- Email: [email protected]
52. Roles of the mTOR signaling pathway in hibernating ground squirrels, a differential suppression of active protein synthesis
356
Abstracts / Cryobiology 66 (2013) 343–356
For many small mammals, survival over the winter months is a serious challenge because of low environmental temperatures and limited food availability. The solution for many species, such as thirteen-lined ground squirrels (Ictidomys tridecemlineatus), is hibernation, an altered physiological state characterized by metabolic rate depression to achieve major energy savings, and a coordinated depression of non-essential ATP-expensive functions such as protein synthesis. This study examines the regulation of PTEN phosphatase, a negative regulator of the insulin receptor network, over the torpor–arousal cycle of hibernation in the skeletal muscle of I. tridecemlineatus. Immunoblots showed that the ratio of non-phosphorylated PTEN (Ser380,Thr382/383) to total PTEN levels were significantly elevated in (by 1.4-fold) during late torpor compared to euthermic controls; this was coupled with a significant decrease in Km for PIP3 (by 59%) in late torpor. Pulse-proteolysis analysis of purified PTEN protein showed a decrease in structural stability of PTEN during late torpor compared to euthermic controls (decreased I50 urea by 21%). Furthermore, the increase in PTEN activity observed was correlated with a decrease in the ratio of phosphorylated PDK-1(Ser241) to total PDK-1 in late torpor, suggesting a downstream effect of PTEN activation during torpor. Transcriptional analysis showed that mRNA expression of both PTEN and PDK-1 was unchanged during the course of the hibernation cycle. Overall, the results indicate that post-translational modifications, more specifically phosphorylation, play a crucial role in regulating the enzymatic activity and half-life of PTEN, a major regulator of the insulin signaling pathway, during torpor. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey.
http://dx.doi.org/10.1016/j.cryobiol.2013.02.055
50. Muscle disuse atrophy: The expression of Myocyte Enhancer Factor 2 in the skeletal muscle of Spermophilus tridecemlineatus during hibernation. Shannon Tessier, Kenneth B. Storey, Carleton University, Institute of Biochemistry & Department of Biology, Ottawa, ON, Canada Thirteen-lined ground squirrels, Spermophlus tridecemlineatus, are true hibernators that survive the winter by hibernating in underground burrows from August through to March. During hibernation, squirrels experience little or no muscle atrophy despite a reduced work load. Atrophy is minimized by changing the composition of muscle proteins to provide an optimal mix of isoforms. This study analyzed protein levels of Myocyte Enhancer Factor-2 (MEF-2) in skeletal muscle. MEF-2 proteins are transcription factors that regulate the expression of genes responsible for muscle remodeling in skeletal muscles. Western immunoblotting showed enhanced levels of MEF-2 and an increase in its nuclear localization in muscle of hibernating animals. Finally, Western blots of MEF-2 downstream genes were analyzed. An increase in protein levels of downstream genes supports the activation of MEF-2 transcription factors and the subsequent transcription of genes necessary for muscle remodeling. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey.
52. Roles of the mTOR signaling pathway in hibernating ground squirrels, a differential suppression of active protein synthesis. Cheng-Wei Wu, Kenneth B. Storey, Carleton University, Institute of Biochemistry & Department of Biology, Ottawa, ON Canada For many small mammals, survival during the winter months is a serious challenge because of cold environmental temperatures and limited food availability. The solution can be hibernation, an altered physiological state characterized by seasonal heterothermy and entry into long periods of torpor that are interspersed with short arousals back to euthermia. During torpor, metabolic rate is strongly depressed, often to just 1–5% of resting metabolic rate in euthermia. The thirteen-lined ground squirrel, Spermophilus tridecemlineatus, is a primary model species for hibernation research. A critical part of torpor is the coordinated strong suppression of nonessential ATP-expensive cell functions such as protein synthesis to achieve major energy savings. The mTOR signalling pathway is a critical component of the insulin receptor network and the primary cellular process that is responsible for regulating protein synthesis, through its direct interaction with P70S6K and 4E-BP, two regulators of ribosome assembly. Activity of the mTOR signalling pathway in skeletal and cardiac muscle was assessed at six different time points over the torpor-arousal cycle: active cold room (ACR), entrance of torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IBA). Western blotting showed that phosphorylation of mTOR at the Ser2448 residue was strongly reduced in skeletal muscle (87%) during LT indicating reduced mTOR activity; however, phosphorylation of this residue in cardiac muscle was enhanced during EN and EA stages (by 2.9 and 3.2-fold, respectively), while remaining relatively constant during torpor. Evaluation of TSC2, an upstream inhibitor of the mTOR complex in its inactive form, showed reduced phosphorylation of TSC2Thr1462 in skeletal muscle (66%) during LT. Suppression of mTOR activity also lead to inactivation of selected downstream substrates; e.g. phospho-4EBPThr46 and phospho-70S6KThr389contents decreased in skeletal muscle by 74% and 45% respectively during LT. Meanwhile, levels of phospho-S6 proteinSer235, a component of the 40S ribosome in its active form, were also selectively reduced skeletal muscle (62%) during LT. Differential regulation of mTOR signalling during torpor suggests its alternative roles in tissue dependent responses. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey. http://dx.doi.org/10.1016/j.cryobiol.2013.02.058
53. Cryosurgery of brain tumors with ultrasound neuronavigation control. Sergey Vasiliev 1, S. Pesnya-Prasolov 1, V. Krylov 2, A. Zuev 1, S. Kungurcev 3, 1 Russian Research Center of Surgery, 119992 Department of Neurosurgery, 2, Abrikosovsky pereulok, Moscow, Russia, 2 Scientific research Institute of Emergency Assistance Sklifosfskiy Moscow, Russia, 3 Joint Institute for Nuclear Research, Dubna, Russia
Apoptosis (programmed cell death) is an essential natural biological process. A synergy between proapoptotic and antiapoptotic proteins, interacting at several signalling crossroads determines a cell’s commitment to mitochondria-activated apoptosis. We proposed that changes in expression of antiapoptotic proteins may aid cytoprotection during hibernation of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) to cope with events including low body temperatures, changes in metabolic requirements, ischemia–reperfusion, muscle disuse, etc. Immunoblotting was used to analyse expression of proteins associated with apoptosis in three tissues. In brown adipose Bcl-2, cIAP and xIAP decreased significantly during torpor (compared with euthermia) whereas p-Bcl-2 (Thr 56), p-Bcl-2 (Ser 70), Bcl-XL, Bcl-3, Mcl-1, and BI-1 were unaltered. In skeletal muscle only xIAP levels were altered during hibernation. In heart, most proteins increased significantly, except for Bcl-XL (significant decrease) and xIAP/BI-1 (unchanged). The data show that antiapoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey.
Goal of study: To estimate the results of brain tumor surgery with cryodestruction usage. Materials and methods: The cryodestruction of neuroepithelial tumors was performed in 18 patients. We used a new cryosurgical apparatus. This method of working is based on the passive liquid nitrogen supply and the active evacuation of nascent gaseous one. Tumor stereobiopsy was performed before the cryoprobe introduction. Cryosurgery was performed by introducing the cryoprobe into the tumor with ultrasound neuronavigation control. MRI of the brain was performed before the operation and 1, 3 and 7 days after it on all patients. The efficiency of the cryodestruction and the diagnostic value of ultrasound navigation to control ice-ball generation and postoperative changes in cryodestruction and surrounding zones were estimated. Results: The ultrasound neuronavigation enabled clear visualization of the tumor, the immersion of cryoprobe and ice-ball formation. With ultrasonography a cryoprobe looked like a hyperechoic structure with an acoustic shadow as in all cases with metal equipment. An iceball looked like a hypoechoic tissue near the cryoprobe with hyperechoic contour with 2–3 mm thickness. Preand postoperative MRI permitted a comparison of the zones of tumor and cryodestruction. The postoperative MRI showed that there was intracerebral cyst formation in the zone of cryodestruction of the tumor. This cyst had plain clear borders. There was no residual tumor tissue. Haemorrhage of cryodestruction and surrounding zones was not detected. According to MRI the clearest visualization of cryodestruction zone was up to 3 days. There was no extension of perifocal edema within first 3 days, however, an insignificant one was found by the 7th postoperative day. The contrast accumulation at the border of destruction zone was marked by the 5th day, it was the effect of delimiting gliosis formation. Conclusion: Cryodestruction enables the destruction of brain tumors efficiently in a given volume. Ultrasound neuronavigation is an effective method of monitoring ice-ball formation in the brain during freezing and thawing in real time. This method allows destruction of the tumors which are located in functionally significant brain areas and attempts at removal is associated with a high risk of disability and death.
http://dx.doi.org/10.1016/j.cryobiol.2013.02.057
http://dx.doi.org/10.1016/j.cryobiol.2013.02.059
http://dx.doi.org/10.1016/j.cryobiol.2013.02.056
51. Antiapoptotic signaling as a cytoprotection mechanism during Mammalian hibernation. Andrew N. Rouble, Shannon N. Tessier, Kenneth B. Storey, Carleton University, Institute of Biochemistry & Department of Biology, Ottawa, ON, Canada
Abstracts / Cryobiology 66 (2013) 343–356
For many small mammals, survival over the winter months is a serious challenge because of low environmental temperatures and limited food availability. The solution for many species, such as thirteen-lined ground squirrels (Ictidomys tridecemlineatus), is hibernation, an altered physiological state characterized by metabolic rate depression to achieve major energy savings, and a coordinated depression of non-essential ATP-expensive functions such as protein synthesis. This study examines the regulation of PTEN phosphatase, a negative regulator of the insulin receptor network, over the torpor–arousal cycle of hibernation in the skeletal muscle of I. tridecemlineatus. Immunoblots showed that the ratio of non-phosphorylated PTEN (Ser380,Thr382/383) to total PTEN levels were significantly elevated in (by 1.4-fold) during late torpor compared to euthermic controls; this was coupled with a significant decrease in Km for PIP3 (by 59%) in late torpor. Pulse-proteolysis analysis of purified PTEN protein showed a decrease in structural stability of PTEN during late torpor compared to euthermic controls (decreased I50 urea by 21%). Furthermore, the increase in PTEN activity observed was correlated with a decrease in the ratio of phosphorylated PDK-1(Ser241) to total PDK-1 in late torpor, suggesting a downstream effect of PTEN activation during torpor. Transcriptional analysis showed that mRNA expression of both PTEN and PDK-1 was unchanged during the course of the hibernation cycle. Overall, the results indicate that post-translational modifications, more specifically phosphorylation, play a crucial role in regulating the enzymatic activity and half-life of PTEN, a major regulator of the insulin signaling pathway, during torpor. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey.
http://dx.doi.org/10.1016/j.cryobiol.2013.02.055
50. Muscle disuse atrophy: The expression of Myocyte Enhancer Factor 2 in the skeletal muscle of Spermophilus tridecemlineatus during hibernation. Shannon Tessier, Kenneth B. Storey, Carleton University, Institute of Biochemistry & Department of Biology, Ottawa, ON, Canada Thirteen-lined ground squirrels, Spermophlus tridecemlineatus, are true hibernators that survive the winter by hibernating in underground burrows from August through to March. During hibernation, squirrels experience little or no muscle atrophy despite a reduced work load. Atrophy is minimized by changing the composition of muscle proteins to provide an optimal mix of isoforms. This study analyzed protein levels of Myocyte Enhancer Factor-2 (MEF-2) in skeletal muscle. MEF-2 proteins are transcription factors that regulate the expression of genes responsible for muscle remodeling in skeletal muscles. Western immunoblotting showed enhanced levels of MEF-2 and an increase in its nuclear localization in muscle of hibernating animals. Finally, Western blots of MEF-2 downstream genes were analyzed. An increase in protein levels of downstream genes supports the activation of MEF-2 transcription factors and the subsequent transcription of genes necessary for muscle remodeling. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey.
52. Roles of the mTOR signaling pathway in hibernating ground squirrels, a differential suppression of active protein synthesis. Cheng-Wei Wu, Kenneth B. Storey, Carleton University, Institute of Biochemistry & Department of Biology, Ottawa, ON Canada For many small mammals, survival during the winter months is a serious challenge because of cold environmental temperatures and limited food availability. The solution can be hibernation, an altered physiological state characterized by seasonal heterothermy and entry into long periods of torpor that are interspersed with short arousals back to euthermia. During torpor, metabolic rate is strongly depressed, often to just 1–5% of resting metabolic rate in euthermia. The thirteen-lined ground squirrel, Spermophilus tridecemlineatus, is a primary model species for hibernation research. A critical part of torpor is the coordinated strong suppression of nonessential ATP-expensive cell functions such as protein synthesis to achieve major energy savings. The mTOR signalling pathway is a critical component of the insulin receptor network and the primary cellular process that is responsible for regulating protein synthesis, through its direct interaction with P70S6K and 4E-BP, two regulators of ribosome assembly. Activity of the mTOR signalling pathway in skeletal and cardiac muscle was assessed at six different time points over the torpor-arousal cycle: active cold room (ACR), entrance of torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IBA). Western blotting showed that phosphorylation of mTOR at the Ser2448 residue was strongly reduced in skeletal muscle (87%) during LT indicating reduced mTOR activity; however, phosphorylation of this residue in cardiac muscle was enhanced during EN and EA stages (by 2.9 and 3.2-fold, respectively), while remaining relatively constant during torpor. Evaluation of TSC2, an upstream inhibitor of the mTOR complex in its inactive form, showed reduced phosphorylation of TSC2Thr1462 in skeletal muscle (66%) during LT. Suppression of mTOR activity also lead to inactivation of selected downstream substrates; e.g. phospho-4EBPThr46 and phospho-70S6KThr389contents decreased in skeletal muscle by 74% and 45% respectively during LT. Meanwhile, levels of phospho-S6 proteinSer235, a component of the 40S ribosome in its active form, were also selectively reduced skeletal muscle (62%) during LT. Differential regulation of mTOR signalling during torpor suggests its alternative roles in tissue dependent responses. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey. http://dx.doi.org/10.1016/j.cryobiol.2013.02.058
53. Cryosurgery of brain tumors with ultrasound neuronavigation control. Sergey Vasiliev 1, S. Pesnya-Prasolov 1, V. Krylov 2, A. Zuev 1, S. Kungurcev 3, 1 Russian Research Center of Surgery, 119992 Department of Neurosurgery, 2, Abrikosovsky pereulok, Moscow, Russia, 2 Scientific research Institute of Emergency Assistance Sklifosfskiy Moscow, Russia, 3 Joint Institute for Nuclear Research, Dubna, Russia
Apoptosis (programmed cell death) is an essential natural biological process. A synergy between proapoptotic and antiapoptotic proteins, interacting at several signalling crossroads determines a cell’s commitment to mitochondria-activated apoptosis. We proposed that changes in expression of antiapoptotic proteins may aid cytoprotection during hibernation of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) to cope with events including low body temperatures, changes in metabolic requirements, ischemia–reperfusion, muscle disuse, etc. Immunoblotting was used to analyse expression of proteins associated with apoptosis in three tissues. In brown adipose Bcl-2, cIAP and xIAP decreased significantly during torpor (compared with euthermia) whereas p-Bcl-2 (Thr 56), p-Bcl-2 (Ser 70), Bcl-XL, Bcl-3, Mcl-1, and BI-1 were unaltered. In skeletal muscle only xIAP levels were altered during hibernation. In heart, most proteins increased significantly, except for Bcl-XL (significant decrease) and xIAP/BI-1 (unchanged). The data show that antiapoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart. Funded by NSERC Canada. For more information go to: www.carleton.ca/kbstorey.
Goal of study: To estimate the results of brain tumor surgery with cryodestruction usage. Materials and methods: The cryodestruction of neuroepithelial tumors was performed in 18 patients. We used a new cryosurgical apparatus. This method of working is based on the passive liquid nitrogen supply and the active evacuation of nascent gaseous one. Tumor stereobiopsy was performed before the cryoprobe introduction. Cryosurgery was performed by introducing the cryoprobe into the tumor with ultrasound neuronavigation control. MRI of the brain was performed before the operation and 1, 3 and 7 days after it on all patients. The efficiency of the cryodestruction and the diagnostic value of ultrasound navigation to control ice-ball generation and postoperative changes in cryodestruction and surrounding zones were estimated. Results: The ultrasound neuronavigation enabled clear visualization of the tumor, the immersion of cryoprobe and ice-ball formation. With ultrasonography a cryoprobe looked like a hyperechoic structure with an acoustic shadow as in all cases with metal equipment. An iceball looked like a hypoechoic tissue near the cryoprobe with hyperechoic contour with 2–3 mm thickness. Preand postoperative MRI permitted a comparison of the zones of tumor and cryodestruction. The postoperative MRI showed that there was intracerebral cyst formation in the zone of cryodestruction of the tumor. This cyst had plain clear borders. There was no residual tumor tissue. Haemorrhage of cryodestruction and surrounding zones was not detected. According to MRI the clearest visualization of cryodestruction zone was up to 3 days. There was no extension of perifocal edema within first 3 days, however, an insignificant one was found by the 7th postoperative day. The contrast accumulation at the border of destruction zone was marked by the 5th day, it was the effect of delimiting gliosis formation. Conclusion: Cryodestruction enables the destruction of brain tumors efficiently in a given volume. Ultrasound neuronavigation is an effective method of monitoring ice-ball formation in the brain during freezing and thawing in real time. This method allows destruction of the tumors which are located in functionally significant brain areas and attempts at removal is associated with a high risk of disability and death.
http://dx.doi.org/10.1016/j.cryobiol.2013.02.057
http://dx.doi.org/10.1016/j.cryobiol.2013.02.059
http://dx.doi.org/10.1016/j.cryobiol.2013.02.056
51. Antiapoptotic signaling as a cytoprotection mechanism during Mammalian hibernation. Andrew N. Rouble, Shannon N. Tessier, Kenneth B. Storey, Carleton University, Institute of Biochemistry & Department of Biology, Ottawa, ON, Canada