Poster Q5 - Modulation of the β-cell Biomechanical Environment alters β-cell Function and Actin Remodeling

Introduction: It is well-established that cells respond to mechanical cues via cytoskeletal remodeling. However, it is not known whether the mechanical environment influences β-cell maturity and function. The coordination between β-cells, and potentially between other endocrine cells, is necessary for proper oscillatory insulin release in the presence of elevated glucose. Here we examine how mechanical stimuli impact coordinated β-cell Ca2+ oscillatory dynamics, cytoskeletal remodeling of F-actin, and expression of β-cell maturity markers.

Materials and

Methods: To examine how mechanical cues influence the β-cell, we used biocompatible substrates with tunable mechanical properties synthesized with elastic moduli ranging from ~2kPa–33 kPa (Fig.1A). The surface was coated with extracellular matrix and MIN6 β-like cells were seeded. We also examined islet cells from mice, and extracted mice islets were first gently dissociated, and then individual islet cells were seeded atop substrates with varied stiffness. For all conditions in MIN6 cells and dissociated islets, we imaged Ca2+ dynamics via Fluo4 at low (2mM) and high glucose (11, 20mM). To independently examine a link between β-cell function and remodeling of F-actin, we treated cells with Latrunculin A (LAT-A), a reagent that perturbs the polymerization of actin filaments in the cell and quantified the resulting changes in Ca2+ oscillatory dynamics. qPCR was conducted on MIN6 cells (6, 22 kPa) to examine expression of genes linked to β-cell function and maturity.

Results, Conclusions, and Discussions: Results and

Discussion: With increased substrate stiffness, MIN6 cell Ca2+ oscillations were more robust, including a significant increase in duty cycle (p < 0.01) and were more coordinated, consistent with increased excitability (Fig. 1B-D). With increasing stiffness, there was significantly decreased expression of Sur1, Kir6.2, Ins2, Pdx1, Fltp, and Ecad (Fig. 1E, p < 0.01). Thus, the dynamics and coordination of Ca2+ activity is mechanoresponsive and may result from altered β-cell maturity. With increased substrate stiffness, there was evidence of cytoskeletal actin reorganization as cells exhibited distinct actin protrusions (Fig. 1F). For cells treated with LAT-A, which depolymerizes actin filaments, Ca2+ oscillations were less robust and coordinated compared to control cells (Fig. 1G). Additionally, perturbations to actin polymerization via LAT-A resulted in a marked decreased in fluorescent intensity (Fig. 1H). Taken together, we show evidence that with increased substrate stiffness, cells are more excitable and may have altered maturity. Additionally, substrate stiffness serves as a mechanical cue for actin remodeling which may impact proper β-cell oscillatory dynamics.

Conclusions: In conclusion, our results suggest there may be a relationship between β-cell function and cytoskeletal actin remodeling. Further, that the maturity and function of β-cells is responsive to the mechanical environment.

Acknowledgements (Optional): Funding was provided by: Aldeen Grant for Junior Faculty (KV), BDC ACORN post-doctoral fellowship (KV) and NIH/NDDK grants –# R01 DK102950 and R01 DK106412 (RB). Additionally, we thank the BDC islet core for assistance with pancreas isolation.