(490c) Shining a Light on Pancreatic ?-Cells: Optogenetic Intensification of Insulin Secretion

The primary mode of treatment for Type 1 Diabetes patients, namely insulin injections via syringe or pump, has numerous pitfalls including cost, reliance on the patient to self-administer correct dosages, and pump failure [1]. Upcoming alternative treatments include cell therapies involving the implantation of insulin-producing cells designed to help auto-regulate blood glucose levels [2]. Our previous research has shown that it is possible to enhance insulin production from rodent pancreatic β-cells optogenetically by increasing cyclic adenosine monophosphate (cAMP) production via photoactivatable adenylyl cyclase [3, 4]. With these studies, we wish to investigate the possibility of further enhancing glucose stimulated insulin secretion (GSIS) in human β-cells by optogenetic engineering. Furthermore, we are investigating whether glucose stimulated insulin secretion is tuned in a physiologically more relevant manner by co-culturing β-cells with glucagon producing pancreatic α-cells.

Insulin-producing human EndoC-βH3 cells were engineered via adenoviral transduction to express a photoactivatable adenylyl cyclase (PAC) from the bacterium Beggiatoa (bPAC). In the presence of 20 mM glucose, the adenovirus (AdbPAC)-transduced cell monolayers exhibited a 3-fold increase in intracellular cAMP (p=5x10^-4, n=3) and 1.8 times greater insulin release than non-transduced (NT) cells (p=5x10^-3, n=3). At 0.5 mM glucose, bPAC-expressing human β-cells secreted 1.4 times more insulin than non-transduced cells (p=0.0213, n=3). At 20 mM glucose, beta-cell clusters termed pseudoislets (PIs) also secreted twice as much insulin per hour than NT cell PIs (p=0.012, n=3). The insulin secretion rate of bPAC⁺ cells was 2.7-fold greater than that of cells transduced with an adenoviral vector carrying the enhanced green fluorescent protein (eGFP) gene (AdeGFP) (p=4x10^-4, n=3). Cells expressing bPAC but kept in the dark did not show appreciable variation of GSIS vs. NT cells or AdeGFP-infected cells, indicating that the activity of bPAC was only observed after exposure to blue light without detectable effects stemming from the use of a viral vehicle. Despite the increased GSIS, oxygen consumption by human β-cells with or without bPAC expression at 20 mM glucose and illumination, was similar. Our findings support the engineering of human β-cells with photomodulation of cAMP to enhance GSIS without the use of pharmacological agents.

In parallel with the optogenetic engineering of human β-cells an alginate-based hydrogel was designed and constructed as a niche to further preserve the cell viability and function. To increase light transmissibility through the hydrogels, a requirement for use with optogenetically active cells, we crosslinked alginate with 50-200 mM CaCO₃ and glucono-δ-lactone (GDL). The resulting scaffolds displayed a Young’s modulus of 1.7±0.46 kPa at 50 mM to 8.83±0.86 kPa at 200 mM of CaCO₃, mimicking the stiffness of native pancreatic tissue.

Additionally, we investigated the effect of glucagon-secreting α-cells on the GSIS of bPAC-expressing β-cells. First, a co-culture system was established for forming mixed α-/β-cell PIs. Each cell type was stained with DiD and DiO membrane dyes before seeding in planar cultures at specific α-cell/β-cell ratios. At given time points, aggregates were dissociated, and cells were tested via flow cytometry to identify resulting ratios, possibly affected by proliferation. The release of insulin and glucagon was determined by enzyme linked immunosorbent assay (ELISA). Images of rodent MIN6-β/αTC1-6 suspended aggregates show naturally occurring self-assembly. αTC1-6 cells segregate to the periphery of pseudo-islets, replicating results from previously published data growing surface-adhered pseudo-islets [5]. Flow cytometry data shows that aggregate cell ratios tend to stabilize after 48 hours of growth, with an initial seeding ratio of 80/20 MIN6-β/αTC1-6 resulting in a desired 75/25 ratio after 2 days. Future experiments will include measuring baseline insulin/glucagon secretion from co-cultured MIN6-β/αTC1-6 or human β-cells/α-cell aggregates in suspension, followed by testing different optogenetic controls on β-cells to address if the presence of α-cells has a synergistic role in enhancing light-induced GSIS in β-cells.

References:

[1] C. Berget, et al. "A clinical overview of insulin pump therapy for the management of diabetes: past, present, and future of intensive therapy" Diabetes Spectrum, 32: 194-204, 2019.

[2] X.-X. Wan, et al., "Stem cell transplantation in the treatment of type 1 diabetes mellitus: from insulin replacement to beta-cell replacement" Frontiers in Endocrinology, 13: 859638, 2022.

[3] F. Zhang, E.S. Tzanakakis, "Amelioration of diabetes in a murine model upon transplantation of pancreatic β-cells with optogenetic control of cyclic adenosine monophosphate" ACS Synthetic Biology, 8: 2248-55, 2019.

[4] F. Zhang, E.S. Tzanakakis, "Optogenetic regulation of insulin secretion in pancreatic β-cells" Scientific Reports, 7: 9357, 2017.

[5] H. Brereton, et al., "Islet alpha-cells do not influence insulin secretion from beta-cells through cell-cell contact", Endocrine, 31: 61-5, 2007.