RESUMEN
BACKGROUND: Maternal diabetes exposes embryos to periods of hyperglycemia. Glucose is important for normal cardiogenesis, and Glut-1 is the predominant glucose transporter in the embryo. METHODS: Pregnant mice were exposed to 6 or 12 hr hyperglycemia during organogenesis using intraperitoneal (IP) injections of D-glucose on gestational day (GD) 9.5 (plug = GD 0.5). Embryos were examined for morphology and total cardiac protein, and embryonic hearts were evaluated for Glut-1 protein and mRNA expression immediately after treatment (GD 9.75, GD 10.0), as well as on GD 10.5 and GD 12.5. RESULTS: IP glucose injections were effective in producing sustained maternal hyperglycemia. Maternal hyperglycemia for 6 or 12 hr on GD 9.5, followed by normoglycemia, produced a decrease in overall size and total cardiac protein in embryos evaluated on GD 10.5 but no difference on GD 12.5. Cardiac Glut-1 expression was immediately upregulated in embryos exposed to 6 or 12 hr maternal hyperglycemia. On GD 10.5, cardiac Glut-1 expression was not different in embryos exposed to maternal hyperglycemia for 6 hr but was downregulated in embryos exposed for 12 hr. On GD 12.5, cardiac Glut-1 expression in embryos exposed to maternal hyperglycemia on GD 9.5 for 6 or 12 hr, followed by normoglycemia, was not different from controls. The temporal pattern was the same for Glut-1 protein and mRNA expression. CONCLUSIONS: Hyperglycemia-induced alterations in Glut-1 expression likely interfere with balance of glucose available to the embryonic heart that may affect cardiac morphogenesis.
Asunto(s)
Glucosa/metabolismo , Corazón/embriología , Hiperglucemia/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Animales , Femenino , Expresión Génica/fisiología , Transportador de Glucosa de Tipo 1 , Ratones , Proteínas de Transporte de Monosacáridos/genética , Embarazo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Hypoglycemia is a side effect of diabetes therapy and causes abnormal heart development. Embryonic heart cells are largely resistant to teratogen-induced apoptosis. METHODS: Hypoglycemia was tested for effects on cell death and cell proliferation in embryonic heart cells by exposing mouse embryos on embryonic day (E) 9.5 (plug = E0.5) to hypoglycemia (30-50 mg/dl glucose) in vivo or in vitro for 24 hr. Long-term effects of in vivo exposure on conceptus viability were evaluated at E18.5. Cell death was evaluated on E10.5 by: 1) two TUNEL assays in sectioned embryos to demonstrate DNA fragmentation; 2) confocal microscopy in whole embryos stained with Lysotracker; 3) flow cytometry in dispersed heart cells stained for TUNEL and myosin heavy chain (MHC) to quantify and characterize cell type susceptibility; and 4) immunohistochemistry (IHC) and Western analysis in sectioned embryos to evaluate potential involvement of caspase-3 active subunit and p53. Effects on cell proliferation were evaluated by IHC and Western analysis of proliferating cell nuclear antigen (PCNA). RESULTS: In vivo hypoglycemic exposure on E9.5 reduced viability in conceptuses examined on E18.5. Hearts examined on E10.5 demonstrated increased TUNEL and Lysotracker staining. In hearts of embryos exposed to hypoglycemia, flow cytometry demonstrated increased TUNEL-positive cells and cells dual-labeled for TUNEL and MHC. Protein expression of caspase-3 active subunit and p53 was increased and PCNA was markedly reduced in hearts of embryos exposed to hypoglycemia. CONCLUSIONS: Hypoglycemia reduces embryonic viability, induces significant cell death, and reduces cell proliferation in the E9.5 mouse heart, and these processes may involve active caspase-3 and p53.
Asunto(s)
Muerte Celular , División Celular , Corazón/embriología , Hipoglucemia/patología , Organogénesis , Animales , Caspasa 3 , Caspasas/metabolismo , Citometría de Flujo , Hipoglucemia/metabolismo , Inmunohistoquímica , Ratones , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Pregnancy increases the risk of listeriosis, a systemic disease caused by Listeria monocytogenes. However, there is incomplete agreement on the reasons for this increased risk. We examined two features of listeriosis in gravid and nongravid female mice following intragastric (gavage) inoculation, namely, (i) disease severity (measured by lethality) and (ii) listerial infectivity (measured by liver and spleen colonization levels up to 120 h postinoculation). Two listerial strains of differing serotype (1/2a and 4nonb) were initially employed. Neither strain produced a lethal infection in nonpregnant female mice (dose range, 10(6) to 10(9) CFU/mouse), and only the 4nonb strain produced lethalities in pregnant mice (dose range, 10(6) to 10(8) CFU/mouse). The 4nonb strain also produced a higher level of liver and spleen colonization than the 1/2a strain following gavage administration. (The two strains showed similar levels of colonization if parenterally administered.) Both strains were equally capable of binding to and forming plaques upon cultured mouse enterocytes. The ability of the 4nonb strain to produce a lethal infection in pregnant animals did not correlate with an increased incidence or level of liver and spleen colonization over that in nonpregnant females. However, the lethality rate did correlate well with the rate at which embryos and their surrounding decidual covering became infected, suggesting that intrauterine infection could be responsible for the increased disease severity in the gravid females.
Asunto(s)
Listeriosis/complicaciones , Complicaciones Infecciosas del Embarazo/etiología , Animales , Secuencia de Bases , Colon/microbiología , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Enterocitos/microbiología , Femenino , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/patogenicidad , Listeriosis/etiología , Hígado/microbiología , Ratones , Mutagénesis Insercional , Embarazo , Factores de Riesgo , Bazo/microbiología , Virulencia/genéticaRESUMEN
BACKGROUND: Glucose metabolites can be detected in embryonic mouse tissues using 13C-NMR spectroscopy. The advantage of this method is in its chemical specificity and the ability to follow metabolic changes. METHODS: In this study, CD-1 mice were mated and embryos excised on gestational day (GD) 10.5 (plug = GD 0.5). Hearts were isolated and cultured in 150 mg/dl glucose (normoglycemic medium) or 40 mg/dl glucose (hypoglycemic medium) for 6 hr. 13C-labeled glucose comprised 62%-64% of total glucose in the culture medium. Pre- and postculture media were treated with deuterated water (D2O), and 13C spectra were obtained using a Bruker Avance 500 MHz spectrometer operating at 11.744 tesla (125.7 MHz for 13C). NMR spectra demonstrated resonances for 13C-glucose in preculture normoglycemic and hypoglycemic media. Postculture spectra for normoglycemic and hypoglycemic media demonstrated 13C-glucose signals as well as a signal for 13C-lactate. Area under the curve (AUC) was measured for the [1-(13)C-glucose] resonance from preculture media and the [3-(13)C-lactate] resonance from postculture media. The ratios of AUC for postculture [3-(13)C-lactate] to preculture [1-(13)C-glucose] were calculated and found to be higher in hypoglycemic than in normoglycemic media. RESULTS: Our results confirm earlier findings using radiolabeled substrates and suggest that 13C-NMR spectroscopy can be used to study glucose metabolism in isolated embryonic hearts exposed to hypoglycemia. CONCLUSIONS: NMR effectively measures glucose and its metabolite, lactate, in the same spectrum and thus determines metabolic flux in the isolated embryonic heart after exposure to hypoglycemia and normoglycemia. This method could evaluate glucose metabolism in embryonic tissues following other teratogenic exposures.
Asunto(s)
Glucólisis/fisiología , Corazón/embriología , Hipoglucemia/embriología , Hipoglucemia/metabolismo , Espectroscopía de Resonancia Magnética , Miocardio/metabolismo , Animales , Isótopos de Carbono , Medios de Cultivo , Glucosa/metabolismo , Ratones , Ratones Endogámicos , Técnicas de Cultivo de ÓrganosRESUMEN
Mirex is a pesticide that is environmentally stable, accumulates in body tissues, and is embryo- and feto-toxic at high concentrations in vivo. This study is the first to evaluate the effects of mirex on organogenesis-stage embryos in vitro. Mouse embryos were exposed on gestation day 8.5 for 24 h in whole-embryo culture to mirex at 100, 200, or 400 microg/ml dissolved in xylene and compared with xylene-treated controls (1, 2, or 4 microl/ml, respectively) and untreated controls. Embryos were evaluated for malformations, somite number, total protein content, and visceral yolk sac circulation. Potential embryotoxic mechanisms were evaluated by using PCNA stain for cell proliferation and the TUNEL assay for apoptotic cell death. Mirex-exposed embryos demonstrated increased malformation rates and decreased total embryonic protein contents at > or =200 microg/ml mirex, and decreased somite numbers and VYS circulation at > or =100 microg/ml mirex, compared with xylene-treated controls. There was no difference in PCNA levels or TUNEL staining in mirex-treated embryos compared with xylene-treated controls or untreated controls. Thus, mirex is embryotoxic in vitro to early organogenesis stage mouse embryos at concentrations > or =100 microg/ml, but the effects do not appear to be mediated by changes in cell proliferation or apoptotic cell death.
Asunto(s)
Anomalías Inducidas por Medicamentos/etiología , Embrión de Mamíferos/efectos de los fármacos , Insecticidas/toxicidad , Mírex/toxicidad , Anomalías Inducidas por Medicamentos/embriología , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Embrión de Mamíferos/ultraestructura , Desarrollo Embrionario y Fetal/efectos de los fármacos , Femenino , Proteínas Fetales/biosíntesis , Edad Gestacional , Técnicas In Vitro , Ratones , Antígeno Nuclear de Célula en Proliferación/análisis , Somitos/efectos de los fármacos , Saco Vitelino/irrigación sanguínea , Saco Vitelino/efectos de los fármacosRESUMEN
BACKGROUND: Tolbutamide is a sulfonylurea oral hypoglycemic agent widely used for the treatment of non insulin-dependent diabetes mellitus. Tolbutamide produces dysmorphogenesis in rodent embryos and becomes concentrated in the embryonic heart after maternal oral dosing. Tolbutamide increases glucose metabolism in extra-pancreatic adult tissues, but this has not previously been examined in embryonic heart. METHODS: CD-1 mouse embryos were exposed on GD 9.5 to tolbutamide (0, 100, 250, or 500 microg/ml) for 6, 12, or 24 hr in whole-embryo culture. Isolated hearts were evaluated for (3)H-2DG uptake and conversion of (14)C-glucose to (14)C-lactate. Glut-1, HKI, and GRP78 protein levels were determined by Western analysis, and Glut-1 mRNA was measured by RT-PCR. RESULTS: Cardiac (3)H-2DG uptake increased after exposure to 500 microg/ml tolbutamide for 6 hr, and 100, 250, or 500 microg/ml tolbutamide for 24 hr, compared to controls. Glycolysis increased after exposure to 500 microg/ml tolbutamide for 6 or 24 hr compared to controls. Glut-1 protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 12 or 24 hr, and Glut-1 mRNA increased in hearts exposed to 500 microg/ml tolbutamide for 24 hr compared to controls. HKI protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 6 hr, but not 12 or 24 hr. There was no effect on GRP78 protein levels in hearts exposed to tolbutamide for 6, 12, or 24 hr. CONCLUSIONS: Tolbutamide stimulates glucose uptake and metabolism in the embryonic heart, as occurs in adult extra-pancreatic tissues. Glut-1 and HKI, but not GRP78, are likely involved in tolbutamide-induced cardiac dysmorphogenesis.
Asunto(s)
Glucosa/metabolismo , Corazón/efectos de los fármacos , Teratógenos/toxicidad , Tolbutamida/toxicidad , Animales , Técnicas de Cultivo , Desoxiglucosa/metabolismo , Chaperón BiP del Retículo Endoplásmico , Femenino , Expresión Génica , Transportador de Glucosa de Tipo 1 , Glucólisis/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Corazón/embriología , Corazón/fisiología , Hexoquinasa/metabolismo , Marcaje Isotópico , Ácido Láctico/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Transporte de Monosacáridos/metabolismo , ARN MensajeroRESUMEN
Abnormal embryonic development is a complication of the diabetic pregnancy, and heart defects are among the most common and detrimental congenital malformations of the diabetic embryopathy. Hypoglycemia is a common side effect of diabetes therapy and is a potential teratogen. An association between hypoglycemia and congenital defects has been difficult to demonstrate in humans, but in vivo and in vitro animal studies have illustrated the importance of glucose as a substrate for normal development. Hypoglycemia alters embryonic heart morphology, producing abnormal looping and chamber expansion, decreased myocardial thickness, disorganized layers, and decreased overall size. Hypoglycemia decreases embryonic heart rate and vascularity, and it alters embryonic heart metabolism by increasing glucose uptake and glycolysis. Hypoglycemia also affects protein expression in the embryonic heart, increasing the expression of glucose regulated proteins, hexokinase, and glucose transport protein. Thus, hypoglycemia interferes with normal cardiogenesis and alters morphology, function, metabolism, and expression of certain proteins in the developing heart. It is likely that these factors contribute to heart defects observed in the diabetic embryopathy, but the definitive link has yet to be made. Future studies are expected to further elucidate mechanisms mediating hypoglycemia-induced cardiac dysmorphogenesis.
Asunto(s)
Corazón Fetal/embriología , Corazón Fetal/fisiopatología , Hipoglucemia/embriología , Hipoglucemia/fisiopatología , Embarazo en Diabéticas/embriología , Embarazo en Diabéticas/fisiopatología , Animales , Desarrollo Embrionario y Fetal , Femenino , Corazón Fetal/enzimología , Corazón Fetal/metabolismo , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/fisiopatología , Humanos , EmbarazoRESUMEN
5-Aza-2'-deoxycytidine (d-AZA) causes temporally-related defects in the mouse. At 1.0 mg/kg on gestational day (GD) 10, d-AZA causes hindlimb phocomelia. Sonic hedgehog (Shh) plays a significant role in the normal development of limbs in rodent species. Sonic hedgehog peptides, found in the posterior mesenchyme of limb buds, are involved in patterning functions and in the regulation of both anterior-posterior polarity and proximal-distal outgrowth of the limb. The objective of the present study was to analyze alterations in Shh expression subsequent to d-AZA exposure. Pregnant mice were treated with d-AZA via intraperitonlal injection on GD 10. Controls were untreated. The reverse transcription-polymerase chain reaction (RT-PCR), whole mount in situ hybridization (ISH), and whole mount immunohistochemistry (WMI) were used to analyze expression patterns of Shh . For RT-PCR, embryonic hindlimb buds (buds) were taken 0, 4, 8, 12, or 24 hr after exposure. Cyclophilin was used as the baseline monitor. RNA was transcribed to cDNA and used as template with Shh specific primers for amplification. Whole embryos were collected 12 and 24 hr posttreatment for ISH. An antisense primer specific for Shh was used in an oligo-based ISH protocol. Whole embryos were collected 36 and 48 hr posttreatment for WMI. The antibody corresponding to the amino terminal subunit of the Shh peptide was used. There was a treatment related up-regulation of Shh transcripts by 12 and 24 hr posttreatment. The protein response of up-regulation was detectable by 36 and 48 hr posttreatment. Our data suggest that 5-aza-2'-deoxycytidine-induced hindlimb defects may be associated with alterations in the level of Shh expression. This may be part of a cascade of signaling events involved in d-AZA-induced hindlimb defects. Work is ongoing to determine the relationship of other gene species that may cooperate with Shh in the induction of the hindlimb defects.