RESUMEN
Schizophrenia (SCZ) is a complex neuropsychiatric disorder associated with altered bioenergetic pathways and mitochondrial dysfunction. Antipsychotic medications, both first and second-generation, are commonly prescribed to manage SCZ symptoms, but their direct impact on mitochondrial function remains poorly understood. In this study, we investigated the effects of commonly prescribed antipsychotics on bioenergetic pathways in cultured neurons. We examined the impact of risperidone, aripiprazole, amisulpride, and clozapine on gene expression, mitochondrial bioenergetic profile, and targeted metabolomics after 24-h treatment, using RNA-seq, Seahorse XF24 Flux Analyser, and gas chromatography-mass spectrometry (GC-MS), respectively. Risperidone treatment reduced the expression of genes involved in oxidative phosphorylation, the tricarboxylic acid cycle, and glycolysis pathways, and it showed a tendency to decrease basal mitochondrial respiration. Aripiprazole led to dose-dependent reductions in various mitochondrial function parameters without significantly affecting gene expression. Aripiprazole, amisulpride and clozapine treatment showed an effect on the tricarboxylic acid cycle metabolism, leading to more abundant metabolite levels. Antipsychotic drug effects on mitochondrial function in SCZ are multifaceted. While some drugs have greater effects on gene expression, others appear to exert their effects through enzymatic post-translational or allosteric modification of enzymatic activity. Understanding these effects is crucial for optimising treatment strategies for SCZ. Novel therapeutic interventions targeting energy metabolism by post-transcriptional pathways might be more effective as these can more directly and efficiently regulate energy production.
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Exercise stimulates mitochondrial biogenesis and increases mitochondrial respiratory function and content. However, during high-intensity exercise muscle pH can decrease below pH 6.8 with a concomitant increase in lactate concentration. This drop in muscle pH is associated with reduced exercise-induced mitochondrial biogenesis, while increased lactate may act as a signaling molecule to affect mitochondrial biogenesis. Therefore, in this study we wished to determine the impact of altering pH and lactate concentration in L6 myotubes on genes and proteins known to be involved in mitochondrial biogenesis. We also examined mitochondrial respiration in response to these perturbations. Differentiated L6 myotubes were exposed to normal (pH 7.5)-, low (pH 7.0)-, or high (pH 8.0)-pH media with and without 20 mM sodium l-lactate for 1 and 6 h. Low pH and 20 mM sodium l-lactate resulted in decreased Akt (Ser473) and AMPK (T172) phosphorylation at 1 h compared with controls, while at 6 h the nuclear localization of histone deacetylase 5 (HDAC5) was decreased. When the pH was increased both Akt (Ser473) and AMPK (T172) phosphorylation was increased at 1 h. Overall increased lactate decreased the nuclear content of HDAC5 at 6 h. Exposure to both high- and low-pH media decreased basal mitochondrial respiration, ATP turnover, and maximum mitochondrial respiratory capacity. These data indicate that muscle pH affects several metabolic signaling pathways, including those required for mitochondrial function.
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Histona Desacetilasas/metabolismo , Mitocondrias/metabolismo , Células Musculares/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Respiración de la Célula/fisiología , Células Cultivadas , Ejercicio Físico/fisiología , Humanos , Concentración de Iones de Hidrógeno , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Biogénesis de Organelos , Fosforilación/fisiología , Transducción de Señal/fisiologíaRESUMEN
There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus-a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata, a known pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC50) = 26.6 ± 0.74 µM) and L4 motility (IC50 = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC50 ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.
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Alcaloides/farmacología , Antihelmínticos/farmacología , Haemonchus/efectos de los fármacos , Alcaloides/química , Animales , Antihelmínticos/química , Australia , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Poríferos/química , RatasRESUMEN
AIM: To determine the effect of Scriptaid, a compound that can replicate aspects of the exercise adaptive response through disruption of the class IIa histone deacetylase (HDAC) corepressor complex, on muscle insulin action in obesity. MATERIALS AND METHODS: Diet-induced obese mice were administered Scriptaid (1 mg/kg) via daily intraperitoneal injection for 4 weeks. Whole-body and skeletal muscle metabolic phenotyping of mice was performed, in addition to echocardiography, to assess cardiac morphology and function. RESULTS: Scriptaid treatment had no effect on body weight or composition, but did increase energy expenditure, supported by increased lipid oxidation, while food intake was also increased. Scriptaid enhanced the expression of oxidative genes and proteins, increased fatty acid oxidation and reduced triglycerides and diacylglycerides in skeletal muscle. Furthermore, ex vivo insulin-stimulated glucose uptake by skeletal muscle was enhanced. Surprisingly, heart weight was reduced in Scriptaid-treated mice and was associated with enhanced expression of genes involved in oxidative metabolism in the heart. Scriptaid also improved indices of both diastolic and systolic cardiac function. CONCLUSION: These data show that pharmacological targeting of the class IIa HDAC corepressor complex with Scriptaid could be used to enhance muscle insulin action and cardiac function in obesity.
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Cardiotónicos/uso terapéutico , Metabolismo Energético/efectos de los fármacos , Corazón/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Hidroxilaminas/uso terapéutico , Músculo Esquelético/efectos de los fármacos , Obesidad/tratamiento farmacológico , Quinolinas/uso terapéutico , Animales , Fármacos Antiobesidad/efectos adversos , Fármacos Antiobesidad/uso terapéutico , Cardiotónicos/efectos adversos , Dieta Alta en Grasa/efectos adversos , Ecocardiografía , Ecocardiografía Doppler , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Corazón/diagnóstico por imagen , Corazón/fisiopatología , Histona Desacetilasa 2/antagonistas & inhibidores , Histona Desacetilasa 2/metabolismo , Inhibidores de Histona Desacetilasas/efectos adversos , Hidroxilaminas/efectos adversos , Resistencia a la Insulina , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Miocardio/patología , Obesidad/etiología , Obesidad/patología , Obesidad/fisiopatología , Tamaño de los Órganos , Quinolinas/efectos adversosRESUMEN
BACKGROUND: The prevalence of type 2 diabetes is rapidly increasing world-wide and insulin resistance is central to the aetiology of this disease. The biology underpinning the development of insulin resistance is not completely understood and the role of impaired mitochondrial function in the development of insulin resistance is controversial. SCOPE OF REVIEW: This review will provide an overview of the major processes regulated by mitochondria, before examining the evidence that has investigated the relationship between mitochondrial function and insulin action. Further considerations aimed at clarifying some controversies surrounding this issue will also be proposed. MAJOR CONCLUSIONS: Controversy on this issue is fuelled by our lack of understanding of some of the basic biological interactions between mitochondria and insulin regulated processes in the context of insults thought to induce insulin resistance. Aspects that have not yet been considered are tissue/cell type specific responses, mitochondrial responses to site-specific impairments in mitochondrial function and as yet uncharacterised retrograde signalling from mitochondria. GENERAL SIGNIFICANCE: Further investigation of the relationship between mitochondria and insulin action could reveal novel mechanisms contributing to insulin resistance in specific patient subsets. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.
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Diabetes Mellitus Tipo 2/etiología , Resistencia a la Insulina , Mitocondrias/fisiología , Animales , Apoptosis/fisiología , Señalización del Calcio/fisiología , Metabolismo Energético/fisiología , Humanos , Insulina/fisiologíaRESUMEN
Emerging evidence indicates that skeletal muscle lipid droplets are an important control point for intracellular lipid homeostasis and that regulating fatty acid fluxes from lipid droplets might influence mitochondrial capacity. We used pharmacological blockers of the major triglyceride lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase, to show that a large proportion of the fatty acids that are transported into myotubes are trafficked through the intramyocellular triglyceride pool. We next tested whether increasing lipolysis from intramyocellular lipid droplets could activate transcriptional responses to enhance mitochondrial and fatty acid oxidative capacity. ATGL was overexpressed by adenoviral and adenoassociated viral infection in C2C12 myotubes and the tibialis anterior muscle of C57Bl/6 mice, respectively. ATGL overexpression in C2C12 myotubes increased lipolysis, which was associated with increased peroxisome proliferator-activated receptor (PPAR)-∂ activity, transcriptional upregulation of some PPAR∂ target genes, and enhanced mitochondrial capacity. The transcriptional responses were specific to ATGL actions and not a generalized increase in fatty acid flux in the myotubes. Marked ATGL overexpression (20-fold) induced modest molecular changes in the skeletal muscle of mice, but these effects were not sufficient to alter fatty acid oxidation. Together, these data demonstrate the importance of lipid droplets for myocellular fatty acid trafficking and the capacity to modulate mitochondrial capacity by enhancing lipid droplet lipolysis in vitro; however, this adaptive program is of minor importance when superimposing the normal metabolic stresses encountered in free-moving animals.
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Lipasa/fisiología , Metabolismo de los Lípidos/genética , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Triglicéridos/metabolismo , Animales , Células Cultivadas , Ácidos Grasos/metabolismo , Lipólisis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción , Receptores Activados del Proliferador del Peroxisoma/metabolismoRESUMEN
Altered metabolism in tissues such as the liver, skeletal muscle and adipose tissue is observed in metabolic diseases characterized by nutrient excess and energy imbalance, such as obesity and type 2 diabetes. These alterations in metabolism can include resistance to the hormone insulin, lipid accumulation, mitochondrial dysfunction and transcriptional remodelling of major metabolic pathways. The underlying assumption has been that these same alterations in metabolism are fundamental to the pathogenesis of metabolic diseases. An alternative view is that these alterations in metabolism occur to protect cell and tissue viability in the face of constant positive energy balance. This speculative review presents evidence that many of the metabolic adaptations that occur in metabolic diseases characterized by nutrient excess can be viewed as protective in nature, rather than pathogenic per se for disease progression. Finally, we also briefly discuss the usefulness and potential pitfalls of therapeutic approaches that attempt to correct these same metabolic defects when energy balance is not altered, and the potential links between metabolic survival responses and other chronic diseases such as cancer.
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Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético/fisiología , Resistencia a la Insulina/fisiología , Obesidad/metabolismo , Hipernutrición/metabolismo , Animales , Diabetes Mellitus Tipo 2/diagnóstico , Humanos , Obesidad/diagnóstico , Hipernutrición/diagnósticoRESUMEN
There are epidemiological associations between obesity and type 2 diabetes, cardiovascular disease and Alzheimer's disease. The role of amyloid beta 42 (Aß42) in these diverse chronic diseases is obscure. Here we show that adipose tissue releases Aß42, which is increased from adipose tissue of male mice with obesity and is associated with higher plasma Aß42. Increasing circulating Aß42 levels in male mice without obesity has no effect on systemic glucose homeostasis but has obesity-like effects on the heart, including reduced cardiac glucose clearance and impaired cardiac function. The closely related Aß40 isoform does not have these same effects on the heart. Administration of an Aß-neutralising antibody prevents obesity-induced cardiac dysfunction and hypertrophy. Furthermore, Aß-neutralising antibody administration in established obesity prevents further deterioration of cardiac function. Multi-contrast transcriptomic analyses reveal that Aß42 impacts pathways of mitochondrial metabolism and exposure of cardiomyocytes to Aß42 inhibits mitochondrial complex I. These data reveal a role for systemic Aß42 in the development of cardiac disease in obesity and suggest that therapeutics designed for Alzheimer's disease could be effective in combating obesity-induced heart failure.
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Enfermedad de Alzheimer , Diabetes Mellitus Tipo 2 , Masculino , Ratones , Animales , Péptidos beta-Amiloides , Diabetes Mellitus Tipo 2/complicaciones , Anticuerpos Neutralizantes , Obesidad/complicaciones , Glucosa , Fragmentos de PéptidosRESUMEN
UNLABELLED: Caveolin-1 (CAV1) is a structural protein of caveolae involved in lipid homeostasis and endocytosis. Using newly generated pure Balb/C CAV1 null ((Balb/C)CAV1-/-) mice, CAV1-/- mice from Jackson Laboratories ((JAX)CAV1-/-), and CAV1-/- mice developed in the Kurzchalia Laboratory ((K)CAV1-/-), we show that under physiological conditions CAV1 expression in mouse tissues is necessary to guarantee an efficient progression of liver regeneration and mouse survival after partial hepatectomy. Absence of CAV1 in mouse tissues is compensated by the development of a carbohydrate-dependent anabolic adaptation. These results were supported by extracellular flux analysis of cellular glycolytic metabolism in CAV1-knockdown AML12 hepatocytes, suggesting cell autonomous effects of CAV1 loss in hepatic glycolysis. Unlike in (K)CAV1-/- livers, in (JAX)CAV1-/- livers CAV1 deficiency is compensated by activation of anabolic metabolism (pentose phosphate pathway and lipogenesis) allowing liver regeneration. Administration of 2-deoxy-glucose in (JAX)CAV1-/- mice indicated that liver regeneration in (JAX)CAV1-/- mice is strictly dependent on hepatic carbohydrate metabolism. Moreover, with the exception of regenerating (JAX)CAV1-/- livers, expression of CAV1 in mice is required for efficient hepatic lipid storage during fasting, liver regeneration, and diet-induced steatosis in the three CAV1-/- mouse strains. Furthermore, under these conditions CAV1 accumulates in the lipid droplet fraction in wildtype mouse hepatocytes. CONCLUSION: Our data demonstrate that lack of CAV1 alters hepatocyte energy metabolism homeostasis under physiological and pathological conditions.
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Caveolina 1/metabolismo , Metabolismo Energético/fisiología , Glucosa/metabolismo , Regeneración Hepática/fisiología , Análisis de Varianza , Animales , Análisis Químico de la Sangre , Proliferación Celular , Cromatografía en Capa Delgada/métodos , Desoxiglucosa/farmacología , Modelos Animales de Enfermedad , Femenino , Hepatectomía , Hepatocitos/metabolismo , Hepatocitos/fisiología , Homeostasis , Metabolismo de los Lípidos/fisiología , Regeneración Hepática/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle, resulting in increased non-oxidative glucose metabolism. This metabolic reprogramming was also associated with impaired apoptosis and ferroptosis responses, and preserved muscle cell viability in response to lipotoxicity. Mechanistically, increased HDAC4 and 5 decreased acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis. Redox drivers of ferroptosis derived from oxidative metabolism were also reduced. The relevance of this pathway was demonstrated by overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice, which enhanced oxidative metabolic capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of skeletal muscle oxidative metabolism, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.
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Histona Desacetilasas , Células Musculares , Ratones , Animales , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Procesamiento Proteico-Postraduccional , Muerte CelularRESUMEN
Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from 'total GSH'. The linear range and limit of detection for both analytes were 7.5 × 10(-7) to 1 × 10(-5) M, and 5 × 10(-7) M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL(-1)), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL(-1) glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL(-1) (p < 0.05), which was rationalised by considering measurements of H(2)O(2) and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL(-1) glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.
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Cromatografía Líquida de Alta Presión/métodos , Disulfuro de Glutatión/análisis , Glutatión/análisis , Mediciones Luminiscentes/métodos , Permanganato de Potasio/química , Animales , Línea Celular , Análisis de Inyección de Flujo , Glucosa Oxidasa/metabolismo , Peróxido de Hidrógeno/análisis , Ratones , Mioblastos/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVES: To investigate the actions of lithium, valproate, lamotrigine and quetiapine on bioenergetic pathways in cultured NT2-N neuronal-like cells and C8-B4 microglial cells. METHODS: NT2-N and C8-B4 cells were cultured and treated with lithium (2.5 mM), valproate (0.5 mM), quetiapine (0.05 mM) or lamotrigine (0.05 mM) for 24 hours. Gene expression and the mitochondrial bioenergetic profile were measured in both cell lines. RESULTS: In NT2-N cells, valproate increased oxidative phosphorylation (OXPHOS) gene expression, mitochondrial uncoupling and maximal respiratory capacity, while quetiapine decreased OXPHOS gene expression and respiration linked to ATP turnover, as well as decreasing the expression of genes in the citric acid cycle. Lamotrigine decreased OXPHOS gene expression but had no effect on respiration, while lithium reduced the expression of genes in the citric acid cycle. In C8-B4 cells, valproate and lithium increased OXPHOS gene expression, and valproate increased basal respiratory rate and maximal and spare respiratory capacities. In contrast, quetiapine significantly reduced basal respiratory rate and maximal and spare respiratory capacities. CONCLUSIONS: Overall our data suggest that some drugs used to treat neuropsychiatric and affective disorders have actions on a range of cellular bioenergetic processes, which could impact their effects in patients.
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Metabolismo Energético , Fosforilación Oxidativa , Humanos , Psicotrópicos , Fumarato de Quetiapina/farmacología , Ácido Valproico/farmacologíaRESUMEN
A series of 1-methyl-1H-pyrazole-5-carboxamides were synthesized as potent inhibitors of the parasitic nematode of sheep, Haemonchus contortus. These compounds did not show overt cytotoxicity to a range of mammalian cell lines under standard in vitro culture conditions, had high selectivity indices, and were progressed to an acute toxicity study in a rodent model. Strikingly, acute toxicity was observed in mice. Experiments measuring cellular respiration showed a dose-dependent inhibition of mitochondrial respiration. Under these conditions, potent cytotoxicity was observed for these compounds in rat hepatocytes suggesting that the potent acute mammalian toxicity of this chemotype is most likely associated with respiratory inhibition. In contrast, parasite toxicity was not correlated to acute toxicity or cytotoxicity in respiring cells. This paper highlights the importance of identifying an appropriate in vitro predictor of in vivo toxicity early on in the drug discovery pipeline, in particular assessment for in vitro mitochondrial toxicity.
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Antiprotozoarios/farmacología , Haemonchus/efectos de los fármacos , Pirazoles/química , Animales , Antiprotozoarios/química , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Pirazoles/farmacología , Ratas , Ovinos/parasitología , Relación Estructura-ActividadRESUMEN
OBJECTIVE: Protein kinase D (PKD) signaling has been implicated in stress-induced cardiac remodeling and function as well as metabolic processes including contraction-mediated cardiac glucose uptake. PKD has recently emerged as a nutrient-sensing kinase that is activated in high-lipid environments, such as in obesity. However, the role of PKD signaling in cardiac glucose metabolism and cardiac function in both normal and obese conditions remains unknown. METHODS: A cardiac-specific and inducible dominant negative (DN) PKD mouse model was developed. Echocardiography was used to assess cardiac function, while metabolic phenotyping was performed, including stable isotope metabolomics on cardiac tissue in mice fed either regular chow or a high-fat diet (43% calories from fat). RESULTS: Cardiac PKD activity declined by â¼90% following DN PKD induction in adult mice. The mice had diminished basal cardiac glucose clearance, suggesting impaired contraction-mediated glucose uptake, but normal cardiac function. In obesity studies, systolic function indices were reduced in control mice, but not in cardiac DN PKD mice. Using targeted stable isotope metabolomic analyses, no differences in glucose flux through glycolysis or the TCA cycle were observed between groups. CONCLUSIONS: The data show that PKD contributes to cardiac dysfunction in obesity and highlight the redundancy in cardiac glucose metabolism that maintains cardiac glucose flux in vivo. The data suggest that impairments in contraction-mediated glucose uptake are unlikely to drive cardiac dysfunction in both normal and metabolic disease states.
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Glucosa/metabolismo , Miocardio/metabolismo , Proteína Quinasa C/metabolismo , Animales , Dieta Alta en Grasa , Femenino , Técnicas de Sustitución del Gen/métodos , Corazón/fisiología , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Obesidad/metabolismo , Obesidad/fisiopatología , Fosforilación , Proteína Quinasa C/genética , Transducción de SeñalRESUMEN
A vast amount of public RNA-sequencing datasets have been generated and used widely to study transcriptome mechanisms. These data offer precious opportunity for advancing biological research in transcriptome studies such as alternative splicing. We report the first large-scale integrated analysis of RNA-Seq data of splicing factors for systematically identifying key factors in diseases and biological processes. We analyzed 1,321 RNA-Seq libraries of various mouse tissues and cell lines, comprising more than 6.6 TB sequences from 75 independent studies that experimentally manipulated 56 splicing factors. Using these data, RNA splicing signatures and gene expression signatures were computed, and signature comparison analysis identified a list of key splicing factors in Rett syndrome and cold-induced thermogenesis. We show that cold-induced RNA-binding proteins rescue the neurite outgrowth defects in Rett syndrome using neuronal morphology analysis, and we also reveal that SRSF1 and PTBP1 are required for energy expenditure in adipocytes using metabolic flux analysis. Our study provides an integrated analysis for identifying key factors in diseases and biological processes and highlights the importance of public data resources for identifying hypotheses for experimental testing.
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Factores de Empalme de ARN , RNA-Seq , Adipocitos/metabolismo , Empalme Alternativo , Animales , Línea Celular , Frío , Conjuntos de Datos como Asunto , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ratones , Proteína de Unión al Tracto de Polipirimidina/genética , Síndrome de Rett/genética , Factores de Empalme Serina-Arginina/genética , Termogénesis/genética , TranscriptomaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
BACKGROUND: Increased flux through both glycolytic and oxidative metabolic pathways is a hallmark of breast cancer cells and is critical for their growth and survival. As such, targeting this metabolic reprograming has received much attention as a potential treatment approach. However, the heterogeneity of breast cancer cell metabolism, even within classifications, suggests a necessity for an individualised approach to treatment in breast cancer patients. METHODS: The metabolic phenotypes of a diverse panel of human breast cancer cell lines representing the major breast cancer classifications were assessed using real-time metabolic flux analysis. Flux linked to ATP production, pathway reserve capacities and specific macromolecule oxidation rates were quantified. Suspected metabolic vulnerabilities were targeted with specific pathway inhibitors, and relative cell viability was assessed using the crystal violet assay. Measures of AMPK and mTORC1 activity were analysed through immunoblotting. RESULTS: Breast cancer cells displayed heterogeneous energy requirements and utilisation of non-oxidative and oxidative energy-producing pathways. Quantification of basal glycolytic and oxidative reserve capacities identified cell lines that were highly dependent on individual pathways, while assessment of substrate oxidation relative to total oxidative capacity revealed cell lines that were highly dependent on individual macromolecules. Based on these findings, mild mitochondrial inhibition in ESH-172 cells, including with the anti-diabetic drug metformin, and mild glycolytic inhibition in Hs578T cells reduced relative viability, which did not occur in non-transformed MCF10a cells. The effects on viability were associated with AMPK activation and inhibition of mTORC1 signalling. Hs578T were also found to be highly dependent on glutamine oxidation and inhibition of this process also impacted viability. CONCLUSIONS: Together, these data highlight that systematic flux analysis in breast cancer cells can identify targetable metabolic vulnerabilities, despite heterogeneity in metabolic profiles between individual cancer cell lines.
RESUMEN
A wealth of epidemiological data has found that patients with type 2 diabetes have a greater risk of developing breast cancer. The molecular mechanisms underpinning this relationship are yet to be elucidated; however, this review examines the available evidence suggesting that the metabolic abnormalities observed in type 2 diabetes can predispose to the development of breast cancer. Alterations in substrate availability and the hormonal milieu, particularly hyperinsulinemia, not only create a favorable metabolic environment for tumorigenesis, but also induce metabolic reprogramming events that are required for the transformation of breast cancer cells. In addition, the dysfunction and hypoxia of adipose tissue surrounding the breast cancer niche is another putative link that will be discussed. Finally, the mechanisms by which breast cancer cells evade checkpoints associated with nutrient overload will be examined. Experimentally validating these potential links will be important for prediction and treatment of breast cancer in patients with type 2 diabetes.
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Neoplasias de la Mama/etiología , Reprogramación Celular , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Susceptibilidad a Enfermedades/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Microambiente Celular/fisiología , Diabetes Mellitus Tipo 2/patología , Femenino , Humanos , Hiperinsulinismo/complicaciones , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patología , Obesidad/complicaciones , Obesidad/metabolismoRESUMEN
The antioxidant Selenoprotein S (Seps1, Selenos) is an endoplasmic reticulum (ER)-resident protein associated with metabolic and inflammatory disease. While Seps1 is highly expressed in skeletal muscle, its mechanistic role as an antioxidant in skeletal muscle cells is not well characterized. In C2C12 myotubes treated with palmitate for 24 h, endogenous Seps1 protein expression was upregulated twofold. Two different siRNA constructs were used to investigate whether decreased levels of Seps1 exacerbated lipid-induced oxidative and ER stress in C2C12 myotubes and myoblasts, which differ with regards to cell cycle state and metabolic phenotype. In myoblasts, Seps1 protein knockdown of ~50% or ~75% exacerbated cellular stress responses in the presence of palmitate; as indicated by decreased cell viability and proliferation, higher H2 O2 levels, a lower reduced to oxidized glutathione (GSH:GSSG) ratio, and enhanced gene expression of ER and oxidative stress markers. Even in the absence of palmitate, Seps1 knockdown increased oxidative stress in myoblasts. Whereas, in myotubes in the presence of palmitate, a ~50% knockdown of Seps1 was associated with a trend toward a marginal (3-5%) decrease in viability (P = 0.05), decreased cellular ROS levels, and a reduced mRNA transcript abundance of the cellular stress marker thioredoxin inhibitory binding protein (Txnip). Furthermore, no enhancement of gene markers of ER stress was observed in palmitate-treated myotubes in response to Seps1 knockdown. In conclusion, reduced Seps1 levels exacerbate nutrient-induced cellular stress responses to a greater extent in glycolytic, proliferating myoblasts than in oxidative, differentiated myotubes, thus demonstrating the importance of cell phenotype to Seps1 function.
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Estrés del Retículo Endoplásmico , Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Estrés Oxidativo , Selenoproteínas/metabolismo , Animales , Línea Celular , Proliferación Celular , Proteínas de la Membrana/genética , Ratones , Fibras Musculares Esqueléticas/fisiología , Mioblastos/fisiología , Selenoproteínas/genéticaRESUMEN
Drugs that recapitulate aspects of the exercise adaptive response have the potential to provide better treatment for diseases associated with physical inactivity. We previously observed reduced skeletal muscle class IIa HDAC (histone deacetylase) transcriptional repressive activity during exercise. Here, we find that exercise-like adaptations are induced by skeletal muscle expression of class IIa HDAC mutants that cannot form a corepressor complex. Adaptations include increased metabolic gene expression, mitochondrial capacity, and lipid oxidation. An existing HDAC inhibitor, Scriptaid, had similar phenotypic effects through disruption of the class IIa HDAC corepressor complex. Acute Scriptaid administration to mice increased the expression of metabolic genes, which required an intact class IIa HDAC corepressor complex. Chronic Scriptaid administration increased exercise capacity, whole-body energy expenditure and lipid oxidation, and reduced fasting blood lipids and glucose. Therefore, compounds that disrupt class IIa HDAC function could be used to enhance metabolic health in chronic diseases driven by physical inactivity.