AMP Deaminase 3 overexpression regulates cellular energetics and signaling for PGC-1? activation.

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Spencer Miller (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: 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.

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Language: English

Date: 2017

Keywords

AMPK, PGC-1?, ATP

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