Humanin Protects Beta Cells in vitro and in vivo

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 758-775-Beta Cells, Glucose Control & Complications
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-758
Kelsey Retting*1, Calvin Vu2, Florence Brunel2, Joseph Stock2, Nancy Levin3 and Kathleen Marie Ogilvie4
1Pfizer, San Diego, CA, 2Pfizer, 3CovX Research, Pfizer WRD, San Diego, CA, 4CovX, San Diego, CA
Humanin is a 24 amino acid peptide previously shown to exert cytoprotective effects in multiple cell types, but its role as a cytoprotective peptide in the beta cell remains unclear.  In this study, we address the role of humanin and its analogs in beta cell protection and function.  Humanin (1uM) exerted protective effects against apoptosis induced by serum starvation in murine MIN-6, NIT-1, and BTC-3 β-cell lines when stimulated for 24 hours following 24 hour incubation in serum free media.  Humanin (1uM) also protected MIN-6 cells against cytokine-induced apoptosis when co-stimulated with TNFα (20ng/ml).  Apoptosis was measured by Caspase 3/7 and DNA fragmentation assays.  The cytoprotective effects however, were not as potent in comparison to exendin-4, which exhibited anti-apoptotic effects at 1nM.  Exendin-4 stimulation for 1 hour induced cAMP elevation (EC50<1nM) in MIN-6, NIT-1, BTC-3, INS-1E, and RINm5F murine and rat β-cells.  No cAMP changes were observed with humanin titration (EC50>1mM) under the same conditions, suggesting that the cytoprotective effects of the peptide are not mediated through Gs/Gi-coupling.   In contrast to exendin (1uM; 2 hours), no glucose-stimulated insulin secretion was observed in response to humanin (1uM; 2hours) in MIN-6, BTC-3, and INS-1E cells.  We also examined whether the beta cell protection observed in vitro resulted in anti-diabetic efficacy in both type 1 (T1D) and type 2 (T2D) rodent models of diabetes.   A humanin analog was administered twice-daily (1.5mpk, IP) for six weeks to db/db mice, a T2D mouse model characterized by glucose intolerance associated with beta cell apoptosis.  No significant effect on acute glucose lowering measured hourly for 4 hrs post initial humanin injection, and no subchronic glucose lowering by IPGTT at 3 and 6 weeks post injection was observed.  When administered daily (0.7mpk, IP) for six weeks in NOD/ShiLtJ mice, a model for T1D associated with islet inflammation, humanin demonstrated a reduction in the incidence of insulinitis by histological examination in pancreas tissue, suggesting an anti-inflammatory effect of the peptide.  The reduction of islet infiltration, however, was not associated with an improvement in glucose tolerance at six weeks by IPGTT.  Together these data suggest that humanin protects beta cells from apoptosis in vitro and in vivo, but these protective effects are not associated with improved glucose homeostasis.

Nothing to Disclose: KR, CV, FB, JS, NL, KMO

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