kumc presentation
TRANSCRIPT
Investigation of the Role of Sortilin-1 in Insulin/mTOR
Signaling in Muscle C2C12 cellsMichael Epp
Baker UniversityLi Lab
Introduction
Obesity and Insulin Resistance: A Health Concern
*Western Diet
Insulin Resistance and Obesity
Free Fatty Acid Mobilization
Increased Blood Lipids
Fatty Infiltration of the Liver
Type II Diabetes
Hyperlipidemia Heart Disease
Non-Alcoholic Fatty Liver Disease
3 Million U.S. cases per year—9.3% of U.S. population
4th leading cause of death in the U.S. between age 45-54.
No. 1 cause of death worldwide
Sortilin-1• Multi-ligand trans-
membrane receptor
• localized in the trans-Golgi network and early endosome
• Sorts various functionally unrelated proteins through endosome and lysosome pathways
Why Do We Care?
• G.W.A.S suggest strong association with serum cholesterol, triglyceride, and heart disease risk in humans
• Past studies also indicate involvement in glucose metabolism
• Sortilin 1 is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes.
Relevant Studies
Jun Shi and Konstantin V. Kandor. Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes. Developmental Cell. (2005).
https://www.boundless.com/biology/textbooks/boundless-biology-textbook/cellular-respiration-7/regulation-of-cellular-respiration-79/regulatory-mechanisms-for-cellular-respiration-369-11595/
Sort-1 Deletion
Hyperglycemia
Impaired Glucose Uptake
• Sortilin deficiency improves the metabolic phenotype and reduces hepatic steatosis of mice subjected to diet induced obesity by regulating ceramide production.
Sort-1 Deletion
Decreased Fat in Liver and Increased Insulin Sensitivity
Relevant Studies
Rabinowich et al. Sortilin deficiency improves the metabolic phenotype and reduces hepatic steatosis of mice subjected to diet induced obesity by regulating ceramide production. Journal of Hepatology. (2015).
Decreased Ceramide Production
Preliminary Results
Global Sort1 KO Gain Less Weight When Challenged with Western Diet Than WT
20
25
30
35
40
45
50
0 wk 3 wks 5 wks 7 wks 9 wks 11 wks
Body
Wei
ght (
g)
Time On Western Diet Provided by Jibiao LI
**
* **
Sort1 KO Experience 40% Less Fat Accumulation in Liver Than WT
Provided by Jibiao Li
WT+CHOW KO+CHOW
WT+WD KO+WD
Global Sort1 KO Fed Western Diet Are More Insulin Sensitive Than WT
Provided by Jibiao Li
0
100
200
300
400
500
600
700
0 min 30 min 60 min 90 min 120 min
Time After Treatment
[Glu
cose
] (m
g/dL
)
**
*
Preliminary Finding
Which tissue Sort-1 deletion is responsible for increased insulin sensitivity?
Question
Sort-1 KO mice show protection against diet-induced obesity and insulin resistance.
Tissue Expression Levels of Sortilin-1 in Mice
Provided by Jibiao Li
60µg of protein loaded into each well
Sort-1 deletion improves insulin sensitivity in muscle cells.
Hypothesis
Approach
Establish Cell Lines
(C2C12 Mouse Muscle)
Sort-1
Tubulin
WT KD
Western BlotInsulin
Membrane
IRS PI3K AKT
mTORC1
P4EBP-1S6K
S6
Results
Insulin (10 nM) 0 15m 30m 1h 2h 4h 0 15m 30m 1h 2h 4h
WT KD
T-AKT
P-AKT
Actin
Sort1 Knockdown Reduces Insulin –Induced AKT Phosphorylation in C2C12 Cells
P-AKT
P-S6
T-AKT
T-S6
WT (24h TNFα) KD (24h TNFα)Insulin (10nM) 0 15m 30m 0 15m 30m
Sort1 Knockdown Reduces Insulin –Induced AKT Phosphorylation & Increases Phosphorylation
of S6 in TNFα Treated C2C12 Cells
P-S6
T-S6
WT KD 0 2m 15m 30m 1h 90m 0 2m 15m 30m 1h 90m
Amino Acids
Sort1 Knockdown Increases Amino Acid –Induced S6 Phosphorylation in C2C12 Cells
Conclusion/Future Direction• Observations: Sort1 knockdown decreases insulin activation of
AKT and increases activation of S6 in C2C12 muscle cells
• Conclusions: Based on our in-vitro study, results do not support a direct role of muscle Sortilin-1 deletion in improving whole body insulin sensitivity observed in western diet fed Sort-1 knockout mice
• Future Direction: Improved metabolic phenotype may be result of decreased weight gain in Sort-1 KO mice—further studies will investigate the role of sortilin-1 deletion in adipose tissue in regulating body weight
Acknowledgments Li Lab• Tiangang Li, PhD• Jibiao Li, PhD• Yifeng Wang, MS• David Matye, BS
KUMC• Department of Pharmacology,
Toxicology and Therapeutics
Funding• NIH R01DK102487• NIH COBRE
ASPET • Summer undergraduate research
fellowship (SURF) program