Sarah Sivinski- ESS 2015- Final

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

Let's try to get a picture in here as the background (so faded out) or something just to make this a little more interestingReduce the font size on the authors and affiliations and then shift everything down a little more

OutlineI. Introduction

II. Objective

III. Hypothesis

IV. Study Design & Methods

V. Results

VI. Discussion

VII. Future Studies

VIII. Conclusion

IX. Acknowledgements


Protein Accretion and Depletion


• 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 SynthesisProtein Degradation

I understand that you're trying to use as few words as possible, but you need to use short phrases or something to lead your audience through the content a little. For example:- Protein accretion is dependent on the balance between rates of protein synthesis and degradation (and then you can go on to explain that if synthesis is greater than degradation, you will have a gain of protein)- The initiation of protein synthesis is regulated through the mechanistic target of rapamycin signaling pathways, which has upstream factors that activate it and inhibit it- AMPK activation results in an inhibition of mTOR signaling

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)


Remove "Current study" from title. It should be obvious that this data will be relating to your current study. Perhaps call it "Role of AMPK in the regulation of muscle protein accretion"


Move this into the slide 3 position


Move this into the slide 3 position

mTOR


Meal stimulus

mTOR

Protein

synthesis

You need to put your mTOR pathway slide (what you currently have as slide 8) as your next slide, then these next two slides (which could potentially be removed, but let's wait and see how it works when you practice)

Fasting Treatment Effects


Fasting treatment

Protein degradation

factors

Protein degradation

Fasting treatment

mTOR

Protein Synthesis

Effects of Age and Feeding Status on Measures of Muscle Protein Synthesis


Key:

Fasted

Refed

(Wagner and

Urschel 2012)

- Retitle this slide to something like: effects of age and feeding status on measures of muscle protein synthesis

Objective

To determine the effects of feeding and age on the phosphorylation of AMPK in the gluteus medius muscle of the horse


Remove "The present study will..." and start with To determine... also, it is the phosphorylation of AMPK in the gluteus medius muscle of the horse

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.


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)


Move this into your materials and methods section. It is still the methods for your study, even if you weren't the one to actually do this.

Study Design


-18 h

Feed

at 0 and 30 min

(Postprandial)

Withhold Feed

(Post-absorptive)

0 h 90 min

Blood Samples

Muscle BiopsyMuscle Biopsy

Move this into your materials and methods section. It is still the methods for your study, even if you weren't the one to actually do this.

Materials and Methods


Quantification (ImageJ Software)

X-ray Imaging (VersadocTM

Imager)

ECL Prime Detection

Western Immunoblot

- total and phosphorylated (Thr172

) AMPK

Put the animal/sampling materials and methods slides in before this slide

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


Results


Treatment: P = 0.33

Age: P = 0.73

Treatment X Age: P = 0.49

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


http://www.ker.com/research/

facility/2.html

Protein SynthesisProtein Degradation

(Kumar, Atherton et al. 2009)

Retitle to "Discussion: reasons for the lack of feeding effect on AMPK phosphorylation"

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


-18 h

Feed

at 0 and 30 min

(Postprandial)

Withhold Feed

(Post-absorptive)

0

h

90 min

Blood Samples

Muscle Biopsy

Again, re-title

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)


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)


Just call this slide "Conclusions"Try to reduce the number of words on the slide

Acknowledgements

Members of Dr. Urschel’s Lab

Maine Chance Farm Staff


Don't need to acknowledge your co-authors. Remove the website link for the logo. Not needed

Documents

Sarah Sivinski- ESS 2015- Final