AMP Deaminase 3 overexpression regulates cellular energetics and signaling for PGC-1α activation.
Skeletal muscles undergoing atrophy have decreased [ATP], PGC-1[alpha] expression, and mitochondrial content. This combination of findings is unexpected because decreased [ATP] is often associated with greater [AMP] and subsequent activation of AMPK, a known inducer of PGC-1[alpha] activity and mitochondrial biogenesis. A possible explanation is increased enzyme activity of AMP Deaminase 3 (AMP [rightwards arrow] IMP + NH3), which is highly overexpressed in atrophic muscles and functions to prevent increases in [AMP]. We tested the hypothesis that AMPD3 overexpression would significantly attenuate DNP (mitochondria uncoupler) induced phosphorylation of AMPK(Thr172) and PGC-1[alpha] promotor activity through regulation of intracellular energetics (ATP, ADP, AMP). Methods: Myotubes were transduced with adenoviruses encoding AMPD3 or GFP (control) and then treated for 1 h with 0.6mM 2,4 dinitrophenol (DNP). Nucleotides, amino acids, and proteins were extracted immediately after DNP treatment and measured by UPLC and Western Blot. To confirm DNP was not toxic, myotubes were washed, and samples were collected 1 h later for nucleotides and amino acids. To determine the effect of AMPD3 overexpression on PGC-1[alpha] promotor activity we transfected myoblasts with a 2kb PGC-1[alpha] promotor-luciferase reporter plasmid. After 5 days of AMPD3 or GFP overexpression and a 4-day 100[mu]M DNP treatment, we measured luciferase activity. We also stained myotubes for mitochondria using a green fluorescent dye (MitoTracker green FM) and quantified the percentage of pixels positive for florescence. Results: DNP treatment resulted in a 40% decline in [ATP], and increased [ADP] (1.4-fold), [AMP] (13.8-fold), AMP:ATP ratio (24-fold), and [IMP] (from undetectable) (p<0.001). DNP treatment also significantly increased phosphorylated AMPK(Thr172) (6.1-fold), and phosphorylation of downstream AMPK targets, ACC (Ser79, 4.8-fold) and ULK1 (Ser555, 2-fold) (p<0.001). Aspartic acid levels increased 7.1-fold (p<0.001), suggesting decreased activity of the purine nucleotide cycle (IMP + aspartic acid [rightwards arrow][rightwards arrow] AMP). As expected, myotubes that were overexpressing AMPD3 had significantly attenuated increases in [ADP] (1.1-fold), [AMP] (5.3-fold), and the AMP:ATP ratio (9.6-fold) (p<0.001), and this was reflected by significantly less phosphorylated AMPK(Thr172) (p<0.05). No changes were measured in aspartic acid or phosphorylation of ACC(Ser79) and ULK1(Ser555) between myotubes overexpressing AMPD3 versus GFP. After a 1 h recovery [AMP], AMP:ATP ratio, and [aspartic acid] were no different than vehicle treated, demonstrating recovery of energetics and cell viability. Long-term treatment with DNP significantly increased PGC-1[alpha] promotor activity (1.4-fold, p<0.001) compared to vehicle groups, while 5 days of AMPD3 overexpression significantly decreased PGC-1[alpha] promotor activity (1.3-fold, p<0.005) compared to GFP. Long-term DNP treatment increased the percentage of pixels positive for green florescence (p<0.05), however, myotubes overexpressing AMPD3 had significantly less than controls (p<0.01). Conclusions: Since activation of AMPK and PGC-1[alpha] are critical for increasing mitochondrial biogenesis, our results suggest that overexpression of AMPD3, such as occurs during muscle atrophy, is an important contributor to reductions in mitochondrial content.
Miller, Spencer. (June 2017). AMP Deaminase 3 overexpression regulates cellular energetics and signaling for PGC-1α activation. (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/6376.)
Miller, Spencer. AMP Deaminase 3 overexpression regulates cellular energetics and signaling for PGC-1α activation.. Master's Thesis. East Carolina University, June 2017. The Scholarship. http://hdl.handle.net/10342/6376. August 14, 2020.
Miller, Spencer, “AMP Deaminase 3 overexpression regulates cellular energetics and signaling for PGC-1α activation.” (Master's Thesis., East Carolina University, June 2017).
Miller, Spencer. AMP Deaminase 3 overexpression regulates cellular energetics and signaling for PGC-1α activation. [Master's Thesis]. Greenville, NC: East Carolina University; June 2017.
East Carolina University