Differential Responses to Leucine Activated mTOR in Heart, Gastrocnemius, Spleen, and Liver Tissues of Mice

Kelsey Kerr
Department of Human Nutrition, Foods, and Exercise, Virginia Tech, Blacksburg, VA

ABSTRACT
Leucine is an essential branched chain amino acid (BCAA) that plays important roles in protein synthesis. In addition to being a building block of proteins, leucine promotes protein synthesis through the mammalian target of rapamycin (mTOR) complex 1. mTOR is a protein kinase that senses nutrient and growth factors to ultimately regulate cell growth, proliferation, and protein synthesis. Knocking out the mitochondrial branched chain aminotransferase (BCATm) enzyme, which catalyzes the first step of BCAA metabolism, results in constitutively elevated leucine in the bloodstream. BCATm KO mice exhibit elevated BCAA levels, hypertrophy of the spleen and heart, and a lean phenotype, despite higher food consumption. Interestingly, no noticeable hypertrophy of skeletal muscle in BCATm KO mice is present, suggesting tissue dependence of mTORC1 response to leucine. On the other hand, hypertrophy of the spleen and heart was absent in the BCATm KO mice consuming rapamycin, a specific inhibitor of mTORC1. To shed more light on the contribution of mTORC1, we decided to investigate the effects of elevated leucine levels on mTORC1 activation in gastrocnemius, heart, spleen, and liver tissues by treating the wild type and BCATm KO mice with rapamycin. The activation of mTORC1 was measured using western blotting to determine the phosphorylation states of signaling proteins involved in the pathway. As expected, total mTORC1 activity was highest in the BCATm KO mice, and the activity was lowered with rapamycin. While this trend was observed in all the tissues, the heart was the most responsive, while the liver was the least responsive. The results indicate that over activation of mTORC1 by leucine is sufficient to induce hypertrophy in certain tissues and that mTORC1 is differentially regulated.

RESULTS
Heart Results
Band Intensities, Relative to Control

CONCLUSIONS
Over activation of mTORC1 by leucine is sufficient to produce hypertrophy in some tissues mTORC1 responds similarly to leucine and rapamycin in different tissues, however, the degree of response varies from tissue to tissue.
Jamshid Davoodi

Figure 3: Organ weight for the wild type and BCATm KO mice fed with either NBCAA or IBCAA diet containing sham capsules or encapsulated rapamycin for a period of 13 days. Jamshid Davoodi

mTOR

P-mTOR

P-mTOR/mTOR
Jamshid Davoodi

S6

P-S6

P-S6/S6

Figure 4

Figure 5

Gastrocnemius Results
Band Intensities, Relative to Control

BACKGROUND
mTOR is a part of a complex pathway that controls cell growth, proliferation, and protein synthesis Leucine activates mTOR,while rapamycin inhibits it When AMPK is phosphylated, and thus activated, it inhibits mTOR When activated, mTOR phosphorylates S6 and 4EBP1 to promote cell growth and proliferation When BCATm is knocked out, it prevents the metabolism of leucine, a BCAA.
Simplified mTORC1 Pathway

AMPK

P-AMPK

P-AMPK/AMPK

mTOR

P-mTOR

P-mTOR/mTOR

S6

P-S6

P-S6/S6

Figure 6

Figure 7

Figure 8

The heart is more sensitive to mTORC1 regulation through leucine and rapamycin than the spleen, gastrocnemius, and liver. This is demonstrated by increased levels of phosphorylated protein in the mTORC1 pathway as a result of elevated leucine, as well as more significant decreases in protein in response to rapamycin. mTORC1 is differentially regulated throughout the body

Figures 6,9,12: The band intensities represent the amount of relative protein for AMPK and P-AMPK. AMPK provides total protein concentration, while P-AMPK accounts only for the active form, which inhibits mTOR.

Figures 4,7,10,13: The band intensities represent the amount of relative protein for mTOR and PmTOR. mTOR provides total protein concentration, while P-mTOR accounts only for active mTOR.

Figures 5,8,11,14: The band intensities represent the amount of relative protein for S6 and P-S6. S6 provides total protein concentration, while P-S6 accounts only for active S6, which corresponds to active mTOR.

Spleen Results
Band Intensities, Relative to Control

ACKNOWLEDGEMENTS
A special thanks to Dr. Susan Hutson, Dr. Jamshid Davoodi, Meghan Jones, Dr. Adele Addington, Dr. Elistsa Ananieva, Dr. Marzieh
AMPK P-AMPK P-AMPK/AMPK

In pathway: AMP= adenosine monophosphate ATP= adenosine triphosphate AMPK- adenosine monophosphate-activated protein kinase S6K1- S6 kinase 1 4EBP1- 4E binding protein 1

mTOR

P-mTOR

P-mTOR/mTOR

S6

P-S6

P-S6/S6

Figure 9

Figure 10

Figure 11

METHODS
Tissues from WT, KO, WT + Rapamycin, and KO + Rapamycin mice were homogenized A protein assay was used to determine protein concentration Western blotting was used to determine protein levels and phosphorylation states ImageJ was used to quantify the bands on the western blot
WT KO WT + Rap KO + Rap

Liver Results
Band Intensities, Relative to Control

Taghavi, Dr. Deborah Good, and Christina McIntyre Fralin Life Sciences Institute- Dr. Dennis Dean Department of Human Nutrition, Foods, and Exercise- Dr. Susan Hutson College of Agriculture and Life Sciences- Dean Susan Sumner USDA Higher Education Challenge Grant, CSREES #2007-38411-18146

AMPK

P-AMPK

P-AMPK/AMPK

Figure 2: Example of a western blot showing mTOR in the liver

Figure 12

mTOR

P-mTOR

P-mTOR/mTOR

S6

P-S6

P-S6/S6

Figure 13

Figure 14