1. An Equal Opportunity University
Sarah E. Sivinski1, Ashley L. Wagner1, Sara L.
Tanner1, Tammy Brewster-Barnes1, Kristine L.
Urschel1
1Animal and Food Sciences, Univ. of Kentucky, Lexington KY
Effects of horse age and
feeding status on AMPK
activation in horses

2. Outline
I. Introduction
II. Objective
III. Hypothesis
IV. Study Design & Methods
V. Results
VI. Discussion
VII. Future Studies
VIII. Conclusion
IX. Acknowledgements
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3. Protein Accretion and Depletion
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• Protein metabolism depends on the ratio of the rates of protein
synthesis : protein degradation (Kumar, Atherton et al. 2009)
• The mechanistic target of rapamycin (mTOR) signaling pathways
regulate the initiation of protein synthesis
• Upstream activation and inhibition factors (Goodman, Mayhew et al. 2011)
• AMP-activated protein kinase (AMPK) results in the inhibition of
protein synthesis (Mihaylova and Shaw 2011)
Protein
Synthesis Protein
Degradation

4. Role of AMPK in the Regulation
of Muscle Protein Accretion
• AMP-activated protein kinase (AMPK)
– Activated by AMP and ADP to regulate growth (Bolster, Crozier et al. 2002)
• Inhibits protein synthesis by:
– Activating tuberous sclerosis complex 2 (TSC2) and therefore
inhibiting ras homologue enriched in brain (RHEB) (Mihaylova and Shaw 2011)
– Activating regulatory-associated protein (raptor) (Gwinn, Shackelford et al. 2008,
Mihaylova and Shaw 2011)
• Activates protein degradation by:
– Activating Forkhead box containing proteins (FoxO) (Goodman, Mayhew et al.
2011)
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7. mTOR
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Meal
stimulus
mTOR
Protein
synthesis

8. Fasting Treatment Effects
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Fasting
treatment
Protein
degradation
factors
Protein
degradation
Fasting
treatment
mTOR
Protein
Synthesis

9. Effects of Age and Feeding Status on Measures
of Muscle Protein Synthesis
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Key:
Fasted
Refed
(Wagner and
Urschel 2012)

10. Objective
To determine the effects of feeding and
age on the phosphorylation of AMPK in
the gluteus medius muscle of the horse
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11. Research Hypothesis
AMPK will become phosphorylated during
the fasting treatment, and phosphorylation
will be greatest in mature horses,
moderate in two-year-olds, and least in
yearlings.
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12. Study Design
• 3 age groups,18 horses:
– 6 yearlings
– 6 two-year-olds
– 6 mature horses
• 2 treatments:
– Postprandial (i.e. refeeding)
– Post-absorptive (i.e. fasted)
• Diets:
– Daily: alfalfa cubes, ration
balancer pellets, and protein
and mineral dietary product
(pelleted)
– Postprandial treatment:
protein and mineral dietary
product (pelleted)
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13. Study Design
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-18 h
Feed
at 0 and 30 min
(Postprandial)
Withhold Feed
(Post-absorptive)
0 h 90 min
Blood Samples
Muscle Biopsy
Muscle Biopsy

14. Materials and Methods
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Quantification (ImageJ Software)
X-ray Imaging (VersadocTM Imager)
ECL Prime Detection
Western Immunoblot
- total and phosphorylated (Thr172) AMPK

15. Statistical Analysis
• Mixed procedure of SAS version 9.3
– Fixed effects: age, feeding state, and the
interaction between age and feeding state
– Random effect: horse nested in treatment
– Significance: P < 0.05
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16. Results
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Treatment: P = 0.33
Age: P = 0.73
Treatment X Age: P = 0.49

17. Discussion
Other stimuli besides
fasting/refeeding
(Wolfe 2006, Drummond, Zhao, Brault et al. 2007, Dreyer et
al. 2008, Kumar, Atherton et al. 2009)
Response to feeding could
depend mostly on
activating stimulatory
pathways rather than
inhibiting stimulatory
pathways
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http://www.ker.com/research/f
acility/2.html
Protein
Synthesis Protein
Degradation
(Kumar, Atherton et al. 2009)

18. Discussion
Insufficient time allotted to
affect AMPK
phosphorylation
– 18-h feed withholding
– 90-min period post-feeding
Protein degradation
pathways more influential
in feeding/fasting response
than inhibitory factors
– FoxO
– Atrogin-1
– MuRF-1
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-18
h
Feed
at 0 and 30 min
(Postprandial)
Withhold Feed
(Post-absorptive)
0
h
90 min
Blood
Samples
Muscle Biopsy

19. Future Studies
• FoxO
– Causes transcription of autophagy-
related (Atg) gene promoters (Zhao,
Brault et al. 2007)
– Activates atrogin-1 and MuRF-1
(Goodman, Mayhew et al. 2011)
• Atrogin-1
– ubiquitin ligase (Sacheck, Ohtsuka et al. 2004)
• MuRF-1
– ubiquitin ligase (Sandri, Sandri et al. 2004)
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20. Conclusions
• Additional research on the effects of feeding on the
signaling pathways regulating muscle protein
synthesis and degradation is needed in the horse
• No significant effect on the ratio of phosphorylated to
total AMPK due to fasting or refeeding treatment or
due to horse age
• Further studies on more downstream protein
degradation activators (Goodman, Mayhew et al. 2011)
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21. Acknowledgements
Members of Dr. Urschel’s Lab
Maine Chance Farm Staff
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