1. Regulation of miRNA 132 and miRNA 212 expresion. Clearly they are up-regulated as a result of obesity. B6 > BTBR

2. miRNA 132 and 212 affect Glucose-stimulated insulin secretion(GSIS). Collaborators at Merck

3. GLP-1 treatment induces the expression of miRNA 132 and 212. GLP-1's insulinotropic effect is mediated in part by miR-132/212 in pancreatic β-cells The gene coding for GLP-1 receptor has a coding SNP between B6 and BTBR.

4. Glucagon-like peptide 1 (GLP-1) GLP-1 is an incretin hormone. GLP-1 analogues and drugs inhibiting DPP-4 emerged as new therapeutic agents for the treatment of type 2 diabetes.

5. Mode of action of the miRNAs 1.mRNA degradation – Co-immunoprecipitation (Co-IP) of Arganaute. 2.Translation repression – Quantitive proteomic study.

6. Arganaute Co-IP Pull down Ago2 using antibody for Ago2. Purify mRNA that comes down in the IP. Check expression levels of all the mRNA and check for DE genes as a result of miRNA over-expression. This will give us Primary targets of the miRNA.

7. Isobaric Technique for relative and absolute quantification (iTRAQ)

8. Genome-wide mRNA profiling following miRNA 132 or 212 over-expression in β-cells. Direct and indirect effects. Only degraded mRNA can be identified.

9. Priority criteria for candidate gene list.

10. Experimental methods/concerns Adenovirus transfection – Experiment: AD-GFP-132 – Negative control: Ad-eGFP – Plating  24hrs  infection  48hrs  GSIS. Issues – Small window size – Toxicity effect of adeno virus – Variability of effect size – Difficulty to replicate in primary cells

11. 12/17/09 Approx. 4.5 cycles change in miR132 expression

12. Approx. 6 cycles change in miR132 expression

13. 10/29/09 Approx. ~6.5 cycles difference

14. 12/23/09 3 cycle change on miR132 expreesion

15. Approx. 6 cycles change in miR132 expression

16. FFA FA-CoA FA-Carn CPT1 CoA Carn Slc25a20 FA-Carn CPT2 Carn CoA FA-CoA β-oxidation Etomoxir ACS Triascin C miR-132/212 GSIS Mitochondria Cytosol

17. β- oxidation is not required to see Fatty acid’s effect on GSIS. Association of fatty acids with CoA is required to see the potentiation of GSIS.