RESUMO
BACKGROUND & AIMS: A high-fat diet (HFD) can cause serious health problems, including alteration of gastrointestinal transit, the exact mechanism of which is not clear. Several microRNAs (miRNAs) are involved in energy homeostasis, lipid metabolism, and HFD-induced weight gain. We investigated the role of miRNAs in HFD-induced damage to the enteric nervous system. METHODS: Male mice were fed a HFD (60% calories from fat) or regular diets (18% calories from fat) for 11 weeks. Mice on regular diets and HFDs were given intraperitoneal injections of Mir375 inhibitor or a negative control. Body weights, food intake, stool indices, and gastrointestinal transit (following Evans blue gavage) were measured. An enteric neuronal cell line (immorto-fetal enteric neuronal) and primary enteric neurons were used for in vitro studies. RESULTS: HFD delayed intestinal transit, which was associated with increased apoptosis and loss of colonic myenteric neurons. Mice fed a low-palmitate HFD did not develop a similar phenotype. Palmitate caused apoptosis of enteric neuronal cells associated with mitochondrial dysfunction and endoplasmic reticulum stress. Palmitate significantly increased the expression of Mir375 in vitro; transfection of cells with a Mir375 inhibitor prevented the palmitate-induced enteric neuronal cell apoptosis. Mir375 expression was increased in myenteric ganglia of mice fed HFD and associated with decreased levels of Mir375 target messenger RNAs, including Pdk1. Systemic injection of a Mir375 inhibitor for 5 weeks prevented HFD-induced delay in intestinal transit and morphologic changes. CONCLUSIONS: HFDs delay colonic transit, partly by inducing apoptosis in enteric neuronal cells. This effect is mediated by Mir375 and is associated with reduced levels of Pdk1. Mir375 might be targeted to increase survival of enteric neurons and gastrointestinal motility.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/efeitos adversos , Sistema Nervoso Entérico/patologia , Trânsito Gastrointestinal/fisiologia , MicroRNAs/metabolismo , Neurônios/patologia , Palmitatos/efeitos adversos , Animais , Apoptose/fisiologia , Biomarcadores/metabolismo , Linhagem Celular , Colo/inervação , Colo/patologia , Colo/fisiopatologia , Sistema Nervoso Entérico/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/administração & dosagem , MicroRNAs/antagonistas & inibidores , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Distribuição Aleatória , Estresse FisiológicoRESUMO
BACKGROUND: Diarrhea is present in up to 30-50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. METHODS: Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. RESULTS: SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. CONCLUSIONS: Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Camundongos , Animais , SARS-CoV-2/metabolismo , Trocador 3 de Sódio-Hidrogênio , Tunicamicina , Células CACO-2 , Meios de Cultivo Condicionados , Proteômica , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Diarreia , Chaperona BiP do Retículo Endoplasmático , Neurônios/metabolismoRESUMO
Glial cell line-derived neurotrophic factor (GDNF) is a factor produced by glial cells that is required for the development of the enteric nervous system. In transgenic mice that overexpress GDNF in the pancreas, GDNF has been shown to enhance beta-cell mass and improve glucose control, but the transcriptional and cellular processes involved are not known. In this study we examined the influence of GDNF on the expression of neurogenin3 (Ngn3) and other transcription factors implicated in early beta-cell development, as well as on beta-cell proliferation during embryonic and early postnatal mouse pancreas development. Embryonic day 15.5 (E15.5) mouse pancreatic tissue when exposed to GDNF for 24 h showed higher Ngn3, pancreatic and duodenal homeobox gene 1 (Pdx1), neuroD1/beta(2), paired homeobox gene 4 (Pax4), and insulin mRNA expression than tissue exposed to vehicle only. Transgenic expression of GDNF in mouse pancreata was associated with increased numbers of Ngn3-expressing pancreatic cells and higher beta-cell mass at embryonic day 18 (E18), as well as higher beta-cell proliferation and Pdx1 expression in beta-cells at E18 and postnatal day 1. In the HIT-T15 beta-cell line, GDNF enhanced the expression of Pax6. This response was, however, blocked in the presence of Pdx1 small interfering RNA (siRNA). Chromatin immunoprecipitation studies using the HIT-T15 beta-cell line demonstrated that GDNF can influence Pdx1 gene expression by enhancing the binding of Sox9 and neuroD1/beta(2) to the Pdx1 promoter. Our data provide evidence of a mechanism by which GDNF influences beta-cell development. GDNF could be a potential therapeutic target for the treatment and prevention of diabetes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Cricetinae , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Organogênese , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Pâncreas/embriologia , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transativadores/metabolismo , Fatores de Transcrição HES-1 , Ativação Transcricional , Transfecção , Regulação para CimaAssuntos
Pâncreas/citologia , Pâncreas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Animais , Biomarcadores/metabolismo , Separação Celular/métodos , Diabetes Mellitus Tipo 1/cirurgia , Quinases Semelhantes a Duplacortina , Humanos , Transplante de Células-TroncoRESUMO
BACKGROUND: Development of pretransplantation islet culture strategies that preserve or enhance ß-cell viability would eliminate the requirement for the large numbers of islets needed to restore insulin independence in type 1 diabetes patients. We investigated whether glial cell line-derived neurotrophic factor (GDNF) could improve human islet survival and posttransplantation function in diabetic mice. METHODS: Human islets were cultured in medium supplemented with or without GDNF (100 ng/mL) and in vitro islet survival and function assessed by analyzing ß-cell apoptosis and glucose stimulated insulin release. In vivo effects of GDNF were assessed in streptozotocin-induced diabetic nude mice transplanted under the kidney capsule with 2000 islet equivalents of human islets precultured in medium supplemented with or without GDNF. RESULTS: In vitro, human islets cultured for 2 to 10 days in medium supplemented with GDNF showed lower ß-cell death, increased Akt phosphorylation, and higher glucose-induced insulin secretion than islets cultured in vehicle. Human islets precultured in medium supplemented with GDNF restored more diabetic mice to normoglycemia and for a longer period after transplantation than islets cultured in vehicle. CONCLUSIONS: Our study shows that GDNF has beneficial effects on human islet survival and could be used to improve islet posttransplantation survival.