RESUMO
OBJECTIVE: Faecal microbiota transplantation (FMT) in germ-free (GF) mice is a common approach to study the causal role of the gut microbiota in metabolic diseases. Lack of consideration of housing conditions post-FMT may contribute to study heterogeneity. We compared the impact of two housing strategies on the metabolic outcomes of GF mice colonised by gut microbiota from mice treated with a known gut modulator (cranberry proanthocyanidins (PAC)) or vehicle. DESIGN: High-fat high-sucrose diet-fed GF mice underwent FMT-PAC colonisation in sterile individual positive flow ventilated cages under rigorous housing conditions and then maintained for 8 weeks either in the gnotobiotic-axenic sector or in the specific pathogen free (SPF) sector of the same animal facility. RESULTS: Unexpectedly, 8 weeks after colonisation, we observed opposing liver phenotypes dependent on the housing environment of mice. Mice housed in the GF sector receiving the PAC gut microbiota showed a significant decrease in liver weight and hepatic triglyceride accumulation compared with control group. Conversely, exacerbated liver steatosis was observed in the FMT-PAC mice housed in the SPF sector. These phenotypic differences were associated with housing-specific profiles of colonising bacterial in the gut and of faecal metabolites. CONCLUSION: These results suggest that the housing environment in which gnotobiotic mice are maintained post-FMT strongly influences gut microbiota composition and function and can lead to distinctive phenotypes in recipient mice. Better standardisation of FMT experiments is needed to ensure reproducible and translatable results.
Assuntos
Habitação , Microbiota , Animais , Camundongos , Qualidade Habitacional , Obesidade/metabolismo , Transplante de Microbiota Fecal , Fenótipo , Dieta Hiperlipídica/efeitos adversos , Vida Livre de Germes , Camundongos Endogâmicos C57BLRESUMO
Overconsumption of added sugars is now largely recognized as a major culprit in the global situation of obesity and metabolic disorders. Previous animal studies reported that maple syrup (MS) is less deleterious than refined sugars on glucose metabolism and hepatic health, but the mechanisms remain poorly studied. Beyond its content in sucrose, MS is a natural sweetener containing several bioactive compounds, such as polyphenols and inulin, which are potential gut microbiota modifiers. We aimed to investigate the impact of MS on metabolic health and gut microbiota in male C57Bl/6J mice fed a high-fat high-sucrose (HFHS + S) diet or an isocaloric HFHS diet in which a fraction (10% of the total caloric intake) of the sucrose was substituted by MS (HFHS + MS). Insulin and glucose tolerance tests were performed at 5 and 7 wk into the diet, respectively. The fecal microbiota was analyzed by whole-genome shotgun sequencing. Liver lipids and inflammation were determined, and hepatic gene expression was assessed by transcriptomic analysis. Maple syrup was less deleterious on insulin resistance and decreased liver steatosis compared with mice consuming sucrose. This could be explained by the decreased intestinal α-glucosidase activity, which is involved in carbohydrate digestion and absorption. Metagenomic shotgun sequencing analysis revealed that MS intake increased the abundance of Faecalibaculum rodentium, Romboutsia ilealis, and Lactobacillus johnsonii, which all possess gene clusters involved in carbohydrate metabolism, such as sucrose utilization and butyric acid production. Liver transcriptomic analyses revealed that the cytochrome P450 (Cyp450) epoxygenase pathway was differently modulated between HFHS + S- and HFHS + MS-fed mice. These results show that substituting sucrose for MS alleviated dysmetabolism in diet-induced obese mice, which were associated with decreased carbohydrate digestion and shifting gut microbiota.NEW & NOTEWORTHY The natural sweetener maple syrup has sparked much interest as an alternative to refined sugars. This study aimed to investigate whether the metabolic benefits of substituting sucrose with an equivalent dose of maple syrup could be linked to changes in gut microbiota composition and digestion of carbohydrates in obese mice. We demonstrated that maple syrup is less detrimental than sucrose on metabolic health and possesses a prebiotic-like activity through novel gut microbiota and liver mechanisms.
Assuntos
Acer , Microbioma Gastrointestinal , Masculino , Animais , Camundongos , Sacarose , Camundongos Obesos , Fígado/metabolismo , Dieta Hiperlipídica , Edulcorantes , Digestão , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Promising results in improvement of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH) have been identified following probiotic (PRO) treatment. OBJECTIVES: To evaluate PRO supplementation on hepatic fibrosis, inflammatory and metabolic markers, and gut microbiota in NASH patients. METHODS: In a double-blind, placebo-controlled clinical trial, 48 patients with NASH with a median age of 58 y and median BMI of 32.7 kg/m2 were randomly assigned to receive PROs (Lactobacillus acidophilus 1 × 109 colony forming units and Bifidobacterium lactis 1 × 109 colony forming units) or a placebo daily for 6 mo. Serum aminotransferases, total cholesterol and fractions, C-reactive protein, ferritin, interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, and leptin were assessed. To evaluate liver fibrosis, Fibromax was used. In addition, 16S rRNA gene-based analysis was performed to evaluate gut microbiota composition. All assessments were performed at baseline and after 6 mo. For the assessment of outcomes after treatment, mixed generalized linear models were used to evaluate the main effects of the group-moment interaction. For multiple comparisons, Bonferroni correction was applied (α = 0.05/4 = 0.0125). Results for the outcomes are presented as mean and SE. RESULTS: The AST to Platelet Ratio Index (APRI) score was the primary outcome that decreased over time in the PRO group. Aspartate aminotransferase presented a statistically significant result in the group-moment interaction analyses, but no statistical significance was found after the Bonferroni correction. Liver fibrosis, steatosis, and inflammatory activity presented no statistically significant differences between the groups. No major shifts in gut microbiota composition were identified between groups after PRO treatment. CONCLUSIONS: Patients with NASH who received PRO supplementation for 6 mo presented improvement in the APRI score after treatment. These results draw attention to clinical practice and suggest that supplementation with PROs alone is not sufficient to improve enzymatic liver markers, inflammatory parameters, and gut microbiota in patients with NASH. This trial was registered at clinicaltrials.gov as NCT02764047.
Assuntos
Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica , Probióticos , Humanos , Hepatopatia Gordurosa não Alcoólica/terapia , RNA Ribossômico 16S , Cirrose Hepática , Probióticos/uso terapêutico , Método Duplo-CegoRESUMO
Subgingival microbiome dysbiosis promotes the development of periodontitis, an irreversible chronic inflammatory disease associated with metabolic diseases. However, studies regarding the effects of a hyperglycemic microenvironment on host-microbiome interactions and host inflammatory response during periodontitis are still scarce. Here, we investigated the impacts of a hyperglycemic microenvironment on the inflammatory response and transcriptome of a gingival coculture model stimulated with dysbiotic subgingival microbiomes. HGF-1 cells overlaid with U937 macrophage-like cells were stimulated with subgingival microbiomes collected from four healthy donors and four patients with periodontitis. Pro-inflammatory cytokines and matrix metalloproteinases were measured while the coculture RNA was submitted to a microarray analysis. Subgingival microbiomes were submitted to 16s rRNA gene sequencing. Data were analyzed using an advanced multi-omics bioinformatic data integration model. Our results show that the genes krt76, krt27, pnma5, mansc4, rab41, thoc6, tm6sf2, and znf506 as well as the pro-inflammatory cytokines IL-1ß, GM-CSF, FGF2, IL-10, the metalloproteinases MMP3 and MMP8, and bacteria from the ASV 105, ASV 211, ASV 299, Prevotella, Campylobacter and Fretibacterium genera are key intercorrelated variables contributing to periodontitis-induced inflammatory response in a hyperglycemic microenvironment. In conclusion, our multi-omics integration analysis unveiled the complex interrelationships involved in the regulation of periodontal inflammation in response to a hyperglycemic microenvironment.
Assuntos
Microbiota , Periodontite , Humanos , Multiômica , Disbiose/microbiologia , RNA Ribossômico 16S/genética , Células U937 , Periodontite/microbiologia , Microbiota/genética , Bactérias/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a RNARESUMO
A daily consumption of cranberry juice (CJ) is linked to many beneficial health effects due to its richness in polyphenols but could also awake some intestinal discomforts due to its organic acid content and possibly lead to intestinal inflammation. Additionally, the impact of such a juice on the gut microbiota is still unknown. Thus, this study aimed to determine the impacts of a daily consumption of CJ and its successive deacidification on the intestinal inflammation and on the gut microbiota in mice. Four deacidified CJs (DCJs) (deacidification rates of 0, 40, 60, and 80%) were produced by electrodialysis with bipolar membrane (EDBM) and administered to C57BL/6J mice for four weeks, while the diet (CHOW) and the water were ad libitum. Different parameters were measured to determine intestinal inflammation when the gut microbiota was profiled. Treatment with a 0% DCJ did not induce intestinal inflammation but increased the gut microbiota diversity and induced a modulation of its functions in comparison with control (water). The effect of the removal of the organic acid content of CJ on the decrease of intestinal inflammation could not be observed. However, deacidification by EDBM of CJ induced an additional increase, in comparison with a 0% DCJ, in the Lachnospiraceae family which have beneficial effects and functions associated with protection of the intestine: the lower the organic acid content, the more bacteria of the Lachnospiraceae family and functions having a positive impact on the gut microbiota.
Assuntos
Ácidos/efeitos adversos , Sucos de Frutas e Vegetais/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Vaccinium macrocarpon/efeitos adversos , Ácidos/química , Ácidos/isolamento & purificação , Fenômenos Fisiológicos da Nutrição Animal , Animais , Biodiversidade , Diálise/métodos , Feminino , Sucos de Frutas e Vegetais/análise , Concentração de Íons de Hidrogênio , Inflamação/etiologia , Inflamação/patologia , Intestinos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Vaccinium macrocarpon/químicaRESUMO
mTOR complex 1 (mTORC1) and p70 S6 kinase (S6K1) are both involved in the development of obesity-linked insulin resistance. Recently, we showed that the S6K1 inhibitor PF-4708671 (PF) increases insulin sensitivity. However, we also reported that PF can increase glucose metabolism even in the absence of insulin in muscle and hepatic cells. Here we further explored the potential mechanisms by which PF increases glucose metabolism in muscle and liver cells independent of insulin. Time course experiments revealed that PF induces AMP-activated protein kinase (AMPK) activation before inhibiting S6K1. However, PF-induced glucose uptake was not prevented in primary muscle cells from AMPK α1/2 double KO (dKO) mice. Moreover, PF-mediated suppression of hepatic glucose production was maintained in hepatocytes derived from AMPK α1/2-dKO mice. Remarkably, PF could still reduce glucose production and activate AMPK in hepatocytes from S6K1/2 dKO mice. Mechanistically, bioenergetics experiments revealed that PF reduces mitochondrial complex I activity in both muscle and hepatic cells. The stimulatory effect of PF on glucose uptake was partially reduced by expression of the Saccharomyces cerevisiae NADH:ubiquinone oxidoreductase in L6 cells. These results indicate that PF-mediated S6K1 inhibition is not required for its effect on insulin-independent glucose metabolism and AMPK activation. We conclude that, although PF rapidly activates AMPK, its ability to acutely increase glucose uptake and suppress glucose production does not require AMPK activation. Unexpectedly, PF rapidly inhibits mitochondrial complex I activity, a mechanism that partially underlies PF's effect on glucose metabolism.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Glucose/metabolismo , Imidazóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/genética , Insulina/farmacologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
Blueberry consumption can prevent obesity-linked metabolic diseases, and it has been proposed that the polyphenol content of blueberries may contribute to these effects. Polyphenols have been shown to favorably impact metabolic health, but the role of specific polyphenol classes and whether the gut microbiota is linked to these effects remain unclear. We aimed to evaluate the impact of whole blueberry powder and blueberry polyphenols on the development of obesity and insulin resistance and to determine the potential role of gut microbes in these effects by using fecal microbiota transplantation (FMT). Sixty-eight C57BL/6 male mice were assigned to one of the following diets for 12 wk: balanced diet (Chow); high-fat, high-sucrose diet (HFHS); or HFHS supplemented with whole blueberry powder (BB), anthocyanidin (ANT)-rich extract, or proanthocyanidin (PAC)-rich extract. After 8 wk, mice were housed in metabolic cages, and an oral glucose tolerance test (OGTT) was performed. Sixty germ-free mice fed HFHS diet received FMT from one of the above groups biweekly for 8 wk, followed by an OGTT. PAC-treated mice were leaner than HFHS controls although they had the same energy intake and were more physically active. This observation was reproduced in germ-free mice receiving FMT from PAC-treated mice. PAC- and ANT-treated mice showed improved insulin responses during OGTT, and this finding was also reproduced in germ-free mice following FMT. These results show that blueberry PAC and ANT polyphenols can reduce diet-induced body weight and improve insulin sensitivity and that at least part of these beneficial effects are explained by modulation of the gut microbiota.
Assuntos
Antocianinas/farmacologia , Mirtilos Azuis (Planta) , Frutas , Microbioma Gastrointestinal/efeitos dos fármacos , Resistência à Insulina , Obesidade/metabolismo , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Sacarose Alimentar , Transplante de Microbiota Fecal , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/microbiologiaRESUMO
Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers of the colon, lung and prostate and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) by binding at the A-769662 drug binding site on the AMPK ß1-subunit, a mechanism that is distinct from metformin which disrupts the adenylate charge of the cell. A hallmark of many cancers is high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is currently unknown whether targeting the AMPK-ACC-lipogenic pathway using salicylate and/or metformin may be effective for inhibiting cancer cell survival. Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations achievable following the ingestion of aspirin (<1.0 mM); effects not observed in prostate (PNT1A) and lung (MRC-5) epithelial cell lines. Salicylate concentrations of 1 mM increased the phosphorylation of ACC and suppressed de novo lipogenesis and these effects were enhanced with the addition of clinical concentrations of metformin (100 µM) and eliminated in mouse embryonic fibroblasts (MEFs) deficient in AMPK ß1. Supplementation of media with fatty acids and/or cholesterol reverses the suppressive effects of salicylate and metformin on cell survival indicating the inhibition of de novo lipogenesis is probably important. Pre-clinical studies evaluating the use of salicylate based drugs alone and in combination with metformin to inhibit de novo lipogenesis and the survival of prostate and lung cancers are warranted.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Hipoglicemiantes/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Metformina/farmacologia , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Salicilato de Sódio/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Anti-Inflamatórios não Esteroides/agonistas , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sinergismo Farmacológico , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Hipoglicemiantes/agonistas , Lipogênese , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Metformina/agonistas , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Salicilato de Sódio/agonistasRESUMO
The LKB1 tumour suppressor protein kinase functions to activate two isoforms of AMPK (AMP-activated protein kinase) and 12 members of the AMPK-related family of protein kinases. The highly conserved C-terminal residues of LKB1 are phosphorylated (Ser431) by PKA (cAMP-dependent protein kinase) and RSK (ribosomal S6 kinase) and farnesylated (Cys433) within a CAAX motif. To better define the role that these post-translational modifications play, we created homozygous LKB1S431A/S431A and LKB1C433S/C433S knockin mice. These animals were viable, fertile and displayed no overt phenotypes. Employing a farnesylation-specific monoclonal antibody that we generated, we established by immunoprecipitation that the vast majority, if not all, of the endogenous LKB1 is prenylated. Levels of LKB1 localized at the membrane of the liver of LKB1C433S/C433S mice and their fibroblasts were reduced substantially compared with the wild-type mice, confirming that farnesylation plays a role in mediating membrane association. Although AMPK was activated normally in the LKB1S431A/S431A animals, we unexpectedly observed in all of the examined tissues and cells taken from LKB1C433S/C433S mice that the basal, as well as that induced by the AMP-mimetic AICAR (5-amino-4-imidazolecarboxamide riboside), AMPK activation, phenformin and muscle contraction were significantly blunted. This resulted in a reduced ability of AICAR to inhibit lipid synthesis in primary hepatocytes isolated from LKB1C433S/C433S mice. The activity of several of the AMPK-related kinases analysed [BRSK1 (BR serine/threonine kinase 1), BRSK2, NUAK1 (NUAK family, SNF1-like kinase 1), SIK3 (salt-inducible kinase 3) and MARK4 (MAP/microtubule affinity-regulating kinase 4)] was not affected in tissues derived from LKB1S431A/S431A or LKB1C433S/C433S mice. Our observations reveal for the first time that farnesylation of LKB1 is required for the activation of AMPK. Previous reports have indicated that a pool of AMPK is localized at the plasma membrane as a result of myristoylation of its regulatory AMPKß subunit. This raises the possibility that LKB1 farnesylation and myristoylation of AMPKß might promote the interaction and co-localization of these enzymes on a two-dimensional membrane surface and thereby promote efficient activation of AMPK.
Assuntos
Adenilato Quinase/metabolismo , Cisteína/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Proteínas Quinases Ativadas por AMP , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Prenilação de Proteína , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Silver-based antibacterial coatings limit the spread of hospital-acquired infections. Indeed, the use of silver and silver oxide nanoparticles (Ag and AgO NPs) incorporated in amorphous hydrogenated carbon (a-C:H) as a matrix demonstrates a promising approach to reduce microbial contamination on environmental surfaces. However, its success as an antibacterial coating hinges on the control of Ag+ release. In this sense, if a continuous release is required, an additional barrier is needed to extend the release time of Ag+. Thus, this research investigated the use of a plasma fluoropolymer (CFx) as an additional top layer to elongate Ag+ release and increase the antibacterial activity due to its high hydrophobic nature. Herein, a porous CFx film was deposited on a-C:H containing Ag and AgO NPs using pulsed afterglow low pressure plasma polymerization. The chemical composition, surface wettability and morphology, release profile, and antibacterial activity were analyzed. Overall, the combination of a-C:H:Ag (12.1 at. % of Ag) and CFx film (120.0°, F/C = 0.8) successfully inactivated 88% of E. coli and delayed biofilm formation after 12 h. Thus, using a hybrid approach composed of Ag NPs and a hydrophobic polymeric layer, it was possible to increase the overall antibacterial activity of the coating.
RESUMO
Overconsumption of added sugars has been pointed out as a major culprit in the increasing rates of obesity worldwide, contributing to the rising popularity of non-caloric sweeteners. In order to satisfy the growing demand, industrial efforts have been made to purify the sweet-tasting molecules found in the natural sweetener stevia, which are characterized by a sweet taste free of unpleasant aftertaste. Although the use of artificial sweeteners has raised many concerns regarding metabolic health, the impact of purified stevia components on the latter remains poorly studied. The objective of this project was to evaluate the impact of two purified sweet-tasting components of stevia, rebaudioside A and D (RebA and RebD), on the development of obesity, insulin resistance, hepatic health, bile acid profile, and gut microbiota in a mouse model of diet-induced obesity. Male C57BL/6 J mice were fed an obesogenic high-fat/high-sucrose (HFHS) diet and orally treated with 50 mg/kg of RebA, RebD or vehicle (water) for 12 weeks. An additional group of chow-fed mice treated with the vehicle was included as a healthy reference. At weeks 10 and 12, insulin and oral glucose tolerance tests were performed. Liver lipids content was analyzed. Whole-genome shotgun sequencing was performed to profile the gut microbiota. Bile acids were measured in the feces, plasma, and liver. Liver lipid content and gene expression were analyzed. As compared to the HFHS-vehicle treatment group, mice administered RebD showed a reduced weight gain, as evidenced by decreased visceral adipose tissue weight. Liver triglycerides and cholesterol from RebD-treated mice were lower and lipid peroxidation was decreased. Interestingly, administration of RebD was associated with a significant enrichment of Faecalibaculum rodentium in the gut microbiota and an increased secondary bile acid metabolism. Moreover, RebD decreased the level of lipopolysaccharide-binding protein (LBP). Neither RebA nor RebD treatments were found to impact glucose homeostasis. The daily consumption of two stevia components has no detrimental effects on metabolic health. In contrast, RebD treatment was found to reduce adiposity, alleviate hepatic steatosis and lipid peroxidation, and decrease LBP, a marker of metabolic endotoxemia in a mouse model of diet-induced obesity.
Assuntos
Adiposidade , Diterpenos do Tipo Caurano , Glicosídeos , Resistência à Insulina , Masculino , Camundongos , Animais , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Obesidade/metabolismo , Triglicerídeos , Dieta Hiperlipídica , Sacarose/metabolismo , Ácidos e Sais Biliares/metabolismo , Metabolismo dos LipídeosRESUMO
BACKGROUND & AIMS: Aspirin reduces the incidence of and mortality from colorectal cancer (CRC) by unknown mechanisms. Cancer cells have defects in signaling via the mechanistic target of rapamycin (mTOR), which regulates proliferation. We investigated whether aspirin affects adenosine monophosphate-activated protein kinase (AMPK) and mTOR signaling in CRC cells. METHODS: The effects of aspirin on mTOR signaling, the ribosomal protein S6, S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) were examined in CRC cells by immunoblotting. Phosphorylation of AMPK was measured; the effects of loss of AMPKα on the aspirin-induced effects of mTOR were determined using small interfering RNA (siRNA) in CRC cells and in AMPK(α1/α2-/-) mouse embryonic fibroblasts. LC3 and ULK1 were used as markers of autophagy. We analyzed rectal mucosa samples from patients given 600 mg aspirin, once daily for 1 week. RESULTS: Aspirin reduced mTOR signaling in CRC cells by inhibiting the mTOR effectors S6K1 and 4E-BP1. Aspirin changed nucleotide ratios and activated AMPK in CRC cells. mTOR was still inhibited by aspirin in CRC cells after siRNA knockdown of AMPKα, indicating AMPK-dependent and AMPK-independent mechanisms of aspirin-induced inhibition of mTOR. Aspirin induced autophagy, a feature of mTOR inhibition. Aspirin and metformin (an activator of AMPK) increased inhibition of mTOR and Akt, as well as autophagy in CRC cells. Rectal mucosal samples from patients given aspirin had reduced phosphorylation of S6K1 and S6. CONCLUSIONS: Aspirin is an inhibitor of mTOR and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism. These could contribute to its protective effects against development of CRC.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Knockout , Fenformin/farmacologia , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína S6 Ribossômica/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Metabolic diseases and low-grade chronic inflammation are interconnected: obese persons are at higher risk of developing periodontitis. However, the molecular mechanisms involved in the development and progression of periodontitis in an obesogenic microenvironment in response to periodontopathogens are still lacking. This study aims to investigate the combined effects of palmitate and Porphyromonas gingivalis on the secretion of pro-inflammatory cytokines and on transcriptional landscape modifications in macrophage-like cells. U937 macrophage-like cells were treated with palmitate and stimulated with P. gingivalis for 24h. Cytokines IL-1ß, TNF-α and IL-6 were measured by ELISA in the culture medium and cell extracted RNA was submitted to a microarray analysis followed by Gene Ontology analyses. P. gingivalis, in presence of palmitate, potentiated IL-1ß and TNF-α secretion in comparison to palmitate alone. Gene Ontology analyses also revealed that the combination palmitate-P. gingivalis potentiated the number of gene molecular functions implicated in the regulation of immune and inflammatory pathways compared to macrophages treated with palmitate alone. Our results provide the first comprehensive mapping of gene interconnections between palmitate and P. gingivalis during inflammatory responses in macrophage-like cells. These data highlight the importance of considering systemic conditions, specifically obesogenic microenvironment, in the management of periodontal disease in obese patients.
Assuntos
Porphyromonas gingivalis , Fator de Necrose Tumoral alfa , Humanos , Células U937 , Citocinas , Macrófagos , Obesidade/genética , Palmitatos/farmacologiaRESUMO
Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)γ agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPARγ agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPARγ activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPARγ activation.
Assuntos
Tecido Adiposo/metabolismo , Metabolismo dos Lipídeos , PPAR gama/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tecido Adiposo/efeitos dos fármacos , Adiposidade/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Interações Medicamentosas , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Masculino , PPAR gama/agonistas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Rosiglitazona , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Triglicerídeos/metabolismoRESUMO
Animal models of human diseases are classically fed purified diets that contain casein as the unique protein source. We show that provision of a mixed protein source mirroring that found in the western diet exacerbates diet-induced obesity and insulin resistance by potentiating hepatic mTORC1/S6K1 signaling as compared to casein alone. These effects involve alterations in gut microbiota as shown by fecal microbiota transplantation studies. The detrimental impact of the mixed protein source is also linked with early changes in microbial production of branched-chain fatty acids (BCFA) and elevated plasma and hepatic acylcarnitines, indicative of aberrant mitochondrial fatty acid oxidation. We further show that the BCFA, isobutyric and isovaleric acid, increase glucose production and activate mTORC1/S6K1 in hepatocytes. Our findings demonstrate that alteration of dietary protein source exerts a rapid and robust impact on gut microbiota and BCFA with significant consequences for the development of obesity and insulin resistance.
Assuntos
Proteínas Alimentares/efeitos adversos , Ácidos Graxos/metabolismo , Microbioma Gastrointestinal/fisiologia , Resistência à Insulina , Obesidade/etiologia , Ração Animal/efeitos adversos , Animais , Linhagem Celular Tumoral , Dieta Hiperlipídica/efeitos adversos , Dieta Ocidental/efeitos adversos , Sacarose Alimentar/efeitos adversos , Modelos Animais de Doenças , Transplante de Microbiota Fecal , Vida Livre de Germes , Gluconeogênese , Hepatócitos , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Obesidade/metabolismo , Obesidade/patologia , Ratos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αßγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKß1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated. METHODS: ApoE-/- and LDLr-/- mice with or without (-/-) germline or bone marrow AMPKß1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow-derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKß1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI). RESULTS: Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE-/- mice, but not ApoE-/- AMPKß1-/- mice. Similarly, salsalate reduced atherosclerosis in LDLr-/- mice receiving wild-type but not AMPKß1-/- bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis. CONCLUSIONS: These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKß1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aterosclerose/metabolismo , Salicilatos/metabolismo , Proteínas Quinases Ativadas por AMP/deficiência , Animais , Células Cultivadas , Camundongos , Camundongos KnockoutRESUMO
The search for bioactive compounds from enzymatic hydrolysates has increased in the last few decades. Fish by-products have been shown to be rich in these valuable molecules; for instance, herring milt is a complex matrix composed of lipids, nucleotides, minerals, and proteins. However, limited information is available on the potential health benefits of this by-product. In this context, three industrial products containing herring milt hydrolysate (HMH) were tested in both animal and cellular models to measure their effects on obesity-related metabolic disorders. Male C57Bl/6J mice were fed either a control chow diet or a high-fat high-sucrose (HFHS) diet for 8 weeks and received either the vehicle (water) or one of the three HMH products (HMH1, HMH2, and HMH3) at a dose of 208.8 mg/kg (representing 1 g/day for a human) by daily oral gavage. The impact of HMH treatments on insulin and glucose tolerance, lipid homeostasis, liver gene expression, and the gut microbiota profile was studied. In parallel, the effects of HMH on glucose uptake and inflammation were studied in L6 myocytes and J774 macrophages, respectively. In vivo, daily treatment with HMH2 and HMH3 improved early time point glycemia during the oral glucose tolerance test (OGTT) induced by the HFHS diet, without changes in weight gain and insulin secretion. Interestingly, we also observed that HMH2 consumption partially prevented a lower abundance of Lactobacillus species in the gut microbiota of HFHS diet-fed animals. In addition to this, modulations of gene expression in the liver, such as the upregulation of sucrose nonfermenting AMPK-related kinase (SNARK), were reported for the first time in mice treated with HMH products. While HMH2 and HMH3 inhibited inducible nitric oxide synthase (iNOS) induction in J774 macrophages, glucose uptake was not modified in L6 muscle cells. These results indicate that milt herring hydrolysates reduce some metabolic and inflammatory alterations in cellular and animal models, suggesting a possible novel marine ingredient to help fight against obesity-related immunometabolic disorders.
Assuntos
Produtos Pesqueiros , Intolerância à Glucose/dietoterapia , Inflamação , Macrófagos/imunologia , Obesidade/complicações , Animais , Glicemia/metabolismo , Linhagem Celular , Dieta da Carga de Carboidratos , Dieta Hiperlipídica , Sacarose Alimentar/administração & dosagem , Microbioma Gastrointestinal , Glucose/metabolismo , Intolerância à Glucose/etiologia , Teste de Tolerância a Glucose , Insulina/metabolismo , Fígado/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , RNA-SeqRESUMO
We investigated cardiac hypertrophy elicited by rosiglitazone treatment at the level of protein synthesis/degradation, mTOR, MAPK and AMPK signalling pathways, cardiac function and aspects of carbohydrate/lipid metabolism. Hearts of rats treated or not with rosiglitazone (15 mg/kg day) for 21 days were evaluated for gene expression, protein synthesis, proteasome and calpain activities, signalling pathways, and function by echocardiography. Rosiglitazone induced eccentric heart hypertrophy associated with increased expression of ANP, BNP, collagen I and III and fibronectin, reduced heart rate and increased stroke volume. Rosiglitazone robustly increased heart glycogen content ( approximately 400%), an effect associated with increases in glycogenin and UDPG-PPL mRNA levels and glucose uptake, and a reduction in glycogen phosphorylase expression and activity. Cardiac triglyceride content, lipoprotein lipase activity and mRNA levels of enzymes involved in fatty acid oxidation were also reduced by the agonist. Rosiglitazone-induced cardiac hypertrophy was associated with an increase in myofibrillar protein content and turnover (increased synthesis and an enhancement of calpain-mediated myofibrillar degradation). In contrast, 26S beta5 chymotryptic proteasome activity and mRNA levels of 20S beta2 and beta5 and 19S RPN 2 proteasome subunits along with the ubiquitin ligases atrogin and CHIP were all reduced by rosiglitazone. These morphological and biochemical changes were associated with marked activation of the key growth-promoting mTOR signalling pathway, whose pharmacological inhibition with rapamycin completely blocked cardiac hypertrophy induced by rosiglitazone. The study demonstrates that both arms of protein balance are involved in rosiglitazone-induced cardiac hypertrophy, and establishes the mTOR pathway as a novel important mediator therein.
Assuntos
Hipoglicemiantes/farmacologia , Miofibrilas/metabolismo , Proteínas Quinases/metabolismo , Tiazolidinedionas/farmacologia , Animais , Fator Natriurético Atrial/genética , Western Blotting , Peso Corporal/efeitos dos fármacos , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Ecocardiografia , Glucosiltransferases/genética , Glicogênio/metabolismo , Glicogênio Fosforilase/genética , Glicogênio Sintase/genética , Glicoproteínas/genética , Hemodinâmica/efeitos dos fármacos , Lipase Lipoproteica/genética , Masculino , Peptídeo Natriurético Encefálico/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Sprague-Dawley , Rosiglitazona , Serina-Treonina Quinases TOR , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.
Assuntos
Acetil-CoA Carboxilase , Carcinoma Hepatocelular/metabolismo , Lipogênese , Neoplasias Hepáticas/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/fisiologia , Animais , Células Hep G2 , Humanos , Masculino , Camundongos , Fosforilação , Ratos , Ratos WistarRESUMO
The AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK ß1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK ß1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK ß1 accelerates the appearance of a T-cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin-1ß (IL1ß), reductions in acetyl-CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK ß1 subunit accelerate the development of T-cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53-mutant tumors.