Uptake And Metabolism Of 5’-AMP In The Erythrocyte Play Key Roles In The 5’-AMP Induced Model Of Deep Hypometabolism

UPTAKE AND METABOLISM OF 5’-AMP IN THE ERYTHROCYTE PLAY KEY ROLES IN THE 5’-AMP INDUCED MODEL OF DEEP HYPOMETABOLISM Publication No. ________ Isadora Susan Daniels, B.A. Supervisory Professor: Cheng Chi Lee, Ph.D. Mechanisms that initiate and control the natural hypometabolic states of mammals are poorly understood. The laboratory developed a model of deep hypometabolism (DH) initiated by uptake of 5’-adenosine monophosphate (5’-AMP) into erythrocytes. Mice enter DH when given a high dose of 5’-AMP and the body cools readily. Influx of 5’-AMP appears to inhibit thermoregulatory control. In a 15°C environment, mice injected with 5’-AMP (0.5 mg/gw) enter a Phase I response in which oxygen consumption (VO2) drops rapidly to 1/3rd of euthermic levels. The Phase I response appears independent of body temperature (Tb). This is followed by gradual body temperature decline that correlates with VO2 decline, called Phase II response. Within 90 minutes, mouse Tb approaches 15°C, and VO2 is 1/10th of normal. Mice can remain several hours in this state, before gradually and safely recovering. The DH state translates to other mammalian species. Our studies show uptake and metabolism of 5’-AMP in erythrocytes causes biochemical changes that initiate DH. Increased AMP shifts the adenylate equilibrium toward ADP formation, consequently decreasing intracellular ATP. In turn, glycolysis slows, indicated by increased glucose and decreased lactate. 2,3-bisphosphoglycerate levels rise, allosterically reducing oxygen affinity for hemoglobin, and deoxyhemoglobin rises. Less oxygen transport to tissues likely triggers the DH model. The major intracellular pathway for AMP catabolism is catalyzed by AMP deaminase (AMPD). Multiple AMPD isozymes are expressed in various tissues, but erythrocytes only have AMPD3. Mice lacking AMPD3 were created to study control of the DH model, specifically in erythrocytes. Telemetric measurements demonstrate lower Tb and difficulty maintaining Tb under moderate metabolic stress. A more dramatic response to lower dose of 5’-AMP suggests AMPD activity in the erythrocyte plays an important role in control of the DH model. Analysis of adenylates in erythrocyte lysate shows 3-fold higher levels of ATP and ADP but similar AMP levels to wild-type. Taken together, results indicate alterations in energy status of erythrocytes can induce a hypometabolic state. AMPD3 control of AMP catabolism is important in controlling the DH model. Genetically reducing AMP catabolism in erythrocytes causes a phenotype of lower Tb and compromised ability to maintain temperature homeostasis.

Ablation of suprachiasmatic nucleus alters timing of hibernation in ground squirrels.

Hibernation patterns were monitored continuously for 2.5 years in female squirrels that were neurologically intact or in which the hypothalamic suprachiasmatic nucleus (SCN) was completely ablated (SCNx). The number of hibernation bouts in SCNx squirrels increased by 159%, total hibernation time increased by 58%, and periodic arousals from hibernation were 47% longer in SCNx than in control squirrels; the duration of individual torpor bouts was 2 days shorter and far more variable in SCNx than in control animals. Some SCNx squirrels cycled through bouts of torpor continuously for nearly 2 years. The SCN appears to be part of the mechanism that controls the duration of the hibernation season and the temporal structure of individual torpor bouts.

Investigating the Intrinsic and Extrinsic Drivers of Primate Heterothermy

Seasonal heterothermy—an orchestrated set of extreme physiological responses—is directly responsible for the over-winter survival of many mammalian groups living in seasonal environments. Historically, it was thought that the use of seasonal heterothermy (i.e. daily torpor and hibernation) was restricted to cold-adapted species; it is now known that such thermoregulatory strategies are used by more species than previously appreciated, including many tropical species. The dwarf and mouse lemurs (family Cheirogaleidae) are among the few primates known to use seasonal heterothermy to avoid Madagascar’s harsh and unpredictable environments. These primates provide an ideal study system for investigating a common mechanism of mammalian seasonal heterothermy. The overarching theme of this dissertation is to understand both the intrinsic and extrinsic drivers of heterothermy in three species of the family Cheirogaleidae. By using transcriptome sequencing to characterize gene expression in both captive and natural settings, we identify unique patterns of differential gene expression that are correlated with extreme changes in physiology in two species of dwarf lemurs: C. medius under captive conditions at the Duke Lemur Center and C. crossleyi studied under field conditions in Madagascar. Genes that are differentially expressed appear to be critical for maintaining the health of these animals when they undergo prolonged periods of metabolic depression concurrent with the hibernation phenotype. Further, a comparative analysis of previously studied mammalian heterotherms identifies shared genetic mechanisms underlying the hibernation phenotype across the phylogeny of mammals. Lastly, conducting a diet manipulation study with a captive colony of mouse lemurs (Microcebus murinus) at the Duke Lemur Center, we investigated the degree to which dietary effects influence torpor patterns. We find that tropical primate heterotherms may be exempt from the traditional paradigms governing cold-adapted heterothermy, having evolved different dietary strategies to tolerate circadian changes in body temperature.