OPTOGENETIC MODULATION OF INSULIN FUNCTION IN PANCREATIC BETA-CELLS

摘要

Diabetes is characterized by elevated blood glucose (BG) due to autoimmune destruction of insulin-producing β-cells or extensive β-cell apoptosis owed to insulin resistance. Over 29 million people in the US suffer 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 serious side effects, are incompatible with abnormal kidney or liver function and their molecular targets have low tissue specificity (e.g. sulfonylurea-targeted ATP-dependent K~+ channels). Daily administration of insulin is essential for the survival of T1D and advanced T2D patients but imposes significant lifestyle restrictions, may cause hypoglycemia, and does not reverse the long-term crippling effects of the disease. Additionally, agents promoting pancreatic endocrine cell regeneration remain elusive. 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 p-cells and primary islets to express a photoactivatable adenylyl cyclase (PAC). PAC-expressing cells exhibited a greater than 5 times rise in cAMP within 5 min of photoactivation and a rapid drop (~12 min) upon termination of illumination. This led to 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. 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 Ca~(2+) 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 (termed 'pseudoislets'; Pls). The function of Pis carrying PAC was tested in vivo. Encapsulated Pis were delivered subcutaneously to mice, which were rendered diabetic upon treatment with streptozotocin (STZ;). 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, a bioartificial pancreas device is developed integrating p-cells amenable to optogenetic regulation of GSIS, glucose biosensing and remotely powered and controlled microLEDs for GSIS enhancement.