Genexpression während des täglichen Torpors: Molekulare Mechanismen der Stoffwechseldepression

Djungarian hamsters display daily torpor spontaneously as part of their seasonal acclimation to winter conditions, providing a strategy to cope with limited food availability and cold. The torpid state during the daily period of rest is characterized by a decrease of metabolic rate resulting in a reduction of energy expenditure. Similar to hibernation, the entrance into the hypometabolic state involves an active metabolic depression that precedes the development of hypothermia. The present thesis is focused on analyses of gene expression during the entrance into daily torpor, in order to identify differentially expressed genes as well as changes in the global transcriptional and translational activity contributing to the observed metabolic depression. Metabolic rate was measured continuously using indirect calorimetry. Tissues were sampled in defined metabolic states: during normometabolism (controls), in the torpid state (when minimal metabolic rate was first attained during entrance into torpor), and after arousal from torpor. Transcriptional run-on assays were employed to investigate the status of transcriptional initiation as a function of the torpid state. The determination of the in vitro transcription rate in isolated nuclei reflects a snapshot of global transcriptional activity in a given physiological state. Nuclei were isolated from liver tissue of normometabolic, torpid and aroused hamsters and subjected to nuclear run-on assays at a temperature of 25°C. A ~40% decrease in transcriptional initiation was observed in liver nuclei of hamsters which had attained minimal metabolic rate during torpor as compared to nuclei from normometabolic hamsters. During arousal from torpor, the transcriptional run-on activity recovered to the normometabolic level. Polysome profile analysis of liver tissue was used to determine the proportion of actively translating polysomes. Profiles of liver samples from torpid animals show a disaggregation of polysomes compared to profiles from normometabolic hamsters, which indicates that, in addition to transcription, protein synthesis decreases during torpor. These results indicate that during torpor a specific inhibition of the energetically costly processes of RNA and protein synthesis contribute to the overall metabolic depression. As candidates for differentially expressed genes during torpor, the mRNA expression of three isoenzymes of the pyruvate dehydrogenase kinase (PDK1, 2 and 4) were analysed in the heart of normometabolic and torpid hamsters using Northernblots. The kinases inactivate the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDC catalyzes the conversion of pyruvate to acetyl-CoA and is therefore a key enzyme responsible for the entry of carbon units into the tricarboxylic acid cycle. Inactivation of PDC leads to an inhibition of glucose oxidation and favours the utilization of lipids as a metabolic fuel. A previous study revealed a positive correlation between PDC activity and metabolic rate, suggesting a role of pyruvate dehydrogenase complex (PDC) inactivation in the selection of metabolic fuel during daily torpor. A weak increase in the concentration of PDK4 mRNA in the heart of torpid hamsters was observed as compared to normometabolic controls. However, this increase did not result in an altered amount of PDK4 protein in the heart of torpid hamsters. An ~10-fold increase in the mRNA encoding PDK4 occurred in the heart of hamsters after 48 h of food deprivation. The expression of PDK4 during fasting is comparable to the seasonal increase of PDK4 in the heart of hibernating ground squirrels, suggesting that this effect is due to long-term food deprivation during hibernation. Winter acclimated Djungarian hamsters display daily torpor spontaneously, without anticipatory development of starvation symptoms, even when fed ad libitum. Despite the energy savings due to daily torpor, they still rely on continuous feeding during winter. Short-term regulation of PDK activity during daily torpor could be based on allosteric effectors more than on the level of gene expression. Two alternative methods were applied to identify differentially expressed genes in the heart during daily torpor. The cDNA-RDA is based on a subtractive hybridization and a subsequent selective amplification of cDNA-fragments representing differentially expressed transcripts. In addition, mouse Unigene filterarrays were hybridized with cDNA from normometabolic and torpid hamsters. Both experiments resulted in a set of candidate genes that appear to differ between the normometabolic and the torpid state, but none could be confirmed as differentially expressed on Northernblots. The entrance into the torpid state seems not to be the result of major changes in gene expression. Rapid and reversible mechanisms like protein phosphorylation are more likely responsible for the metabolic depression during torpor, enabling cells to resume normal function after arousal from torpor.