Effects of Treadmill Exercise on the Expression Level of BAX, BAD, BCL-2, BCL-XL, TFAM, and PGC-1α in the Hippocampus of Thimerosal-Treated Rats.

Thimerosal (THIM) induces neurotoxic changes including neuronal death and releases apoptosis inducing factors from mitochondria to cytosol. THIM alters the expression level of factors involved in apoptosis. On the other hand, the anti-apoptotic effects of exercise have been reported. In this study, we aimed to discover the effect of three protocols of treadmill exercise on the expression level of mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), BCL-2-associated death (BAD), BCL-2-associated X (BAX), BCL-XL, and BCL-2 (a pro-survival BCL-2 protein) in the hippocampus of control and THIM-exposed rats. Male Wistar rats were used in this research. Real-time PCR was applied to assess genes expression. The results showed that THIM increased the expression of pro-apoptotic factors (BAD and BAX), decreased the expression of anti-apoptotic factors (BCL-2 and BCL-XL), and decreased the expression of factors involved in mitochondrial biogenesis (TFAM and PGC-1α). Treadmill exercise protocols reversed the effect of THIM on all genes. In addition, treadmill exercise protocols decreased the expression of BAD and BAX, increased the expression of BCL-2, and increased the expression of TFAM and PGC-1α in control rats. In conclusion, THIM induced a pro-apoptotic effect and disturbed mitochondrial biogenesis and stability, whereas treadmill exercise reversed these effects.

Design, expression, purification and crystallization of human 14-3-3ζ protein chimera with phosphopeptide from proapoptotic protein BAD.

14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.

Expression of Apoptosis Regulator Proteins Bcl-2 and Bad in Rat Ovarian Follicular Apparatus during Recovery after Extreme Hypothermia.

The expression of molecular and cellular regulators of apoptosis (proapoptotic protein Bad and antiapoptotic protein Bcl-2) was measured in the follicular apparatus of rat ovaries during the recovery period (days 7 and 14) after hyperthermia (up to rectal temperature 43.5°C). The Bcl-2/Bad index was calculated. The expression of Bcl-2 in the follicular apparatus of rat ovaries increased on day 7 after the exposure. The Bcl-2/Bad index also increased, which suggests that the development of apoptosis by the mitochondrial pathway in follicles was limited at this term after hyperthermia. On day 14 after hyperthermia, the area of immunohistochemical staining for the antiapoptotic protein Bcl-2 significantly decreased in cells of the ovarian follicular epithelium, but the expression of the proapoptotic protein Bad significantly increased; these changes led to a decrease in Bcl-2/Bad index, which attested to weakening of the antiapoptotic defense and activation of oocyte apoptosis by the mitochondrial pathway.

Rosiglitazone attenuates cell apoptosis through antioxidative and anti-apoptotic pathways in the hippocampi of spontaneously hypertensive rats.

Oxidative stress serves an important role in hypertensive brain damage. Peroxisome proliferator­activated receptor γ (PPAR­Î³) agonists possess antioxidative and anti­apoptotic effects. The present study verified the possibility that rosiglitazone serves a neuroprotective role by alleviating oxidative stress and cell apoptosis in the hippocampi of spontaneously hypertensive rats (SHRs). SHRs and age­matched Wistar­Kyoto (WKY; both 56 weeks old) rats received gavage administration of vehicle or rosiglitazone (5 mg/kg/day) for eight weeks. Systolic blood pressure (SBP) was measured by the indirect tail­cuff method. The expression ratio of activated astrocytes was analyzed by glial fibrillary acidic protein immunohistochemistry. PPAR­Î³, inducible nitric oxide synthase (iNOS), gp47phox, B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax) and caspase­3 expression were investigated by quantitative polymerase chain reaction and western blot analysis. The number of apoptotic cells in the hippocampus of four groups was detected using the terminal deoxynucleotidyl transferase­mediated dUTP end­labeling (TUNEL) method. Compared with the WKY group, the SHR group exhibited decreased Bcl­2 and PPAR­Î³ expression, increased SBP, increased ratio of activated astrocytes and TUNEL­positive cells, increased expression of iNOS, gp47phox, caspase­3 and Bax. Rosiglitazone administration increased Bcl­2 and PPAR­Î³ expression, decreased the ratio of activated astrocytes and TUNEL­positive cells, decreased iNOS, gp47phox, caspase­3 and Bax expression in the hippocampi of SHRs. However, rosiglitazone did not significantly decreased SBP in the SHR group. Therefore, rosiglitazone exerts neuroprotective effect through antioxidative and anti­apoptotic pathways, which was independent of blood pressure control.

Proliferation of prostate epithelia induced by IL-6 from stroma reacted with Trichomonas vaginalis.

Benign prostatic hyperplasia (BPH) is characterized by the proliferation of stromal and epithelial cell types in the prostate, and interactions between the two types of cells. We demonstrated previously that proliferation of prostate stromal cells was induced by BPH epithelial cells in response to Trichomonas vaginalis (Tv) infection via crosstalk with mast cells. In this study, we investigated whether IL-6 released by the proliferating stromal cells in turn induce the BPH epithelial cells to multiply. When culture supernatants of the proliferating prostate stromal cells were added to BPH epithelial cells, the latter multiplied, and expression of cyclin D1, FGF2 and Bcl-2 increased. Blocking the IL-6 signalling pathway with anti-IL-6R antibody or JAK1/2 inhibitor inhibited the proliferation of the BPH epithelial cells and reduced the expression of IL-6, IL-6R and STAT3. Also, epithelial-mesenchymal transition was detected in the proliferating BPH epithelial cells. In conclusion, IL-6 released from proliferating prostate stromal cells induced by BPH epithelial cells infected with Tv in turn induces multiplication of the BPH epithelial cells. This result provides first evidence that the inflammatory microenvironment of prostate stromal cells resulting from Tv infection induces the proliferation of prostate epithelial cells by stromal-epithelial interaction.

The RhoJ-BAD signaling network: An Achilles' heel for BRAF mutant melanomas.

Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.

Suture compression induced midpalatal suture chondrocyte apoptosis with increased caspase-3, caspase-9, Bad, Bak, Bax and Bid expression.

OBJECTIVE: Previous studies found bone resorption and chondrocytes loss in mouse models of mid-palatal suture when given continuous compressive force, although chondrocytes response remained unknown. Herein, we design this study to determine how continuous compression force induces chondrocytes apoptosis. METHODS: Thirty C57BL/6 male mice (aged 6 weeks) were randomly assigned into controls (not ligated to a spring), blank controls (ligated with no compression) and the compression group (ligated with 20-g compression). After 4 d, palatal tissues were sampled and stained by TB and safranin-O. Tunel staining measured the percentage of apoptotic chondrocytes, and immunohistochemistry was performed to label apoptosis-associated proteins (e.g., Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, caspase-3, caspase-8 and caspase-9). Intergroup comparison was made by the rank sum test, and P < 0.05 was defined as statistical significance. RESULTS: After 7d of induction, TB and safranin-O staining revealed that the cartilage area in the compression group was significantly decreased, while the control group remained largely unaltered. Tunel staining showed that apoptotic cell numbers in the mid-palatal suture were significantly higher than the control group. Immunohistochemistry showed that mice in the compression group had significantly increased expression of caspase-3, caspase-9, Bad, Bak, Bax and Bid; However, caspase-8 remained unaltered. No expression of Bcl-2 and Bcl-xl was detected. CONCLUSIONS: Continuous compression force induces chondrocytes apoptosis in the mid-palatal suture. This process might be associated with the mitochondrial pathway.

RNAi-mediated downregulation of cyclin Y to attenuate human breast cancer cell growth.

Cyclin Y (CCNY) is a newly identified PFTK1 interacting protein and has been found to be associated with the proliferation and tumorigenesis of human non-small cell lung cancer. In the present study, we analyzed the expression levels of CCNY in 65 cases of breast cancer (BC) tissues and in four BC cell lines, BT-474, MDA-MB-231, T-47D and MCF-7. Lentivirus-mediated short hairpin RNA (shRNA) was employed to knock down CCNY expression in MCF-7 and MDA-MB-231 cells. The effects of CCNY depletion on cell growth were examined by MTT, colony formation and flow cytometry assays. The results showed that immunohistochemical expression of CCNY in tumor tissues is stronger than that in normal tissues. CCNY was also expressed in all four BC cells. The knockdown of CCNY resulted in a significant reduction in cell proliferation and colony formation ability. Cell cycle analysis showed that CCNY knockdown arrested MDA-MB­231 cells in the G0/G1 phase. Furthermore, depletion of CCNY inhibited BC cell growth via the activation of Bad and GSK3ß, as well as cleavages of PARP and caspase-3 in a p53-dependent manner. Therefore, we believe that CCNY has biological effect in BC development, and its inhibition via an RNA interference lentiviral system may provide a therapeutic option for BC.

Naltrexone at low doses upregulates a unique gene expression not seen with normal doses: Implications for its use in cancer therapy.

It has been reported that lower doses of the opioid antagonist naltrexone are able to reduce tumour growth by interfering with cell signalling as well as by modifying the immune system. We have evaluated the gene expression profile of a cancer cell line after treatment with low-dose naltrexone (LDN), and assessed the effect that adapting treatment schedules with LDN may have on enhancing efficacy. LDN had a selective impact on genes involved with cell cycle regulation and immune modulation. Similarly, the pro-apoptotic genes BAD and BIK1 were increased only after LDN. Continuous treatment with LDN had little effect on growth in different cell lines; however, altering the treatment schedule to include a phase of culture in the absence of drug following an initial round of LDN treatment, resulted in enhanced cell killing. Furthermore, cells pre-treated with LDN were more sensitive to the cytotoxic effects of a number of common chemotherapy agents. For example, priming HCT116 with LDN before treatment with oxaliplatin significantly increased cell killing to 49±7.0 vs. 14±2.4% in cultures where priming was not used. Interestingly, priming with NTX before oxaliplatin resulted in just 32±1.8% cell killing. Our data support further the idea that LDN possesses anticancer activity, which can be improved by modifying the treatment schedule.

Polyphenols from Korean prostrate spurge Euphorbia supina induce apoptosis through the Fas-associated extrinsic pathway and activation of ERK in human leukemic U937 cells.

The Korean prostrate spurge Euphorbia supina (Euphorbiaceae family) has been used as a folk medicine in Korea against a variety of ailments such as bronchitis, hemorrhage, jaundice and multiple gastrointestinal diseases. Polyphenols from Korean E. supina (PES) which include quercetin and kaempferol derivatives have anticancer properties. Hence, we investigated the anticancer effects of PES on U937 human leukemic cells. Firstly, PES significantly inhibited the proliferation of U937 cells in a dose-dependent manner. PES induced accumulation of the sub-G1 DNA content (apoptotic cell population), apoptotic bodies and chromatin condensation and DNA fragmentation in the U937 cells. PES also induced activation of caspase-3, -8 and -9, subsequent cleavage of PARP, and significantly suppressed XIAP, cIAP-1 and cIAP-2 in a dose-dependent manner. Furthermore, PES activated Bid, and induced the loss of mitochondrial membrane potential (MMP, ΔΨm) along with upregulation of pro-apoptotic proteins (Bax and Bad), and downregulation of anti-apoptotic proteins (Bcl-2 and Bcl-xL) and cytochrome c release. The Fas receptor was upregulated by PES in a dose-dependent manner, suggesting that the extrinsic pathway was also involved in the PES-induced apoptosis. Moreover, the PES-induced apoptosis was at least in part associated with extracellular signal-regulated kinase (ERK) activation in the U937 human leukemic cells. This study provides evidence that PES may be useful in the treatment of leukemia.

Ruxolitinib synergizes with DMF to kill via BIM+BAD-induced mitochondrial dysfunction and via reduced SOD2/TRX expression and ROS.

We determined whether the myelofibrosis drug ruxolitinib, an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could interact with the multiple sclerosis drug dimethyl-fumarate (DMF) to kill tumor cells; studies used the in vivo active form of the drug, mono-methyl fumarate (MMF). Ruxolitinib interacted with MMF to kill brain, breast, lung and ovarian cancer cells, and enhanced the lethality of standard of care therapies such as paclitaxel and temozolomide. MMF also interacted with other FDA approved drugs to kill tumor cells including Celebrex® and Gilenya®. The combination of [ruxolitinib + MMF] inactivated ERK1/2, AKT, STAT3 and STAT5; reduced expression of MCL-1, BCL-XL, SOD2 and TRX; increased BIM expression; decreased BAD S112 S136 phosphorylation; and enhanced pro-caspase 3 cleavage. Expression of activated forms of STAT3, MEK1 or AKT each significantly reduced drug combination lethality; prevented BAD S112 S136 dephosphorylation and decreased BIM expression; and preserved TRX, SOD2, MCL-1 and BCL-XL expression. The drug combination increased the levels of reactive oxygen species in cells, and over-expression of TRX or SOD2 prevented drug combination tumor cell killing. Over-expression of BCL-XL or knock down of BAX, BIM, BAD or apoptosis inducing factor (AIF) protected tumor cells. The drug combination increased AIF : HSP70 co-localization in the cytosol but this event did not prevent AIF : eIF3A association in the nucleus.

Glucagon-like peptide-1 protects cardiomyocytes from advanced oxidation protein product-induced apoptosis via the PI3K/Akt/Bad signaling pathway.

Cardiomyocyte apoptosis is a major event in the pathogenesis of diabetic cardiomyopathy. Currently, no single effective treatment for diabetic cardiomyopathy exists. The present study investigated whether advanced oxidative protein products (AOPPs) have a detrimental role in the survival of cardiomyocytes and if glucagon-like peptide-1 (GLP-1) exerts a cardioprotective effect under these circumstances. The present study also aimed to determine the underlying mechanisms. H9c2 cells were exposed to increasing concentrations of AOPPs in the presence or absence of GLP-1, and the viability and apoptotic rate were detected using a cell counting kit-8 assay and flow cytometry, respectively. In addition, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor, LY294002, was employed to illustrate the mechanism of the antiapoptotic effect of GLP-1. The expression levels of the apoptotic-associated proteins, Akt, B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter (Bad), Bcl-2-associated X protein (Bax) and caspase-3 were measured by western blotting. It was revealed that GLP-1 significantly attenuated AOPP-induced cell toxicity and apoptosis. AOPPs inactivated the phosphorylation of Akt, reduced the phosphorylation of Bad, decreased the expression of Bcl-2, increased the expression of Bax and the activation of caspase-3 in H9c2 cells. GLP-1 reversed the above changes induced by AOPPs and the protective effects of GLP-1 were abolished by the PI3K inhibitor, LY294002. In conclusion, the present data suggested that GLP-1 protected cardiomyocytes against AOPP-induced apoptosis, predominantly via the PI3K/Akt/Bad pathway. These results provided a conceivable mechanism for the development of diabetic cardiomyopathy and rendered a novel application of GLP-1 exerting favorable cardiac effects for the treatment of diabetic cardiomyopathy.

Protective effects of HGF gene-expressing human mesenchymal stem cells in acetaminophen-treated hepatocytes.

Mesenchymal stem cells (MSC) secrete a great variety of cytokines that have beneficial paracrine actions. Hepatocyte growth factor (HGF) promotes proliferation in several cell types. The aim of the present study was to investigate the protective effect of HGF gene-transfected MSC (HGF-MSC) in acetaminophen (AAP)-treated hepatocytes. We transfected the HGF gene into MSCs and confirmed HGF expression by RT-PCR and western blot. The concentration of HGF in HGF-MSC conditioned media (HGFCM) was upregulated compared with that in control MSCCM samples. Cell viability was increased in HGFCM-treated hepatocytes. Expression of Mcl-1, an anti-apoptosis protein, was increased and expression of pro-apoptosis proteins (Bad, Bik and Bid) was decreased in HGFCM-treated hepatocytes. HGF-MSC had protective effects on AAP-induced hepatocyte damage by enhancing proliferation. These results suggest that HGF-expressing MSCs may provide regenerative potential for liver cell damage.

Glucose-regulated protein 78 mediates the therapeutic efficacy of 17-DMAG in colon cancer cells.

Glucose-regulated protein 78 (GRP78) is expressed as part of the molecular response to endoplasmic reticulum (ER) stress and mediates protein folding within the cell. GRP78 is also an important biomarker of cancer progression and the therapeutic response of patients with different cancer types. However, the role of GRP78 in the cytotoxic effect of 17-DMAG in colon cancer cells remains unclear. GRP78 expression was knocked down by small interfering RNA (siRNA). The anticancer effects of 17-DMAG were assessed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometric cell-cycle analysis, and an Annexin V-propidium iodide (PI) apoptotic assay. We found that HT-29 cells expressed a lower level of GRP78 compared with DLD-1 cells. The MTT assay revealed that HT-29 cells were more sensitive to 17-DMAG treatment than DLD-1 cells. GRP78 knock down (GRP78KD) cells demonstrated an increased sensitivity to 17-DMAG treatment compared with the scrambled control cells. Based on the cell-cycle analysis and Annexin V-PI apoptotic assay, apoptosis dramatically increased in GRP78KD cells compared with scrambled control DLD-1 cells after these cells were treated with 17-DMAG. Finally, we observed a decrease in the level of Bcl-2 and an increase in the levels of Bad and Bax in GRP78KD cells treated with 17-DMAG. These results are consistent with an increased sensitivity to 17-DMAG after knock down of GRP78. The level of GRP78 expression may determine the therapeutic efficacy of 17-DMAG against colon cancer cells.

Recombinant hirudin suppresses the viability, adhesion, migration and invasion of Hep-2 human laryngeal cancer cells.

Recombinant hirudin (rH) is a highly potent and specific inhibitor of thrombin, and has been shown to inhibit the growth and metastasis of several types of cancers in experimental tumor models. The objective of this study was to evaluate the antitumor effects and explore the underlying mechanisms of rH in Hep-2 human laryngeal carcinoma (LC) cells. Hep-2 cells were treated with various concentrations of rH for 24 h. The cell viability was evaluated by a water-soluble tetrazolium salt (WST) assay. The adhesion ability of the cells was evaluated by cell adhesion to fibronectin. Cell migration and invasion were measured with the Boyden chamber assay. Cell apoptosis was detected by Hoechst 33324 fluorescence staining. A chicken chorioallantoic membrane (CAM) assay was used to assess the effects of rH on angiogenesis in vivo. Western blotting was used to detect the expression levels of vascular endothelial growth factor receptor (VEGF-R), focal adhesion kinase (FAK), Bcl-2-associated agonist of cell death (Bad) and B-cell CLL/lymphoma 2 (Bcl-2) proteins. rH significantly inhibited the cell viability and induced apoptosis in LC Hep-2 cells in a dose-dependent manner, as compared with phosphate-buffered saline (PBS) as control. These results were accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bad. Moreover, rH dose-dependently inhibited the adhesion, migration and invasion of the Hep-2 cells, compared to the vehicle PBS. In addition, rH robustly suppressed angiogenesis in the CAM assay. Importantly, the expression of adhesion and angiogenesis-associated proteins FAK and VEGF-R was significantly downregulated by rH in a dose-dependent manner. The present findings demonstrate that rH exerts antitumor effects in Hep-2 human laryngeal cancer cells via multiple mechanisms and suggests that targeting thrombin by rH is a potential strategy for the treatment of LC.

The downregulation of Bcl-xL/Bcl-2-associated death promoter indicates worse outcomes in patients with small cell lung carcinoma.

It is well known that lung cancer is the 1st leading cause of death worldwide. Many reports have demonstrated that Bad, the Bcl-xL/Bcl-2-associated death promoter plays a crucial role in the intrinsic apoptosis pathway. The aim of this study was to explore the expression of Bad and its clinical significance in small cell lung carcinoma (SCLC) By analyzing the expression of Bad in 147 SCLC patient specimen, we found that Bad expression was remarkably decreased in 55.8% (82/147) cases, compared with the neighboring non-tumor tissues. Further study showed that Bad expression was correlated with adverse clinical characters such as clinical stage (P = 0.001), tumor size (P = 0.036) and tumor recurrence (P = 0.030). Furthermore, the results of Kaplan-Meier analysis showed that low Bad expression was significantly correlated to overall survival (P < 0.0001) and disease-free survival (P = 0.017) of patients with SCLC. Moreover, multivariate analyses revealed that Bad was an independent indicator of overall survival in SCLC (hazard ration = 0.620, 95% confidence interval: 0.389-0.987, P < 0.001). In summary, we can conclude that patients with SCLC represent downregulation of Bad and the latter could be served as a useful biomarker for the outcomes of SCLC.

Recombinant Human Keratinocyte Growth Factor Induces Akt Mediated Cell Survival Progression in Emphysematous Mice.

INTRODUCTION: Emphysema has been associated with decreased VEGF and VEGFR-2 expression and the presence of high numbers of apoptotic alveolar cells. Keratinocyte growth factor stimulates VEGF synthesis which in turn confers normal lung structure maintenance via the Akt pathway. In this study the potential role of rHuKGF in the improvement of deregulated Akt mediated cell survival pathway in emphysematous mice was investigated. METHODS: Three experimental groups, i.e., emphysema, treatment and control groups, were prepared. Lungs of mice were treated on 3 occasions by oropharyngeal instillation of 10mg rHuKGF per kg body weight after induction of emphysema with porcine pancreatic elastase. Subsequently, lung tissues from mice were collected for histopathology and molecular biology studies. RESULTS AND DISCUSSION: Histopathology photomicrographs and destructive index analysis have shown that elastase-induced airspace enlargement and loss of alveoli recovered in the treatment group. rHuKGF stimulates VEGF production which in turn induces the Akt mediated cell survival pathway in emphysematous lungs. mRNA expression of VEGF, VEGFR, PI3K and Akt was significantly increased while Pten, Caspase-9 and Bad was notably decreased in treatment group when compared with emphysema group, being comparable with the control group. Moreover, VEGF protein expression was in accordance with that found for mRNA. CONCLUSION: Therapeutic rHuKGF supplementation improves the deregulated Akt pathway in emphysema, resulting in alveolar cell survival through activation of the endogenous VEGF-dependent cell survival pathway. Hence rHuKGF may prove to be a potential drug in the treatment of emphysema.

Fasting mediated increase in p-BAD(ser155) and p-AKT(ser473) in the prefrontal cortex of mice.

BAD-deficient mice and fasting have several common functional roles in seizures, beta-hydroxybutyrate (BHB) uptake in brain and alteration in counterregulatory hormonal regulation during hypoglycemia. Neuronal specific insulin receptor knockout (NIRKO) mice display impaired counterregulatory hormonal responses during hypoglycemia. In this study we investigated the fasting mediated expression of p-BAD(ser155) and p-AKT(ser473) in different regions of brain (prefrontal cortex, hippocampus, midbrain and hypothalamus). Fasting specifically increases p-BAD(ser155) and p-AKT(ser473) in prefrontal cortex and decreases in other regions of brain. Our results suggest that fasting may increase the uptake BHB by decreasing p-BAD(ser155) in the brain during hypoglycemia except prefrontal cortex and it uncovers specific functional area of p-BAD(ser155) and p-AKT(ser473) that may regulates counter regulatory hormonal response. Overall in support with previous findings, fasting mediated hypoglycemia activates prefrontal cortex insulin signaling which influences the hypothalamic paraventricular nucleus mediated activation of sympathoadrenal hormonal responses.

Specific epiblast loss and hypoblast impairment in cattle embryos sensitized to survival signalling by ubiquitous overexpression of the proapoptotic gene BAD.

Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day 13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.