Acidity Enhances the Ability of 5-Aminoimidazole-4-carboxamide Ribonucleotide to Increase Respiration and Lipid Metabolism in Daphnia magna

Abstract

5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), a structural analog of adenosine monophosphate (AMP), promotes oxidative remodeling in muscle cells. AICAR activates AMP-dependent protein kinase (AMPK), thus increasing lipid metabolism, respiration, and mitochondrial counts. This process is called oxidative remodeling, which enhances the physical endurance of mice. To test this drug on an invertebrate that is genetically similar to humans, we used the small water crustacean Daphnia magna, which is sensitive to changes in water conditions. We tested the effects of pH on the efficacy of AICAR using two methods. One method measured oxygen consumption of Daphnia in oxygen chambers. The other method determined lipid levels of Daphnia through fluorescent tagging of lipids. The results showed that when exposed to AICAR at pH 6.58, D. magna consumed more oxygen and had lower overall levels of lipids, which is consistent with the expected effects of AICAR, such as increased respiration and lipid metabolism.

Figure 1. Percentage change of bpm in D. magna increases as AICAR concentration increases at varying pH. The average resting heart rate of six Daphnia at each pH served as a standard to calculate the percentage change of bpm from the resting bpm. This final dose-response curve was used to determine the optimal sublethal concentration of AICAR. For all pH levels, increased AICAR concentrations generally increased the percentage change relative to the average resting bpm.

Figure 2. Deposition of lipids increases outside of vital organs following exposure to AICAR at acidic pH. After at least 1 h of exposure to Nile Red dye following AICAR treatment, lipids within the D. magna were observed. Nile Red is lipophilic, thus the lipids within the organisms emit orange/yellow fluorescence. Panels A–E, pH 6.58; F–J, pH 7.25;, and K-O, pH 8.51. Panels A, F, and K show the controls. D. magna were observed at exposure times of 15 s, 2 min, 5 min, and 10 min. As the exposure time increased at each pH, fluorescence increased. Fluorescence intensities at pH 8.51 and pH 7.25 were significantly higher compared with that at pH 6.58 pH. At lower pH, the lipids were less widely distributed, instead accumulating around the brain, digestive gland, esophagus, midgut, and upper end of the gut. The fluorescence intensities were highest at pH 8.51. Lipid droplets that were observed in the controls and in only in some AICAR-treated D. magna that emitted, dim yellow-green fluorescence.

Figure 3. Oxygen consumption immediately increases significantly following exposure to AICAR at pH 6.58. AICAR-treated Daphnia at pH 6.58 consumed high levels of oxygen upon immediate exposure. Untreated Daphnia consumed oxygen at a relatively constant rate. AICAR-treated Daphnia eventually returned to normal rates of oxygen consumption. The rate of oxygen consumption was higher at pH 6.58 compared with that at pH 8.51.

Figure 4. Oxygen consumption immediately and slightly increases after exposure to AICAR at pH 7.25. AICAR-treated Daphnia at a pH 7.25 consumed oxygen at a constant rate compared with untreated Daphnia at the same pH. Although the rate of oxygen consumption was slightly greater during the first 15 s of exposure, there was little variance from normal oxygen consumption at that pH. Oxygen consumption by AICAR-treated Daphnia decreased further as exposure increased, similar to that of untreated Daphnia.

Figure 5. Oxygen consumption significantly increases immediately after exposure to AICAR at pH 8.51. D. magna consumed high levels of oxygen immediately (15 s) after exposure to AICAR. In contrast, the control showed very little change in oxygen consumption. Oxygen consumption by AICAR-treated Daphnia returned to normal after prolonged exposure (>15 s).