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

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

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

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.

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.

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

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.

Isobaric Technique for relative and absolute quantification (iTRAQ)

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

Priority criteria for candidate gene list.

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

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

Approx. 6 cycles change in miR132 expression

10/29/09 Approx. ~6.5 cycles difference

12/23/09 3 cycle change on miR132 expreesion

Approx. 6 cycles change in miR132 expression

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

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.