1. Micro-RNA 132 and 212 mediated regulation of fatty acid metabolism and its effect on insulin secretion.
Prelim Mock Talk by
-Mufaddal S Soni
Attie Lab

2. Outline Introduction Preliminary Data Specific Aims Conclusion
Diabetes model
Micro-RNAs
Preliminary Data
Specific Aims
Conclusion
The Economist, 12/13/03

3. Obesity-diabetes dichotomy
Lean
Lepob/ob B6
BTBR
Obese
Diabetic
Age (weeks) 3

4. What are micro-RNAs?
Chang, Z. C. 2005

5. Micro-RNA 132 and 212 enhances insulin secretion.

6. CACT (Slc25a20) : Most down regulated target of miRNA 132 and 212

7. CACT mediates translocation of FA-Carn into the mitochondria for β-oxidation.

8. CACT mediates translocation of FA-Carn into the mitochondria for β-oxidation.

9. CACT mediates translocation of FA-Carn into the mitochondria for β-oxidation.

10. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion.
Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion.
Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation.
Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished.
Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

11. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion.
Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion.
Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation.
Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished.
Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

12. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion.
Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion.
Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation.
Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished.
Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

13. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion.
Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion.
Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation.
Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished.
Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

14. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion.
Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion.
Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation.
Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished.
Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

15. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion.
Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion.
Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation.
Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished.
Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

16. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion.
Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion.
Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation.
Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished.
Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

17. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.
Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation.
Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion.
Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion.
FFA

18. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.
Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation.
Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion.
Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Ro
PMA
UNC-01
siRNA

19. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.
Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation.
Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion.
Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion.

20. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.
Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation.
Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion.
Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion.
Measure increase in cAMP levels
Probe PKA and Epac pathways

21. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.
Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation.
Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion.
Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion.

22. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.
Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation.
Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion.
Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion.
Wortmannin
PI3K inhibitor

23. Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.
Prof. Randall Mynatt, LSU.

24. Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.
Prof. Randall Mynatt, LSU.

25. Amplification Pathway
Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.
Experiment 2: Determine the phase of insulin secretion altered in CrAT mice.
Amplification Pathway
(2nd Phase)
Triggering Pathway
(1st Phase)

26. Experiment 3: Metabolic profiling of the βCrAT mouse islets.
Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.
Experiment 3: Metabolic profiling of the βCrAT mouse islets.
Fat animals have elevated carnitine pools in the muscle.
I will profile the carnitine metabolites in the βCrAT mouse islets to determine the correlation between insulin secretion and carnitine levels.
Koves, R. T. et.al. 2007

27. Thank You
Summary

29. biotin–HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide