metabolism , Myocardium/metabolism , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinases , Amino Acid Substitution/genetics , Animals , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Disease Models, Animal , Enzyme Activation/genetics , Glycogen Storage Disease/enzymology , Glycogen Storage Disease/genetics , Humans , Mice , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/genetics , Oxidative Stress/genetics , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Substrate Cycling/genetics , Up-Regulation/geneticsABSTRACT
AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5-7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and (31)P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr(172) on AMPK-alpha, and phosphorylation of Ser(79) on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.
Subject(s)
Adenosine Monophosphate/metabolism , Adenylate Kinase/metabolism , Cytosol/metabolism , Metformin/administration & dosage , Myocardium/metabolism , Phenformin/administration & dosage , Signal Transduction/physiology , Animals , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Hypoglycemic Agents/administration & dosage , Male , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effectsABSTRACT
Exercise increases AMPK (AMP-activated protein kinase) activity in human and rat adipocytes, but the underlying molecular mechanisms and functional consequences of this activation are not known. Since adrenaline (epinephrine) concentrations increase with exercise, in the present study we hypothesized that adrenaline activates AMPK in adipocytes. We show that a single bout of exercise increases AMPKalpha1 and alpha2 activities and ACC (acetyl-CoA carboxylase) Ser79 phosphorylation in rat adipocytes. Similarly to exercise, adrenaline treatment in vivo increased AMPK activities and ACC phosphorylation. Pre-treatment of rats with the beta-blocker propranolol fully blocked exercise-induced AMPK activation. Increased AMPK activity with exercise and adrenaline treatment in vivo was accompanied by an increased AMP/ATP ratio. Adrenaline incubation of isolated adipocytes also increased the AMP/ATP ratio and AMPK activities, an effect blocked by propranolol. Adrenaline incubation increased lipolysis in isolated adipocytes, and Compound C, an AMPK inhibitor, attenuated this effect. Finally, a potential role for AMPK in the decreased adiposity associated with chronic exercise was suggested by marked increases in AMPKalpha1 and alpha2 activities in adipocytes from rats trained for 6 weeks. In conclusion, both acute and chronic exercise are significant regulators of AMPK activity in rat adipocytes. Our findings suggest that adrenaline plays a critical role in exercise-stimulated AMPKalpha1 and alpha2 activities in adipocytes, and that AMPK can function in the regulation of lipolysis.