1. iRNA-132 by ark, ary & uffi M

2. miRNA 132 and 212 affect Glucose- stimulated insulin secretion(GSIS). Yun-Ping

3. 12/17/09

4. 12/22/09 Approx. 4.5 cycles change in miR132 expression

6. Approx. 6 cycles change in miR132 expression

7. 0409 mouse islets miR132(Merck) 0409 mouse islets GFP(Merck) 1209 rat islets GFP(Merck) 1209 rat islets miR132(Merck) ViraQuest GFP Confocal fluorescence images to show arterial delivery of different adenoviruses

9. 10/29/09

10. MiR-132 Expression levels after rat islets in-vitro adeno virus delivery Approx. ~6.5 cycles difference

11. 12/23/09

12. MiR-132 Expression levels after rat islets arterial adeno virus delivery 3 cycle change on miR132 expreesion

13. Sigma-Lenti-GFP titration

14. microRNA and Beta Cells Markus Stoffel, Nature 432, 226-230 (2004) “A pancreatic islet-specific microRNA regulates insulin secretion” - miR-375, shows effect on GSIS, validated V-1/Mtpn as a target gene Romano Regazzi, JBC 281, 26932-26942 (2006) “MicrRNA-9 controls the expression of Granuphilin/Slp4 and the secretory Response of Insulin-producing cells” Shows effect on GSIS, by measuring hGH

15. “Over-expression of miR-375 in MIN6 cells at a multiplicity of infection (MOI) of 50 led to an,2.5-fold increase in expression and resulted in an,40% reduction in insulin secretion induced by 25mMglucose compared to cells infected with Ad- eGFP (Fig. 2b). The defect in insulin secretion did not result from defective insulin synthesis because total insulin content was equivalent in Ad-375- and Ad- eGFPinfected MIN6 cells (data not shown).” Stoffel: MIN6 cells have endogenously high levels of miR-375, but even with just a 2.5-fold increase in expression levels he got the effect on GSIS. GSIS was performed 48 hours after adeno infection. It does not state how long they exposed the cells to the MOI 50 adeno.

16. Regazzi: They found that raising the level of mir-9, a miRNA expressed in neurons, and in the rat -cell line INS-1E, results in a decrease in glucose-stimulated insulin release. “This perturbation of the secretory functions was associated with an increase in the level of Granuphilin/Slp4, a Rab3/Rab27 effector playing a negative modulatory role on insulin exocytosis (6, 25).” “To evaluate the role of mir-9 on the secretory capacity, we co-transfected INS-1E cells with pSup-mir-9 and with a plasmid encoding hGH. We have previously shown that hGH is targeted to secretory granules and is co-released with insulin during exocytosis (4). This approach allows a direct assessment of the secretory activity of transiently transfected cells independently of their capacity to produce insulin”

17. miR-132 Continue working with the adenovirus, to try to get a consistent 2-4 fold effect on GSIS? Make a lentivirus? Make it inducible so we can allow cells to “recover” from the infection, then induce the micro to look at primary target genes Do we consider inducible lenti for other projects (stxbp5l)?

18. Concerns We have not been able to obtain a consistent enhancement of GSIS in either mouse islets, rat islets or Ins-1 cells in response to adenovirus mediated over-expression of miR-132 or 212. We are concerned about a general toxic effect of adenoviruses on Ins-1 cell viability. The study yielding the highest level of miR-132 over-expression (10/29 rat islet experiment) did not show enhanced GSIS. What is the relationship between the level of miR-132 expression and enhanced GSIS? Why is Amaxa-based transfection yielding results not reproduced by adeno- mediated over-expression? The Amaxa study provided by Jin Shang (1/7) raises 2 questions: –Why does it take so much miR-132 over-expression to yield a relatively small effect on GSIS? –Similar to adenoviruses and reagent-mediated transfection approaches, electroporation appears to yield toxic side-effects; how does the magnitude decrease resulting from zapping compare to the magnitude increase due to the high expression of miRNAs? We are considering an Ago2 immunoprecipitation study to identify “direct” targets for miR-132, but need to first establish conditions of its over- expression that consistently enhance GSIS.

19. To be continued next week with the Carnitine story

20. Background on the Carnitine story. Acute treatment (2hrs) of INS-1 cells with L-Carnitine, Acetyl-l-carnitine and Palmitoyl-l-carnitine enhances GSIS. Slc25a20 knock down enhances GSIS.

21. Inside the Cell Ref. Mark A. Herman and Barbara B. Kahn

22. Latest addition to the Carnitine story