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Heparin and heparan sulfate (HS) are naturally occurring mammalian glycosaminoglycans, and their synthetic and semi-synthetic mimetics have attracted significant interest as potential therapeutics. However, understanding the mechanism of action by which HS, heparin, and HS mimetics have a biological effect is difficult due to their highly charged nature, broad protein interactomes, and variable structures. To address this, a library of novel single-entity dendritic mimetics conjugated to BODIPY, Fluorine-19 (19 F), and biotin was synthesized for imaging and localization studies. The novel dendritic scaffold allowed for the conjugation of labeling moieties without reducing the number of sulfated capping groups, thereby better mimicking the multivalent nature of HS-protein interactions. The 19 F labeled mimetics were assessed in phantom studies and were detected at concentrations as low as 5â mM. Flow cytometric studies using a fluorescently labeled mimetic showed that the compound associated with immune cells from tumors more readily than splenic counterparts and was directed to endosomal-lysosomal compartments within immune cells and cancer cells. Furthermore, the fluorescently labeled mimetic entered the central nervous system and was detectable in brain-infiltrating immune cells 24â hours after treatment. Here, we report the enabling methodology for rapidly preparing various labeled HS mimetics and molecular probes with diverse potential therapeutic applications.
Assuntos
Biotina , Compostos de Boro , Heparitina Sulfato , Animais , Heparitina Sulfato/química , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Mamíferos/metabolismoRESUMO
The tumor suppressor p53 is involved in the adaptation of hepatic metabolism to nutrient availability. Acute deletion of p53 in the mouse liver affects hepatic glucose and triglyceride metabolism. However, long-term adaptations upon the loss of hepatic p53 and its transcriptional regulators are unknown. Here we show that short-term, but not chronic, liver-specific deletion of p53 in mice reduces liver glycogen levels, and we implicate the transcription factor forkhead box O1 protein (FOXO1) in the regulation of p53 and its target genes. We demonstrate that acute p53 deletion prevents glycogen accumulation upon refeeding, whereas a chronic loss of p53 associates with a compensational activation of the glycogen synthesis pathway. Moreover, we identify fasting-activated FOXO1 as a repressor of p53 transcription in hepatocytes. We show that this repression is relieved by inactivation of FOXO1 by insulin, which likely mediates the upregulation of p53 expression upon refeeding. Strikingly, we find that high-fat diet-induced insulin resistance with persistent FOXO1 activation not only blunted the regulation of p53 but also the induction of p53 target genes like p21 during fasting, indicating overlapping effects of both FOXO1 and p53 on target gene expression in a context-dependent manner. Thus, we conclude that p53 acutely controls glycogen storage in the liver and is linked to insulin signaling via FOXO1, which has important implications for our understanding of the hepatic adaptation to nutrient availability.
Assuntos
Proteína Forkhead Box O1 , Homeostase , Glicogênio Hepático , Fígado , Proteína Supressora de Tumor p53 , Animais , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Deleção de Genes , Glucose/metabolismo , Hepatócitos/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Camundongos , Triglicerídeos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Disruption of the extracellular matrix at the blood-brain barrier (BBB) underpins neuroinflammation in multiple sclerosis (MS). The degradation of extracellular matrix components, such as heparan sulfate (HS) proteoglycans, can be prevented by treatment with HS-mimetics through their ability to inhibit the enzyme heparanase. The heparanase-inhibiting ability of our small dendrimer HS-mimetics has been investigated in various cancers but their efficacy in neuroinflammatory models has not been evaluated. This study investigates the use of a novel HS-mimetic, Tet-29, in an animal model of MS. METHODS: Neuroinflammation was induced in mice by experimental autoimmune encephalomyelitis, a murine model of MS. In addition, the BBB and choroid plexus were modelled in vitro using transmigration assays, and migration of immune cells in vivo and in vitro was quantified by flow cytometry. RESULTS: We found that Tet-29 significantly reduced lymphocyte accumulation in the central nervous system which, in turn, decreased disease severity in experimental autoimmune encephalomyelitis. The disease-modifying effect of Tet-29 was associated with a rescue of BBB integrity, as well as inhibition of activated lymphocyte migration across the BBB and choroid plexus in transwell models. In contrast, Tet-29 did not significantly impair in vivo or in vitro steady state-trafficking under homeostatic conditions. CONCLUSIONS: Together these results suggest that Tet-29 modulates, rather than abolishes, trafficking across central nervous system barriers.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Encefalomielite Autoimune Experimental/metabolismo , Doenças Neuroinflamatórias , Sistema Nervoso Central/metabolismo , Barreira Hematoencefálica/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.
Assuntos
Transdução de Sinais , Proteína Supressora de Tumor p53 , Carcinoma Hepatocelular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Nutrientes , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: The ability of skeletal muscle to respond adequately to changes in nutrient availability, known as metabolic flexibility, is essential for the maintenance of metabolic health and loss of flexibility contributes to the development of diabetes and obesity. The tumour suppressor protein, p53, has been linked to the control of energy metabolism. We assessed its role in the acute control of nutrient allocation in skeletal muscle in the context of limited nutrient availability. METHODS: A mouse model with inducible deletion of the p53-encoding gene, Trp53, in skeletal muscle was generated using the Cre-loxP-system. A detailed analysis of nutrient metabolism in mice with control and knockout genotypes was performed under ad libitum fed and fasting conditions and in exercised mice. RESULTS: Acute deletion of p53 in myofibres of mice activated catabolic nutrient usage pathways even under ad libitum fed conditions, resulting in significantly increased overall energy expenditure (+10.6%; P = 0.0385) and a severe nutrient deficit in muscle characterized by depleted intramuscular glucose and glycogen levels (-62,0%; P < 0.0001 and -52.7%; P < 0.0001, respectively). This was accompanied by changes in marker gene expression patterns of circadian rhythmicity and hyperactivity (+57.4%; P = 0.0068). These metabolic changes occurred acutely, within 2-3 days after deletion of Trp53 was initiated, suggesting a rapid adaptive response to loss of p53, which resulted in a transient increase in lactate release to the circulation (+46.6%; P = 0.0115) from non-exercised muscle as a result of elevated carbohydrate mobilization. Conversely, an impairment of proteostasis and amino acid metabolism was observed in knockout mice during fasting. During endurance exercise testing, mice with acute, muscle-specific Trp53 inactivation displayed an early exhaustion phenotype with a premature shift in fuel usage and reductions in multiple performance parameters, including a significantly reduced running time and distance (-13.8%; P = 0.049 and -22.2%; P = 0.0384, respectively). CONCLUSIONS: These findings suggest that efficient nutrient conservation is a key element of normal metabolic homeostasis that is sustained by p53. The homeostatic state in metabolic tissues is actively maintained to coordinate efficient energy conservation and metabolic flexibility towards nutrient stress. The acute deletion of Trp53 unlocks mechanisms that suppress the activity of nutrient catabolic pathways, causing substantial loss of intramuscular energy stores, which contributes to a fasting-like state in muscle tissue. Altogether, these findings uncover a novel function of p53 in the short-term regulation of nutrient metabolism in skeletal muscle and show that p53 serves to maintain metabolic homeostasis and efficient energy conservation.
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Active thermogenic adipocytes avidly consume energy substrates like fatty acids and glucose to maintain body temperature upon cold exposure. Despite strong evidence for the involvement of brown adipose tissue (BAT) in controlling systemic energy homeostasis upon nutrient excess, it is unclear how the activity of brown adipocytes is regulated in times of nutrient scarcity. Therefore, this study aimed to scrutinize factors that modulate BAT activity to balance thermogenic and energetic needs upon simultaneous fasting and cold stress. For an unbiased view, we performed transcriptomic and miRNA sequencing analyses of BAT from acutely fasted (24 h) mice under mild cold exposure. Combining these data with in-depth bioinformatic analyses and in vitro gain-of-function experiments, we define a previously undescribed axis of p53 inducing miR-92a-1-5p transcription that is highly upregulated by fasting in thermogenic adipocytes. p53, a fasting-responsive transcription factor, was previously shown to control genes involved in the thermogenic program and miR-92a-1-5p was found to negatively correlate with human BAT activity. Here, we identify fructose transporter Slc2a5 as one direct downstream target of this axis and show that fructose can be taken up by and metabolized in brown adipocytes. In sum, this study delineates a fasting-induced pathway involving p53 that transactivates miR-92a-1-5p, which in turn decreases Slc2a5 expression, and suggests fructose as an energy substrate in thermogenic adipocytes.
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Postprandial glycemic responses to meals are inhibited by polyphenol-rich plant foods. Combinations of polyphenols may be particularly effective through complementary mechanisms. A randomized, controlled, double-blinded cross-over trial was conducted in healthy volunteers to test the hypothesis that apple and blackcurrant polyphenol-rich drinks would reduce postprandial blood glucose concentrations. Secondary outcomes included insulin and glucose-dependent insulinotropic polypeptide (GIP) secretion. Twenty men (mean age 26 y, SD 8) and 5 postmenopausal women (mean age 57 y, SD 3) consumed a placebo drink (CON) and 2 polyphenol-rich drinks containing fruit extracts: either 1200 mg apple polyphenols (AE), or 600 mg apple polyphenols+600 mg blackcurrant anthocyanins (AE+BE), in random order with a starch and sucrose meal. Incremental areas under the curve (iAUC) for plasma glucose concentrations were lower following AE+BE over 0-30 and 0-120 min compared with CON; mean differences (95% CI) -32 mmol/L·min (-41, -22, P<.0005) and -52 mmol/L min (-94, -9, P<.05), respectively. AE significantly reduced iAUC 0-30 min (mean difference -26 mmol/L min, -35, -18, P<.0005) compared with CON, but the difference over 120 min was not significant. Postprandial insulin, C-peptide and GIP concentrations were significantly reduced relative to CON. A dose response inhibition of glucose transport was demonstrated in Caco-2 cells, including total and GLUT-mediated transport, and SGLT1-mediated glucose transport was strongly inhibited at all doses in Xenopus oocytes, following 10 min incubation with 0.125-4 mg apple polyphenols/ml. In conclusion, ingestion of apple and blackcurrant polyphenols decreased postprandial glycemia, which may be partly related to inhibition of intestinal glucose transport.
Assuntos
Bebidas , Dieta da Carga de Carboidratos/efeitos adversos , Frutas , Hiperglicemia/prevenção & controle , Malus , Polifenóis/uso terapêutico , Ribes , Adulto , Células CACO-2 , Estudos Cross-Over , Método Duplo-Cego , Enterócitos/metabolismo , Feminino , Frutas/química , Glucose/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/metabolismo , Hipoglicemiantes/metabolismo , Hipoglicemiantes/uso terapêutico , Incretinas/sangue , Incretinas/metabolismo , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Absorção Intestinal , Masculino , Malus/química , Extratos Vegetais/metabolismo , Extratos Vegetais/uso terapêutico , Polifenóis/metabolismo , Período Pós-Prandial , Ribes/químicaRESUMO
Dietary inclusion of fish and fish supplements as a means to improve cancer prognosis and prevent tumour growth is largely controversial. Long chain omega-3 polyunsaturated fatty acids (LCn-3 PUFA), eicosapentaenoic acid and docosahexaenoic acid, may modulate the production of inflammatory eicosanoids, thereby influencing local inflammatory status, which is important in cancer development. Although in vitro studies have demonstrated inhibition of tumour cell growth and proliferation by LCn-3 PUFA, results from human studies have been mainly inconsistent. Genes involved in the desaturation of fatty acids, as well as the genes encoding enzymes responsible for eicosanoid production, are known to be implicated in tumour development. This review discusses the current evidence for an interaction between genetic polymorphisms and dietary LCn-3 PUFA in the risk for breast, prostate and colorectal cancers, in regards to inflammation and eicosanoid synthesis.
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The prevalence of type 2 diabetes (T2D) is predicted to reach unprecedented levels in the next few decades. In addition to excess body weight, there may be other overlapping dietary drivers of impaired glucose homeostasis that are associated with an obesogenic diet, such as regular exposure to postprandial spikes in blood glucose arising from diets dominated by highly refined starches and added sugars. Strategies to reduce postprandial hyperglycaemia by optimising the functionality of foods would strengthen efforts to reduce the risk of T2D. Berry bioactives, including anthocyanins, are recognised for their inhibitory effects on carbohydrate digestion and glucose absorption. Regular consumption of berries has been associated with a reduction in the risk of T2D. This review aims to examine the evidence from in vitro, animal and human studies, showing that berries and berry anthocyanins may act in the gut to modulate postprandial glycaemia. Specifically, berry extracts and anthocyanins inhibit the activities of pancreatic α-amylase and α-glucosidase in the gut lumen, and interact with intestinal sugar transporters, sodium-dependent glucose transporter 1 and GLUT2, to reduce the rate of glucose uptake into the circulation. Growing evidence from randomised controlled trials suggests that berry extracts, purées and nectars acutely inhibit postprandial glycaemia and insulinaemia following oral carbohydrate loads. Evidence to date presents a sound basis for exploring the potential for using berries/berry extracts as an additional stratagem to weight loss, adherence to dietary guidelines and increasing physical exercise, for the prevention of T2D.
Assuntos
Antocianinas/análise , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/prevenção & controle , Frutas/química , Alimento Funcional/análise , Extratos Vegetais/análise , Animais , Antocianinas/farmacocinética , Disponibilidade Biológica , Glicemia/metabolismo , Dieta , Modelos Animais de Doenças , Índice Glicêmico , Humanos , Extratos Vegetais/farmacocinética , Período Pós-Prandial , Prevalência , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Increasing demand for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) containing fish oils is putting pressure on fish species and numbers. Fisheries provide fish for human consumption, supplement production and fish feeds and are currently supplying fish at a maximum historical rate, suggesting mass-scale fishing is no longer sustainable. However, the health properties of EPA and DHA long-chain (LC) omega-3 polyunsaturated fatty acids (PUFA) demonstrate the necessity for these oils in our diets. EPA and DHA from fish oils show favourable effects in inflammatory bowel disease, some cancers and cardiovascular complications. The high prevalence of these diseases worldwide indicates the requirement for alternative sources of LC-PUFA. Strategies have included plant-based fish diets, although this may compromise the health benefits associated with fish oils. Alternatively, stearidonic acid, the product of α-linolenic acid desaturation, may act as an EPA-enhancing fatty acid. Additionally, algae oils may be a promising omega-3 PUFA source for the future. Algae are beneficial for multiple industries, offering a source of biodiesel and livestock feeds. However, further research is required to develop efficient and sustainable LC-PUFA production from algae. This paper summarises the recent research for developing prospective substitutes for omega-3 PUFA and the current limitations that are faced.