Optogenetics in Pancreatic Islets: Actuators and Effects Free

The field of optogenetics integrates genetic and optical principles to manipulate cell and animal physiology using light. Through genetic targeting, light-sensitive photoreceptor proteins are expressed in defined cell types so that specific behaviors can be remote controlled with unprecedented spatiotemporal precision. Optogenetics originally emerged as a method to manipulate action potential firing in neural circuits (1,2) but has been increasingly applied in other contexts. For instance, optogenetics has enabled the dissection of the inner workings of cells, such as signaling pathway dynamics, as well as interrogation and correction of disease states in preclinical models, such as in hearing, cardiac pacing, bladder dysfunction, and cancer. In a pioneering clinical study, optogenetics restored a basic form of vision in a patient with blindness (3).

Pancreatic islet physiology relies on cell type–specific functions and the interplay between various islet (and non-islet) cell types (4). β-Cells are insulin-producing cells and essential for glucose homeostasis and metabolism (5). Glucagon-secreting α-cells exert counterregulatory effects, and somatostatin (SST)–secreting δ-cells perform paracrine inhibition of both insulin and glucagon (6,7). Islet cell type function is intimately linked to diabetes, a series of metabolic disorders characterized by chronically high blood glucose levels. In type 1 diabetes (T1D), β-cells are targeted via an autoimmune attack, whereas β-cell dysfunction and insulin resistance are characteristic for type 2 diabetes (T2D). Both forms can be managed through the administration of exogenous insulin. However, glycemic control is suboptimal and still requires challenging glucose monitoring and insulin dosing. Patients with T2D can be provided medications that remove excess glucose from the bloodstream, increase insulin sensitivity, or augment insulin secretion. For example, glucagon-like peptide 1 receptor (GLP-1R) agonists have been used to mimic the action of the incretin hormone GLP-1 (8). Yet, both forms of diabetes often still lead to a host of secondary complications, such as neuropathy, nephropathy, and retinopathy (9). It is evident that islet function, and its integration into body physiology, are both complex and dynamic, calling for experimental approaches that offer high specificity.

The past 5 years have seen a rapid increase in the number of studies that apply optogenetics to islet cells, taking advantage of its high spatiotemporal precision. These applications focused either directly on the metabolically important islet cell types in situ or “designer” replacement cells (e.g., engineered insulin-secreting cells). Here, we first introduce the fundamental working principles of optogenetics and in relation to the established field of neural optogenetics and other genetically encoded actuators deployed into islets. We then cover the form and function of light-sensitive actuators used to modulate islet cell function and signaling (Table 1), as well as effects at the cell, tissue, and organism level (Figs. 1and 2). Insulin production and secretion, cell-cell interactions, and signaling pathways are discussed, along with illumination methods (Table 2 and Fig. 3). Powerful chemogenetic and photopharmacological methods are only briefly touched upon as they have recently been reviewed elsewhere (10–13). Finally, we discuss the applicability of optogenetics in islet biology and conclude with an outlook.

As optogenetic methods rely on a physical stimulation modality (light), they have the potential to overcome some of the limitations inherent to pharmacological or genetic methods. First, unlike small molecules or biologics, light can be applied remotely (i.e., through tissue barriers to some depths [see below]) and withdrawn easily (i.e., without retention in the cellular milieu). Second, unlike genetic modification, signals can be precisely manipulated on very short (e.g., even down to milliseconds) or much longer timescales (e.g., up to days or weeks). Third, and rather uniquely, spatial precision can be achieved by delivering light to only the desired tissues, cells, or even subcellular compartments, reducing off-target effects. Fourth, in many optogenetic experiments, biological systems are studied in their intact state, with spatial information and signaling dynamics being preserved.

Optogenetics is now a staple technique in neuroscience primarily for dissecting brain circuits and behavior (14). There are parallels between applications to islets and neurons. For instance, on the tissue level, it is difficult, but desirable, to decipher the role of individual cell populations (e.g., one specific type of neuron or neurons vs. glia, compared with β-cells vs. α-cells in islets). This commonality has inspired the transfer of transgenic models between the fields. On the cellular level, the presence of voltage-gated ion channels endow β-, δ-, and α-cells with electrical excitability (15). This provides opportunity to control Ca²⁺ influx and hormone secretion dynamics using the same light-sensitive proteins commonly used to stimulate (16) or inhibit (17) neurons. Notably, optogenetic neural manipulation has been used to study islet-specific effects of neural-endocrine cell interactions (18). Thus, neuronal optogenetics has both informed and become integrated into islet biology.

Optogenetics was not the first neuroscience-derived method applied to islet cells. Chemogenetic systems, most prominently designer receptors exclusively activated by designer drugs (DREADDs), achieve orthogonal and temporal control over G-protein–coupled receptor (GPCR) signaling pathways and thereby changes in cell and organism state (19,20). In lieu of their endogenous ligand, DREADDs are engineered to bind a pharmacologically inert drug. DREADDs have been applied in islets to study, for instance, GPCR-mediated signaling and endocrine cell function (13,21). Both DREADDs and optogenetic actuators permit interrogation of cellular processes with some spatial precision through selective expression in transgenic mouse lines or localized viral delivery. However, optogenetics can provide further specificity through confined illumination. The timescales accessible also differ, with chemogenetics suited for medium- or long-term chronic stimulation (i.e., hours to weeks) and optogenetics for shorter stimulation (i.e., milliseconds to minutes, or longer as required) and greater flexibility in stimulation protocols (e.g., pulsed vs. continuous illumination). In terms of stimulus delivery, chemogenetics negates the need for optical devices, and light penetration deep into tissues can be limiting (see below). However, in vivo models, such as islet transplantation into the anterior chamber of the eye (ACE) (22,23), provide light accessibility, and the use of upconversion nanoparticles (UCNPs) achieve minimally invasive, tether-free light delivery deep into tissues (24,25). Finally, most current ligand-orthogonal actuators are GPCRs and ion channels, whereas the optogenetic toolkit has great diversity to control a variety of processes. Thus, the researcher’s choice will largely depend on the process being investigated and the properties of available tools.

Insulin is natively expressed from the INS locus on human chromosome 11 under the control of the highly β-cell–specific insulin promoter. It is first synthesized as a single-chain (pre) proinsulin before being processed into granules and secreted via exocytosis (26). Several light-dependent transcription systems have been developed to produce insulin using transplanted designer cells in animal models. Most of these studies delivered light sources in the cage or through fiber optic implants (Fig. 3A). The LightOn system employs a light-sensitive chimeric transcription factor termed GAVPO that contains the light-oxygen-voltage (LOV)–sensing domain of the fungal protein vivid (VVD), the DNA-binding domain of the transcription factor Gal4, and a p65 transactivation domain (27) (Fig. 1A). Blue light promotes VVD dimerization, allowing Gal4 to bind to galactose-responsive upstream activation sequence (UAS) elements that drive insulin expression on a second locus. Wang et al. (27) delivered this two-component system into streptozotocin (STZ)-treated T1D mice via hydrodynamic plasmid transfection and observed reduced blood glucose levels after 8 h of illumination. More recently, the luminescent version LuminON was generated (28). This facultatively bioluminescence resonance energy transfer–based system contains nanoluciferase (nLuc) fused to GAVPO and can be controlled either by light or the luciferase substrate furimazine. Pulsatile expression of the hormone was achieved in microencapsulated engineered LuminON human embryonic kidney 293 (HEK-293) cells transplanted into STZ-treated T1D mice. However, illumination resulted in shortened antidiabetic efficacy compared with furimazine, likely attributed to poor penetration of blue light.

GBOI is the glucose-blue light chemi-optogenetic cell–implanted therapy system in which insulin production is only triggered in the presence of light and high blood glucose (29) (Fig. 1B). The system harnesses a LOV domain from Arabidopsis thaliana (30) and consists of a LOV-VP16 fusion protein constitutively expressed from the cytomegalovirus (CMV) promoter P_CMV-LOV-VP16, a Gigantea (GI)-Gal4 fusion construct under the control of the glucose-dependent insulinotropic polypeptide (GIP) promoter P_GIP-GI-Gal4, and human preproinsulin under control of the Gal4-responsive UAS promoter P_UAS-insulin. High glucose induces GI-Gal4 expression, and in the presence of blue light, LOV-VP16 and GI-Gal4 form a complex to drive insulin expression. STZ-treated T1D mice subcutaneously implanted with microencapsulated GBOI HEK-293 cells displayed a reduction in blood glucose and increased serum insulin. Glucose tolerance tests (GTTs) under continuous illumination revealed that GBOI-implanted animals also had lower postprandial glucose levels. The GBOI system could be programmed into already-existing blood glucose monitoring devices that would only illuminate cells when blood glucose levels exceed a threshold.

The limited tissue penetration of blue light (e.g., less than a few millimeters for 450–480 nm light of a moderate nontoxic intensity) has prompted the development of actuators sensitive to longer light wavelengths, in particular red light. Reduced light-induced cytotoxicity during long-term illumination is an added advantage of red light stimulation. A far-red light (FRL)–controlled system was engineered to drive insulin expression using the bacterial diguanylate cyclase, BphS (31) (Fig. 1C). Activation with FRL promotes BphS to convert GTP to cyclic diguanylate monophosphate (c-di-GMP), which then binds to a synthetic transactivating element (FRTA) containing the Streptomyces coelicolor–derived transcription factor, BldD. C-di-GMP–promoted dimerization of FRTA leads to its binding to BldD-specific DNA operator sites upstream of insulin. HEK-293 cells containing these elements were subcutaneously implanted in STZ-treated T1D mice. FRL illumination elicited improved blood glucose profiles and tolerance. It also resulted in sustained and detectable levels of insulin in the bloodstream. These genetic components were further combined with a semiautomatic illumination system, comprising smartphone technology, glucose sensing, and a wirelessly powered FRL micro–light-emitting diode (LED) integrated with the cells in a hydrogel (31,32) (Fig. 3B). Most recently, an updated semiautonomous closed-loop bioelectronic system was developed (33) that achieved light-mediated insulin expression for up to 1 month in STZ-treated T1D mice.

The BphS system has also been tested in FRL-activated human islet–like designer (FAID) cells (34). BphS was stably integrated into human mesenchymal stem cells, which are commonly explored in cell therapy. Encapsulated subcutaneous transplantations in STZ-treated T1D mice were illuminated for up to 40 days. This is likely the longest experimental timeline to date using a light-induced engineered insulin-producing cell therapy. FRL illumination improved glucose tolerance and homeostasis, increased weight gain compared with diabetic controls, and improved polyuria. Most importantly, monitoring of nonfasting blood glucose demonstrated that illuminated STZ-FAID mice had fewer fluctuations compared with diabetic mice administered insulin. Lowered hemoglobin A_1c levels were also achieved, indicating longer-term blood glucose control. Interestingly, assessment of diabetes-related complications revealed amelioration of fibrosis as well as renal and cardiac damage.

Another recent red light/FRL-sensitive approach is the miniaturized Δphytochrome A (ΔphyA)–based photoswitch (REDMAP) (35) (Fig. 1D). PhyA is a plant photoreceptor that also utilizes phycocyanobilin as its chromophore. Red light/FRL promotes/reverses nuclear translocation of phyA through interactions with FHY1 and FHY1-like shuttle proteins (36,37). Zhou et al. (35) generated a transcriptional activation system by fusing the truncated photoreceptor to Gal4 (ΔphyA-Gal4) and FHY1 to a VP64 transactivator (FHY1-VP64). Illumination with red light promoted the ΔphyA-FHY1 interaction, nuclear translocation, and transcriptional activation of a target gene downstream of synthetic UAS repeats. For gene inactivation, FRL illumination disrupts the ΔphyA-FHY1 interaction, leading to dissociation of ΔphyA-Gal4 and FHY1-VP64. The system was applied in vivo by transplanting encapsulated REDMAP HEK-293 cells into STZ-treated T1D mice to achieve light-dependent insulin production. Insulin levels were increased along with a reduction in blood glucose levels. Moreover, GTTs revealed that a single pulse of red light was sufficient to elicit substantial improvement in glucose homeostasis. Studies over weeks also showed improved glucose tolerance and reduced blood glucose levels. In contrast to BphS, REDMAP displays efficient and high transcriptional activation (>150-fold) along with rapid (de)activation kinetics (∼1 s compared with hours).

Insulin secretion can be further enhanced through incretin hormones, such as GLP-1, which targets the highly expressed GLP-1R (38). Several groups have generated designer cells that produce and release GLP-1 in a light-dependent manner. The earliest approach was by coexpression of melanopsin (OPN4), a blue light–activated GPCR of the opsin family, and a short variant of GLP-1 (shGLP-1) under control of the nuclear factor–activated T cell (NFAT) promoter (39) (Fig. 1E). OPN4 activation relies on photoisomerization of the chromophore 11-cis retinal, a form of vitamin A (39). When engineered into HEK-293 cells, activated OPN4 drives endogenous phospholipase C (PLC) signaling, leading to Ca²⁺ influx and NFAT-mediated transcription of shGLP-1. This synthetic transcription device combined heterologous and endogenous factors in designer HEK-293 cells and was applied in db/db T2D mice (39). Pulsed blue light illumination increased blood insulin levels and reduced glycemic excursions. The system has also been combined with a light-guiding hydrogel implant (40).

A complementary system termed Glow Control used green light–sensitive cobalamin-binding domains (CBDs) from the Thermus thermophilus CarH protein (TtCBD) (Fig. 1F). These domains bind the 5′-deoxyadenosylcobalamin chromophore, an active vitamin B₁₂ coenzyme, and form tetrameric assemblies in the dark that dissociate into monomers (41,42). Mansouri et al. (43) engineered Glow_hGLP1 HEK-293 cells that expressed membrane-anchored TtCBD as well as a transactivator containing a synthetic transcription factor (VP64, p65, and the transcriptional activator, Rta) fused with a tetracycline repressor (TetR) and TtCBD. Expression of a target gene (e.g., GLP-1) is achieved using synthetic tetracycline-responsive promoter elements. In the dark, the transactivator is sequestered at the plasma membrane via TtCBD oligomerization. Green light promotes transactivator release and TetR binding to the tetracycline-responsive promoter to drive gene expression. Light-dependent GLP-1 production was achieved using subcutaneous implantation in a db/db T2D mouse model. Illumination was performed using an implant that mimicked the green light emitted from smart watches and resulted in lowered fasting glucose levels, attenuated insulin resistance, and reduced body weight by day 12 of the illumination period. However, protection of such sensitive implants from ambient light was highlighted as an important consideration for efficacy.

In addition to insulin production, the BphS FRL system was also used to drive shGLP-1 transcription in Shao et al. using db/db T2D mice, resulting in insulin tolerance, reduced insulin resistance, and restored glucose homeostasis (31). As a further development, a self-powered optogenetic system was established (44). The device converts biomechanical energy from respiration movement in the animal into electricity to power the LED implant. A db/db T2D mouse model illuminated with the self-powered optogenetic system showed fast regeneration of blood glucose levels, insulin tolerance, and blood glucose homeostasis.

Another recent advance has involved coupling glucose levels to illumination control using specialized UCNPs. These are a class of nanomaterials that can convert near infrared (NIR) light into higher-energy visible light. Specialized glucose-sensitive UCNPs have been generated to adaptively tune the intensity of nanoparticle luminescence to blood glucose concentrations (45). Lu et al. (45) demonstrated their applicability in closed-loop blood glucose homeostasis. Glucose-sensing UCNPs were encapsulated in a hydrogel with OPN4 GLP-1 HEK-293 cells (Fig. 3C). The RIN-m5F murine islet line was also included in the hydrogel to accomplish GLP-1–stimulated insulin release in an STZ-treated T1D mouse model.

The insulin secretory pathway is regulated by glucose and modulatory ligands, such as fatty acids, neurotransmitters, and incretin hormones. As β-cells metabolize glucose the elevated ATP/ADP ratio causes the block of K_ATP channels. The resulting depolarization triggers the influx of extracellular Ca²⁺ across the plasma membrane through voltage-gated Ca²⁺ channels, ultimately leading to exocytosis of insulin via secretory granules (6). Additional pathways can enhance insulin release, such as the PLC and cAMP/protein kinase A (PKA) pathways. A range of optogenetic approaches have been used to control these second messengers and have mostly combined designer cell implantation with light sources in the cage or fiber optic implants (Fig. 3A). An OPN4-based approach has been employed to promote rapid Ca²⁺-mediated insulin release from β-cell secretory granules (46) (Fig. 2A). The human INS_vesc cell line was engineered to express OPN4 to create designer iβ-cells. INS_vesc cells are recalcitrant to glucose levels and contain a constitutively expressed proinsulin-nLuc. Activation of OPN4 and downstream intracellular Ca²⁺ signals lead to the rapid on-demand release of proinsulin-nLuc from preloaded vesicular granules. In vitro, this system achieved insulin secretion that more closely mimics native minute timescales compared with the OPN4 transcription-based system (39). Subcutaneous transplantation of microencapsulated iβ-cells into a STZ-treated T1D mouse model revealed that smartphone white light was sufficient to increase blood insulin levels and restore glucose homeostasis.

The influx of Ca²⁺ into β-cells has also been manipulated more directly. The blue light–sensitive ion channel channelrhodopsin-2 (ChR2) derived from Chlamydomonas algae is widely applied in neuronal optogenetics. Opening of ChR2 promotes influx of cations, including Ca²⁺, and consequently, cell depolarization (16) (Fig. 2B). Reinbothe et al. (47) generated a transgenic mouse model expressing ChR2 under control of the insulin promoter to stimulate insulin secretion in β-cells. Patch-clamp analysis of dispersed islets revealed a rapid electrical response to blue light in ChR2-expressing β-cells. Glucose titrations demonstrated light-induced insulin secretion at a range of concentrations in intact islets; however, further potentiation was not observed at high glucose levels, likely due to an already highly stimulated secretory pathway. Interestingly, islets from mice exposed to a high-fat diet (HFD) displayed higher levels of light-dependent insulin secretion compared with control animals, suggesting the involvement of potentiation mechanisms. In a related study, Kushibiki et al. (48) generated ChR2-expressing MIN6 β-cells for use in transplantation experiments. Insulin secretion could be triggered in the absence of glucose in vitro, and subcutaneous engraftment of these cells into STZ-treated T1D mice resulted in a reduction of blood glucose levels.

Although ChR2 activation has been used to induce secretion, its pore is not Ca²⁺ selective (16). Most recently, Choi et al. (49) demonstrated light-dependent regulation of the Ca²⁺ release–activated channel (CRAC) Orai1. The authors used monster-opto-stromal interaction molecule 1 (monSTIM1), an optimized, highly blue light–sensitive variant of OptoSTIM1 (50), to regulate intracellular Ca²⁺. This actuator comprises a truncated STIM1 fused to GFP and the photolyase homology region domain of the flavin-binding plant photoreceptor cryptochrome 2 (CRY2) (51). Light-induced oligomerization of monSTIM1 (mediated through CRY2) leads to activation of endogenous CRAC and Ca²⁺ influx (52) (Fig. 2C). The monSTIM1 construct was introduced into human pluripotent stem cells (hPSCs) via a CRISPR-Cas9–mediated genomic knock-in approach. Rapid, reversible, and repetitive Ca²⁺ influx was achieved both before and after cells were differentiated into pancreatic islet–like organoids (PIOs). Insulin secretion was also demonstrated in STZ-treated T1D mice subcutaneously transplanted with monSTIM1 PIOs. Illumination promoted reduction in GTTs and C-peptide levels 3–4 days after transplantation. However, illuminated monSTIM1 PIOs presented delayed insulin secretion under high-glucose conditions. The underlying mechanism is unknown, but endogenous STIM1 and monSTIM1 may be working competitively in CRAC activation.

Adenylyl cyclase (AC) downstream of GLP-1R synthesizes cAMP from ATP and thereby activates PKA, leading to enhanced Ca²⁺ influx and secretion of insulin (Fig. 2D). A blue light–sensitive bacterial photoactivatable AC (bPAC) from Beggiatoa has been applied to regulate cAMP signaling in β-cells. In Zhang and Tzanakakis (53), illumination of an engineered MIN6 β-cell line showed cAMP elevation and enhanced insulin release on the scale of minutes (thus on similar timescales as β-cells when exposed to varying glucose levels). Notably, the actuator was comparable to known secretagogues, such as forskolin, and its action was Ca²⁺-dependent. Similar effects were observed in virally transduced pseudoislets formed from MIN6 monolayer cultures and murine islets. Insulin secretion was augmented under both low- and high-glucose conditions; however, in the absence of glucose, illuminated bPAC-MIN6 cells exhibited increased cAMP but unchanged insulin secretion. Thus, this approach is different from ChR2 activation, which can stimulate insulin secretion in the absence of glucose and may pose a potential risk of hypoglycemic events. In a follow-up study, Zhang and Tzanakakis (54) transplanted MIN6-bPAC–engineered pseudoislets in an STZ-treated T1D mouse model. Animals displayed improved glucose tolerance, lower hyperglycemia, and greater plasma insulin concentrations. Importantly, the number of engineered bPAC-expressing cells required for transplantation was 2.5 times lower than in the case of control pseudoislets, demonstrating the potential for cell therapy with reduced cell numbers. More recently, the bPAC system was applied to engineered human EndoC-βH3 pseudoislets transplanted into STZ-treated T1D mice (55). However, in both in vivo studies, euglycemia was not achieved, which was attributed to the reduced viability of the cells 3–5 days post-transplantation.

Most recently, synthetic protease–based platforms enabled rapid secretion of proteins in response to light (56,57). For example, the protein of interest is fused to an endoplasmic reticulum (ER) retention motif, synthetic transmembrane domain, furin cleavage site, and protease cleavage site. This strategy allows for localization of pretranslated synthetic insulin within the ER membrane. The second key component is a light-sensitive split protease in which each half of the enzyme is fused to a specific light-sensitive magnet domain (positive magnet or negative magnet) (58). These engineered LOV domains heterodimerize in the presence of blue light to reconstitute the functional protease leading to protein release from the ER and rapid on-demand secretion (Fig. 2E). Wang et al. (57) applied their optical protease-based rapid protein secretion system (optoPASS) to secrete insulin in a STZ-treated T1D mouse model. Subcutaneous transplantation of engineered microencapsulated optoPASS_insulin HEK-293 cells and acute blue light illumination led to an increase in serum insulin levels by 15 min and resulted in nondiabetic glucose levels after 1 h.

Islets are multicellular microorgans that exhibit paracrine signaling. As mentioned above, δ-cells produce and release SST, which inhibits glucagon secretion from α-cells and insulin from β-cells. Insulin is also an inhibitor of glucagon secretion. Along with paracrine interactions, β-cell-to-β-cell interactions occur through cell adhesion molecules (59). Optogenetic actuators have been applied to dissect these complex relationships in intact islets with preserved spatial organization and temporal interaction dynamics. These studies typically used transgenic mouse models combined with microscopy or fiber-optic stimulation (Table 2 and Fig. 3A).

Individual β-cells within an islet display significant variability in several properties, including metabolic activity, electrical dynamics, insulin secretion, and glucose sensitivity (60). However, little is known about how this cell heterogeneity impacts islet function. β-Cells are electrically coupled via connexin 36 gap junctions, which enable highly coordinated and synchronized Ca²⁺ waves across intact islets in response to glucose. Johnston et al. (61) used high-speed Ca²⁺ imaging in whole islets to find that rare and highly connected β-cell subregions exist (termed hubs). In their firing, hubs repeatedly both advance and outlast the general β-cell population. In an elegant follow-up experiment (61), hubs were interrogated by optogenetic hyperpolarization and silencing of individual cells in islets from transgenic mice expressing an enhanced version of the yellow light–sensitive chloride (Cl⁻) pump halorhodopsin (NpHR) (17) (Fig. 2F). Strikingly, intraislet responses to high-glucose conditions as well as insulin release were disrupted upon silencing of hubs. Further characterization revealed that hub cells are highly metabolic and contain features of both mature and immature β-cells. Finally, islets were challenged with cytokines, which are implicated in both forms of diabetes and often used in cell models to mimic proinflammatory conditions and trigger islet dysfunction. This resulted in a reduced number of hubs and their connections, suggesting that hub failure may contribute to disease pathogenesis. In addition to NpHRs, several other light-gated ion channels and pumps have been used to inhibit neuronal activity and could potentially be applied to endocrine cells (62–64). Alternatively, silencing or inhibition can be achieved with methods that produce reactive oxygen species upon illumination, leading to cell ablation (65). This was recently demonstrated with the LOV domain–containing small fluorescent mini singlet oxygen generator 2 (miniSOG2) in the peri-islets of zebrafish (66).

In lieu of silencing, Westacott et al. (67) stimulated different proportions of islets in a murine β-cell–specific-ChR2 expression model to understand how subpopulations control Ca²⁺ responses and insulin secretion. Visualization of these responses identified islet subregions that more readily undergo light-dependent depolarization and propagate Ca²⁺ signals. Furthermore, specific regions assume a pacemaker-like role in initiating Ca²⁺ wave propagation. Taken together, these optogenetic inhibition and activation studies demonstrate how precise manipulation of Ca²⁺ dynamics in spatially defined cells can reveal islet-level functional interactions emerging from β-cell subpopulations.

Along with β-cells, the greater islet paracrine milieu is known to be affected in T2D. For example, oversecretion of glucagon from α-cells contributes to hyperglycemia. However, the precise mechanisms underlying glucagon secretion (and α-cell function more broadly) have been difficult to interrogate. Briant et al. (68) used β-cell–specific ChR2 expression to elucidate the paracrine regulation of glucagon secretion. Optogenetic activation of β-cells caused hyperpolarization of α-cells, with a marked reduction in action potential frequency and glucagon secretion. In contrast, β-cell activation led to rapid elevation of electrical activity in δ-cells, as well as increased secretion of SST. α-Cells express SST-2 receptors, and their pharmacological inhibition abolished the observed effects. It was concluded that SST secretion from δ-cells affects membrane potential and electrical activity of α-cells. In addition, electrical coupling via gap junctions was confirmed between β- and δ-cells. These findings point to a possible mechanism contributing to hyperglycemia in T2D, whereby islets develop reduced contacts between β- and δ-cells.

Paracrine regulation in the opposite direction, i.e., from δ- to β-cells, has been studied with the help of a δ-cell–specific ChR2 mouse model (SST-ChR2) (69). Islets were subsequently virally transduced with a β-cell–specific Ca²⁺ reporter and transplanted into the ACE of wild-type mice as an in situ model. The direct δ-cell activation led to a rapid reduction in β-cell Ca²⁺ oscillations as well as in the frequency and amplitude of their electrical activity (69). The work also demonstrated that δ-cells contain dynamic filopodia structures that comprise SST secretory machinery and allow a δ-cell to contact multiple β-cells. In a HFD prediabetic mouse model, δ-cell function and filopodia were altered, aligning with previous reports on altered SST responses in islets from diabetic animals (70,71).

Complementary to depolarization in endocrine cells, ChR2 has been applied to islet-innervating neurons. Neurons from the dorsal motor nucleus of the vagus nerve project to the pancreas and modulate glucose homeostasis. For example, insulin secretion has previously been observed to be regulated by electrical parasympathetic stimulation (72,73). However, the majority of parasympathetic axons innervate the abdominal and thoracic areas of the body, making it difficult to target pancreas-specific axons. Optogenetics has been used to overcome this limitation toward a more targeted understanding of islet-innervating neuronal function. As mentioned above, Yang et al. (66) showed that selective light-dependent inhibition of peri-islet neurons leads to decreased electrical coupling between β-cells and other endocrine cell types in zebrafish. Two further mechanistic studies have been performed using a transgenic mouse model comprising cholinergic cell-specific expression of the ChR2 gene under the control of the choline acetyltransferase promoter. Fontaine et al. (74) first demonstrated that light-dependent stimulation of vagus nerves in the pancreas using an optical cannula in anesthetized mice promotes glucose-stimulated insulin secretion as well as increase blood flow within the pancreatic vasculature. Kawana et al. (18) used a different illumination approach that relied on UCNPs in live animals. UCNPs were delivered via pancreatic intraductal injection, and animals were subsequently subjected to external NIR illumination for chronic stimulation (2–8 weeks) (Fig. 3C). Along with increased insulin secretion and blood flow, chronic stimulation promoted β-cell proliferation, β-cell mass expansion, and suppression of STZ-induced hyperglycemia. These results demonstrate the strong influence of vagal signals in regulating β-cell physiology and function.

An emerging application of optogenetics is to dissect the inner workings of cells, in particular the wiring and dynamics of their signaling pathways. As mentioned above, membrane potential alterations are intricately linked to intracellular Ca²⁺ signals and, consequently, insulin release (Fig. 2). In addition, dynamic Ca²⁺ levels regulate genetic programs, in many cases through calcineurin and NFAT. Miranda et al. (75) used ChR2 to examine the links between β-cell electrical activity, Ca²⁺ oscillations, and NFAT-driven gene regulation. The authors first demonstrated that elevated glucose increased NFAT activity through a membrane potential– and Ca²⁺-dependent process. Temporal light patterns then induced Ca²⁺ bouts to dissect the signaling network. Notably, this analysis revealed that only relatively slow light patterns, akin to natural β-cell activity profiles, elicited NFAT activation. Thus β-cells, unlike other cell types (76), acted with the characteristics of a low-pass filter rather than a detector of cumulative Ca²⁺. The authors next investigated to what extent ChR2 activation induced transcriptional changes using transcriptomics. ChR2 stimulation resulted in up/downregulation of a smaller number of genes compared with glucose stimulation. These critical differences may reflect either a requirement for a different temporal NFAT activation pattern that was not recapitulated or effects of glucose through other pathways.

Signaling processes other than Ca²⁺ also play a role in β-cells. For instance, mitogenic and prosurvival activities are exerted through peptide growth factors and their cognate receptors, such as fibroblast growth factors acting on fibroblast growth factor receptors (FGFRs). Our group has used optogenetics to control the mitogen-activated protein kinases/extracellular signal–regulated kinase and phosphatidylinositol-3 kinase/Akt pathways downstream of growth factors (77,78). In INS-1E β-cells, the optogenetic method was a highly engineered fusion receptor consisting of a red light–sensitive phytochrome of cyanobacterial origin and the catalytic tyrosine kinase domain of murine FGFR1 (rOpto-FGFR1) (79). Due to the efficient tissue penetration of red light, and because of the exceptional light sensitivity of phytochromes, signaling could be activated even through several millimeters of synthetic tissue, comparable to the distance separating the pancreas from skin in mice. This proof-of-principle study demonstrated the potential of red light to manipulate activity in kinase-dependent signaling pathways deep in a mammalian tissue.

Notably, mechanistic signaling studies in islets have reached beyond endocrine cells. Pericytes are contractile mural cells that line capillaries and regulate vasculature stability and blood flow. The regulation of blood flow likely impacts the delivery of glucose to islets and in turn the release of glucoregulatory hormones into the circulation. Two recent studies used optogenetics to interrogate pericyte activity. In the first study, Tamayo et al. (80) expressed ChR2 in islet pericytes that were grafted into the retinas of STZ-treated diabetic mice. Pericyte activity was controlled using focused laser light (during in vivo imaging of islet blood flow) or whole-cage LED illumination (during long-term illumination of awake animals). Although only 50% of the identifiable pericyte population expressed ChR2, marked reversible reductions in capillary diameter and blood flow were observed. Importantly, these changes manifested in reduced islet glucose sensing and hormone secretion, leading ultimately to hyperglycemia. In a further study, Michau et al. (81) expressed ChR2 in pericytes in the intact, but exposed pancreas of anesthetized mice. Stimulation with blue light from an optical fiber also decreased blood flow, which was delayed in the case of diabetic animals that had been fed a HFD. Collectively, these two studies provided cell type–specific evidence on the involvement of pericytes in acute metabolic processes and, in turn, on how metabolic stress impacts pericyte function.

At the inception of the field in the mid-2000s, optogenetic methods have been applied in a handful of core laboratories. Ever since, several major waves of evolution have resulted in broader access to and uptake of optogenetics. First, the many available transgenic rodents and advanced viral systems designed to express actuators have been adapted to generate new, e.g., islet cell–specific models. Second, diverse light sources are now available to stimulate cells ex vivo and in vivo (Table 2 and Fig. 3). Ex vivo, these include dedicated LED illuminators, the most advanced of which even allow single-well control in multiwell plates (82,83), as well as widefield and confocal microscopes that may already be available in many laboratories. In vivo, these include the methods summarized above, the most advanced of which allow wireless, noninvasive light delivery (Table 2 and Fig. 3). Third, a wide range of cellular processes can now be controlled using light, including ionic signals, protein secretion, signal transduction, transcription, protein stability, and subcellular localization.

While impressive outcomes have been achieved as summarized in this review, it is worthwhile noting potential limitations of the technique. First, there always is the potential for cellular toxicity and undesired immune responses as a consequence of actuator delivery or expression (84,85). Second, expression of actuators can result in functional alterations in the targeted cells and tissues in the absence of light. The prominent causes of this dark activity are contaminating light from light sources other than the stimulation source or the intrinsic activity of some actuators in unstimulated states. Adequate controls and multiple independent assays can assist in limiting this (86,87). Third, while light signals can be controlled with high temporal precision (essentially limited by the performance of the light source and software), responses can act tardily. This is due to the residence time of some actuators in the light-activated state even after light has been withdrawn. Active state lifetimes can vary between seconds to many minutes or hours depending on the method applied (88–90). Fourth, some actuators are subject to peculiarities that need to be understood to collect reliable data toward meaningful conclusions. For instance, a paradoxical sporadic excitation of electrical activity can be observed in NpHR-expressing cells after extensive periods of light-induced inhibition (91,92). As a further example, some opsins suffer from decreased current activity during continuous stimulation or decreased responsiveness to repeated stimulation attempts (93,94). Furthermore, not all actuators are compatible with multiphoton excitation that provides highly confined deep tissue stimulation (95–97).

As an alternative to genetically encoded actuators, photopharmacology negates the need for gene delivery by using light-responsive drugs often constructed around photochromic moieties (e.g., azobenzenes). Photopharmacology has been used in studies of β-cell function and is particularly suitable for interrogation of endogenous receptor systems and signaling pathways (11,98). Photopharmacology calls for its own set of considerations and limitations, including a need to effectively deliver the photoactive drug and the undesired activation of all cell types that are exposed to the compound and express its target receptor.

Irrespective of the molecular nature of the modality, the use of light can be associated with undesirable side effects. Several studies have reported that light, in the absence of actuators, can induce cellular responses through either endogenously expressed opsins, heat, or photochemical effects (99–101). Such effects have been documented in other tissues and can be managed using controls (i.e., naive cells), red-shifted excitation light, dedicated cell culture media (102,103), and carefully engineered optical devices.

Pancreatic islets are complex and essential multicellular structures. In the past 5 years, primary functionalities of islet cells, their internal wiring, as well as their cell-cell interactions have been interrogated using optogenetics. Leaving from here, what are future perspectives for the field?

Optogenetic actuators have been harnessed from an array of nonmetazoan organisms, and the repertoire is impressive. For instance, the comprehensive OptoBase database (104) lists >40 light-sensitive core domains, many of which have been combined with signaling effectors. Remarkably, many actuator classes have not yet been used in islets, including light-activated transcription factors and genome modifiers (105), GPCRs other than OPN4 (106), or kinases (78,107). Consequently, many interesting and medically relevant processes are yet to be interrogated with optogenetics. Furthermore, development of novel actuators has become more achievable in recent years through new resources (108,109).

Optogenetics has now matured to the point where applications to humans are within reach. The retina has been the first target for optogenetic therapies, with currently several ongoing clinical trials (84,110,111). These all rely on the delivery of ChRs using adeno-associated viruses, and a recent first clinical report revealed some visual restoration in one patient with advanced retinitis pigmentosa (3). In light of this progress, it is important to consider possible translational barriers in the context of islet cells. For light-sensitive designer cells (e.g., replacement β-cells), challenges are well known from other cell replacement strategies. Most studies to date used HEK-293 cells or β-cell lines to optimize the light-sensitive method. However, due to immunogenicity, these and other cell types may require encapsulation toward further applications. Encapsulation is also important in protecting these cells from the host immune system and general environment while being permeable to oxygen and nutrients (112). More clinically applicable chassis, such as human mesenchymal stem cells (34) and induced pluripotent stem cells (49), are increasingly being pursued in genetic engineering and cell therapy. Other challenges include finding an appropriate implantation site, limiting fibrosis, improving vascularization methods using stable biocompatible materials that support cell survival and function, and ensuring that the materials are not immunogenic (113). A wide range of strategies are being explored; however, the most optimal for cellular diabetes therapy is yet to be established (112,113).

In addition to cell type selection and general cell therapy challenges, the optical hardware needs to fit the purpose (Table 2). At present, many studies in rodents have used external light sources (Fig. 3). More advanced coencapsulation of designer cells with LED implants has been achieved (44), as have closed-loop systems with continuous glucose sensing (31,33,45). However, optimal materials and conditions for safe and long-term optogenetic therapy are unknown. Further applications will likely require improved integration of hardware, cells, glucose monitoring, and encapsulation. Key considerations may involve reduction of heat; incorporation of lightweight, flexible, and stable materials that are light-permeable and limit an immune response from the hardware itself; and minimization of local tissue damage (114,115). Alternatively, light-transducing materials, such as UCNPs, that negate the need for implants, can also be coencapsulated with cells (45) and achieve light delivery deep into tissues (114). However, the particle concentration sufficient for visible light generation and actuator stimulation is critical, and the slow excitation response and long luminescence decay times of some UCNP systems may not be appropriate for controlling rapid processes. Finally, potential cytotoxicity of UCNPs in vivo needs to be assessed to determine clinical applicability (114). In general, direct stimulation of islets residing in the human pancreas using an illumination device will likely be very difficult because of its relatively large size and location in the peritoneal cavity.

While we have focused on islet cells and metabolic consequences of (dys)regulation, it is noteworthy that optogenetics has also been applied to other (patho)physiological processes related to the pancreas. For example, a recent study demonstrated the optical control of the nuclear factor-κB signaling pathway in a cancer cell line of pancreatic ductal origin to study disease development and progression (116). In combination with optical reporters, such as luciferases, light-sensitive models may also allow the identification of new active agents in optogenetics-assisted drug screens as demonstrated previously (117,118). These final examples demonstrate the breadth of opportunities that are offered by eliciting effects using light-sensitive actuators in pancreatic islets.

Acknowledgments. The authors thank their colleagues for critical advice on this manuscript.

Funding. This study was supported by a JDRF Australia PhD Top-up Scholarship (to C.G.G.), Australian Research Council grants FT200100519 and DP200102093 (to H.J.), and National Health and Medical Research Council grant APP1187638 (to H.J.). The Australian Regenerative Medicine Institute is supported by State Government of Victoria and the Australian Government grants. The European Molecular Biology Laboratory Australia Partnership Laboratory is supported by the National Collaborative Research Infrastructure Strategy of the Australian Government.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.G.G. generated the figures and tables. C.G.G. and H.J. reviewed the current literature and contributed to the writing and editing of the manuscript. C.G.G. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Data and Resource Availability. No data sets and applicable resources were generated or analyzed for this article. All previously published data are referenced.