RESUMO
Accurate measurements of metabolic fluxes in living cells are central to metabolism research and metabolic engineering. The gold standard method is model-based metabolic flux analysis (MFA), where fluxes are estimated indirectly from mass isotopomer data with the use of a mathematical model of the metabolic network. A critical step in MFA is model selection: choosing what compartments, metabolites, and reactions to include in the metabolic network model. Model selection is often done informally during the modelling process, based on the same data that is used for model fitting (estimation data). This can lead to either overly complex models (overfitting) or too simple ones (underfitting), in both cases resulting in poor flux estimates. Here, we propose a method for model selection based on independent validation data. We demonstrate in simulation studies that this method consistently chooses the correct model in a way that is independent on errors in measurement uncertainty. This independence is beneficial, since estimating the true magnitude of these errors can be difficult. In contrast, commonly used model selection methods based on the χ2-test choose different model structures depending on the believed measurement uncertainty; this can lead to errors in flux estimates, especially when the magnitude of the error is substantially off. We present a new approach for quantification of prediction uncertainty of mass isotopomer distributions in other labelling experiments, to check for problems with too much or too little novelty in the validation data. Finally, in an isotope tracing study on human mammary epithelial cells, the validation-based model selection method identified pyruvate carboxylase as a key model component. Our results argue that validation-based model selection should be an integral part of MFA model development.
Assuntos
Análise do Fluxo Metabólico , Modelos Biológicos , Isótopos de Carbono/metabolismo , Humanos , Marcação por Isótopo/métodos , IncertezaRESUMO
The metabolism of branched-chain amino acids (BCAA) has recently been implicated in the growth of several cancer cell types. Gabapentin, a synthetic amino acid, is commonly used in high concentrations in this context to inhibit the cytosolic branched-chain amino acid transferase (BCAT1) enzyme. Here, we report that 10 mM gabapentin reduces the growth of HCT116 cells, which have an active branched-chain amino acid transferase but express very low levels of BCAT1, and presumably rely on the mitochondrial BCAT2 enzyme. Gabapentin did not affect transamination of BCAA to branched-chain keto acids (BCKA) in HCT116 cells, nor the reverse formation of BCAA from BCKA, indicating that the branched-chain amino acid transaminase is not inhibited. Moreover, the growth-inhibitory effect of gabapentin could not be rescued by supplementation with BCKA, and this was not due to the lack of uptake of BCKA, indicating that other effects of gabapentin are important. An untargeted LC-MS analysis of gabapentin-treated cells revealed a marked depletion of branched-chain carnitines. These results demonstrate that gabapentin at high concentrations can inhibit cell proliferation without affecting BCAT1 and may affect mitochondrial BCKA catabolism.
Assuntos
Proliferação de Células/efeitos dos fármacos , Gabapentina/farmacologia , Transaminases/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Citosol/enzimologia , Células HCT116 , Humanos , Cetoácidos/metabolismo , Cinética , Mitocôndrias/metabolismoRESUMO
PP5 is a ubiquitously expressed Ser/Thr protein phosphatase. High levels of PP5 have been observed in human cancers, and constitutive PP5 overexpression aids tumor progression in mouse models of tumor development. However, PP5 is highly conserved among species, and the roles of PP5 in normal tissues are not clear. Here, to help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. In marked contrast to the early embryonic lethality associated with the genetic disruption of other PPP family phosphatases (e.g. PP2A and PP4), intercrosses with mouse lines that ubiquitously express Cre recombinase starting early in development (e.g. MeuCre40 and ACTB-Cre) produced viable and fertile PP5-deficient mice. Phenotypic differences caused by the total disruption of PP5 were minor, suggesting that small molecule inhibitors of PP5 will not have widespread systemic toxicity. Examination of roles for PP5 in fibroblasts generated from PP5-deficient embryos (PP5(-/-) mouse embryonic fibroblasts) confirmed some known roles and identified new actions for PP5. PP5(-/-) mouse embryonic fibroblasts demonstrated increased sensitivity to UV light, hydroxyurea, and camptothecin, which are known activators of ATR (ataxia-telangiectasia and Rad3-related) kinase. Further study revealed a previously unrecognized role for PP5 downstream of ATR activation in a UV light-induced response. The genetic disruption of PP5 is associated with enhanced and prolonged phosphorylation of a single serine (Ser-345) on Chk1, increased phosphorylation of the p53 tumor suppressor protein (p53) at serine 18, and increased p53 protein levels. A comparable role for PP5 in the regulation of Chk1 phosphorylation was also observed in human cells.
Assuntos
Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/metabolismo , Raios Ultravioleta , Actinas/genética , Alelos , Animais , Blastocisto/metabolismo , Cruzamento , Linhagem Celular , Quinase 1 do Ponto de Checagem , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Células HeLa , Humanos , Hidroxiureia/farmacologia , Integrases/genética , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/efeitos da radiação , Regiões Promotoras Genéticas/genética , Proteínas Quinases/química , Receptores de Neuropeptídeo Y/metabolismo , Serina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fosfatases cdc25/metabolismoRESUMO
Diabetes is a strong risk factor for premature and severe stroke. The GLP-1R (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic GotoKakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 µg/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 24 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-1R agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Peptídeos/uso terapêutico , Receptores de Glucagon/agonistas , Acidente Vascular Cerebral/tratamento farmacológico , Peçonhas/uso terapêutico , Animais , Isquemia Encefálica/patologia , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Avaliação Pré-Clínica de Medicamentos , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hiperglicemia/tratamento farmacológico , Masculino , Microglia/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Ratos , Volume Sistólico/efeitos dos fármacosRESUMO
The recently developed deep labeling method allows for large-scale profiling of metabolic activities in human cells or tissues using isotope tracing with a highly 13C enriched culture medium in combination with liquid chromatography-high resolution mass spectrometry. This method generates mass spectrometry data sets where endogenous cellular products can be identified, and active pathways can be determined from observed 13C mass isotopomers of the various metabolites measured. Here we describe in detail the experimental procedures for deep labeling experiments in cultured mammalian cells, including synthesis of the deep labeling medium, experimental considerations for cell culture, metabolite extractions and sample preparation, and liquid chromatography-mass spectrometry. We also outline a workflow for the downstream data analysis using publicly available software.
Assuntos
Isótopos de Carbono/química , Meios de Cultura/metabolismo , Metabolômica/métodos , Animais , Técnicas de Cultura de Células , Células Cultivadas , Cromatografia Líquida/métodos , Humanos , Marcação por Isótopo/métodos , Mamíferos/metabolismo , Software , Espectrometria de Massas em Tandem/métodosRESUMO
Studying metabolic activities in living cells is crucial for understanding human metabolism, but facile methods for profiling metabolic activities in an unbiased, hypothesis-free manner are still lacking. To address this need, we here introduce the deep-labeling method, which combines a custom 13C medium with high-resolution mass spectrometry. A proof-of-principle study on human cancer cells demonstrates that deep labeling can identify hundreds of endogenous metabolites as well as active and inactive pathways. For example, protein and nucleic acids were almost exclusively de novo synthesized, while lipids were partly derived from serum; synthesis of cysteine, carnitine, and creatine was absent, suggesting metabolic dependencies; and branched-chain keto acids (BCKAs) were formed and metabolized to short-chain acylcarnitines, but did not enter the tricarboxylic acid cycle. Remarkably, BCKAs could substitute for essential amino acids to support growth. The deep-labeling method may prove useful to map metabolic phenotypes across a range of cell types and conditions.
Assuntos
Metaboloma , Metabolômica/métodos , Neoplasias/metabolismo , Espectrometria de Massas em Tandem/métodos , Aminoácidos de Cadeia Ramificada/metabolismo , Isótopos de Carbono/metabolismo , Ciclo do Ácido Cítrico , Células HCT116 , Humanos , Marcação por Isótopo/métodos , Células MCF-7RESUMO
OBJECTIVE: Renal conservation (retention) of fluid might affect the outcome of hospital care and can be indicated by increased urinary concentrations of metabolic waste products. We obtained a reference material for further studies by exploring the prevalence of fluid retention in a healthy population. METHODS: Spot urine sampling was performed in 300 healthy hospital workers. A previously validated algorithm summarized the urine-specific gravity, osmolality, creatinine, and color to a fluid retention index (FRI), where 4.0 is the cut-off for fluid retention consistent with dehydration. In 50 of the volunteers, we also studied the relationships between FRI, plasma osmolality, and water-retaining hormones. RESULTS: The cut-off for fluid retention (FRI ≥ 4.0) was reached by 38% of the population. No correlation was found between the FRI and the time of the day of urine sample collection, and the FRI was only marginally correlated with the time period spent without fluid intake. Volunteers with fluid retention were younger, generally men, and more often had albuminuria (88% vs. 34%, P < 0.001). Plasma osmolality and plasma sodium were somewhat higher in those with a high FRI (mean 294.8 vs. 293.4 mosmol/kg and 140.3 vs. 139.9 mmol/l). Plasma vasopressin was consistently below the limit of detection, and the plasma cortisol, aldosterone, and renin concentrations were similar in subjects with a high or low FRI. The very highest FRI values (≥ 5.0, N = 61) were always accompanied by albuminuria. CONCLUSION: Fluid retention consistent with moderate dehydration is common in healthy staff working in a Swedish hospital.
Assuntos
Líquidos Corporais/química , Desidratação/urina , Urinálise , Adulto , Albuminúria/patologia , Aldosterona/sangue , Líquidos Corporais/metabolismo , Creatinina/urina , Estudos Transversais , Feminino , Pessoal de Saúde/estatística & dados numéricos , Humanos , Hidrocortisona/sangue , Masculino , Análise Multivariada , Concentração Osmolar , Renina/sangue , Gravidade Específica , Vasopressinas/sangueRESUMO
The prevalence of diabetes is increasing rapidly worldwide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing ß-cells, impaired glucose-sensitive insulin secretion from the ß-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to the stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating the phosphorylation of proteins involved in the insulin secretory process by the ß-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases. Herein, we review extant data implicating serine/threonine and tyrosine phosphatases in various aspects of healthy and diabetic islet biology, ranging from control of hormonal stimulus-secretion coupling to mitogenesis and apoptosis.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Modelos Biológicos , Fosfoproteínas Fosfatases/classificação , FosforilaçãoRESUMO
Regulation of mRNA translation is of vital importance for a cell to adapt to environmental changes. To serve this purpose, intricate mechanisms controlling mRNA translation have evolved, of which the eukaryotic initiation factors eIF2 and eIF4 represent essential regulatory nodes for both stress sensing and signal transduction. Stress sensing by eIF2 α subunit (eIF2α) kinases, translation regulation by eIF2α subunit phosphorylation and subsequent dephosphorylation constitute a core molecular switch for stress adaptation and rapid metabolic regulation. It is not surprising; therefore, that dysfunction of such a pathway is implicated in human disease, especially metabolic syndrome and diabetes. In theory, therapeutic intervention to target the eIF2α phosphorylation pathway provides a promising therapeutic solution to tackle this debilitating syndrome. Careful evaluation of such therapies is crucially needed considering the central role of eIF2α pathway in cellular function for every organ.
RESUMO
Glucocorticoid excess is associated with glucose intolerance and diabetes. In addition to inducing insulin resistance, glucocorticoids impair ß-cell function and cause ß-cell apoptosis. In this study we show that dexamethasone activates mitogen-activated protein kinases (MAPKs) signaling in MIN6 ß-cells, as evident by enhanced phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK). In contrast, the integrated stress response pathway was inhibited by dexamethasone. A p38 MAPK inhibitor attenuated dexamethasone-induced apoptosis in ß-cells and isolated islets and decreased glucocorticoid receptor phosphorylation at S220. In contrast, a JNK inhibitor augmented DNA fragmentation and dexamethasone-induced formation of cleaved caspase 3. We also show that inhibition of protein phosphatase 5 (PP5) augments apoptosis in dexamethasone-exposed islets and ß-cells, with a concomitant activation of p38 MAPK. In conclusion, our data provide evidence that in islets and ß-cells, p38 MAPK and JNK phosphorylation work in concert with PP5 to regulate the cytotoxic effects exerted by glucocorticoids.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Regulação da Expressão Gênica , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Estresse Fisiológico/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
BACKGROUND: Green tea was suggested as a therapeutic agent for the treatment of diabetes more than 70 years ago, but the mechanisms behind its antidiabetic effect remains elusive. In this work, we address this issue by feeding a green tea extract (TEAVIGO™) with a high content of epigallocatechin gallate (EGCG) or the thiazolidinedione PPAR-γ agonist rosiglitazone, as positive control, to db/db mice, an animal model for diabetes. METHODS: Young (7 week-old) db/db mice were randomized and assigned to receive diets supplemented with or without EGCG or rosiglitazone for 10 weeks. Fasting blood glucose, body weight and food intake was measured along the treatment. Glucose and insulin levels were determined during an oral glucose tolerance test after 10 weeks of treatment. Pancreata were sampled at the end of the study for blinded histomorphometric analysis. Islets were isolated and their mRNA expression analyzed by quantitative RT-PCR. RESULTS: The results show that, in db/db mice, EGCG improves glucose tolerance and increases glucose-stimulated insulin secretion. EGCG supplementation reduces the number of pathologically changed islets of Langerhans, increases the number and the size of islets, and heightens pancreatic endocrine area. These effects occurred in parallel with a reduction in islet endoplasmic reticulum stress markers, possibly linked to the antioxidative capacity of EGCG. CONCLUSIONS: This study shows that the green tea extract EGCG markedly preserves islet structure and enhances glucose tolerance in genetically diabetic mice. Dietary supplementation with EGCG could potentially contribute to nutritional strategies for the prevention and treatment of type 2 diabetes.