1. Micro-RNAs, Fatty Acids and 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. Mature miRNA: Mechanism of action
Shivdasani, R.A. Blood 2006

5. Micro-RNA 132 and 212 up-regulated due to obesity in islets
~13 fold in B6 (Diabetes resistant)
and
~3 fold in BTBR mice (Diabetes susceptible)

6. Micro-RNA 132 enhances insulin secretion in β-cells
Micro-RNA 132 and 212 up-regulated due to obesity in islets
Micro-RNA 132 enhances insulin secretion in β-cells
Note: miRNA 212 is also shown to enhance insulin secretion.

7. slc25a20 is the most down-regulated gene
slc25A20 = CACT
Carnitine Acyl-Carnitine Translocase
(CACT)

8. CACT Insulin Secretion in β-cells
slc25a20 is the most down-regulated gene
(CACT)

9. CACT mediates translocation of FA-Carn into the mitochondria for β-oxidation
miRNA 132
ATP

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

11. Preliminary Data & Hypothesis..

12. Conditions that I have shown to enhance insulin secretion
ATP

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.
ATP

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.
ATP

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.
ATP

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.
ATP

17. 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.
ATP

18. 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.
ATP

19. 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.
ATP

20. Aim 2: Identify the underlying signaling pathway using which 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

21. Aim 2: Identify the underlying signaling pathway using which 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

22. Aim 2: Identify the underlying signaling pathway using which 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

23. Aim 2: Identify the underlying signaling pathway using which 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

24. Aim 2: Identify the underlying signaling pathway using which 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.

25. Aim 2: Identify the underlying signaling pathway using which 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.

26. Aim 2: Identify the underlying signaling pathway using which 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.

27. Aim 2: Identify the underlying signaling pathway using which 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.

28. Aim 2: Identify the underlying signaling pathway using which 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.

29. Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.
Glucose Tolerance Test
ATP
β-cell specific CrAT KO mouse
Glucose stimulated insulin secretion
Prof. Randall Mynatt, LSU.

30. Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.
Glucose Tolerance Test
Factors leading to defective glucose clearance:
Reduced insulin secretion.
Insulin resistance.
Experiment 1: Measure insulin secretion and glucose clearance of β-cell CrAT KO mice.
Repeat the “Glucose Tolerance Test” and also track insulin levels using C-peptide measurements.
2. Perform an “Insulin Tolerance Test” to measure insulin sensitivity of the CrAT KO mice.

31. 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.
Perifusion study
Amplification Pathway
(2nd Phase)
Triggering Pathway
(1st Phase)

32. 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.
Carnitine metabolites of the βCrAT mouse islets will be profiled.
A correlation between acyl carnitine levels and insulin secretion will be obtained.

33. Conclusion Aim 1: Determine the active species
Aim 2: Determine the signaling pathway involved in LC Carnintine mediated insulin secretion.
Aim 3: Identify the function of CrAT in insulin secretion.

34. Thank You!!!
Questions??

35. FFA enhances insulin secretion LC-Carn enhances insulin secretion
FFA is the active species
Both use independent mechanisms
LC-Carn is the active species
Koves, R. T. et.al. Cell Metabolism, 2007