RESUMO
BACKGROUND: Obesity and type 2 diabetes are two interrelated metabolic disorders characterized by insulin resistance and a mild chronic inflammatory state. We previously observed that leptin (ob/ob) and leptin receptor (db/db) knockout mice display a distinct inflammatory tone in the liver and adipose tissue. The present study aimed at investigating whether alterations in these tissues of the molecules belonging to the endocannabinoidome (eCBome), an extension of the endocannabinoid (eCB) signaling system, whose functions are important in the context of metabolic disorders and inflammation, could reflect their different inflammatory phenotypes. RESULTS: The basal eCBome lipid and gene expression profiles, measured by targeted lipidomics and qPCR transcriptomics, respectively, in the liver and subcutaneous or visceral adipose tissues, highlighted a differentially altered eCBome tone, which may explain the impaired hepatic function and more pronounced liver inflammation remarked in the ob/ob mice, as well as the more pronounced inflammatory state observed in the subcutaneous adipose tissue of db/db mice. In particular, the levels of linoleic acid-derived endocannabinoid-like molecules, of one of their 12-lipoxygenase metabolites and of Trpv2 expression, were always altered in tissues exhibiting the highest inflammation. Correlation studies suggested the possible interactions with some gut microbiota bacterial taxa, whose respective absolute abundances were significantly different between ob/ob and the db/db mice. CONCLUSIONS: The present findings emphasize the possibility that bioactive lipids and the respective receptors and enzymes belonging to the eCBome may sustain the tissue-dependent inflammatory state that characterizes obesity and diabetes, possibly in relation with gut microbiome alterations.
Assuntos
Canais de Cálcio/genética , Diabetes Mellitus Tipo 2/genética , Leptina/genética , Obesidade/genética , Receptores para Leptina/genética , Canais de Cátion TRPV/genética , Tecido Adiposo/metabolismo , Animais , Araquidonato 12-Lipoxigenase/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Endocanabinoides/genética , Microbioma Gastrointestinal/genética , Regulação da Expressão Gênica/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/microbiologia , Camundongos Obesos/genética , Camundongos Obesos/microbiologia , Obesidade/metabolismo , Obesidade/patologia , Transcriptoma/genéticaRESUMO
Sexual dimorphism exists in the onset and development of type 1 diabetes (T1D), but its potential pathological mechanism is poorly understood. In the present study, we examined sex-specific changes in the gut microbiome and host metabolome of T1D mice via 16S rRNA gene sequencing and nuclear magnetic resonance (NMR)-based metabolomics approach, and aimed to investigate potential mechanism of the gut microbiota-host metabolic interaction in the sexual dimorphism of T1D. Our results demonstrate that female mice had a greater shift in the gut microbiota than male mice during the development of T1D; however, host metabolome was more susceptible to T1D in male mice. The correlation network analysis indicates that T1D-induced host metabolic changes may be regulated by the gut microbiota in a sex-specific manner, mainly involving short-chain fatty acids (SCFAs) metabolism, energy metabolism, amino acid metabolism, and choline metabolism. Therefore, our study suggests that sex-dependent "gut microbiota-host metabolism axis" may be implicated in the sexual dimorphism of T1D, and the link between microbes and metabolites might contribute to the prevention and treatment of T1D.
Assuntos
Diabetes Mellitus Tipo 1/genética , Ácidos Graxos Voláteis/genética , Microbioma Gastrointestinal/genética , Metaboloma/genética , Animais , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Ácidos Graxos Voláteis/metabolismo , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/metabolismo , Caracteres SexuaisRESUMO
Ageing-related bone impairment due to exposure to hyperglycemic environment is scarcely researched. The aim was to confirm the improvement effects of undenatured type II collagen (UC II) on bone impairment in ageing db/db mice, and the ageing model was established by normal feeding for 48-week-old. Then, the ageing db/db mice were randomly assigned to UC II intervention, the ageing model, and the chondroitin sulfate + glucosamine hydrochloride control groups. After 12 weeks of treatment, femoral microarchitecture and biomechanical parameters were observed, biomarkers including bone metabolism, inflammatory cytokines, and oxidative stress were measured, and the gastrocnemius function and expressions of interleukin (IL) 1ß, receptor activator of nuclear factor (NF)-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) were analyzed. The results showed that the mice in the UC II intervention group showed significantly superior bone and gastrocnemius properties than those in the ageing model group, including bone mineral density (287.65 ± 72.77 vs. 186.97 ± 32.2 mg/cm3), gastrocnemius index (0.46 ± 0.07 vs. 0.18 ± 0.01%), muscle fiber diameter (0.0415 ± 0.005 vs. 0.0330 ± 0.002 mm), and cross-sectional area (0.0011 ± 0.00007 vs. 0.00038 ± 0.00004 mm2). The UC II intervention elevated bone mineralization and formation and decreased bone resorption, inflammatory cytokines, and the oxidative stress. In addition, lower protein expression of IL-1ß, RANKL, and TRAP in the UC II intervention group was observed. These findings suggested that UC II improved bones impaired by T2DM during ageing, and the likely mechanism was partly due to inhibition of inflammation and oxidative stress.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Colágeno Tipo II/farmacologia , Interleucina-1beta/genética , Ligante RANK/genética , Fosfatase Ácida Resistente a Tartarato/genética , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Envelhecimento/patologia , Animais , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/etiologia , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Sulfatos de Condroitina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosamina/farmacologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Endogâmicos NOD/genética , Estresse Oxidativo/efeitos dos fármacosRESUMO
Extracellular vesicles (EVs) have an important role in mediating intercellular signaling in inflammation and affect the kinetics of wound healing, however, an understanding of the mechanisms regulating these responses remains limited. Therefore, we have focused on the use of cutaneous injury models in which to study the biology of EVs on the inflammatory phase of wound healing. For this, the foreign body response using sterile subcutaneous polyvinylalcohol (PVA) sponges is ideally suited for the parallel analysis of immune cells and EVs without the need for tissue dissociation, which would introduce additional variables. We have previously used this model to identify mediators of EV biogenesis, establishing that control of how EVs are made affects their payload and biological activity. These studies in normal mice led us to consider how conditions such as immunodeficiency and obsesity affect the profile of immune cells and EVs in this model using genetically defined mutant mice. Since EVs are intrinsically heterogenous in biological fluids, we have focused our studies on a novel technology, vesicle flow cytometry (vFC) to quantify changes in EVs in mouse models. Here, we show that myeloid-derived immune cells and EVs express proteins relevant in antigen presentation in PVA sponge implants that have distinct profiles in wildtype, immune-deficient (NOD scid) vs. diabetic (Leprdb) mice. Together, these results establish a foundation for the parallel analysis of both immune cells and EVs with technologies that begin to address the heterogeneity of intercellular communication in the wound bed.
Assuntos
Antígenos CD/imunologia , Vesículas Extracelulares/fisiologia , Pele/lesões , Pele/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Diabetes Mellitus Experimental/imunologia , Modelos Animais de Doenças , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/patologia , Cinética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/imunologia , Camundongos Obesos/imunologia , Células Mieloides/imunologia , Álcool de Polivinil , Cicatrização/imunologia , Cicatrização/fisiologiaRESUMO
Type 1 diabetes (T1D) results from the autoimmune destruction of ß cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of ß cells. It is known that ß cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of ß cell regeneration in autoimmune nonobese diabetic (NOD) mice remains unclear. Here, we show that, after reversal of autoimmunity by induction of haploidentical mixed chimerism, administration of gastrin plus epidermal growth factor augments ß cell regeneration and normalizes blood glucose in the firmly established diabetic NOD mice. Using transgenic NOD mice with inducible lineage-tracing markers for insulin-producing ß cells, Sox9+ ductal progenitors, Nestin+ mesenchymal stem cells, and glucagon-producing α cells, we have found that both reactivation of dysfunctional low-level insulin expression (insulinlo) ß cells and neogenesis contribute to the regeneration, with the latter predominantly coming from transdifferentiation of α cells. These results indicate that, after reversal of autoimmunity, reactivation of ß cells and transdifferentiation of α cells can provide sufficient new functional ß cells to reach euglycemia in firmly established T1D.
Assuntos
Diabetes Mellitus Tipo 1/genética , Células Secretoras de Insulina/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Regeneração/genética , Animais , Autoimunidade/genética , Glicemia/efeitos dos fármacos , Transdiferenciação Celular/genética , Quimerismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Gastrinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucagon/biossíntese , Células Secretoras de Glucagon/metabolismo , Insulina/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos NOD/genética , Células Precursoras de Linfócitos B/efeitos dos fármacosRESUMO
Catalpol was tested for various disorders including diabetes mellitus. Numerous molecular mechanisms have emerged supporting its biological effects but with little information towards its insulin sensitizing effect. In this study, we have investigated its effect on skeletal muscle mitochondrial respiration and insulin signaling pathway. Type-2 diabetes (T2DM) was induced in male C57BL/6 by a high fat diet (60% Kcal) and streptozotocin (50 mg/kg, i.p.). Diabetic mice were orally administered with catalpol (100 and 200 mg/kg), metformin (200 mg/kg), and saline for four weeks. Fasting blood glucose (FBG), HbA1c, plasma insulin, oral glucose tolerance test (OGTT), insulin tolerance test (ITT), oxygen consumption rate, gene (IRS-1, Akt, PI3k, AMPK, GLUT4, and PGC-1α) and protein (AMPK, GLUT4, and PPAR-γ) expression in muscle were measured. Catalpol (200 mg/kg) significantly (p < 0.05) reduced the FBG, HbA1C, HOMA_IR index, and AUC of OGTT whereas, improved the ITT slope. Gene (IRS-1, Akt, PI3k, GLUT4, AMPK, and PGC-1α) and protein (AMPK, p-AMPK, PPAR-γ and GLUT4) expressions, as well as augmented state-3 respiration, oxygen consumption rate, and citrate synthase activity in muscle was observed in catalpol treated mice. The antidiabetic activity of catalpol is credited with a marked improvement in insulin sensitivity and mitochondrial respiration through the insulin signaling pathway and AMPK/SIRT1/PGC-1α/PPAR-γ activation in the skeletal muscle of T2DM mice.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucosídeos Iridoides/farmacologia , PPAR gama/genética , Proteínas Quinases/genética , Sirtuína 1/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Humanos , Insulina/genética , Insulina/metabolismo , Resistência à Insulina/genética , Camundongos Endogâmicos NOD/genética , Doenças Mitocondriais , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Methionine restrictive diet may alleviate ischaemia/reperfusion (I/R)-induced myocardial injury, but its underlying mechanism remains unclear. HE staining was performed to evaluate the myocardial injury caused by I/R and the effect of methionine-restricted diet (MRD) in I/R mice. IHC and Western blot were carried out to analyse the expression of CSE, CHOP and active caspase3 in I/R mice and hypoxia/reoxygenation (H/R) cells. TUNEL assay and flow cytometry were used to assess the apoptotic status of I/R mice and H/R cells. MTT was performed to analyse the proliferation of H/R cells. H2S assay was used to evaluate the concentration of H2S in the myocardial tissues and peripheral blood of I/R mice. I/R-induced mediated myocardial injury and apoptosis were partially reversed by methionine-restricted diet (MRD) via the down-regulation of CSE expression and up-regulation of CHOP and active caspase3 expression. The decreased H2S concentration in myocardial tissues and peripheral blood of I/R mice was increased by MRD. Accordingly, in a cellular model of I/R injury established with H9C2 cells, cell proliferation was inhibited, cell apoptosis was increased, and the expressions of CSE, CHOP and active caspase3 were dysregulated, whereas NaHS treatment alleviated the effect of I/R injury in H9C2 cells in a dose-dependent manner. This study provided a deep insight into the mechanism underlying the role of MRD in I/R-induced myocardial injury.
Assuntos
Injúria Renal Aguda/metabolismo , Diabetes Mellitus Experimental/dietoterapia , Metionina/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Injúria Renal Aguda/complicações , Injúria Renal Aguda/dietoterapia , Animais , Apoptose/genética , Caspase 3/genética , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Humanos , Rim , Metionina/genética , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/metabolismo , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/dietoterapia , Traumatismo por Reperfusão Miocárdica/etiologia , Miocárdio/metabolismo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/dietoterapia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/genética , Sulfitos/farmacologia , Fator de Transcrição CHOP/genéticaRESUMO
The gut microbiome plays an important role in obesity and Type 2 diabetes (T2D); however, it remains unclear whether the gut microbiome could clarify the dietary versus genetic origin of these ailments. Moreover, studies examining the gut microbiome in diet- versus genetically induced obesity/T2D in the same experimental set-up are lacking. We herein characterized the gut microbiomes in three of the most widely used mouse models of obesity/T2D, i.e., genetically induced (leptin-deficient i.e., Lepob/ob; and leptin-receptor-deficient i.e., Lepdb/db) and high-fat diet (HFD)-induced obese (DIO)/T2D mice, with reference to their normal chow-fed (NC) and low-fat-diet-fed (LF) control counterparts. In terms of ß-diversity, Lepob/ob and Lepdb/db mice showed similarity to NC mice, whereas DIO and LF mice appeared as distinct clusters. The phylum- and genus-level compositions were relatively similar in NC, Lepob/ob, and Lepdb/db mice, whereas DIO and LF mice demonstrated distinct compositions. Further analyses revealed several unique bacterial taxa, metagenomic functional features, and their correlation patterns in these models. The data revealed that obesity/T2D driven by diet as opposed to genetics presents distinct gut microbiome signatures enriched with distinct functional capacities, and indicated that these signatures can distinguish diet- versus genetically induced obesity/T2D and, if extrapolated to humans, might offer translational potential in devising dietary and/or genetics-based therapies against these maladies.
Assuntos
Diabetes Mellitus Tipo 2/microbiologia , Microbioma Gastrointestinal/genética , Leptina/genética , Obesidade/microbiologia , Receptores para Leptina/genética , Animais , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica/efeitos adversos , Humanos , Leptina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos NOD/genética , Obesidade/genética , Obesidade/patologia , Receptores para Leptina/deficiênciaRESUMO
Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.
Assuntos
Dobesilato de Cálcio/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Células Endoteliais/efeitos dos fármacos , Heparitina Sulfato/metabolismo , Humanos , Cinética , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/crescimento & desenvolvimento , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are a novel class of potential biomarkers emerging in many diseases, including type 1 diabetes. Here, we aim to analyse a panel of circulating miRNAs in non-obese diabetic (NOD) mice and individuals with type 1 diabetes. METHODS: We adopted standardised methodologies for extracting miRNAs from small sample volumes to evaluate a profiling panel of mature miRNAs in paired plasma and laser-captured microdissected immune-infiltrated islets of recently diabetic and normoglycaemic NOD mice. Moreover, we validated the findings during disease progression and remission after anti-CD3 therapy in NOD mice, as well as in individuals with type 1 diabetes. RESULTS: Plasma levels of five miRNAs were downregulated in diabetic vs normoglycaemic mice. Of those, miR-409-3p was also downregulated in situ in the immune islet infiltrates of diabetic mice, suggesting an association with disease pathogenesis. Target-prediction tools linked miR-409-3p to immune- and metabolism-related signalling molecules. In situ miR-409-3p expression correlated with insulitis severity, and CD8+ central memory T cells were found to be enriched in miR-409-3p. Plasma miR-409-3p levels gradually decreased during diabetes development and improved with disease remission after anti-CD3 antibody therapy. Finally, plasma miR-409-3p levels were lower in people recently diagnosed with type 1 diabetes compared with a non-diabetic control group, and levels were inversely correlated with HbA1c levels. CONCLUSIONS/INTERPRETATION: We propose that miR-409-3p may represent a new circulating biomarker of islet inflammation and type 1 diabetes severity.
Assuntos
Diabetes Mellitus Tipo 1/genética , Camundongos Endogâmicos NOD/genética , MicroRNAs/genética , Animais , Biomarcadores/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Sodiumglucose cotransporter 2 inhibitors were developed for the treatment of diabetes mellitus. Although recent studies have indicated that sodiumglucose cotransporter 2 inhibitors have suppressive effects on several types of cancer, their effects against colorectal cancer remain unknown. The purpose of the present study was to investigate the effects of tofogliflozin, a sodiumglucose cotransporter 2 inhibitor, on the development of colorectal cancer in diabetic and obese mice. The direct effects of tofogliflozin on the proliferation of colorectal cancer cells were also evaluated. C57BL/KsJdb/db mice were injected with azoxymethane to induce colorectal premalignancy and they received drinking water with or without tofogliflozin. At the end of the study, administration of tofogliflozin was revealed to significantly suppress the development of colorectal neoplastic lesions and ßcatenin accumulated crypts. In the tofogliflozintreated mice, the levels of blood glucose and serum TNFα, as well as mRNA expression of the proinflammatory markers in the white adipose tissue, were reduced. Furthermore, macrophage infiltrations in the white adipose tissues were also reduced significantly. The proliferation of the sodiumglucose cotransporter 2expressing human colorectal cancer cells was not altered by tofogliflozin. These results indicated that tofogliflozin ameliorated chronic inflammation and hyperglycemic condition leading to prevention of colorectal tumorigenesis in a diabetes and obesityrelated colorectal cancer model.
Assuntos
Compostos Benzidrílicos/farmacologia , Carcinogênese/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Glucosídeos/farmacologia , Transportador 2 de Glucose-Sódio/genética , Animais , Azoximetano/toxicidade , Glicemia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Obesos/genética , Obesidade/tratamento farmacológico , Obesidade/genética , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Angiogenesis is a critical process in repair of tissue injury that is regulated by a delicate balance between pro- and antiangiogenic factors. In disease states associated with impaired angiogenesis, we identified that miR-135a-3p is rapidly induced and serves as an antiangiogenic microRNA (miRNA) by targeting endothelial cell (EC) p38 signaling in vitro and in vivo. MiR-135a-3p overexpression significantly inhibited EC proliferation, migration, and network tube formation in matrigel, whereas miR-135-3p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3'-UTR reporter and miRNA ribonucleoprotein complex -immunoprecipitation assays, and small interfering RNA dependency studies revealed that miR-135a-3p inhibits the p38 signaling pathway in ECs by targeting huntingtin-interacting protein 1 (HIP1). Local delivery of miR-135a-3p inhibitors to wounds of diabetic db/db mice markedly increased angiogenesis, granulation tissue thickness, and wound closure rates, whereas local delivery of miR-135a-3p mimics impaired these effects. Finally, through gain- and loss-of-function studies in human skin organoids as a model of tissue injury, we demonstrated that miR-135a-3p potently modulated p38 signaling and angiogenesis in response to VEGF stimulation by targeting HIP1. These findings establish miR-135a-3p as a pivotal regulator of pathophysiological angiogenesis and tissue repair by targeting a VEGF-HIP1-p38K signaling axis, providing new targets for angiogenic therapy to promote tissue repair.-Icli, B., Wu, W., Ozdemir, D., Li, H., Haemmig, S., Liu, X., Giatsidis, G., Cheng, H. S., Avci, S. N., Kurt, M., Lee, N., Guimaraes, R. B., Manica, A., Marchini, J. F., Rynning, S. E., Risnes, I., Hollan, I., Croce, K., Orgill, D. P., Feinberg, M. W. MicroRNA-135a-3p regulates angiogenesis and tissue repair by targeting p38 signaling in endothelial cells.
Assuntos
Células Endoteliais/patologia , MicroRNAs/genética , Neovascularização Patológica/genética , Transdução de Sinais/genética , Cicatrização/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.
Assuntos
Mapeamento Cromossômico , Loci Gênicos , Genoma , Haplótipos , Camundongos Endogâmicos/genética , Animais , Animais de Laboratório , Mapeamento Cromossômico/veterinária , Haplótipos/genética , Camundongos , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos C3H/genética , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos CBA/genética , Camundongos Endogâmicos DBA/genética , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos/classificação , Anotação de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Especificidade da EspécieRESUMO
The present study investigated whether insulinlike growth factor1 (IGF1) exerts a protective effect against neuropathy in diabetic mice and its potential underlying mechanisms. Mice were divided into four groups: Db/m (control), db/db (diabetes), IGF1treated db/db and IGF1picropodophyllin (PPP)treated db/db. Behavioral studies were conducted using the hot plate and von Frey methods at 6 weeks of age prior to treatment. The motor nerve conduction velocity (NCV) of the sciatic nerve was measured using a neurophysiological method at 8 weeks of age. The alterations in the expression levels of IGF1 receptor (IGF1R), cJun Nterminal kinase (JNK), extracellular signalregulated kinase (ERK), p38 and effect of IGF1 on the sciatic nerve morphology were observed by western blotting and electron microscopy. Compared with the control group, the diabetes group developed hypoalgesia after 12 weeks, and neurological lesions improved following an intraperitoneal injection of recombinant (r)IGF1. The sciatic NCV in the diabetes group was significantly lower compared with the control group. The sciatic NCV improved following rIGF1 intervention; however, was impaired following administration of the IGF1 receptor antagonist, PPP. The myelin sheath in the sciatic nerve of the diabetes group was significantly more impaired compared with the control group. The myelin sheath in the sciatic nerves of the rIGF1treated group was significantly improved compared with the diabetes group; whereas, they were significantly impaired following administration of the IGF1R inhibitor. In addition, the expression of IGF1R, phosphorylated (p)JNK and pERK of sciatic nerves in the db/db mice was significantly increased following treatment with IGF1. The expression levels of these proteins were significantly lower in the IGF1PPP group compared with the IGF1 group; however, no significant difference was observed in the expression levels of pp38 following treatment with IGF1. The results of the present study demonstrated that IGF1 may improve neuropathy in diabetic mice. This IGF1induced neurotrophic effect may be associated with the increased phosphorylation levels of JNK and ERK, not p38; however, it was attenuated by administration of an IGF1R antagonist.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Neuropatias Diabéticas/tratamento farmacológico , Fator de Crescimento Insulin-Like I/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like I/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Endogâmicos NOD/genética , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/genética , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/genética , Fosforilação , Podofilotoxina/administração & dosagem , Podofilotoxina/análogos & derivados , Proteínas Recombinantes/genética , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/fisiopatologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Parent-of-origin effects (POE) in mammals typically arise from maternal effects or imprinting. In some instances, such POE have been associated with psychiatric disorders, as well as with changes in a handful of animal behaviors. However, POE on complex traits such as behavior remain largely uncharacterized. Moreover, although both behavior and epigenetic effects are known to be modified by perinatal environmental exposures such as nutrient deficiency, the architecture of such environment-by-POE is mostly unexplored. To study POE and environment-by-POE, we employ a relatively neglected but especially powerful experimental system for POE-detection: reciprocal F1 hybrids (RF1s). We exposed female NOD/ShiLtJ×C57Bl/6J and C57Bl/6J×NOD/ShiLtJ mice, perinatally, to one of four different diets, then after weaning recorded a set of behaviors that model psychiatric disease. Whole-brain microarray expression data revealed an imprinting-enriched set of 15 genes subject to POE. The most-significant expression POE, on the non-imprinted gene Carmil1 (a.k.a. Lrrc16a), was validated using qPCR in the same and in a new set of mice. Several behaviors, especially locomotor behaviors, also showed POE. Bayesian mediation analysis suggested Carmil1 expression suppresses behavioral POE, and that the imprinted gene Airn suppresses POE on Carmil1 expression. A suggestive diet-by-POE was observed on percent center time in the open field test, and a significant diet-by-POE was observed on one imprinted gene, Mir341, and on 16 non-imprinted genes. The relatively small, tractable set of POE and diet-by-POE detected on behavior and expression here motivates further studies examining such effects across RF1s on multiple genetic backgrounds.
Assuntos
Comportamento Animal , Dieta , Impressão Genômica , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos NOD/genética , Animais , Encéfalo/metabolismo , Feminino , Masculino , Estresse Psicológico , Análise Serial de TecidosRESUMO
OBJECTIVE: The NOD/SCID/IL2Rγ- /- (NSG) mouse strain is the most widely used immunodeficient strain for xenograft transplantation. However, the existing SCID mutation is a spontaneous mutation of the Prkdc gene, which leads to leaky T cell developmental block and difficulty in genotyping. It is therefore important to develop a new strain of NSG mice with targeted disruption of Prkdc and IL2Rγ genes. METHODS: Targeted disruption of Prkdc and IL2Rγ genes was achieved using the CRISPR/Cas9 system. By intercrossing the knockout and NOD mice, we obtained a novel strain of NOD/SCID/IL2Rγ- /-(NSG) mice, denoted as cNSG (Chinese NSG) mice. RESULTS: In addition to the NOD mutation, cNSG mice exhibited a complete absence of T cells, B cells and NK cells. cNSG mice allowed more efficient engraftment of human cancer cells than the commonly used immunodeficient nude mice. CONCLUSION: cNSG mice will provide an important xenotransplantation model for biomedical research.
Assuntos
Sistemas CRISPR-Cas , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos Endogâmicos NOD/genética , Camundongos SCID/genética , Proteínas Nucleares/genética , Transplante Heterólogo , Animais , Linfócitos B , Células Matadoras Naturais , Camundongos , Camundongos Knockout , Camundongos Nus , Modelos Animais , Seleção Artificial/genética , Especificidade da Espécie , Linfócitos TRESUMO
Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non-obese diabetic)-derived mouse strains, such as NSG (NOD-Scid-il2Rg) or NRG (NOD-Rag1-il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell-derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre-implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata-2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%-100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD-based mouse models with the aim to improve the existing xenograft models.
Assuntos
Sistemas CRISPR-Cas , Linhagem Celular , Células-Tronco Embrionárias , Camundongos Endogâmicos NOD/genética , Animais , Fertilização in vitro , Fator de Transcrição GATA2/genética , Marcação de Genes , Hospedeiro Imunocomprometido/genética , Camundongos , Camundongos Endogâmicos NOD/imunologia , Modelos BiológicosRESUMO
AIMS: Inhibition of microRNA-92a (miR-92a) is reported to suppress endothelial inflammation and delay atherogenesis. We hypothesize that miR-92a inhibition protects endothelial function through suppressing oxidative stress in diabetic db/db mice. RESULTS: In this study, we found elevated expression of miR-92a in aortic endothelium from db/db mice and in renal arteries from diabetic subjects. Endothelial cells (ECs) exposed to advanced glycation end products (AGEs) and oxidized low-density lipoprotein express higher level of miR-92a. Overexpression of miR-92a impairs endothelium-dependent relaxations (EDRs) in C57BL/6 mouse aortas. Overexpression of miR-92a suppresses expression of heme oxygenase-1 (HO-1), a critical cytoprotective enzyme, whereas inhibition of miR-92a increases HO-1 expression in human umbilical vein ECs (HUVECs) and db/db mouse aortas. Importantly, miR-92a inhibition by Ad-anti-miR-92a improved EDRs and reduced reactive oxygen species (ROS) production in db/db mouse aortas. HO-1 inhibition by SnMP or HO-1 knockdown by shHO-1 reversed the suppressive effect of miR-92a inhibition on ROS production induced by AGE treatment in C57BL/6 mouse aortas. In addition, SnMP reversed miR-92a inhibition-induced improvement of EDRs in AGE-treated C57BL/6 mouse aortas and in db/db mouse aortas. INNOVATION: Expression of miR-92a is increased in diabetic aortic endothelium and inhibition of miR-92a exerts vasoprotective effect in diabetic mice through HO-1 upregulation in ECs. CONCLUSION: MiR-92a expression is elevated in diabetic ECs. MiR-92a overexpression impairs endothelial function and suppresses HO-1 expression in ECs. Inhibition of miR-92a attenuates oxidative stress and improves endothelial function through enhancing HO-1 expression and activity in db/db mouse aortas. Antioxid. Redox Signal. 28, 358-370.
Assuntos
Aterosclerose/genética , Heme Oxigenase-1/genética , MicroRNAs/genética , Estresse Oxidativo/genética , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Regulação da Expressão Gênica/genética , Produtos Finais de Glicação Avançada/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/metabolismo , MicroRNAs/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismoRESUMO
Diabetic gastroparesis is a common complication of diabetes mellitus (DM) that is characterized by decreased serum insulin and insulin-like growth factor-1 (IGF-1). Despite the fact that insulin treatment not glycemic control potently accelerated gastric emptying in type 1 DM patients, the role of insulin/InsR and IGF-1/IGF-1R signaling in diabetic gastroparesis remains incompletely elucidated. In the present study, type 1 DM mice were established and treated with insulin or Voglibose for 8 weeks. The gastric emptying was delayed from DM week 4 when the gastric InsR and IGF-1R were declined. Meanwhile, the gastric choline acetyltransferase (ChAT) was significantly reduced and the myenteric cholinergic neurones and their fibers were significantly diminished. The production of stem cell factor (SCF) was dramatically repressed in the gastric smooth muscles in DM week 6. TWereafter, interstitial cells of Cajal (ICC) were clearly lost and their networks were impaired in DM week 8. Significantly, compared with Voglibose, an 8-week treatment with insulin more efficiently delayed diabetic gastroparesis development by protecting the myenteric cholinergic neurones and ICC. In conclusion, diabetic gastroparesis was an aggressive process due to the successive damages of myenteric cholinergic neurones and ICC by impairing the insulin/InsR and IGF-1/IGF-1R signaling. Insulin therapy in the early stage may delay diabetic gastroparesis.
Assuntos
Antígenos CD/genética , Complicações do Diabetes/genética , Gastroparesia/genética , Fator de Crescimento Insulin-Like I/genética , Insulina/administração & dosagem , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Animais , Antígenos CD/metabolismo , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/metabolismo , Complicações do Diabetes/patologia , Modelos Animais de Doenças , Gastroparesia/tratamento farmacológico , Gastroparesia/metabolismo , Gastroparesia/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inositol/administração & dosagem , Inositol/análogos & derivados , Insulina/genética , Fator de Crescimento Insulin-Like I/metabolismo , Células Intersticiais de Cajal/metabolismo , Células Intersticiais de Cajal/patologia , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/metabolismo , Músculo Liso/metabolismo , Músculo Liso/patologia , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Fator de Células-Tronco/genéticaRESUMO
A global nontargeted longitudinal metabolomics study was carried out in male and female NOD mice to characterize the time-profile of the changes in the metabolic signature caused by onset of type 1 diabetes (T1D) and identify possible early biomarkers in T1D progressors. Metabolomics profiling of samples collected at five different time-points identified 676 and 706 biochemicals in blood and feces, respectively. Several metabolites were expressed at significantly different levels in progressors at all time-points, and their proportion increased strongly following onset of hyperglycemia. At the last time-point, when all progressors were diabetic, a large percentage of metabolites had significantly different levels: 57.8% in blood and 27.8% in feces. Metabolic pathways most strongly affected included the carbohydrate, lipid, branched-chain amino acid, and oxidative ones. Several biochemicals showed considerable (>4×) change. Maltose, 3-hydroxybutyric acid, and kojibiose increased, while 1,5-anhydroglucitol decreased more than 10-fold. At the earliest time-point (6-week), differences between the metabolic signatures of progressors and nonprogressors were relatively modest. Nevertheless, several compounds had significantly different levels and show promise as possible early T1D biomarkers. They include fatty acid phosphocholine derivatives from the phosphatidylcholine subpathway (elevated in both blood and feces) as well as serotonin, ribose, and arabinose (increased) in blood plus 13-HODE, tocopherol (increased), diaminopimelate, valerate, hydroxymethylpyrimidine, and dulcitol (decreased) in feces. A combined metabolic signature based on these compounds might serve as an early predictor of T1D-progressors.