Expression of glucose-regulated proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.     

In vivo hyperglycemia and its effect on Glut-1 expression in the embryonic heart

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.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Sonic hedgehog is required for cardiac outflow tract and neural crest cell development

The Hedgehog signaling pathway is critical for a significant number of developmental patterning events. In this study, we focus on the defects in pharyngeal arch and cardiovascular patterning present in Sonic hedgehog (Shh) null mouse embryos. Our data indicate that, in the absence of Shh, there is general failure of the pharyngeal arch development leading to cardiac and craniofacial defects. The cardiac phenotype results from arch artery and outflow tract patterning defects, as well as abnormal development of migratory neural crest cells (NCCs). The constellation of cardiovascular defects resembles a severe form of the human birth defect syndrome tetralogy of Fallot with complete pulmonary artery atresia. Previous studies have demonstrated a role for Shh in NCC survival and proliferation at later stages of development. Our data suggest that SHH signaling does not act directly on NCCs as a survival factor, but rather acts to restrict the domains that NCCs can populate during early stages (e8.5-10.5) of cardiovascular and craniofacial development.     

Variability of responses across training levels to maximal treadmill exercise

The variability of peak VO2 (ml/min, ml.kg-1.min-1), time on treadmill (TMILLTM), maximal heart rate (HRmax), respiratory exchange ratio at peak VO2 (Rmax), rate of respiration at peak VO2 (FREQ), and exercise-induced changes in plasma lactate concentration (LACDIF) was measured across three maximal treadmill runs in five highly trained, seven moderately trained, and five untrained males. No effect of training level on the variability of any of the parameters was found. Test-retest correlation coefficients for peak VO2 (r = 0.95, run 1 with run 2; r = 0.92, run 1 with run 3; r = 0.92, run 2 with run 3) were similar to previously reported values. Variance component distributions suggested that the underlying physiological mechanisms of response for peak VO2, TMILLTM, and HRmax were different from those of FREQ, Rmax, and LACDIF. Minimum detectable differences for peak VO2 (ml.kg-1.min-1, n = 5, minimum detectable within subject difference, 11.5%; minimum detectable among subject effects, 21.3%) indicated a need for careful attention to research design in future studies.     

Prolonged whole-body cold water immersion: fluid and ion shifts

To characterize fluid and ion shifts during prolonged whole-body immersion, 16 divers wearing dry suits completed four whole-body immersions in 5 degrees C water during each of two 5-day air saturation dives at 6.1 msw. One immersion was conducted at 1000 (AM) and one at 2200 (PM) so that diurnal variations could be evaluated. Fifty-four hours separated the immersions, which lasted up to 6 h; 9 days separated each air saturation dive. Blood was collected before and after immersion; urine was collected for 12 h before, during, and after immersion for a total of 24 h. Plasma volume decreased significantly and to the same extent (approximately 17%) during both AM and PM immersions. Urine flow increased by 236.1 +/- 38.7 and 296.3 +/- 52.0%, urinary excretion of Na increased by 290.4 +/- 89.0 and 329.5 +/- 77.0%, K by 245.0 +/- 73.4 and 215.5 +/- 44.6%, Ca by 211.0 +/- 31.4 and 241.1 +/- 50.4%, Mg by 201.4 +/- 45.9 and 165.3 +/- 287%, and Zn by 427.8 +/- 93.7 and 301.9 +/- 75.4% during AM and PM immersions, respectively, compared with preimmersion. Urine flow and K excretion were significantly higher during the AM than PM. In summary, when subjects are immersed in cold water for prolonged periods, combined with a slow rate of body cooling afforded by thermal protection and enforced intermittent exercise, there is diuresis, decreased plasma volume, and increased excretions of Na, K, Ca, Mg, and Zn.     

13C-NMR study of hypoglycemia-induced glycolytic changes in embryonic mouse heart

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.