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

Author

Isadora Daniels, The University of Texas Graduate School of Biomedical Sciences at HoustonFollow

Date of Graduation

8-2011

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Cheng Chi Lee, Ph.D.

Committee Member

Diane Bick, Ph.D.

Committee Member

Michael R. Blackburn, Ph.D.

Committee Member

Rodney Kellems, Ph.D.

Committee Member

Ann-Bin Shyu, Ph.D.

Abstract

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 1/3^rd of euthermic levels. The Phase I response appears independent of body temperature (T_b). This is followed by gradual body temperature decline that correlates with VO₂ decline, called Phase II response. Within 90 minutes, mouse T_b approaches 15°C, and VO₂ is 1/10^th 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 T_b and difficulty maintaining T_b 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 T_b and compromised ability to maintain temperature homeostasis.

Keywords

5'-adenosine monophosphate, hypometabolism, amp deaminase, erythrocyte, body temperature, hypothermia, torpor, 2, 3-bisphosphoglycerate, glycolysis, mouse