Neuroprotective properties of ketosis may be related to the up-regulation of hypoxia inducible factor 1 (HIF-1α), a primary constituent associated with hypoxic angiogenesis and a regulator of neuroprotective responses. The rationale that the utilization of ketones by brain results in elevation of intracellular succinate, a known inhibitor of prolyl-hydroxylase (the enzyme responsible for the degradation of HIF-1α) was deemed as a potential mechanism of ketosis on the up-regulation of HIF-1α. The neuroprotective effect of diet-induced ketosis (3 weeks of feeding a ketogenic diet), as pretreatment, on infarct volume, following reversible middle cerebral artery occlusion (MCAO) and the up-regulation of HIF-1α was investigated. The effect of beta-hydroxybutyrate (BHB), as a pretreatment via intraventricular infusion (4 days of infusion prior to stroke) was also investigated following MCAO. HIF-1α and Bcl-2 (anti-apoptotic protein) protein levels, and succinate content were measured. A 55–70% reduction in infarct volume was observed with BHB infusion or diet-induced ketosis, respectively. HIF-1α and Bcl-2 protein levels increased 3-fold with diet-induced ketosis; BHB infusions resulted in increases in these proteins. As hypothesized, succinate content increased by 55% with diet-induced ketosis and 4-fold with BHB infusion. We conclude, the biochemical link between ketosis and the stabilization of HIF-1α is through the elevation of succinate, and both HIF-1α stabilization and Bcl-2 up-regulation play a role in ketone induced neuroprotection in brain.

SUMMARYTesticular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19 MOLF/Ei ), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation.

BackgroundCertain mutations in the Deadend1 (Dnd1) gene are the most potent modifiers of testicular germ cell tumor (TGCT) susceptibility in mice and rats. In the 129 family of mice, the Dnd1Ter mutation significantly increases occurrence of TGCT-affected males. To test the hypothesis that he Dnd1Ter allele is a loss-of-function mutation; we characterized the consequences of a genetically-engineered loss-of-function mutation in mice, and compared these results with those for Dnd1Ter.ResultsWe found that intercrossing Dnd1+/KO heterozygotes to generate a complete loss-of-function led to absence of Dnd1KO/KO homozygotes and significantly reduced numbers of Dnd1+/KO heterozygotes. Further crosses showed that Dnd1Ter partially rescues loss of Dnd1KO mice. We also found that loss of a single copy of Dnd1 in Dnd1KO/+ heterozygotes did not affect baseline occurrence of TGCT-affected males and that Dnd1Ter increased TGCT risk regardless whether the alternative allele was loss-of-function (Dnd1KO) or wild-type (Dnd1+). Finally, we found that the action of Dnd1Ter was not limited to testicular cancer, but also significantly increased polyp number and burden in the Apc+/Min model of intestinal polyposis.ConclusionThese results show that Dnd1 is essential for normal allelic inheritance and that Dnd1Ter has a novel combination of functions that significantly increase risk for both testicular and intestinal cancer.

The endoplasmic reticulum (ER) is emerging as a contributory component of cell death after ischemia. Since caspase-12 has been localized to the ER and is a novel signal for apoptosis, we examined the message levels and protein expression of caspase-12 after cerebral ischemia in vivo. Animals underwent permanent middle cerebral artery occlusion (MCAO) and were sacrificed 24 h after ischemia. Protein analysis revealed a significant increase in caspase-12 and a corresponding up-regulation of caspase-12 mRNA in the ischemia group compared with that in the sham group. Immunohistochemical analysis revealed diffuse positive immunostaining of caspase-12 throughout the striatum and cerebral cortex in animals that underwent ischemia, with more intense caspase-12 immunostaining in the striatum than in the cortex after ischemia. These results demonstrate that cerebral ischemia initiates an ER-based stress response that results in the transcriptional up-regulation and corresponding increased expression of caspase-12 protein, and may provide a new area for therapeutic intervention to ameliorate outcomes following stroke.