(445b) Engineering of Pancreatic Beta-Cells for Optogenetic Modulation of Insulin Function

Diabetes is characterized by elevated blood glucose (BG) due to autoimmune destruction of insulin-producing β-cells (type 1; T1D) or extensive β-cell apoptosis owed to insulin resistance (type 2; T2D). Over 9% of the US population suffers from diabetes and its complications including blindness, kidney failure and stroke making this the single most expensive disease with total costs of $327 billion in 2017. Pharmacological interventions are linked to side effects and target molecules with low tissue. Daily administration of insulin is essential for the survival of T1D and advanced T2D patients but imposes significant lifestyle restrictions, requires patient compliance, may cause hypoglycemia, and does not reverse the long-term crippling effects of the disease. Therefore, novel strategies to achieve BG homeostasis in a glucose-dependent fashion without side effects are highly desirable.

Optogenetic technologies allow the drug-free manipulation of diverse cellular functions through modulation of molecular moieties with unique spatiotemporal precision. One such moiety is cyclic AMP (cAMP), which is an amplifier of glucose-stimulated insulin secretion (GSIS) in β-cells. To this end, we engineered pancreatic β-cells and primary islets to express a photoactivatable adenylyl cyclase (PAC) [1]. PAC-expressing cells exhibited a greater than five times rise in cAMP within 5 min of photoactivation and a rapid drop (~12 min) upon termination of illumination. This resulted in a 2- to 3-fold increase of GSIS in PAC-positive β-cells or islets exposed to blue light vs. those kept in dark. Cells exhibited consistently enhanced GSIS over multiple rounds of photoactivation without changes in viability. The pronounced response was comparable to that by β-cells treated with known secretagogues including adenylyl cyclase activators or phosphodiesterase inhibitors [1]. No difference was observed in insulin release with or without illumination in the absence of glucose, further justifying the optogenetic targeting of cAMP, which augments but does not induce hormone secretion. The dependence of GSIS on Ca²⁺ signaling remained intact in engineered β-cells. Light-stimulated secretion of insulin was an order of magnitude greater when PAC-expressing β-cells formed clusters (‘pseudoislets’; PIs). The function of PIs carrying PAC was tested in vivo [2]. Encapsulated PIs were delivered subcutaneously to mice, which were rendered diabetic upon treatment with streptozotocin. The animals were subjected to a glucose tolerance test (GTT) following a 6-h fasting and intraperitoneal injection of glucose. Blood was sampled before (0 min) and after glucose injection (30, 60, 90 and 120 min) and BG was determined using a glucometer. The mice were exposed to blue light for 3 h starting 1 h before the GTT. Animals receiving PAC-expressing β-cells exhibited impaired glucose tolerance in the absence of illumination while the BG returned to baseline in mice with engineered cells undergoing photostimulation.

Currently, we are exploring the use of a PAC that is engineered for activation by near-infrared window light [3]. Red light exhibits greater depth of penetration into tissues. This suggests that a cell delivery design can be realized without the need of integrating the light source for optogenetic stimulation thereby reducing the device complexity and size. Our work will enable technologies combining cellular engineering and optogenetics for the treatment of diabetes.

Acknowledgements:

This work has been partially supported by a grant (CBET-1951104) to ET by the National Science Foundation (NSF).

References:

1. Zhang, E.S. Tzanakakis, Optogenetic regulation of insulin secretion in pancreatic beta-cells, Sci. Rep., 7(1):9357, 2017.
2. Zhang, E.S. Tzanakakis, Amelioration of diabetes in a murine model upon transplantation of pancreatic beta-cells with optogenetic control of cyclic AMP, ACS Synth. Biol., 8:2248, 2019.
3. Fomicheva, C. Zhou, Q-Q Sun, M. Gomelsky, Engineering adenylate cyclase activated by near-infrared window light for mammalian optogenetic applications, ACS Synth. Biol., 8:1314, 2019.