RESUMO
Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.
Assuntos
Encéfalo , Colina , Proteínas de Membrana Transportadoras , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Colina/metabolismo , Microscopia Crioeletrônica , Técnicas In Vitro , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Modelos MolecularesRESUMO
Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.
Assuntos
Ácido Ascórbico , Purinas , Animais , Transporte Biológico , Purinas/metabolismo , Mutação , Sítios de Ligação , Ácido Ascórbico/metabolismo , Mamíferos/metabolismoRESUMO
Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter's central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.
Assuntos
Artemisininas/uso terapêutico , Cloroquina/uso terapêutico , Resistência a Múltiplos Medicamentos , Proteínas de Membrana Transportadoras/genética , Piperazinas/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Quinolinas/uso terapêutico , Alelos , Animais , Antimaláricos/uso terapêutico , Simulação por Computador , Humanos , Malária Falciparum/parasitologiaRESUMO
Brown adipose tissue (BAT) dysfunction in aging and obesity has been related to chronic unresolved inflammation, which could be mediated by an impaired production of specialized proresolving lipid mediators (SPMs), such as Lipoxins-LXs, Resolvins-Rvs, Protectins-PDs, and Maresins-MaRs. Our aim was to characterize the changes in BAT SPMs signatures and their association with BAT dysfunction during aging, especially under obesogenic conditions, and their modulation by a docosahexaenoic acid (DHA)-rich diet. Lipidomic, functional, and molecular studies were performed in BAT of 2- and 18-month-old lean (CT) female mice and in 18-month-old diet-induced obese (DIO) mice fed with a high-fat diet (HFD), or a DHA-enriched HFD. Aging downregulated Prdm16 and UCP1 levels, especially in DIO mice, while DHA partially restored them. Arachidonic acid (AA)-derived LXs and DHA-derived MaRs and PDs were the most abundant SPMs in BAT of young CT mice. Interestingly, the sum of LXs and of PDs were significantly lower in aged DIO mice compared to young CT mice. Some of the SPMs most significantly reduced in obese-aged mice included LXB4 , MaR2, 4S,14S-diHDHA, 10S,17S-diHDHA (a.k.a. PDX), and RvD6. In contrast, DHA increased DHA-derived SPMs, without modifying LXs. However, MicroPET studies showed that DHA was not able to counteract the impaired cold exposure response in BAT of obese-aged mice. Our data suggest that a defective SPMs production could underlie the decrease of BAT activity observed in obese-aged mice, and highlight the relevance to further characterize the physiological role and therapeutic potential of specific SPMs on BAT development and function.
Assuntos
Tecido Adiposo Marrom/metabolismo , Envelhecimento/patologia , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Lipídeos/análise , Obesidade/fisiopatologia , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/patologia , Animais , Dieta Hiperlipídica , Feminino , Metabolismo dos Lipídeos , Lipidômica , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
GLUT12 was cloned from the mammary cancer cell line MCF-7, but its physiological role still needs to be elucidated. To gain more knowledge of GLUT12 function in the intestine, we investigated GLUT12 subcellular localization in the small intestine and its regulation by sugars, hormones, and intracellular mediators in Caco-2 cells and mice. Immunohistochemical methods were used to determine GLUT12 subcellular localization in human and murine small intestine. Brush border membrane vesicles were isolated for western blot analyses. Functional studies were performed in Caco-2 cells by measuring α-methyl-d-glucose (αMG) uptake in the absence of sodium. GLUT12 is located in the apical cytoplasm, below the brush border membrane, and in the perinuclear region of murine and human enterocytes. In Caco-2 cells, GLUT12 translocation to the apical membrane and α-methyl- d-glucose uptake by the transporter are stimulated by protons, glucose, insulin, tumor necrosis factor-α (TNF-α), protein kinase C, and AMP-activated protein kinase. In contrast, hypoxia decreases GLUT12 expression in the apical membrane. Upregulation of TNF-α and hypoxia-inducible factor-1α ( HIF-1α) genes is found in the jejunal mucosa of diet-induced obese mice. In these animals, GLUT12 expression in the brush border membrane is slightly decreased compared with lean animals. Moreover, an intraperitoneal injection of insulin does not induce GLUT12 translocation to the membrane, as it occurs in lean animals. GLUT12 rapid translocation to the enterocytes' apical membrane in response to glucose and insulin could be related to GLUT12 participation in sugar absorption during postprandial periods. In obesity, in which insulin sensitivity is reduced, the contribution of GLUT12 to sugar absorption is affected.
Assuntos
Colo/metabolismo , Enterócitos/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Absorção Intestinal , Intestino Delgado/metabolismo , Metilglucosídeos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células CACO-2 , Hipóxia Celular , Colo/citologia , Colo/efeitos dos fármacos , Modelos Animais de Doenças , Enterócitos/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Insulina/farmacologia , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/metabolismo , Proteína Quinase C/metabolismo , Transporte Proteico , Ratos Wistar , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We have previously demonstrated in Caco-2 cells that tumor necrosis factor-α (TNF-α) inhibits sugar uptake, acting from the apical membrane, by decreasing the expression of the Na+ -glucose cotransporter SGLT1 in the brush border membrane. The goal was to investigate the hypothesis that TNF-α from abdominal adipose tissue (adipocytes and macrophages) would decrease sugar and amino acid transport acting from the basolateral membrane of the enterocytes. TNF-α placed in the basal compartment of Caco-2 cells decreased α-methyl- d-glucose (αMG) and glutamine uptake. The apical medium derived from these Caco-2 cells apically placed in another set of cells, also reduced sugar and glutamine transport. Reverse-transcription polymerase chain reaction analysis demonstrated upregulation of TNF-α, IL-1ß, and MCP1 expression in Caco-2 cells exposed to basal TNF-α. Similarly, αMG uptake was inhibited after Caco-2 cells were incubated, in the basal compartment, with medium from visceral human mesenchymal stem cells-derived adipocytes of overweight individuals. The apical medium collected from those Caco-2 cells, and placed in the upper side of other set of cells, also decreased sugar uptake. Basal presence of medium derived from lipopolysaccharide-activated macrophages and nonactivated macrophages decreased αMG uptake as well. Diet-induced obese mice showed an increase in the visceral adipose tissue surrounding the intestine. In this physiological condition, there was a reduction on αMG uptake in jejunal everted rings. Altogether, these results suggest that basolateral TNF-α, which can be produced by adipocytes and macrophages during obesity, would be able to activate TNF-α and other proinflammatory proteins expression in the small intestine and diminish intestinal sugar and amino acids transport.
Assuntos
Adipócitos/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Metilglucosídeos/metabolismo , Comunicação Parácrina , Transportador 1 de Glucose-Sódio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células CACO-2 , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Glutamina/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Via Secretória , Transdução de Sinais , Células THP-1 , Fator de Necrose Tumoral alfa/genéticaRESUMO
Pendrin (SLC26A4) is an anion exchanger that mediates bicarbonate (HCO3-) exchange for chloride (Cl-) and is crucial for maintaining pH and salt homeostasis in the kidney, lung, and cochlea. Pendrin also exports iodide (I-) in the thyroid gland. Pendrin mutations in humans lead to Pendred syndrome, causing hearing loss and goiter. Inhibition of pendrin is a validated approach for attenuating airway hyperresponsiveness in asthma and for treating hypertension. However, the mechanism of anion exchange and its inhibition by drugs remains poorly understood. We applied cryo-electron microscopy to determine structures of pendrin from Sus scrofa in the presence of either Cl-, I-, HCO3- or in the apo-state. The structures reveal two anion-binding sites in each protomer, and functional analyses show both sites are involved in anion exchange. The structures also show interactions between the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) and transmembrane domains, and mutational studies suggest a regulatory role. We also determine the structure of pendrin in a complex with niflumic acid (NFA), which uncovers a mechanism of inhibition by competing with anion binding and impeding the structural changes necessary for anion exchange. These results reveal directions for understanding the mechanisms of anion selectivity and exchange and their regulations by the STAS domain. This work also establishes a foundation for analyzing the pathophysiology of mutations associated with Pendred syndrome.
Assuntos
Perda Auditiva Neurossensorial , Humanos , Bicarbonatos , Cloretos , Microscopia Crioeletrônica , Halogênios , Suínos , AnimaisRESUMO
Obesity constitutes a global health epidemic which worsens the main leading death causes such as type 2 diabetes, cardiovascular diseases, and cancer. Changes in the metabolism in patients with obesity frequently lead to insulin resistance, along with hyperglycemia, dyslipidemia and low-grade inflammation, favoring a more aggressive tumor microenvironment. One of the hallmarks of cancer is the reprogramming of the energy metabolism, in which tumor cells change oxidative phosphorylation to aerobic glycolysis or "Warburg effect". Aerobic glycolysis is faster than oxidative phosphorylation, but less efficient in terms of ATP production. To obtain sufficient ATP, tumor cells increase glucose uptake by the glucose transporters of the GLUT/SLC2 family. The human glucose transporter GLUT12 was isolated from the breast cancer cell line MCF7. It is expressed in adipose tissue, skeletal muscle and small intestine, where insulin promotes its translocation to the plasma membrane. Moreover, GLUT12 over-expression in mice increases the whole-body insulin sensitivity. Thus, GLUT12 has been proposed as a second insulin-responsive glucose transporter. In obesity, GLUT12 is downregulated and does not respond to insulin. In contrast, GLUT12 is overexpressed in human solid tumors such as breast, prostate, gastric, liver and colon. High glucose concentration, insulin, and hypoxia upregulate GLUT12 both in adipocytes and tumor cells. Inhibition of GLUT12 mediated Warburg effect suppresses proliferation, migration, and invasion of cancer cells and xenografted tumors. This review summarizes the up-to-date information about GLUT12 physiological role and its implication in obesity and cancer, opening new perspectives to consider this transporter as a therapeutic target.
RESUMO
IMPORTANCE: Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.
Assuntos
Antimaláricos , Malária Falciparum , Parasitos , Piperazinas , Quinolinas , Animais , Plasmodium falciparum , Quinolinas/farmacologia , Quinolinas/química , Cloroquina/farmacologia , Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Mutação , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Malária Falciparum/parasitologiaRESUMO
This study aimed to characterize the potential beneficial effects of chronic docosahexaenoic acid (DHA) supplementation on restoring subcutaneous white adipose tissue (scWAT) plasticity in obese aged female mice. Two-month-old female C57BL/6J mice received a control (CT) or a high fat diet (HFD) for 4 months. Then, 6-month-old diet-induced obese (DIO) mice were distributed into the DIO and the DIOMEG group (fed with a DHA-enriched HFD) up to 18 months. In scWAT, the DHA-enriched diet reduced the mean adipocyte size and reversed the upregulation of lipogenic genes induced by the HFD, reaching values even lower than those observed in CT animals. DIO mice exhibited an up-regulation of lipolytic and fatty oxidation gene expressions that was reversed in DHA-supplemented mice except for Cpt1a mRNA levels, which were higher in DIOMEG as compared to CT mice. DHA restored the increase of proinflammatory genes observed in scWAT of DIO mice. While no changes were observed in total macrophage F4/80+/CD11b+ content, the DHA treatment switched scWAT macrophages profile by reducing the M1 marker Cd11c and increasing the M2 marker CD206. These events occurred alongside with a stimulation of beige adipocyte specific genes, the restoration of UCP1 and pAKT/AKT ratio, and a recovery of the HFD-induced Fgf21 upregulation. In summary, DHA supplementation induced a metabolic remodeling of scWAT to a healthier phenotype in aged obese mice by modulating genes controlling lipid accumulation in adipocytes, reducing the inflammatory status, and inducing beige adipocyte markers in obese aged mice.
Assuntos
Ácidos Docosa-Hexaenoicos , Obesidade , Feminino , Camundongos , Animais , Camundongos Obesos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Obesidade/metabolismo , Camundongos Endogâmicos C57BL , Tecido Adiposo Branco/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gordura Subcutânea/metabolismo , Suplementos Nutricionais , Tecido Adiposo/metabolismoRESUMO
Obesity exacerbates aging-induced adipose tissue dysfunction. This study aimed to investigate the effects of long-term exercise on inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (iBAT) of aged obese mice. Two-month-old female mice received a high-fat diet for 4 months. Then, six-month-old diet-induced obese animals were allocated to sedentarism (DIO) or to a long-term treadmill training (DIOEX) up to 18 months of age. In exercised mice, iWAT depot revealed more adaptability, with an increase in the expression of fatty acid oxidation genes (Cpt1a, Acox1), and an amelioration of the inflammatory status, with a favorable modulation of pro/antiinflammatory genes and lower macrophage infiltration. Additionally, iWAT of trained animals showed an increment in the expression of mitochondrial biogenesis (Pgc1a, Tfam, Nrf1), thermogenesis (Ucp1), and beige adipocytes genes (Cd137, Tbx1). In contrast, iBAT of aged obese mice was less responsive to exercise. Indeed, although an increase in functional brown adipocytes genes and proteins (Pgc1a, Prdm16 and UCP1) was observed, few changes were found on inflammation-related and fatty acid metabolism genes. The remodeling of iWAT and iBAT depots occurred along with an improvement in the HOMA index for insulin resistance and in glucose tolerance. In conclusion, long-term exercise effectively prevented the loss of iWAT and iBAT thermogenic properties during aging and obesity. In iWAT, the long-term exercise program also reduced the inflammatory status and stimulated a fat-oxidative gene profile. These exercise-induced adipose tissue adaptations could contribute to the beneficial effects on glucose homeostasis in aged obese mice.
Assuntos
Tecido Adiposo Marrom , Tecido Adiposo Branco , Feminino , Camundongos , Animais , Tecido Adiposo Marrom/metabolismo , Camundongos Obesos , Tecido Adiposo Branco/metabolismo , Obesidade/terapia , Obesidade/metabolismo , Glucose/metabolismo , Ácidos Graxos/metabolismo , Termogênese/genética , Camundongos Endogâmicos C57BLRESUMO
Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.
RESUMO
Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance in vitro and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping using 34 recombinant haplotypes, and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.
Assuntos
Malária Falciparum , Parasitos , Animais , Camundongos , Plasmodium falciparum/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/genética , Malária Falciparum/parasitologia , Resistência a Medicamentos/genética , Resistência a Múltiplos Medicamentos , GenômicaRESUMO
Remodeling of the microenvironment by tumor cells can activate pathways that favor cancer growth. Molecular delineation and targeting of such malignant-cell nonautonomous pathways may help overcome resistance to targeted therapies. Herein we leverage genetic mouse models, patient-derived xenografts, and patient samples to show that acute myeloid leukemia (AML) exploits peripheral serotonin signaling to remodel the endosteal niche to its advantage. AML progression requires the presence of serotonin receptor 1B (HTR1B) in osteoblasts and is driven by AML-secreted kynurenine, which acts as an oncometabolite and HTR1B ligand. AML cells utilize kynurenine to induce a proinflammatory state in osteoblasts that, through the acute-phase protein serum amyloid A (SAA), acts in a positive feedback loop on leukemia cells by increasing expression of IDO1-the rate-limiting enzyme for kynurenine synthesis-thereby enabling AML progression. This leukemia-osteoblast cross-talk, conferred by the kynurenine-HTR1B-SAA-IDO1 axis, could be exploited as a niche-focused therapeutic approach against AML, opening new avenues for cancer treatment. SIGNIFICANCE: AML remains recalcitrant to treatments due to the emergence of resistant clones. We show a leukemia-cell nonautonomous progression mechanism that involves activation of a kynurenine-HTR1B-SAA-IDO1 axis between AML cells and osteoblasts. Targeting the niche by interrupting this axis can be pharmacologically harnessed to hamper AML progression and overcome therapy resistance. This article is highlighted in the In This Issue feature, p. 873.
Assuntos
Cinurenina , Leucemia Mieloide Aguda , Animais , Humanos , Cinurenina/metabolismo , Cinurenina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Osteoblastos/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
Changes in dietary habits have occurred concomitantly with a rise of type 2 diabetes (T2D) and obesity. Intestine is the first organ facing nutrient ingestion and has to adapt its metabolism with these dietary changes. HNF-4γ, a transcription factor member of the nuclear receptor superfamily and mainly expressed in intestine, has been suggested to be involved in susceptibility to T2D. Our aim was to investigate the role of HNF-4γ in metabolic disorders and related mechanisms. Hnf4g-/- mice were fed high-fat/high-fructose (HF-HF) diet for 6 weeks to induce obesity and T2D. Glucose homeostasis, energy homeostasis in metabolic cages, body composition and stool energy composition, as well as gene expression analysis in the jejunum were analyzed. Despite an absence of decrease in calorie intake, of increase in locomotor activity or energy expenditure, Hnf4g-/- mice fed with HF-HF are protected against weight gain after 6 weeks of HF-HF diet. We showed that Hnf4g-/- mice fed HF-HF display an increase in fecal calorie loss, mainly due to intestinal lipid malabsorption. Gene expression of lipid transporters, Fatp4 and Scarb1 and of triglyceride-rich lipoprotein secretion proteins, Mttp and ApoB are decreased in gut epithelium of Hnf4g-/- mice fed HF-HF, showing the HNF-4γ role in intestine lipid absorption. Furthermore, plasma GLP-1 and jejunal GLP-1 content are increased in Hnf4g-/- mice fed HF-HF, which could contribute to the glucose intolerance protection. The loss of HNF-4γ leads to a protection against a diet-induced weight gain and to a deregulated glucose homeostasis, associated with lipid malabsorption.
Assuntos
Fator 4 Nuclear de Hepatócito/genética , Absorção Intestinal/genética , Metabolismo dos Lipídeos/genética , Obesidade/genética , Animais , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Feminino , Frutose/efeitos adversos , Deleção de Genes , Intolerância à Glucose/etiologia , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Intestinos/metabolismo , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismo , Triglicerídeos/metabolismo , Aumento de Peso/genéticaRESUMO
Obesity and aging are associated to non-alcoholic fatty liver disease (NAFLD) development. Here, we investigate whether long-term feeding with a docosahexaenoic acid (DHA)-enriched diet and aerobic exercise, alone or in combination, are effective in ameliorating NAFLD in aged obese mice. Two-month-old female C57BL/6J mice received control or high fat diet (HFD) for 4 months. Then, the diet-induced obese (DIO) mice were distributed into four groups: DIO, DIO + DHA (15% dietary lipids replaced by a DHA-rich concentrate), DIO + EX (treadmill running), and DIO + DHA + EX up to 18 months. The DHA-rich diet reduced liver steatosis in DIO mice, decreasing lipogenic genes (Dgat2, Scd1, Srebp1c), and upregulated lipid catabolism genes (Hsl/Acox) expression. A similar pattern was observed in the DIO + EX group. The combination of DHA + exercise potentiated an increase in Cpt1a and Ppara genes, and AMPK activation, key regulators of fatty acid oxidation. Exercise, alone or in combination with DHA, significantly reversed the induction of proinflammatory genes (Mcp1, Il6, Tnfα, Tlr4) in DIO mice. DHA supplementation was effective in preventing the alterations induced by the HFD in endoplasmic reticulum stress-related genes (Ern1/Xbp1) and autophagy markers (LC3II/I ratio, p62, Atg7). In summary, long-term DHA supplementation and/or exercise could be helpful to delay NAFLD progression during aging in obesity.
Assuntos
Envelhecimento/fisiologia , Ácidos Docosa-Hexaenoicos/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Obesidade/complicações , Condicionamento Físico Animal/fisiologia , Animais , Autofagia/genética , Autofagia/fisiologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Inflamação/genética , Metabolismo dos Lipídeos , Lipogênese/genética , Fígado/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Obesidade/etiologia , RNA Mensageiro/análiseRESUMO
Obesity is characterized by excessive fat accumulation and inflammation. Aging has also been characterized as an inflammatory condition, frequently accompanied by accumulation of visceral fat. Beneficial effects of exercise and n-3 long-chain polyunsaturated fatty acids in metabolic disorders have been described. Glucose transporter 12 (GLUT12) is one of the less investigated members of the GLUT family. Glucose, insulin, and tumor necrosis factor alpha (TNF-α) induce GLUT12 translocation to the membrane in muscle, adipose tissue, and intestine. We aimed to investigate GLUT12 expression in obesity and aging, and under diet supplementation with docosahexaenoic acid (DHA) alone or in combination with physical exercise in mice. Aging increased GLUT12 expression in intestine, kidney, and adipose tissue, whereas obesity reduced it. No changes on the transporter occurred in skeletal muscle. In obese 18-month-old mice, DHA further decreased GLUT12 in the 4 organs. Aerobic exercise alone did not modify GLUT12, but the changes triggered by exercise were able to prevent the DHA-diminishing effect, and almost restored GLUT12 basal levels. In conclusion, the downregulation of metabolism in aging would be a stimulus to upregulate GLUT12 expression. Contrary, obesity, an excessive energy condition, would induce GLUT12 downregulation. The combination of exercise and DHA would contribute to restore basal function of GLUT12. Novelty In small intestine, kidney and adipose tissue aging increases GLUT12 protein expression whereas obesity reduces it. Dietary DHA decreases GLUT12 in small intestine, kidney, adipose tissue and skeletal muscle. Exercise alone does not modify GLUT12 expression, nevertheless exercise prevents the DHA-diminishing effect on GLUT12.
Assuntos
Envelhecimento/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Obesidade/metabolismo , Condicionamento Físico Animal , Tecido Adiposo/metabolismo , Animais , Células CACO-2 , Dieta , Feminino , Humanos , Intestino Delgado/metabolismo , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismoRESUMO
Tumor necrosis factor-alfa (TNF-α) is a pro-inflammatory cytokine highly-involved in intestinal inflammation. Omega-3 polyunsaturated fatty acids (n3-PUFAs) show anti-inflammatory actions. We previously demonstrated that the n3-PUFA EPA prevents TNF-α inhibition of sugar uptake in Caco-2 cells. Here, we investigated whether the n3-PUFA DHA and its derived specialized pro-resolving lipid mediators (SPMs) MaR1, RvD1 and RvD2, could block TNF-α inhibition of intestinal sugar and glutamine uptake. DHA blocked TNF-α-induced inhibition of α-methyl-D-glucose (αMG) uptake and SGLT1 expression in the apical membrane of Caco-2 cells, through a pathway independent of GPR120. SPMs showed the same preventive effect but acting at concentrations 1000 times lower. In diet-induced obese (DIO) mice, oral gavage of MaR1 reversed the up-regulation of pro-inflammatory cytokines found in intestinal mucosa of these mice. However, MaR1 treatment was not able to counteract the reduced intestinal transport of αMG and SGLT1 expression in the DIO mice. In Caco-2 cells, TNF-α also inhibited glutamine uptake being this inhibition prevented by EPA, DHA and the DHA-derived SPMs. Interestingly, TNF-α increased the expression in the apical membrane of the glutamine transporter B0AT1. This increase was partially blocked by the n-3 PUFAs. These data reveal DHA and its SPMs as promising biomolecules to restore intestinal nutrients transport during intestinal inflammation.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Glutamina/metabolismo , Lipídeos/química , Açúcares/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Transporte Biológico/efeitos dos fármacos , Biotinilação , Células CACO-2 , Dieta , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Humanos , Inflamação , Mucosa Intestinal/metabolismo , Intestinos/química , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The brain depends on glucose as a source of energy. This implies the presence of glucose transporters, being GLUT1 and GLUT3 the most relevant. Expression of GLUT12 is found in mouse and human brain at low levels. We previously demonstrated GLUT12 upregulation in the frontal cortex of aged subjects that was even higher in aged Alzheimer's disease (AD) patients. However, the cause and the mechanism through which this increase occurs are still unknown. Here, we aimed to investigate whether the upregulation of GLUT12 in AD is related with aging or Aß deposition in comparison with GLUT1, GLUT3, and GLUT4. In the frontal cortex of two amyloidogenic mouse models (Tg2576 and APP/PS1) GLUT12 levels were increased. Contrary, expression of GLUT1 and GLUT3 were decreased, while GLUT4 did not change. In aged mice and the senescence-accelerated model SAMP8, GLUT12 and GLUT4 were upregulated in comparison with young animals. GLUT1 and GLUT3 did not show significant changes with age. The effect of ß-amyloid (Aß) deposition was also evaluated in Aß peptide i.c.v. injected mice. In the hippocampus, GLUT12 expression increased whereas GLUT4 was not modified. Consistent with the results in the amyloidogenic models, GLUT3 and GLUT1 were downregulated. In summary, Aß increases GLUT12 protein expression in the brain pointing out a central role of the transporter in AD pathology and opening new perspectives for the treatment of this neurodegenerative disease.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/administração & dosagem , Animais , Encéfalo/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Injeções Intraventriculares , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
AIM: The facilitative glucose transporter GLUT12 was isolated from the breast cancer cell line MCF-7 by its homology with GLUT4. GLUT12 is expressed in insulin-sensitive tissues such as adipose tissue. The aim of this work was to investigate GLUT12 expression and hormonal regulation in 3T3-L1 adipocytes and in adipose tissue of lean and diet-induced obese mice. METHODS: Uptake studies were performed using radio-labelled sugars; α-methyl-d-glucose (αMG) was used as specific substrate of GLUT12. Expression and localization of GLUT12 in adipocytes were investigated by western blot and immunohistochemical methods. RESULTS: GLUT12 is expressed in the peri-nuclear region of mouse adipocytes. Insulin, by AKT activation, and TNF-α, by AMPK activation, increase αMG uptake by inducing GLUT12 translocation to the membrane. In contrast, leptin and adiponectin decrease GLUT12 activity through its internalization. Under hypoxia conditions GLUT12 expression is upregulated. The response of GLUT12 to TNF-α, leptin, adiponectin and hypoxia is the opposite to that of GLUT4. In diet-induced obese mice and obese subjects, GLUT12 protein is decreased. Intraperitoneal injection of insulin increases AKT phosphorylation and GLUT12 expression, but this effect is lost in obese animals. CONCLUSION: We hypothesize that GLUT12 would contribute to modulate sugar absorption in physiological and pathophysiological situations such as obesity.