RESUMO
Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.
Assuntos
Monoéster Fosfórico Hidrolases , Viviparidade não Mamífera , Animais , Feminino , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Enterócitos/metabolismo , Enterócitos/enzimologia , Peixe Elétrico/fisiologia , Peixe Elétrico/metabolismo , Peixe-Zebra , Potenciais da MembranaRESUMO
Angiotensin-converting enzyme 2 (ACE2) has been identified as a primary receptor for severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2). Here, we investigated the expression regulation of ACE2 in enterocytes under amino acid deprivation conditions. In this study, we found that ACE2 expression was upregulated upon all or single essential amino acid deprivation in human colonic epithelial CCD841 cells. Furthermore, we found that knockdown of general control nonderepressible 2 (GCN2) reduced intestinal ACE2 mRNA and protein levels in vitro and in vivo. Consistently, we revealed two GCN2 inhibitors, GCN2iB and GCN2-IN-1, downregulated ACE2 protein expression in CCD841 cells. Moreover, we found that increased ACE2 expression in response to leucine deprivation was GCN2 dependent. Through RNA-sequencing analysis, we identified two transcription factors, MAFB and MAFF, positively regulated ACE2 expression under leucine deprivation in CCD841 cells. These findings demonstrate that amino acid deficiency increases ACE2 expression and thereby likely aggravates intestinal SARS-CoV-2 infection.
Assuntos
Aminoácidos , Enzima de Conversão de Angiotensina 2 , COVID-19 , Enterócitos , Proteínas Serina-Treonina Quinases , Aminoácidos/deficiência , Aminoácidos/metabolismo , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/enzimologia , COVID-19/genética , COVID-19/virologia , Enterócitos/enzimologia , Enterócitos/metabolismo , Humanos , Leucina/farmacologia , Peptidil Dipeptidase A/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , SARS-CoV-2/metabolismoRESUMO
Lawsonia intracellularis is endemic to swine herds worldwide, however much is still unknown regarding its impact on intestinal function. Thus, this study aimed to characterize the impact of L. intracellularis on digestive function, and how vaccination mitigates these impacts. Thirty-six L. intracellularis negative barrows were assigned to treatment groups (n = 12/trt): (1) nonvaccinated, L. intracellularis negative (NC); (2) nonvaccinated, L intracellularis challenged (PC); and (3) L. intracellularis challenged, vaccinated (Enterisol® Ileitis, Boehringer Ingelheim) 7 weeks pre-challenge (VAC). On days post-inoculation (dpi) 0 PC and VAC pigs were inoculated with L. intracellularis. From dpi 19-21 fecal samples were collected for apparent total tract digestibility (ATTD) and at dpi 21, pigs were euthanized for sample collection. Post-inoculation, ADG was reduced in PC pigs compared with NC (41%, P < 0.001) and VAC (25%, P < 0.001) pigs. Ileal gross lesion severity was greater in PC pigs compared with NC (P = 0.003) and VAC (P = 0.018) pigs. Dry matter, organic matter, nitrogen, and energy ATTD were reduced in PC pigs compared with NC pigs (P ≤ 0.001 for all). RNAscope in situ hybridization revealed abolition of sucrase-isomaltase transcript in the ileum of PC pigs compared with NC and VAC pigs (P < 0.01). Conversely, abundance of stem cell signaling markers Wnt3, Hes1, and p27Kip1 were increased in PC pigs compared with NC pigs (P ≤ 0.085). Taken together, these data demonstrate that reduced digestibility during L. intracellularis challenge is partially driven by abolition of digestive machinery in lesioned tissue. Further, vaccination mitigated several of these effects, likely from lower bacterial burden and reduced disease severity.
Assuntos
Infecções por Desulfovibrionaceae/veterinária , Enterócitos/microbiologia , Lawsonia (Bactéria)/fisiologia , Oligo-1,6-Glucosidase/deficiência , Sacarase/deficiência , Animais , Infecções por Desulfovibrionaceae/enzimologia , Infecções por Desulfovibrionaceae/microbiologia , Infecções por Desulfovibrionaceae/fisiopatologia , Enterócitos/enzimologia , Sus scrofa , Suínos , Doenças dos Suínos/enzimologia , Doenças dos Suínos/microbiologia , Doenças dos Suínos/fisiopatologiaRESUMO
The gut wall consists of many biological elements, including enterocytes. Rapid turnover, a prominent feature of the enterocytes, has generally been ignored in the development of enterocyte-targeting drugs, although it has a comparable rate to other kinetic rates. Here, we investigated the impact of enterocyte turnover on the pharmacodynamics of enterocyte-targeting drugs by applying a model accounting for turnover of enterocytes and target proteins. Simulations showed that the pharmacodynamics depend on enterocyte lifespan when drug-target affinity is strong and half-life of target protein is long. Interindividual variability of enterocyte lifespan, which can be amplified by disease conditions, has a substantial impact on the variability of response. However, our comprehensive literature search showed that the enterocyte turnover causes a marginal impact on currently approved enterocyte-targeting drugs due to their relatively weak target affinities. This study proposes a model-informed drug development approach for selecting enterocyte-targeting drugs and their optimal dosage regimens.
Assuntos
Desenvolvimento de Medicamentos/métodos , Enterócitos/enzimologia , Gastroenteropatias/tratamento farmacológico , Mucosa Intestinal/metabolismo , Administração Oral , Variação Biológica da População , Ensaios Clínicos como Assunto , Simulação por Computador , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/métodos , Enterócitos/efeitos dos fármacos , Meia-Vida , Humanos , Terapia de Alvo Molecular/métodos , Farmacocinética , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Postprandial triglycerides (TGs) are elevated in people with type 2 diabetes (T2D). Glucose-lowering agents, such as glucagon-like peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, also reduce postprandial TG excursion. Although the glucose-lowering mechanisms of DPP-4 have been extensively studied, how the reduction of DPP-4 activity improves lipid tolerance remains unclear. Here, we demonstrate that gut-selective and systemic inhibition of DPP-4 activity reduces postprandial TG excursion in young mice. Genetic inactivation of Dpp4 simultaneously within endothelial cells and hematopoietic cells using Tie2-Cre reduced intestinal lipoprotein secretion under regular chow diet conditions. Bone marrow transplantation revealed a key role for hematopoietic cells in modulation of lipid responses arising from genetic reduction of DPP-4 activity. Unexpectedly, deletion of Dpp4 in enterocytes increased TG excursion in high-fat diet-fed (HFD-fed) mice. Moreover, chemical reduction of DPP-4 activity and increased levels of GLP-1 were uncoupled from TG excursion in older or HFD-fed mice, yet lipid tolerance remained improved in older Dpp4-/- and Dpp4EC-/- mice. Taken together, this study defines roles for specific DPP-4 compartments, age, and diet as modifiers of DPP-4 activity linked to control of gut lipid metabolism.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Enterócitos/enzimologia , Triglicerídeos/metabolismo , Animais , Transplante de Medula Óssea , Dieta Hiperlipídica/efeitos adversos , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/isolamento & purificação , Inibidores da Dipeptidil Peptidase IV/farmacologia , Peptídeo 1 Semelhante ao Glucagon/sangue , Células-Tronco Hematopoéticas/enzimologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Período Pós-Prandial/efeitos dos fármacos , Período Pós-Prandial/fisiologia , Fosfato de Sitagliptina/farmacologiaAssuntos
Doença Celíaca/enzimologia , Enterócitos/enzimologia , Proteínas de Ligação ao GTP/imunologia , Glutens/imunologia , Intestino Delgado/enzimologia , Transglutaminases/imunologia , Animais , Linfócitos B/imunologia , Doença Celíaca/imunologia , Linhagem Celular Tumoral , Epitopos , Proteínas de Ligação ao GTP/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Linfócitos T/imunologia , Transglutaminases/genéticaRESUMO
The coronavirus disease 2019 (COVID-19) was first reported in Wuhan, China and rapidly spread in other countries in December 2019. The infected patients presented with fever, respiratory symptoms, sometimes with digestive and other systemic manifestations, and some progressed with a severe acute respiratory syndrome or even death. Associated digestive symptoms were frequently observed in the patients, with an unknown significance and mechanism. ACE2, as the major known functional receptor of the 2019 novel coronavirus (2019-nCoV) attracted our attention. We collected the clinical data of the 2019-nCoV-infected patients from published studies and extracted the data about the incidence of gastrointestinal symptoms. Furthermore, we used online datasets to analyze ACE2 expression in different human organs, especially in the small intestine, to explore the relationship between ACE2 expression patterns and clinical symptoms. We found that diarrhea accounted for a notable proportion of COVID-19 patients, ranging from 8.0% to 12.9%. The results reveal that ACE2 mRNA and protein are highly expressed in the small intestinal enterocytes but not in the goblet cells or intestinal immune cells. High expression of ACE2 on the surface cells in the digestive tract may lead to gastrointestinal symptoms and inflammation susceptibility. Overall, digestive symptoms were common in the COVID-19 patients. ACE2 expression on surface cells of the small intestine may mediate the invasion and amplification of the virus and activation of gastrointestinal inflammation. It is a possible mechanism of digestive symptoms in the COVID-19 patients and explains the presence of the virus in patients' stool samples. The study also highlights the necessity of taking stool samples for suspected patients to help in early diagnosis and assessment of disease status.
Assuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Diarreia/etiologia , Enterócitos/enzimologia , Gastroenteropatias/etiologia , Intestino Delgado/enzimologia , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2 , COVID-19 , Fezes/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Peptidil Dipeptidase A/genética , SARS-CoV-2RESUMO
The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.
Assuntos
Aminopeptidases/genética , Animais Geneticamente Modificados/genética , Infecções por Coronavirus/genética , Coronavirus/patogenicidade , Aminopeptidases/deficiência , Animais , Animais Geneticamente Modificados/virologia , Sistemas CRISPR-Cas , Coronavirus/genética , Infecções por Coronavirus/virologia , Enterócitos/enzimologia , Enterócitos/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Vírus da Gastroenterite Transmissível/patogenicidadeRESUMO
INTRODUCTION: Cytochrome (CYP) epoxygenases (CYP2C and CYP2J) and soluble epoxide hydrolase (sEH) participate in the metabolism of arachidonic acid and may also have a potential role in enterocyte differentiation. The first critical step in the study of intestinal cell differentiation is the determination of a suitable in vitro model, which must be as similar as possible to the conditions of a living organism. It is known that HT-29 and Caco2 cell lines derived from human colorectal carcinomas can differentiate into enterocyte-like cells in appropriate culture conditions. MATERIAL AND METHODS: We tested 4 different approaches of enterocyte-like differentiation and determined the most appropriate culture conditions for each model. Subsequently, the changes in the expression of CYP epoxygenases and sEH in undifferentiated and differentiated cells were measured by In-Cell ELISA. These results were compared with immunohistochemical profiles of expression of CYP epoxygenases and sEH in samples of human embryonic and fetal intestines as well as adult duodenum and colon. RESULTS: Our results show that sodium butyrate (NaBt)-differentiated HT-29 cells and spontaneously differentiated Caco2 cells resemble CYP epoxygenases and sEH profiles, corresponding with different types of intestines. CONCLUSION: Our study revealed that the most suitable models for the study of the role of CYP epoxygenases and sEH expression in differentiation of intestinal epithelium are NaBt-differentiated HT-29 cells and spontaneously differentiated Caco2 cells.
Assuntos
Diferenciação Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Enterócitos/enzimologia , Epóxido Hidrolases/metabolismo , Mucosa Intestinal , Ácido Araquidônico/metabolismo , Células CACO-2 , Células HT29 , Humanos , Técnicas In Vitro , Mucosa Intestinal/embriologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/embriologiaRESUMO
BACKGROUND: We report here an evaluation of a novel experimental system- cofactorsupplemented permeabilized cryopreserved human enterocytes (MetMax™ cryopreserved human enterocytes (MMHE), patent pending) for applications in the evaluation of enteric drug metabolism. A major advantage of MMHE over Conventional Cryopreserved Human Enterocytes (CCHE) is the simplification of the use procedures including storage at -80°C instead of in liquid nitrogen, and use of the cells immediately after thawing without a need for centrifugation and microscopic evaluation of cell density and viability and cell density adjustment. METHODS: In this study, we compared MMHE and CCHE in key phase 1 oxidation and phase 2 conjugation Drug Metabolism Enzyme (DME) activities that we recently reported for cryopreserved human enterocytes: CYP2C9 (diclofenac 4'- hydroxylation), CYP2C19 (s-mephenytoin hydroxylation), CYP3A4 (midazolam 1'-hydroxylation), CYP2J2 (astemizole O-demethylation), uridine 5'-diphosphoglucuronosyltransferase (UGT; 7-hydroxycoumarin glucuronidation), sulfotransferase (SULT; 7- hydroxycoumarin sulfation), N-acetyl transferase-1 (NAT-1; p-benzoic acid N-acetylation), and carboxyesterase- 2 (CES-2; hydrolysis of irinotecan to SN38). Both CCHE and MMHE were active in all the DME pathways evaluated, with specific activities of MMHE ranged from 142% (CYP2C9) to 1713% (UGT) of that for CCHE. ß-hydroxylation and testosterone 6. RESULT AND CONCLUSION: Our results suggest that the MMHE system represents a convenient and robust in vitro experimental system for the evaluation of enteric drug metabolism.
Assuntos
Carboxilesterase/metabolismo , Criopreservação/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Enterócitos/enzimologia , Glucuronosiltransferase/metabolismo , Preparações Farmacêuticas/metabolismo , Sulfotransferases/metabolismo , Adulto , Biotransformação , Permeabilidade da Membrana Celular , Feminino , Humanos , Técnicas In Vitro , Isoenzimas , Masculino , Pessoa de Meia-IdadeRESUMO
After crossing floxed stearoyl-CoA desaturase-1 (Scd1fl/fl) mice with LDL receptor-null (ldlr-/-) mice, and then Villin Cre (VilCre) mice, enterocyte Scd1 expression in Scd1fl/fl/ldlr-/-/VilCre mice was reduced 70%. On Western diet (WD), Scd1fl/fl/ldlr-/- mice gained more weight than Scd1fl/fl/ldlr-/-/VilCre mice (P < 0.0023). On WD, jejunum levels of lysophosphatidylcholine (LysoPC) 18:1 and lysophosphatidic acid (LPA) 18:1 were significantly less in Scd1fl/fl/ldlr-/-/VilCre compared with Scd1fl/fl/ldlr-/- mice (P < 0.0004 and P < 0.026, respectively). On WD, Scd1fl/fl/ldlr-/-/VilCre mice compared with Scd1fl/fl/ldlr-/- mice had lower protein levels of lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR4), and myeloid differentiation factor-88 (MyD88) in enterocytes and plasma, and less dyslipidemia and systemic inflammation. Adding a concentrate of tomatoes transgenic for the apoA-I mimetic peptide 6F (Tg6F) to WD resulted in reduced enterocyte protein levels of LBP, CD14, TLR4, and MyD88 in Scd1fl/fl/ldlr-/- mice similar to that seen in Scd1fl/fl/ldlr-/-/VilCre mice. Adding LysoPC 18:1 to WD did not reverse the effects of enterocyte Scd1 knockdown. Adding LysoPC 18:1 (but not LysoPC 18:0) to chow induced jejunum Scd1 expression and increased dyslipidemia and plasma serum amyloid A and interleukin 6 levels in Scd1fl/fl/ldlr-/- mice, but not in Scd1fl/fl/ldlr-/-/VilCre mice. We conclude that enterocyte Scd1 is partially responsible for LysoPC 18:1- and WD-induced dyslipidemia and inflammation in ldlr-/- mice.
Assuntos
Enterócitos/enzimologia , Deleção de Genes , Receptores de LDL/deficiência , Receptores de LDL/genética , Estearoil-CoA Dessaturase/metabolismo , Proteínas de Fase Aguda/metabolismo , Animais , Peso Corporal , Proteínas de Transporte/metabolismo , HDL-Colesterol/sangue , Dislipidemias/enzimologia , Dislipidemias/genética , Dislipidemias/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Jejuno/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lisofosfatidilcolinas/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Estearoil-CoA Dessaturase/deficiência , Estearoil-CoA Dessaturase/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Protein abundance and activity of UGT2B17, a highly variable drug- and androgen-metabolizing enzyme, were quantified in microsomes, S9 fractions, and primary cells isolated from human liver and intestine by validated LC-MS/MS methods. UGT2B17 protein abundance showed >160-fold variation (mean⯱â¯SD, 1.7⯱â¯2.7â¯pmol/mg microsomal protein) in adult human liver microsomes (nâ¯=â¯26) and significant correlation (r2â¯=â¯0.77, pâ¯<â¯0.001) with testosterone glucuronide (TG) formation. Primary role of UGT2B17 in TG formation compared to UGT2B15 was confirmed by performing activity assays in UGT2B17 gene deletion samples and with a selective UGT2B17 inhibitor, imatinib. Human intestinal microsomes isolated from small intestine (nâ¯=â¯6) showed on average significantly higher protein abundance (7.4⯱â¯6.6â¯pmol/mg microsomal protein, pâ¯=â¯0.016) compared to liver microsomes, with an increasing trend towards distal segments of the gastrointestinal (GI) tract. Commercially available pooled microsomes and S9 fractions confirmed greater abundance and activity of UGT2B17 in intestinal fractions compared to liver fractions. To further investigate the quantitative role of UGT2B17 in testosterone metabolism in whole cell system, a targeted metabolomics study was performed in hepatocytes (nâ¯=â¯5) and enterocytes (nâ¯=â¯16). TG was the second most abundant metabolite after androstenedione in both cell systems. Reasonable correlation between UGT2B17 abundance and activity were observed in enterocytes (r2â¯=â¯0.69, pâ¯=â¯0.003), but not in hepatocytes. These observational and mechanistic data will be useful in developing physiologically-based pharmacokinetic (PBPK) models for predicting highly-variable first-pass metabolism of testosterone and other UGT2B17 substrates.
Assuntos
Enterócitos/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucuronosiltransferase/metabolismo , Hepatócitos/enzimologia , Microssomos/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Testosterona/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Humanos , Mesilato de Imatinib/farmacologia , Antígenos de Histocompatibilidade Menor/genéticaRESUMO
The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.
Assuntos
Intestino Delgado/metabolismo , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , NADPH Oxidases/metabolismo , Animais , Enterócitos/enzimologia , Enterócitos/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Trato Gastrointestinal/enzimologia , Trato Gastrointestinal/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Intestino Delgado/enzimologia , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , NADPH Oxidase 2/genética , NADPH Oxidase 4/genética , NADPH Oxidases/genética , Fagócitos/enzimologia , Fagócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Decitabine (DAC), a potent DNA methyltransferase (DNMT) inhibitor, has a limited oral bioavailability. Its 5'-amino acid ester prodrugs could improve its oral delivery but the specific absorption mechanism is not yet fully understood. The aim of this present study was to investigate the in vivo absorption and activation mechanism of these prodrugs using in situ intestinal perfusion and pharmacokinetics studies in rats. Although PEPT1 transporter is pH dependent, there appeared to be no proton cotransport in the perfusion experiment with a preferable transport at pH 7.4 rather than pH 6.5. This suggested that the transport was mostly dependent on the dissociated state of the prodrugs and the proton gradient might play only a limited role. In pH 7.4 HEPES buffer, an increase in Peff was observed for L-val-DAC, D-val-DAC, L-phe-DAC and L-trp-DAC (2.89-fold, 1.2-fold, 2.73-fold, and 1.90-fold, respectively), compared with the parent drug. When co-perfusing the prodrug with Glysar, a known substrate of PEPT1, the permeabilities of the prodrugs were significantly inhibited compared with the control. To further investigate the absorption of the prodrugs, L-val-DAC was selected and found to be concentration-dependent and saturable, suggesting a carrier-mediated process (intrinsic Km: 7.80⯱â¯2.61â¯mM) along with passive transport. Determination of drug in intestinal homogenate after perfusion further confirmed that the metabolic activation mainly involved an intestinal first-pass effect. In a pharmacokinetic evaluation, the oral bioavailability of L-val-DAC, L-phe-DAC and L-trp-DAC were nearly 1.74-fold, 1.69-fold and 1.49-fold greater than that of DAC. The differences in membrane permeability and oral bioavailability might be due to the different stability in the intestinal lumen and the distinct PEPT1 affinity which is mainly caused by the stereochemistry, hydrophobicity and steric hindrance of the side chains. In summary, the detailed investigation of the absorption mechanism by in vivo intestinal perfusion and pharmacokinetic studies showed that the prodrugs of DAC exhibited excellent permeability and oral bioavailability, which might be attributed to a hybrid (partly PEPT1-mediated and partly passive) transport mode and a rapid activation process in enterocytes.
Assuntos
Azacitidina/análogos & derivados , Enterócitos/enzimologia , Inibidores Enzimáticos/farmacocinética , Absorção Intestinal , Transportador 1 de Peptídeos/metabolismo , Pró-Fármacos/farmacocinética , Administração Oral , Aminoácidos/química , Animais , Azacitidina/administração & dosagem , Azacitidina/química , Azacitidina/farmacocinética , Disponibilidade Biológica , Permeabilidade da Membrana Celular , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Ésteres/química , Mucosa Intestinal/metabolismo , Intestinos/citologia , Masculino , Modelos Animais , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Ratos , Ratos Sprague-DawleyRESUMO
Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the mechanism of the increase in transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. After dosing JTT-553, transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was due to increased enzyme protein synthesis and was suppressed by inhibiting fatty acid-transport to the enterocytes. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to release of the enzymes from injured cells into the circulation but to be phenomena resulting from enhancement of enzyme protein synthesis in the small intestine due to the pharmacological action of JTT-553 in this organ.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Enterócitos/enzimologia , Intestino Delgado/enzimologia , Intestino Delgado/metabolismo , Oxazinas/farmacologia , Compostos de Espiro/farmacologia , Transaminases/sangue , Administração Oral , Animais , Diacilglicerol O-Aciltransferase/metabolismo , Cães , Ácidos Graxos/metabolismo , Intestino Delgado/citologia , Macaca fascicularis , Oxazinas/administração & dosagem , Ratos Endogâmicos F344 , Compostos de Espiro/administração & dosagem , Fatores de TempoRESUMO
Organoselemium compounds possess strong antioxidant activity as well as protecting cells from DNA damage, mitochondrial injury, lipid peroxidation, protein denaturation and cell death. Herein, we used an in vitro oxidative model to further investigate the antioxidant effects of a novel organoselemium compound, low molecular-weight seleno-aminopolysaccharides (LSA) in intestinal porcine epithelial cells (IPEC-1), and the molecular mechanisms of these effects. Analysis by MTT assay showed that LSA could significantly increase the viability of IPEC-1 cells compared to cells exposed to H2O2. We found that the levels of different antioxidant enzymes could dramatically increase in LSA pretreatment group compared to H2O2 treatment group. Furthermore, LSA significantly increased the gene expression of antioxidant enzymes and phase 2 detoxifying enzymes in IPEC-1 cells, as measured by qRT-PCR. In addition, LSA up-regulated the expression level of intracellular transcription factor NF-E2-related factor 2 (Nrf2) and inhibited the level of kelch-like ECH-associated protein 1 (Keap1) with western blot analysis. Collectively, the present study suggested that LSA has the protective effect of IPEC-1 cells against H2O2-induecd oxidative stress, and its mechanism may be related to activation of Keap1/Nrf2 signaling pathway in intestinal epithelial cells.
Assuntos
Enterócitos/patologia , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/farmacologia , Substâncias Protetoras/farmacologia , Selênio/farmacologia , Animais , Antioxidantes/metabolismo , Catalase/genética , Catalase/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Citoproteção/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Enterócitos/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Inativação Metabólica/efeitos dos fármacos , Inativação Metabólica/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Peso Molecular , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Sus scrofaRESUMO
The guanylate cyclase C (GC-C) receptor regulates electrolyte and water secretion into the gut following activation by the E. coli enterotoxin STa, or by weaker endogenous agonists guanylin and uroguanylin. Our previous work has demonstrated that GC-C plays an important role in controlling initial infection as well as carrying load of non-invasive bacterial pathogens in the gut. Here, we use Salmonella enterica serovar Typhimurium to determine whether GC-C signaling is important in host defense against pathogens that actively invade enterocytes. In vitro studies indicated that GC-C signaling significantly reduces Salmonella invasion into Caco2-BBE monolayers. Relative to controls, GC-C knockout mice develop severe systemic illness following oral Salmonella infection, characterized by disrupted intestinal mucus layer, elevated cytokines and organ CFUs, and reduced animal survival. In Salmonella-infected wildtype mice, oral gavage of GC-C agonist peptide reduced host/pathogen physical interaction and diminished bacterial translocation to mesenteric lymph nodes. These studies suggest that early life susceptibility to STa-secreting enterotoxigenic E. coli may be counter-balanced by a critical role of GC-C in protecting the mucosa from non-STa producing, invasive bacterial pathogens.
Assuntos
Endocitose , Enterócitos/enzimologia , Enterócitos/microbiologia , Receptores de Enterotoxina/metabolismo , Infecções por Salmonella/patologia , Salmonella typhimurium/imunologia , Estruturas Animais/microbiologia , Animais , Carga Bacteriana , Células CACO-2 , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Knockout , Infecções por Salmonella/microbiologia , Análise de SobrevidaRESUMO
The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD) and mucous-cell types was evaluated in the gastrointestinal tract of the half-smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB-PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB-) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS-AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.
Assuntos
Linguados/metabolismo , Trato Gastrointestinal/enzimologia , Animais , Enterócitos/enzimologia , Células Epiteliais/enzimologia , Mucosa Intestinal/enzimologia , Estômago/enzimologiaRESUMO
OBJECTIVE: Intestinal metabolism might play a greater role in regulating whole body metabolism than previously believed. We aimed to enhance enterocyte metabolism in mice and investigate if it plays a role in diet-induced obesity (DIO) and its comorbidities. METHODS: Using the cre-loxP system, we overexpressed the mitochondrial NAD+ dependent protein deacetylase SIRT3 in enterocytes of mice (iSIRT3 mice). We chronically fed iSIRT3 mice and floxed-SIRT3 control (S3fl) mice a low-fat, control diet (CD) or a high-fat diet (HFD) and then phenotyped the mice. RESULTS: There were no genotype differences in any of the parameters tested when the mice were fed CD. Also, iSIRT3 mice were equally susceptible to the development of DIO as S3fl mice when fed HFD. They were, however, better able than S3fl mice to regulate their blood glucose levels in response to exogenous insulin and glucose, indicating that they were protected from developing insulin resistance. This improved glucose homeostasis was accompanied by an increase in enterocyte metabolic activity and an upregulation of ketogenic gene expression in the small intestine. CONCLUSION: Enhancing enterocyte oxidative metabolism can improve whole body glucose homeostasis.
Assuntos
Peso Corporal/fisiologia , Enterócitos/metabolismo , Glucose/metabolismo , Mucosa Intestinal/metabolismo , Sirtuína 3/biossíntese , Animais , Glicemia/metabolismo , Metabolismo Energético , Enterócitos/enzimologia , Intolerância à Glucose/metabolismo , Homeostase , Humanos , Insulina/metabolismo , Resistência à Insulina/fisiologia , Mucosa Intestinal/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Obesidade/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
BACKGROUND: Understanding the specific mechanisms of rare autosomal disorders has greatly expanded insights into the complex processes regulating intestinal fat transport. Sar1B GTPase is one of the critical proteins governing chylomicron secretion by the small intestine, and its mutations lead to chylomicron retention disease, despite the presence of Sar1A paralog. OBJECTIVE: The central aim of this work is to examine the cause-effect relationship between Sar1B expression and chylomicron output and to determine whether Sar1B is obligatory for normal high-density lipoprotein biogenesis. APPROACH AND RESULTS: The SAR1B gene was totally silenced in Caco-2/15 cells using the zinc finger nuclease technique. SAR1B deletion resulted in significantly decreased secretion of triglycerides (≈40%), apolipoprotein B-48 (≈57%), and chylomicron (≈34.5%). The absence of expected chylomicron production collapse may be because of the compensatory SAR1A elevation observed in our experiments. Therefore, a double knockout of SAR1A and SAR1B was engineered in Caco-2/15 cells, which led to almost complete inhibition of triglycerides, apolipoprotein B-48, and chylomicron output. Further experiments with labeled cholesterol revealed the downregulation of high-density lipoprotein biogenesis in cells deficient in SAR1B or with the double knockout of the 2 SAR1 paralogs. Similarly, there was a fall in the movement of labeled cholesterol from cells to basolateral medium containing apolipoprotein A-I, thereby limiting newly synthesized high-density lipoprotein in genetically modified cells. The decreased cholesterol efflux was associated with impaired expression of ABCA1 (ATP-binding cassette subfamily A member 1). CONCLUSIONS: These findings demonstrate that the deletion of the 2 SAR1 isoforms is required to fully eliminate the secretion of chylomicron in vitro. They also underscore the limited high-density lipoprotein production by the intestinal cells in response to SAR1 knockout.