Investigating ZMP Accumulation in C2C12 Myotubes with AICAR and Prodrug-39

Abstract

5’Adenosine monophosphate (AMP) is a signaling molecule and a known regulator of enzymes in glycolysis, glycogen breakdown, and may play a role in regulating protein synthesis. It is also a known activator of AMP-dependent protein kinase (AMPK), a key energy sensor within the cell. Activation of AMPK has been linked to the regulation of mitochondrial biogenesis, upregulation of BCAA catabolism, and increased skeletal muscle glucose uptake and fatty acid oxidation. AMPK can be activated pharmacologically with 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an adenosine analogue. AICAR enters the cell through adenosine transporters and undergoes phosphorylation by Adenosine Kinase forming AICAR monophosphate (ZMP). Unfortunately, the poor bioavailability of AICAR limits its use pharmacologically. Prodrug-39 (P39) is comprised of ZMP with a 4-nitrophenol group to allow membrane permeability and subsequent ZMP formation without the need of membrane transporters. We investigated the ZMP accumulation in C2C12 myotubes following exposure to different concentrations of P39 or AICAR. Based on preliminary data, we hypothesized that AICAR treatments would be more effective at producing ZMP over short periods of time and when treatment concentrations were high, but that P39 treatments would result in greater and more consistent ZMP accumulation over longer periods of time when treatment concentrations were low. We first measured the absorbance of 4-nitrophenol (4NP) within the cell media to determine phosphodiesterase cleavage of P39. Nitrophenol absorbance was 3.9 times higher at 1000 μM compared to 100 μM treatment of P39 after 15 minute incubation, and 13.3 times higher at 11 hours. The nitrophenol absorbance per hour increased linearly between 250 μM to 1000 μM of P39. Adenine nucleotide content and ZMP accumulation in treated cells was determined following organic protein extraction and UV-Vis ultra-performance liquid chromatography (UPLC). After 13 hours of treatment with 500 μM AICAR or P39, ZMP accumulation was 14 fold higher following AICAR treatment compared to P39 (59.7±5.0 vs 4.2±0.1 μmol/g protein). Interestingly, ZMP accumulation was not different between 100 μM treatments (5.6±1.3 vs. 5.7±1.5 μmol/g protein). This suggests that phosphodiesterase cleavage of P39 is likely the limiting factor for ZMP accumulation. Interestingly, while ZMP accumulation following 13 hour incubation with 100 μM AICAR or P39 was not different, ATP content was approximately 50% higher in P39 treated cells (33.7±5.8 vs. 51.0±5.8 μmol/g protein). While the measurement of ZMP accumulation at very low treatment concentrations (10 μM) is very diffcult, P39 exposure for 1 to 1.5 hours resulted in a 3 to 8 fold increase in ZMP content compared to AICAR. This data illustrates the potential for P39 as a drug capable of accumulating greater ZMP than AICAR at very low treatment concentrations, however the capacity of phosphodiesterase activity may limit the application of this drug. This work was funded by a grant from Skylark Biosciences. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.