1. Micro-RNAs, Fatty Acids and Insulin Secretion Midwest Islet Conference -Mufaddal S Soni Attie Lab

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

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

4. Mature miRNA: Mechanism of action Shivdasani, R.A. Blood 2006 4

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) 5

6. 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. 6

7. slc25A20 = CACT Carnitine Acyl-Carnitine Translocase slc25a20 is the most down- regulated gene (CACT) 7 Genes colored in red contain seed regions specific for miR132 and 212.

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

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

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

11. Fatty acyl carnitine enhances insulin secretion 10mM 50µM 600µM

12. Fatty acyl carnitine enhances insulin secretion Cytosolic accumulation of Fatty acyl carnitine enhances insulin secretion 10mM 50µM 600µM 50µM

13. Conclusion β-oxidation negatively correlates with insulin secretion. Fatty-acyl carnitines enhance insulin secretion through accumulation in the cytosol. MiRNA mediated CACT knock-down is responsible for cytosolic accumulation of fatty acyl carnitines 13

14. Future Directions Identify if CACT is the direct target of miR-132 and miR-212. Determine the difference between Fatty-acyl CoA and Fatty- acyl carnitine mediated insulin secretion. Determine the mechanism through which fatty-acyl carnitine enhances insulin secretion. 14

15. Thank You!!! Questions?? 15

16. 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.

17. 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. 17

18. 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. 18