Mechanism and therapeutic potential of BefA, a bacterial protein that promotes pancreatic beta cell proliferation and function - SUMMARY Pancreatic β-cells are the sole source of insulin in the human body and the loss or impairment of these cells leads to diabetes. Current cell replacement strategies to treat diabetes are limited by our capacity to generate metabolically mature β-cells in culture. Based on knowledge about β-cell embryonic development, human pluripotent stem cells can be directed to adopt a β-cell-like state, but these cells lack the full capacity of adult human β-cells to secrete insulin in response to glucose. Shortly after birth, β-cells undergo extensive proliferation and maturation. Concurrent with this critical postnatal period of β-cell development is the establishment of the gastrointestinal microbiota. We have shown that in the absence of this resident microbial community, both mice and zebrafish fail to undergo their normal program of postnatal β-cell proliferation and exhibit hyperglycemia. We discovered a secreted bacterial protein, Beta-Cell Expansion Factor A (BefA) made by certain early life gut colonizers of humans, mice, and zebrafish, which induces β-cells proliferation and restores metabolic health in germ-free animals. BefA is a membrane permeabilizing protein, and this biochemical activity is required for its mitogenic activity. We found that BefA co-localizes with both plasma and mitochondrial membranes and has enhanced permeabilizing activity on membranes containing cardiolipin, a lipid found exclusively in bacterial and mitochondrial membranes. We show that BefA is capable of inducing calcium transients in cultured rodent β-cell and within the β-cells of live larval zebrafish islets. Furthermore, pharmacological inhibition of the calcium- dependent phosphatase Calcineurin, blocks BefA’s mitogenic effect in zebrafish. We also found that treatment of cultured rodent β-cells with BefA causes transient outer mitochondrial membrane permeabilization but longer- term increases in mitochondrial activity through an adaptation process termed mitohormesis. Based on these preliminary data, we hypothesize that BefA can stimulate early life β-cell expansion and maturation through its membrane permeabilizing activities. We will test this hypothesis in three Specific Aims. Aim 1 will test whether immediate effects of BefA on β-cell plasma membranes (on the timescale of seconds) stimulates intracellular calcium signaling that promotes early life β-cell proliferation. Aim 2 will test whether longer-term effects of BefA on β-cell calcium dynamics and mitochondria (on the timescale of hours) stimulates mitochondrial activity and metabolic maturation of β-cells. Experiments in Aims 1 and 2 will be performed in live zebrafish, allowing us to study β-cell calcium and mitochondrial dynamics in the intact islet in the normal vascular, neurological, and immunological environment. We will confirm key findings about BefA’s mechanisms of action in isolated neonatal mouse islets. Aim 3 will test whether BefA can stimulate the proliferation and metabolic maturation of induced human pluripotent stem cell derived β-cells through similar mechanisms of stimulating calcium signaling and mitochondrial activity. Collectively, these studies will provide new insights into the early life development and maturation of β-cells and will inform the production of metabolically mature β-cells for cell replacement therapies.
