RESUMO
BACKGROUND: Due to the shortage of donor organs, an increasing number of transplant organs are harvested after circulatory arrest (donation after circulatory death [DCD]). Using a translational porcine model of DCD, this study developed and evaluated a protocol based on cardioprotection by multidrug postconditioning to optimize resuscitation of DCD hearts during ex situ heart perfusion (ESHP). METHODS: Hearts of female pigs (45.0 ± 4.5 kg) were procured following a clinically identical DCD protocol, consisting of the termination of ventilator support and confirmation of circulatory arrest, followed by a 15-min standoff period. DCD hearts were randomly allocated to ESHP (38.4°C) in the absence (untreated, N = 5) or presence (treated, N = 5) of a postconditioning treatment added to the perfusate, consisting of Intralipid (1%), sevoflurane (2% v/v), and remifentanil (3 nM). All hearts were perfused with blood and Krebs-Henseleit solution (1:1) for 60 min in Langendorff mode and for an additional 300 min in working mode for a total perfusion time of 6 h. Oxidative capacity and detailed left ventricular mechanical function under an increasing workload (left atrial pressure, 6 to 12 mmHg) were assessed hourly. Left ventricular tissue was snap-frozen at the end of ESHP and used for molecular analyses. RESULTS: Left ventricular inotropy (LVdP/dtmax) did not decline over time in treated DCD hearts and was significantly higher at the end of the protocol as compared with untreated DCD hearts (ΔLVdP/dtmax = 440 mmHg/s; P = 0.009). Treated DCD hearts exhibited persistently higher left ventricular stroke work index during the 6-h period of ESHP, whereas untreated DCD hearts displayed a significant decline (change in left ventricular stroke work index = -3.10 ml · mmHg/g; P(time within untreated group) < 0.001). Treated DCD hearts displayed higher metabolic activity as measured by oxygen consumption (ΔO2 = 3.11 ml O2 · min-1 · 100 g-1; P = 0.004) and released lower amounts of cell-free mitochondrial DNA into the perfusate, a marker of potential graft dysfunction. Treated hearts also used fatty acids from Intralipid as an energy source, whereas untreated DCD hearts showed glyceroneogenesis with triglyceride accumulation and depletion of tricarboxylic acid cycle intermediates; reduced mitochondrial complex I, II, and III activities with accumulation of mitochondrial NADH, and signs of ultrastructural damage. CONCLUSIONS: A translationally relevant protective ESHP protocol consisting of treatment with Intralipid, sevoflurane, and remifentanil markedly accelerated functional recovery and improved viability of DCD hearts.
Assuntos
Transplante de Coração , Animais , Suínos , Feminino , Transplante de Coração/métodos , Ressuscitação/métodos , Doadores de Tecidos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/fisiologia , Obtenção de Tecidos e Órgãos/métodos , Modelos Animais de DoençasRESUMO
BACKGROUND: While lipid emulsions in modern formulations for total parenteral nutrition (TPN) provide essential fatty acids and dense calories, they also promote inflammation and immunometabolic disruptions. OBJECTIVES: We aimed to develop a novel lipid emulsion for TPN use with superior immunometabolic actions compared with available standard lipid emulsions. METHODS: A novel lipid emulsion [Vegaven (VV)] containing 30% of 18-carbon n-3 fatty acids (α-linolenic acid and stearidonic acid) was developed for TPN (VV-TPN) and compared with TPN containing soybean oil-based lipid emulsion (IL-TPN) and fish-oil-based lipid emulsion (OV-TPN). In vivo studies were performed in instrumented male C57BL/6 mice subjected to 7-d TPN prior to analysis of cytokines, indices of whole-body and hepatic glucose metabolism, immune cells, lipid mediators, and mucosal bowel microbiome. RESULTS: IL-6 to IL-10 ratios were significantly lower in liver and skeletal muscle of VV-TPN mice when compared with IL-TPN or OV-TPN mice. VV-TPN and OV-TPN each increased hepatic insulin receptor abundance and resulted in similar HOMA-IR values, whereas only VV-TPN increased hepatic insulin receptor substrate 2 and maintained normal hepatic glycogen content, effects that were IL-10-dependent and mediated by glucokinase activation. The percentages of IFN-γ- and IL-17-expressing CD4+ T cells were increased in livers of VV-TPN mice, and liver macrophages exhibited primed phenotypes when compared with IL-TPN. This immunomodulation was associated with successful elimination of the microinvasive bacterium Akkermansia muciniphila from the bowel mucosa by VV-TPN as opposed to standard lipid emulsions. Assay of hepatic lipid mediators revealed a distinct profile with VV-TPN, including increases in 9(S)-hydroxy-octadecatrienoic acid. When co-administered with IL-TPN, hydroxy-octadecatrienoic acids mimicked the VV-TPN immunometabolic phenotype. CONCLUSIONS: We here report the unique anti-inflammatory, insulin-sensitizing, and immunity-enhancing properties of a newly developed lipid emulsion designed for TPN use based on 18-carbon n-3 fatty acids.
Assuntos
Ácidos Graxos Ômega-3 , Nutrição Parenteral Total , Animais , Masculino , Camundongos , Modelos Animais de Doenças , Emulsões , Emulsões Gordurosas Intravenosas/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Interleucina-10 , Camundongos Endogâmicos C57BL , Fenótipo , Óleo de Soja/farmacologiaRESUMO
BACKGROUND: Lipid emulsions are a key component of total parenteral nutrition (TPN) and are administered to patients who are unable to ingest their daily required calories orally. Lipid emulsions rich with n-6 (ω-6) PUFAs are known to cause parenteral nutrition-associated liver disease and have inflammatory side effects, whereas n-3 PUFA-rich emulsions have favourable clinical outcomes. OBJECTIVES: The present study used targeted lipid mediator analysis to investigate the metabolism of a n-3 PUFA-rich lipid emulsion and a n-6 PUFA-rich lipid emulsion in a mouse model of TPN and in primary human monocyte-derived macrophages (MDMs) and CD4+ T cells. RESULTS: Mice given n-3 PUFA-based TPN for 7 d had a less proinflammatory lipid mediator profile compared with those receiving n-6 PUFA-based TPN. This was characterized by higher concentrations of specialized pro-resolving mediators (SPMs) and endocannabinoids, including resolvin D (RvD) 1, maresin (MaR) 1, MaR2, protectin D1 (PD1), protectin DX (PDX), and the endocannabinoids eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) in the liver and RvD1, 17R-RvD1, RvD2, RvD3, RvD5, MaR1, MaR2, PD1, PDX, and EPEA and DHEA in the spleen. The spleen was identified as a source of high lipid mediator and SPM formation as lipid mediator concentrations were on average 25-fold higher than in the liver. Additionally, n-3 PUFA-treated primary human MDMs produced RvD5 and the endocannabinoids EPEA and DHEA, which was associated with an increased IL-10 secretion. In contrast, primary human CD4+ T cells showed only an increase in SPM precursors and an increase in the endocannabinoids EPEA and DHEA, which was associated with reduced cytokine expression. CONCLUSIONS: This demonstrates that lipid mediators, particularly SPMs and endocannabinoids from spleen, could play a key role in facilitating the favorable clinical outcomes associated with the use of n-3 PUFA-rich lipid emulsions in TPN.
Assuntos
Ácidos Graxos Ômega-3 , Animais , Desidroepiandrosterona , Ácidos Docosa-Hexaenoicos , Emulsões , Endocanabinoides , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Insaturados , Humanos , CamundongosRESUMO
BACKGROUND & AIMS: Total parenteral nutrition (TPN) causes gut atrophy, dysbiosis and leakage of the gut barrier. This study aimed to characterize the gut microbiome in response to different TPNs and tested the hypothesis whether increased gut permeability in TPN would lead to changes in the circulating bacterial DNA ("blood microbiome"). METHODS: Male C57BL/6J mice were randomly allocated to the following groups for seven days (1) chow-fed control (C) without jugular vein catheter (JVC, n=6) (2) chow-fed with JVC and infusion of saline (S) (n = 6) (3) Intralipid-based TPN (n-6:n-3 ratio 7:1) (IL, n = 6) (4) Omegaven-based TPN (n-6:n-3 ratio 1:8) (OV, n = 6). Blood was collected by cardiac puncture and feces (stool pellet) were collected from the colon. Blood and stool samples were analyzed by 16S rRNA gene sequencing. RESULTS: TPN administration was associated with a compositional shift in the gut microbial community that involved the expansion of Bacteroidota along with a decrease in gut bacteria belonging to the Firmicutes phylum as compared to chow-fed mice. Gram-negative Verrucomicrobiota and Proteobacteria were also increased in the gut microbiome of mice receiving TPN. Gammaproteobacteria, namely Burkholderiales, were specifically increased in Intralipid-based TPN. On the other hand, Proteobacteria and Actinobacteriota were the dominant taxa in blood samples. The families Comamonadaceae and Burkholderiaceae (both from Burkholderiales order) were increased in the "blood microbiome" of mice with indwelling JVC when compared with chow-fed mice without JVC. The increase in Burkholderiaceae was more pronounced in Intralipid-based TPN. CONCLUSIONS: Profound changes in the gut microbiome of mice subjected to TPN occurred, which were not reflected in the "blood microbiome" suggesting that the gut and "blood microbiome" represent two rather distinct separate microbiotic compartments. The parenteral provision of n-3 fatty acids appears to protect against proinflammatory bacteria in the gut and against the increased presence of JVC-associated bacteria as measured by circulating bacterial DNA.
Assuntos
Ácidos Nucleicos Livres , Microbioma Gastrointestinal , Animais , Bactérias/genética , DNA Bacteriano , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nutrição Parenteral Total , RNA Ribossômico 16SRESUMO
OBJECTIVES: Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma. METHODS: Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion. RESULTS: We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2). CONCLUSIONS: Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice.
Assuntos
Ácidos Graxos Ômega-3 , Espectrometria de Massas em Tandem , Adolescente , Animais , Cromatografia Líquida de Alta Pressão , Eicosanoides , Humanos , Inflamação , Camundongos , Espectrometria de Massas em Tandem/métodosRESUMO
Total parenteral nutrition (TPN) is a life-saving therapy administered to millions of patients. However, it is associated with significant adverse effects, namely liver injury, risk of infections, and metabolic derangements. In this review, the underlying causes of TPN-associated adverse effects, specifically gut atrophy, dysbiosis of the intestinal microbiome, leakage of the epithelial barrier with bacterial invasion, and inflammation are first described. The role of the bile acid receptors farnesoid X receptor and Takeda G protein-coupled receptor, of pleiotropic hormones, and growth factors is highlighted, and the mechanisms of insulin resistance, namely the lack of insulinotropic and insulinomimetic signaling of gut-originating incretins as well as the potentially toxicity of phytosterols and pro-inflammatory fatty acids mainly released from soybean oil-based lipid emulsions, are discussed. Finally, novel approaches in the design of next generation lipid delivery systems are proposed. Propositions include modifying the physicochemical properties of lipid emulsions, the use of lipid emulsions generated from sustainable oils with favorable ratios of anti-inflammatory n-3 to pro-inflammatory n-6 fatty acids, beneficial adjuncts to TPN, and concomitant pharmacotherapies to mitigate TPN-associated adverse effects.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Gastrite/prevenção & controle , Hepatite/prevenção & controle , Lipídeos/administração & dosagem , Nutrição Parenteral Total/efeitos adversos , Ácidos e Sais Biliares/metabolismo , Disbiose/etiologia , Emulsões/administração & dosagem , Emulsões/química , Gastrite/etiologia , Hepatite/etiologia , Humanos , Insulina/metabolismo , Lipídeos/farmacologia , Nutrição Parenteral Total/métodos , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
SCOPE: The aim of this study is to test whether the choice of the lipid emulsion in total parenteral nutrition (TPN), that is, n-3 fatty acid-based Omegaven versus n-6 fatty acid-based Intralipid, determines inflammation in the liver, the incretin profile, and insulin resistance. METHODS AND RESULTS: Jugular vein catheters (JVC) are placed in C57BL/6 mice and used for TPN for 7 days. Mice are randomized into a saline group (saline infusion with oral chow), an Intralipid group (IL-TPN, no chow), an Omegaven group (OV-TPN, no chow), or a chow only group (without JVC). Both TPN elicite higher abundance of lipopolysaccharide binding protein in the liver, but only IL-TPN increases interleukin-6 and interferon-γ, while OV-TPN reduces interleukin-4, monocyte chemoattractant protein-1, and interleukin-1α. Insulin plasma concentrations are higher in both TPN, while glucagon and glucagon-like peptide-1 (GLP-1) were higher in IL-TPN. Gluconeogenesis is increased in IL-TPN and the nuclear profile of key metabolic transcription factors shows a liver-protective phenotype in OV-TPN. OV-TPN increases insulin sensitivity in the liver and skeletal muscle. CONCLUSION: OV-TPN as opposed to IL-TPN mitigates inflammation in the liver and reduces the negative metabolic effects of hyperinsulinemia and hyperglucagonemia by "re-sensitizing" the liver and skeletal muscle to insulin.
Assuntos
Gastrite/etiologia , Hepatite/etiologia , Insulina/metabolismo , Lipídeos/administração & dosagem , Nutrição Parenteral Total/métodos , Animais , Emulsões/administração & dosagem , Emulsões/química , Emulsões/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Óleos de Peixe/farmacologia , Incretinas/metabolismo , Insulina/sangue , Resistência à Insulina , Interferon gama/metabolismo , Interleucina-6/metabolismo , Lipídeos/química , Síndromes de Malabsorção/etiologia , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Nutrição Parenteral Total/efeitos adversos , Fosfolipídeos/administração & dosagem , Fosfolipídeos/farmacologia , Óleo de Soja/administração & dosagem , Óleo de Soja/farmacologia , Triglicerídeos/farmacologiaRESUMO
Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.
Assuntos
Calnexina/metabolismo , Retículo Endoplasmático/metabolismo , Glicólise , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Animais , Camundongos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
BACKGROUND: While Omegaven, an omega-3 (n3) fatty acid-based lipid emulsion, fosters insulin signaling in healthy hearts, it is unknown whether beneficial metabolic effects occur in insulin-resistant diabetic hearts. METHODS: Diabetic hearts from fructose-fed Sprague-Dawley rats were perfused in the working mode for 90 minutes in the presence of 11 mM glucose and 1.2 mM palmitate bound to albumin, the first 30 minutes without insulin followed by 60 minutes with insulin (50 mU/L). Hearts were randomly allocated to Intralipid (25 and 100 µM), Omegaven (25 and 100 µM), or no emulsion (insulin alone) for 60 minutes. Glycolysis, glycogen synthesis, and glucose oxidation were measured with the radioactive tracers [5-H]glucose and [U-C]glucose. Central carbon metabolites, acyl-coenzyme A species (acyl-CoAs), ketoacids, purines, phosphocreatine, acylcarnitines, and acyl composition of phospholipids were measured with mass spectrometry. RESULTS: Diabetic hearts showed no response to insulin with regard to glycolytic flux, consistent with insulin resistance. Addition of either lipid emulsion did not alter this response but unexpectedly increased glucose oxidation (ratio of treatment/baseline, ie, fold change): no insulin 1.3 (0.3) [mean (standard deviation)], insulin alone 1.4 (0.4), insulin + 25 µM Intralipid 1.8 (0.5), insulin + 100 µM Intralipid 2.2 (0.4), P < .001; no insulin 1.3 (0.3), insulin alone 1.4 (0.4), insulin + 25 µM Omegaven 2.3 (0.5) insulin + 100 µM Omegaven 1.9 (0.4), P < .001. Intralipid treatment led to accumulation of acylcarnitines as a result of the released linoleic acid (C18:2-n6) and enhanced its integration into phospholipids, consistent with incomplete or impaired ß-oxidation necessitating a compensatory increase in glucose oxidation. Accumulation of acylcarnitines was also associated with a higher nicotinamide adenine dinucleotide reduced/oxidized (NADH/NAD) ratio, which inhibited pyruvate dehydrogenase (PDH), and resulted in excess lactate production. In contrast, Omegaven-treated hearts showed no acylcarnitine accumulation, low malonyl-CoA concentrations consistent with activated ß-oxidation, and elevated PDH activity and glucose oxidation, together indicative of a higher metabolic rate possibly by substrate cycling. CONCLUSIONS: Omegaven is the preferred lipid emulsion for insulin-resistant diabetic hearts.
Assuntos
Cardiomiopatias Diabéticas/tratamento farmacológico , Metabolismo Energético/efeitos dos fármacos , Óleos de Peixe/farmacologia , Resistência à Insulina , Miócitos Cardíacos/efeitos dos fármacos , Fosfolipídeos/farmacologia , Óleo de Soja/farmacologia , Animais , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Açúcares da Dieta , Modelos Animais de Doenças , Emulsões/farmacologia , Frutose , Masculino , Miócitos Cardíacos/metabolismo , Oxirredução , Ratos Sprague-Dawley , TriglicerídeosRESUMO
BACKGROUND: It is currently unknown whether acute exposure to n3 fatty acid-containing fish oil-based lipid emulsion Omegaven as opposed to the n6 fatty acid-containing soybean oil-based lipid emulsion Intralipid is more favorable in terms of insulin signaling and glucose uptake in the intact beating heart. METHODS: Sprague-Dawley rat hearts were perfused in the working mode for 90 minutes in the presence of 11 mM glucose and 1.2 mM palmitate bound to albumin, the first 30 minutes without insulin followed by 60 minutes with insulin (50 mU/L). Hearts were randomly allocated to 100 µM Intralipid, 100 µM Omegaven, or no emulsion (insulin treatment alone) for 60 minutes. Glycolysis and glycogen synthesis were measured with the radioactive tracer [5-H]glucose, and glucose uptake was calculated. Phosphorylation of protein phosphatase 2A (PP2A), protein kinase Akt, and phosphofructokinase (PFK)-2 was measured by immunoblotting. Glycolytic metabolites were determined by enzymatic assays. Mass spectrometry was used to establish acylcarnitine profiles. Nuclear factor κB (NFκB) nuclear translocation served as reactive oxygen species (ROS) biosensor. RESULTS: Insulin-mediated glucose uptake was decreased by Intralipid (4.9 ± 0.4 vs 3.7 ± 0.3 µmol/gram dry heart weight [gdw]·min; P = .047) due to both reduced glycolysis and glycogen synthesis. In contrast, Omegaven treatment did not affect insulin-mediated glycolysis or glycogen synthesis and thus preserved glucose uptake (5.1 ± 0.3 vs 4.9 ± 0.4 µmol/gdw·min; P = .94). While Intralipid did not affect PP2A phosphorylation status, Omegaven resulted in significantly enhanced tyrosine phosphorylation and inhibition of PP2A. This was accompanied by increased selective threonine phosphorylation of Akt and the downstream target PFK-2 at S483. PFK-1 activity was increased when compared with Intralipid as measured by the ratio of fructose 1,6-bisphosphate to fructose 6-phosphate (Omegaven 0.60 ± 0.11 versus Intralipid 0.47 ± 0.09; P = .023), consistent with increased formation of fructose 2,6-bisphosphate by PFK2, its main allosteric activator. Omegaven lead to accumulation of acylcarnitines and fostered a prooxidant response as evidenced by NFκB nuclear translocation and activation. CONCLUSIONS: Omegaven as opposed to Intralipid preserves glucose uptake via the PP2A-Akt-PFK pathway in intact beating hearts. n3 fatty acids decelerate ß-oxidation causing accumulation of acylcarnitine species and a prooxidant response, which likely inhibits redox-sensitive PP2A and thus preserves insulin signaling and glucose uptake.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Emulsões Gordurosas Intravenosas/farmacologia , Óleos de Peixe/farmacologia , Glucose/metabolismo , Insulina/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Fosfolipídeos/farmacologia , Óleo de Soja/farmacologia , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Emulsões/farmacologia , Preparação de Coração Isolado , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Oxirredução , Fosfofrutoquinase-1/metabolismo , Fosfofrutoquinase-2/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , TriglicerídeosRESUMO
PURPOSE: Intralipid® (ILE), a clinically used lipid emulsion, reduces ischemia-reperfusion (IR) injury in healthy and infarct-remodelled rat hearts. We tested whether ILE is also cardioprotective in large porcine hearts in the context of the donation after circulatory death (DCD) model, where human hearts are procured for transplantation after cardiac arrest and thus are exposed to significant IR injury. METHODS: After induction of anesthesia, surgical preparation, termination of ventilator support, and cardiac arrest, hearts of female pigs were procured following a 15 min standoff period, with an optimized normokalemic crystalloid adenosine-lidocaine cardioplegia. Hearts were then randomly allocated to ex vivo reperfusion (38°C) in the absence (control) or presence of 1% ILE. All hearts were perfused with blood and Krebs-Henseleit solution (1:1) for 30 min in Langendorff mode and for an additional 30 min in working mode to assess mechanical function. Left ventricular (LV) biopsies were obtained after five minutes of reperfusion and LV tissue was preserved at the end of reperfusion for biochemical analyses and immunohistochemistry. RESULTS: Intralipid® postconditioning reduced cell membrane damage as assessed by the mean (standard deviation) leakage of myocardial glutathione disulfide (39 (9) nmol·mg-1 protein vs 19 (7) nmol·mg-1 protein; P = 0.006), protected LV tissue from protein carbonylation (3.4 [0.6] nmol·mg-1 protein vs 5.3 [0.9] nmol·mg-1 protein; P = 0.006), decreased myeloperoxidase activity (35 [8] nmol·min-1·mg-1 protein vs 75 [11] nmol·min-1·mg-1 protein; P < 0.001), and increased inotropy (maximum rate of rise of LV pressure 2001 [345] mmHg·sec-1vs 1584 [192] mmHg·sec-1; P = 0.044). Intralipid® postconditioning triggered reactive oxygen species signalling at early reperfusion and activated protection signalling (Akt, signal transducer and activator of transcription 3, and glycogen synthase kinase 3ß) in LV tissue, recapitulating all features of ILE-mediated protection reported in small rodent hearts. CONCLUSIONS: Our data show that ILE postconditioning elicits protection signalling in large mammalian hearts while mimicking clinical conditions, and is capable of enhancing protection of DCD hearts.
Assuntos
Pós-Condicionamento Isquêmico/métodos , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fosfolipídeos/administração & dosagem , Óleo de Soja/administração & dosagem , Animais , Modelos Animais de Doenças , Emulsões/administração & dosagem , Emulsões Gordurosas Intravenosas/administração & dosagem , Feminino , Parada Cardíaca/fisiopatologia , Transplante de Coração/métodos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , Suínos , Obtenção de Tecidos e ÓrgãosRESUMO
BACKGROUND: Despite an array of cardioprotective interventions identified in preclinical models of ischemia-reperfusion (IR) injury, successful clinical translation has not been achieved. This study investigated whether drugs routinely used in clinical anesthesia influence cardioprotective effectiveness by reducing effects of reactive oxygen species (ROS), upstream triggers of cardioprotective signaling. Effects of propofol, sevoflurane, or remifentanil were compared on postischemic functional recovery induced by ROS-mediated postconditioning with Intralipid. METHODS: Recovery of left ventricular (LV) work, an index of IR injury, was measured in isolated Sprague-Dawley rat hearts subjected to global ischemia (20 minutes) and reperfusion (30 minutes). Hearts were either untreated or were treated with postconditioning with Intralipid (1%, throughout reperfusion). Propofol (10 µM), sevoflurane (2 vol%), remifentanil (3 nM), or combinations thereof were administered peri-ischemically (before and during IR). The effects of anesthetics on ROS production were measured in LV cardiac fibers by Amplex Red assay under phosphorylating and nonphosphorylating conditions. RESULTS: Recovery of LV work (expressed as percentage of the preischemic value ± standard deviation) in untreated hearts was poor (20% ± 7%) and was improved by Intralipid postconditioning (58% ± 8%, P = .001). In the absence of Intralipid postconditioning, recovery of LV work was enhanced by propofol (28% ± 9%, P = .049), sevoflurane (49% ± 5%, P < .001), and remifentanil (51% ± 6%, P < .001). The benefit of Intralipid postconditioning was abolished by propofol (33% ± 10%, P < .001), but enhanced by sevoflurane (80% ± 7%, P < .001) or remifentanil (80% ± 9%, P < .001). ROS signaling in LV fibers was abolished by propofol, but unaffected by sevoflurane or remifentanil. We conclude that propofol abolishes ROS-mediated Intralipid postconditioning by acting as a ROS scavenger. Sevoflurane and remifentanil are protective per se and provide additive cardioprotection to ROS-mediated cardioprotection. CONCLUSIONS: These divergent effects of routinely used drugs in clinical anesthesia may influence the translatability of cardioprotective therapies such as Intralipid postconditioning.
Assuntos
Analgésicos Opioides/administração & dosagem , Anestésicos Inalatórios/administração & dosagem , Anestésicos Intravenosos/administração & dosagem , Pós-Condicionamento Isquêmico/métodos , Traumatismo por Reperfusão Miocárdica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Opioides/metabolismo , Animais , Coração/efeitos dos fármacos , Coração/fisiologia , Preparação de Coração Isolado/métodos , Masculino , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Propofol/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores Opioides/agonistas , Remifentanil/administração & dosagem , Sevoflurano/administração & dosagemRESUMO
Despite the fact that skeletal muscle insulin resistance is the hallmark of type-2 diabetes mellitus (T2DM), inflexibility in substrate energy metabolism has been observed in other tissues such as liver, adipose tissue, and heart. In the heart, structural and functional changes ultimately lead to diabetic cardiomyopathy. However, little is known about the early biochemical changes that cause cardiac metabolic dysregulation and dysfunction. We used a dietary model of fructose-induced T2DM (10% fructose in drinking water for 6 weeks) to study cardiac fatty acid metabolism in early T2DM and related signaling events in order to better understand mechanisms of disease. In early type-2 diabetic hearts, flux through the fatty acid oxidation pathway was increased as a result of increased cellular uptake (CD36), mitochondrial uptake (CPT1B), as well as increased ß-hydroxyacyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase activities, despite reduced mitochondrial mass. Long-chain acyl-CoA dehydrogenase activity was slightly decreased, resulting in the accumulation of long-chain acylcarnitine species. Cardiac function and overall mitochondrial respiration were unaffected. However, evidence of oxidative stress and subtle changes in cardiolipin content and composition were found in early type-2 diabetic mitochondria. Finally, we observed decreased activity of SIRT1, a pivotal regulator of fatty acid metabolism, despite increased protein levels. This indicates that the heart is no longer capable of further increasing its capacity for fatty acid oxidation. Along with increased oxidative stress, this may represent one of the earliest signs of dysfunction that will ultimately lead to inflammation and remodeling in the diabetic heart.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Ácidos Graxos/metabolismo , Miocárdio/metabolismo , Sirtuína 1/metabolismo , Acil-CoA Desidrogenase/metabolismo , Animais , Antígenos CD36/metabolismo , Cardiolipinas/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Frutose/toxicidade , Masculino , Mitocôndrias Musculares/metabolismo , Miocárdio/patologia , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
The clinically used lipid emulsion Intralipid (ILE) reduces ischemia reperfusion injury in healthy rodent hearts. We tested whether ILE is cardioprotective in postinfarct remodeled hearts. Post-infarct remodeled and sham Sprague-Dawley rat hearts were perfused in working mode and subjected to ischemia (15 minutes) and reperfusion (30 minutes). Left ventricular (LV) work was measured in hearts that were untreated or that received ILE (1%) postconditioning administered at the onset of reperfusion, or the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine (10 µM) alone or in combination with ILE. Mitochondrial O2 consumption was measured in LV muscle fibers. Acetyl CoA production was calculated from the oxidation of [U-14C]glucose and [9,10-3H]palmitate. ROS production was assessed by loss of aconitase activity as well as by release of hydrogen peroxide. Phosphorylation of Akt, Erk1/2, and STAT3 were used to evaluate protection signaling. Remodeled hearts exhibited LV dysfunction and signs of hypertrophy consistent with significant postinfarct remodeling. ILE postconditioning enhanced the recovery of postischemic LV function in remodeled hearts, preserved energy metabolism in mitochondria, accelerated palmitate oxidation and acetyl CoA production, and activated Akt/Erk/STAT3 in a ROS-dependent manner. Protection by ILE postconditioning evolved rapidly within the first minutes of reperfusion without evidence of additional cardiotonic effects due to provision of supplementary energy substrates potentially released from ILE during reperfusion. ILE represents a novel and clinically feasible cardioprotective strategy that is highly effective in remodeled hearts. Our data provide a rationale for the clinical evaluation of ILE postconditioning where ILE is administered as a bolus at the onset of reperfusion.
Assuntos
Pós-Condicionamento Isquêmico , Infarto do Miocárdio/complicações , Fosfolipídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Óleo de Soja/farmacologia , Animais , Emulsões/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipertrofia Ventricular Esquerda , Masculino , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Disfunção Ventricular Esquerda , Remodelação VentricularRESUMO
The flux of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER-mitochondria Ca(2+) flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER-mitochondria Ca(2+) flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca(2+), accelerated cytosolic Ca(2+) clearance, and reduced Ca(2+) transfer to mitochondria. Thus, low levels of TMX1 reduce ER-mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER-mitochondria Ca(2+) flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.
Assuntos
Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Compostos de Sulfidrila/metabolismo , Tiorredoxinas/metabolismo , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Metabolismo Energético , Glucose/farmacologia , Células HeLa , Humanos , Lipoilação , Membranas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The IV anesthetic, propofol, when administered as fat emulsion-based formulation (Diprivan) promotes insulin resistance, but the direct effects of propofol and its solvent, Intralipid, on cardiac insulin resistance are unknown. METHODS: Hearts of healthy and type-2 diabetic rats (generated by fructose feeding) were aerobically perfused for 60 minutes with 10 µM propofol in the formulation of Diprivan or an equivalent concentration of its solvent Intralipid (25 µM) ± insulin (100 mUâ¢L). Glucose uptake, glycolysis, and glycogen metabolism were measured using [H]glucose. Activation of Akt, GSK3ß, AMPK, ERK1/2, p38MAPK, S6K1, JNK, protein kinase Cθ (PKCθ), and protein kinase CCßII (PKCßII) was determined using immunoblotting. GLUT4 trafficking and phosphorylations of insulin receptor substrate-1 (IRS-1) at Ser307(h312), Ser1100(h1101), and Tyr608(hTyr612) were measured. Mass spectrometry was used to determine acylcarnitines, phospholipids, and sphingolipids. RESULTS: Diprivan and Intralipid reduced insulin-induced glucose uptake and redirected glucose to glycogen stores in diabetic hearts. Reduced glucose uptake was accompanied by lower GLUT4 trafficking to the sarcolemma. Diprivan and Intralipid inactivated GSK3ß but activated AMPK and ERK1/2 in diabetic hearts. Only Diprivan increased phosphorylation of Akt(Ser473/Thr308) and translocated PKCθ and PKCßII to the sarcolemma in healthy hearts, whereas it activated S6K1 and p38MAPK and translocated PKCßII in diabetic hearts. Furthermore, only Diprivan phosphorylated IRS-1 at Ser1100(h1101) in healthy and diabetic hearts. JNK expression, phosphorylation of Ser307(h312) of IRS-1, and PKCθ expression and translocation were increased, whereas GLUT4 expression was reduced in insulin-treated diabetic hearts. Phosphatidylglycerol, phosphatidylethanolamine, and C18-sphingolipids accumulated in Diprivan-perfused and Intralipid-perfused diabetic hearts. CONCLUSIONS: Propofol and Intralipid promote insulin resistance predominantly in type-2 diabetic hearts.
Assuntos
Anestésicos Intravenosos/toxicidade , Diabetes Mellitus Tipo 2/metabolismo , Emulsões Gordurosas Intravenosas/toxicidade , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Coração/efeitos dos fármacos , Resistência à Insulina , Fosfolipídeos/toxicidade , Propofol/toxicidade , Óleo de Soja/toxicidade , Animais , Citrato (si)-Sintase/metabolismo , Diabetes Mellitus Tipo 2/induzido quimicamente , Emulsões/toxicidade , Frutose , Glucose/metabolismo , Glicogênio/metabolismo , Glicólise/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Insulin resistance and early type-2 diabetes are highly prevalent. However, it is unknown whether Intralipid® and sevoflurane protect the early diabetic heart against ischemia-reperfusion injury. METHODS: Early type-2 diabetic hearts from Sprague-Dawley rats fed for 6 weeks with fructose were exposed to 15 min of ischemia and 30 min of reperfusion. Intralipid® (1%) was administered at the onset of reperfusion. Peri-ischemic sevoflurane (2 vol.-%) served as alternative protection strategy. Recovery of left ventricular function was recorded and the activation of Akt and ERK 1/2 was monitored. Mitochondrial function was assessed by high-resolution respirometry and mitochondrial ROS production was measured by Amplex Red and aconitase activity assays. Acylcarnitine tissue content was measured and concentration-response curves of complex IV inhibition by palmitoylcarnitine were obtained. RESULTS: Intralipid® did not exert protection in early diabetic hearts, while sevoflurane improved functional recovery. Sevoflurane protection was abolished by concomitant administration of the ROS scavenger N-2-mercaptopropionyl glycine. Sevoflurane, but not Intralipid® produced protective ROS during reperfusion, which activated Akt. Intralipid® failed to inhibit respiratory complex IV, while sevoflurane inhibited complex I. Early diabetic hearts exhibited reduced carnitine-palmitoyl-transferase-1 activity, but palmitoylcarnitine could not rescue protection and enhance postischemic functional recovery. Cardiac mitochondria from early diabetic rats exhibited an increased content of subunit IV-2 of respiratory complex IV and of uncoupling protein-3. CONCLUSIONS: Early type-2 diabetic hearts lose complex IV-mediated protection by Intralipid® potentially due to a switch in complex IV subunit expression and increased mitochondrial uncoupling, but are amenable to complex I-mediated sevoflurane protection.
Assuntos
Cardiotônicos/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Emulsões Gordurosas Intravenosas/uso terapêutico , Coração/efeitos dos fármacos , Éteres Metílicos/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Fosfolipídeos/uso terapêutico , Óleo de Soja/uso terapêutico , Animais , Diabetes Mellitus Tipo 2/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Emulsões/uso terapêutico , Frutose/metabolismo , Canais Iônicos/metabolismo , Masculino , Proteínas Mitocondriais/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Sevoflurano , Transdução de Sinais/efeitos dos fármacos , Proteína Desacopladora 3RESUMO
BACKGROUND: Intralipid® administration at reperfusion elicits protection against myocardial ischemia-reperfusion injury. However, the underlying mechanisms are not fully understood. METHODS: Sprague-Dawley rat hearts were exposed to 15 min of ischemia and 30 min of reperfusion in the absence or presence of Intralipid® 1% administered at the onset of reperfusion. In separate experiments, the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine was added either alone or with Intralipid®. Left ventricular work and activation of Akt, STAT3, and ERK1/2 were used to evaluate cardioprotection. ROS production was assessed by measuring the loss of aconitase activity and the release of hydrogen peroxide using Amplex Red. Electron transport chain complex activities and proton leak were measured by high-resolution respirometry in permeabilized cardiac fibers. Titration experiments using the fatty acid intermediates of Intralipid® palmitoyl-, oleoyl- and linoleoylcarnitine served to determine concentration-dependent inhibition of complex IV activity and mitochondrial ROS release. RESULTS: Intralipid® enhanced postischemic recovery and activated Akt and Erk1/2, effects that were abolished by the ROS scavenger N-(2-mercaptopropionyl)glycine. Palmitoylcarnitine and linoleoylcarnitine, but not oleoylcarnitine concentration-dependently inhibited complex IV. Only palmitoylcarnitine reached high tissue concentrations during early reperfusion and generated significant ROS by complex IV inhibition. Palmitoylcarnitine (1 µM), administered at reperfusion, also fully mimicked Intralipid®-mediated protection in an N-(2-mercaptopropionyl)-glycine -dependent manner. CONCLUSIONS: Our data describe a new mechanism of postconditioning cardioprotection by the clinically available fat emulsion, Intralipid®. Protection is elicited by the fatty acid intermediate palmitoylcarnitine, and involves inhibition of complex IV, an increase in ROS production and activation of the RISK pathway.
Assuntos
Cardiotônicos/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/metabolismo , Palmitoilcarnitina/metabolismo , Fosfolipídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Óleo de Soja/farmacologia , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Emulsões/farmacologia , Coração/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Although evidence that type 2 diabetes mellitus (T2DM) is accompanied by mitochondrial dysfunction in skeletal muscle has been accumulating, a causal link between mitochondrial dysfunction and the pathogenesis of the disease remains unclear. Our study focuses on an early stage of the disease to determine whether mitochondrial dysfunction contributes to the development of T2DM. The fructose-fed (FF) rat was used as an animal model of early T2DM. Mitochondrial respiration and acylcarnitine species were measured in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscle. Although FF rats displayed characteristic signs of T2DM, including hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, mitochondrial content was preserved in both muscles from FF rats. The EDL muscle had reduced complex I and complex I and II respiration in the presence of pyruvate but not glutamate. The decrease in pyruvate-supported respiration was due to a decrease in pyruvate dehydrogenase activity. Accumulation of C14:1 and C14:2 acylcarnitine species and a decrease in respiration supported by long-chain acylcarnitines but not acetylcarnitine indicated dysfunctional ß-oxidation in the EDL muscle. In contrast, the soleus muscle showed preserved mitochondrial respiration, pyruvate dehydrogenase activity, and increased fatty acid oxidation, as evidenced by overall reduced acylcarnitine levels. Aconitase activity, a sensitive index of reactive oxygen species production in mitochondria, was reduced exclusively in EDL muscle, which showed lower levels of the antioxidant enzymes thioredoxin reductase and glutathione peroxidase. Here, we show that the glycolytic EDL muscle is more prone to an imbalance between energy supply and oxidation caused by insulin resistance than the oxidative soleus muscle.