1. Micro-RNAs, Fatty Acids and Insulin Secretion Prelim Mock Talk by -Mufaddal S Soni Attie Lab

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

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

4. What are micro-RNAs? 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 and 212 up- regulated due to obesity in islets Micro-RNA 132 enhances insulin secretion in β-cells *miRNA 212 is also shown to enhance insulin secretion.

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

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

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

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

11. What have I shown? & What is my hypothesis?

12. Conditions that I have shown to enhance 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. 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. Aim 1: Determine the molecular fate of LC-Carn when 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. FFA enhances insulin secretion LC-Carn enhances insulin secretion FFA is the active species Both use independent mechanisms LC-Carn is the active species

16. 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. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. ATP

17. 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. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. ATP

18. 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. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. ATP

19. 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. Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. ATP

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. FFA

21. 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 31-8220 PMA UNC-01 siRNA Ro 31-8220 PMA UNC-01 siRNA Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.

22. 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. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.

23. 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 Measure increase in cAMP levels Probe PKA and Epac pathways Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.

24. 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. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.

25. 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 Wortmannin PI3K inhibitor Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion.

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

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

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

29. 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. Cell Metabolism, 2007 Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.

30. Summary 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.

31. Fatty acyl carnitine enhances insulin secretion Figure 6. (A) Effect of CACT knock-down on insulin secretion. Cells were transfected with siRNA against CACT and a scrambled oligonucleotide 24 hrs after plating and then incubated for 48hrs before the insulin secretion assay. Cells were then stimulated with 6mM glucose for 2 hrs, in the presence and absence of Palmitoyl-Carnitine (Palm-Carn). Insulin secreted is plotted as a % of total insulin content. (B) and (C) depicts effect of long and short chain carnitines on insulin secretion. 48hrs after plating, cells were treated with (B) short and long chain carnitines (50µM) and (C) L- Carnitine and acetyl-carnitine (10mM) for 2 hrs during stimulation with 6mM glucose.

32. Figure 7. Effect of CPT-1 inhibition on Palmitoyl-Carm and Palmitate mediated insulin secretion. Ins-1 β-cells were pre-treated with CPT-1 inhibitor etomoxir for 2 hrs, followed by the adding 6mM glucose and varying doses of Palmitoyl-Carnitine (A) or Palmitate (B) for an additional 2 hrs. Insulin was measured using an insulin ELISA and plotted as a percentage of insulin secreted per total insulin cell content.

33. Figure 8. Fatty acyl Carnitine analogs. Figure 9 Fatty acid mediated GPR40 activation. GPR40/CHO stable cells were incubated with Fluo-4-AM in hanks buffer 24 hrs after seeding for 100mins. Fatty acids and small molecule GPR40 activators (e.g. Cpd-A) were added to cells and fluorescence output (Ca 2+ concentration) was measured using a fluorometric imaging plate reader. Cpd-A and linoleate are potent activators of GPR40, while Palmitoyl-Carnitine and Acetyl-Carnitine were incapable of activating a GPR40 mediated Ca 2+ response.

34. Figure 10. Schematic of iTRAQ study. Cellular peptides are bound with iTRAQ reagents and mixed in a 1:1:1:1 ratio for the 4 experimental conditions. Each iTRAQ consist of 3 moieties: a charged reporter group, a neutral mass balance group and a peptide reactive group. The first MS scan separates the peptides as a function of length, whereas the MS2 scan discriminates the same peptide from different experimental conditions based on the condition-specific iTRAQ reporter. MS2 provides amino acid sequence for individual peptides, from which proteins are identified. Relative changes in protein abundance will be compared between the untreated cells and the LC-Carn treated cells.

35. Priorities / Timeline Year Work plan 2011  Aim 1: Experiment 1 and 2,  Publish manuscript describing CACT as the target of miRNAs 132 and 212.  Experiment 1 of Aim 3 as mice become available. 2012  In vitro insulin secretion studies from  CrAT mice.  Aim 1: Experiment 3 and then based on its result experiment 4 will be conducted by our collaborators at Merck.  Profiling will be done for Aim 1, experiment 3; and Aim 3, experiment 3 simultaneously.  Publish manuscript characterizing  CrAT mice. 2013  Aim 2: Experiment 1, PKC inhibitor studies and activity assays;  Aim 2: Experiment 2, cAMP measurement and PKA and Epac inhibition studies.  Aim 2: Experiment 4, Phospho-proteomics to identify phosphorylation targets. 2014  Aim 2: Experiment 3, determine role of PI3K in LC-Carn mediated insulin secretion.  Aim 2: Experiment 5, determine the role of protein acylation in LC-Carn mediated insulin secretion.  Aim 2: Experiment 5, identify acylated proteins post LC-Carn treatment.  Publish manuscript on the mechanism by which FA-Carn stimulates GSIS.

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

37. State-level Estimates of Obesity and diabetes among Adults aged ≥ 20 years 1999 2009 No Data <10% 10%–14% 15%–19% 20%–24% 25%–29% ≥30% 2008 1998 No Data <4.5% 4.5-5.9% 6.0-7.4% 7.5-8.9% >9.0% Obesity Diabetes