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In type1 diabetes (T1D) immune-cell infiltration into islets of Langerhans (insulitis) and β-cell decline occurs years before diabetes presents. There is a lack of validated clinical approaches for detecting insulitis and β-cell decline, to diagnose eventual diabetes and monitor the efficacy of therapeutic interventions. We previously demonstrated contrast-enhanced ultrasound measurements of pancreas perfusion dynamics predicted disease progression in T1D pre-clinical models. Here, we test whether these measurements predict therapeutic prevention of T1D. We performed destruction-reperfusion measurements with size-isolated microbubbles in non-obese diabetic (NOD)-scid mice receiving an adoptive transfer of diabetogenic splenocytes. Mice received vehicle control, or the following treatments: 1) antiCD3 to block T-cell activation, 2) antiCD4 to deplete CD4+ T-cells; 3) Verapamil to reduce β-cell apoptosis or 4) TUDCA to reduce β-cell ER stress. We compared measurements of pancreas perfusion dynamics with subsequent progression to diabetes. AntiCD3, AntiCD4, and verapamil delayed diabetes development. Blood-flow dynamics were significantly altered in treated mice with delayed/absent diabetes development compared to untreated mice. Conversely, blood-flow dynamics in treated mice with unchanged diabetes development was similar to untreated mice. Thus, measuring pancreas perfusion dynamics predicted the successful prevention of diabetes. This strategy may provide a clinically-deployable predictive marker for therapeutic prevention in asymptomatic T1D.

Mapping the localization of multiple proteins in their native three-dimensional (3D) context would be useful across many areas of biomedicine, but multiplexed fluorescence imaging has limited intrinsic multiplexing capability, and most methods for increasing multiplexity can only be applied to thin samples (

Stem cell–derived β-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult β-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human β-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilize an expanding gas surface by employing the lung surfactant replacement, beractant. Lung surfactant contains a suite of lipids and proteins that provide efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and resolidification. We then harnessed this unique functionality to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

Caloric restriction can decrease the incidence of metabolic diseases, such as obesity and Type 2 diabetes mellitus. The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine whether caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high-fat diet (HFD), showing indications of prediabetes after 2 mo, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to 1 mo of 40% caloric restriction (2 g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose, and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

Understanding how cell subpopulations in a tissue impact overall system function is challenging. There is extensive heterogeneity among insulin-secreting β-cells within islets of Langerhans, including their insulin secretory response and gene expression profile, and this heterogeneity can be altered in diabetes. Several studies have identified variations in nutrient sensing between β-cells, including glucokinase (GK) levels, mitochondrial function, or expression of genes important for glucose metabolism. Subpopulations of β-cells with defined electrical properties can disproportionately influence islet-wide free-calcium activity ([Ca2+]) and insulin secretion via gap-junction electrical coupling. However, it is poorly understood how subpopulations of β-cells with altered glucose metabolism may impact islet function. To address this, we utilized a multicellular computational model of the islet in which a population of cells deficient in GK activity and glucose metabolism was imposed on the islet or in which β-cells were heterogeneous in glucose metabolism and GK kinetics were altered. This included simulating GK gene (GCK) mutations that cause monogenic diabetes. We combined these approaches with experimental models in which gck was genetically deleted in a population of cells or GK was pharmacologically inhibited. In each case, we modulated gap-junction electrical coupling. Both the simulated islet and the experimental system required 30-50% of the cells to have near-normal glucose metabolism, fewer than cells with normal KATP conductance. Below this number, the islet lacked any glucose-stimulated [Ca2+] elevations. In the absence of electrical coupling, the change in [Ca2+] was more gradual. As such, electrical coupling allows a large minority of cells with normal glucose metabolism to promote glucose-stimulated [Ca2+]. If insufficient numbers of cells are present, which we predict can be caused by a subset of GCK mutations that cause monogenic diabetes, electrical coupling exacerbates [Ca2+] suppression. This demonstrates precisely how metabolically heterogeneous β-cell populations interact to impact islet function.

One hundred years have passed since the discovery of insulin—an achievement that transformed diabetes from a fatal illness into a manageable chronic condition. The decades since that momentous achievement have brought ever more rapid innovation and advancement in diabetes research and clinical care. To celebrate the important work of the past century and help to chart a course for its continuation into the next, the Canadian Institutes of Health Research’s Institute of Nutrition, Metabolism and Diabetes and the U.S. National Institutes of Health’s National Institute of Diabetes and Digestive and Kidney Diseases recently held a joint international symposium, bringing together a cohort of researchers with diverse interests and backgrounds from both countries and beyond to discuss their collective quest to better understand the heterogeneity of diabetes and thus gain insights to inform new directions in diabetes treatment and prevention. This article summarizes the proceedings of that symposium, which spanned cutting-edge research into various aspects of islet biology, the heterogeneity of diabetic phenotypes, and the current state of and future prospects for precision medicine in diabetes.

Endocrine cells within the pancreatic islets of Langerhans are heterogeneous in terms of transcriptional profile, protein expression and the regulation of hormone release. Even though this heterogeneity has long been appreciated, only within the past 5 years have detailed molecular analyses led to an improved understanding of its basis. Although we are beginning to recognize why some subpopulations of endocrine cells are phenotypically different to others, arguably the most important consideration is how this heterogeneity affects the regulation of hormone release to control the homeostasis of glucose and other energy-rich nutrients. The focus of this Review is the description of how endocrine cell heterogeneity (and principally that of insulin-secreting β-cells) affects the regulation of hormone secretion within the islets of Langerhans. This discussion includes an overview of the functional characteristics of the different islet cell subpopulations and describes how they can communicate to influence islet function under basal and glucose-stimulated conditions. We further discuss how changes to the specific islet cell subpopulations or their numbers might underlie islet dysfunction in type 2 diabetes mellitus. We conclude with a discussion of several key open questions regarding the physiological role of islet cell heterogeneity.

Type 1 diabetes (T1D) results from immune infiltration and destruction of insulin-producing β-cells within the pancreatic islets of Langerhans (insulitis), resulting in loss of glucose homeostasis. Early diagnosis during pre-symptomatic T1D would allow for therapeutic intervention prior to substantial loss of β-cell mass at T1D onset. There are limited methods to track the progression of insulitis and β-cell mass decline in pre-symptomatic T1D. During insulitis, the islet microvasculature increases permeability, such that sub-micron sized particles can extravasate and accumulate within the islet microenvironment. Ultrasound is a widely deployable and cost-effective clinical imaging modality. However, conventional microbubble contrast agents are restricted to the vasculature. Sub-micron sized nanodroplet (ND) phasechange agents can be vaporized into micron-sized bubbles; serving as a circulating microbubble precursor. We tested if NDs extravasate into the immune-infiltrated islet microenvironment. We performed ultrasound contrast-imaging following ND infusion in NOD mice and NOD;Rag1ko controls, and tracked diabetes development. We measured the biodistribution of fluorescently labeled NDs, with histological analysis of insulitis. Ultrasound contrast signal was elevated in the pancreas of 10w NOD mice following ND infusion and vaporization, but was absent in both the non-infiltrated kidney of NOD mice and pancreas of Rag1ko controls. High contrast elevation also correlated with rapid diabetes onset. In pancreata of NOD mice, infiltrated islets and nearby exocrine tissue were selectively labeled with fluorescent NDs. Thus, contrast ultrasound imaging with ND phase-change agents can detect insulitis prior to diabetes onset. This will be important for monitoring disease progression to guide and assess preventative therapeutic interventions for T1D.SignificanceThere is a need for imaging methods to detect type1 diabetes (T1D) progression prior to clinical diagnosis. T1D is a chronic disease that results from autoreactive T cells infiltrating the islet of Langerhans and destroying insulin-producing β-cells. Overt disease takes years to present and is only diagnosed after significant β-cells loss. As such, the possibility of therapeutic intervention to preserve β-cell mass is hampered by an inability to follow pre-symptomatic T1D progression. There are immunotherapies that can delay T1D development. However identifying ‘at risk’ individuals, and tracking whether therapeutic interventions are impacting disease progression, prior to T1D onset, is lacking. A method to detect insulitis and β-cell mass decline would present an opportunity to guide therapeutic treatments to prevent T1D.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

In type 1 diabetes (T1D) autoreactive T-cells secrete cytokines such as tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and interferon-γ (IFNγ) that can initiate β-cell apoptosis. Protein kinase C delta (PKCδ) plays a role in mediating cytokine-induced β-cell apoptosis. The exact mechanisms of PKCδ regulation in β-cell death and role in T1D are unknown. In cancer cells PKCδ mediates apoptosis by translocating to the nucleus and being cleaved by caspase 3. We hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis by translocating to the nucleus and cleavage by caspase 3. Islets from WT or mice with a β-cell specific PKCδ knockout were cultured for 24hr with cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFNγ), caspase 3 inhibitor (Z-DEVD-FMK, 33µg/ml), or adenovirus to produce GFP-PKCδ, GFP with a constitutively active nuclear localization sequence, and a PKCδ mutant that cannot be cleaved by caspase 3 (CM-GFP-PKCδ). Human islets were treated with 10μM PKCδ inhibitor (δV1-1) with cytokines. Apoptosis was determined by fluorescent live/dead staining. PKCδ translocation was determined by GFP colocalization with a nuclear stain and activity was determined with a FRET-based sensor. Decreasing PKCδ activity protected mouse (p Disclosure J. Collins: None. R. K. Benninger: None. N. L. Farnsworth: None. Funding JDRF (D1671-FAC-2020-891-A-N)

Pancreatic islet zinc levels vary widely between species. Very low islet zinc levels in Guinea pigs were thought to be driven by evolution of the INS gene that resulted in the generation of an isoform lacking a histidine at amino acid 10 in the B chain of insulin that is unable to bind zinc. However, we recently showed that the SLC30A8 gene, that encodes the zinc transporter ZnT8, is a pseudogene in Guinea pigs, providing an alternate mechanism to potentially explain the low zinc levels. We show here that the SLC30A8 gene is also inactivated in sheep, cows, chinchillas and naked mole rats but in all four species a histidine is retained at amino acid 10 in the B chain of insulin. Zinc levels are known to be very low in sheep and cow islets. These data suggest that evolution of SLC30A8 rather than INS drives variation in pancreatic islet zinc content in multiple species.

The islets of Langerhans exist as multicellular networks that regulate blood glucose levels. The majority of cells in the islet are excitable, insulin-producing β-cells that are electrically coupled via gap junction channels. β-cells are known to display heterogeneous functionality. However, due to gap junction coupling, β-cells show coordinated [Ca2+] oscillations when stimulated with glucose, and global quiescence when unstimulated. Small subpopulations of highly functional β-cells have been suggested to control [Ca2+] dynamics across the islet. When these populations were targeted by optogenetic silencing or photoablation, [Ca2+] dynamics across the islet were largely disrupted. In this study, we investigated the theoretical basis of these experiments and how small populations can disproportionality control islet [Ca2+] dynamics. Using a multicellular islet model, we generated normal, skewed or bimodal distributions of β-cell heterogeneity. We examined how islet [Ca2+] dynamics were disrupted when cells were targeted via hyperpolarization or populations were removed; to mimic optogenetic silencing or photoablation, respectively. Targeted cell populations were chosen based on characteristics linked to functional subpopulation, including metabolic rate of glucose oxidation or [Ca2+] oscillation frequency. Islets were susceptible to marked suppression of [Ca2+] when ~10% of cells with high metabolic activity were hyperpolarized; where hyperpolarizing cells with normal metabolic activity had little effect. However, when highly metabolic cells were removed from the model, [Ca2+] oscillations remained. Similarly, when ~10% of cells with either the highest frequency or earliest elevations in [Ca2+] were removed from the islet, the [Ca2+] oscillation frequency remained largely unchanged. Overall, these results indicate small populations of β-cells with either increased metabolic activity or increased frequency are unable to disproportionately control islet-wide [Ca2+] via gap junction coupling. Therefore, we need to reconsider the physiological basis for such small β-cell populations or the mechanism by which they may be acting to control normal islet function., Author summary Many biological systems can be studied using network theory. How heterogeneous cell subpopulations come together to create complex multicellular behavior is of great value in understanding function and dysfunction in tissues. The pancreatic islet of Langerhans is a highly coupled structure that is important for maintaining blood glucose homeostasis. β-cell electrical activity is coordinated via gap junction communication. The function of the insulin-producing β-cell within the islet is disrupted in diabetes. As such, to understand the causes of islet dysfunction we need to understand how different cells within the islet contribute to its overall function via gap junction coupling. Using a computational model of β-cell electrophysiology, we investigated how small highly functional β-cell populations within the islet contribute to its function. We found that when small populations with greater functionality were introduced into the islet, they displayed signatures of this enhanced functionality. However, when these cells were removed, the islet, retained near-normal function. Thus, in a highly coupled system, such as an islet, the heterogeneity of cells allows small subpopulations to be dispensable, and thus their absence is unable to disrupt the larger cellular network. These findings can be applied to other electrical systems that have heterogeneous cell populations.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion, expected concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools, however, provide the potential for cell-type specific neural stimulation using genetic targeting and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting parasympathetic (cholinergic) axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to (1) ultrasound image-guided optical stimulation of axon terminals in the pancreas or (2) optical stimulation of axons of the cervical vagus nerve. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and cervical vagal stimulation, while a rapid reduction in glycemic levels were observed under pancreatic stimulation. Additionally, ultrasound-based measurements of blood flow in the pancreas were increased under pancreatic stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest its therapeutic potential for the control of insulin secretion and glucose homeostasis.

Insulin-secreting {beta}-cells are functionally heterogeneous. Subpopulations of {beta}-cells can control islet function and the regulation of hormone release, such as driving the second (oscillatory) phase of free-calcium ([Ca2+]) following glucose elevation. Whether there exists a subpopulation that drives the first-phase response, critical for effective insulin secretion and disrupted early in diabetes, has not been examined. Here, we examine a first responder cell population, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation. We record [Ca2+] dynamics in intact mouse islets, via {beta}-cell specific expression of the [Ca2+] indicator GCamP6s. We identify multiple {beta}-cell subpopulations based on signatures of their [Ca2+] dynamics and investigate the role of first responder cells in islet function by means of 2-photon laser ablation. We further characterize the functional properties of first responder cells by NAD(P)H autofluorescence, fluorescent recovery after photobleaching, glibenclamide stimulation, and network analysis. We also investigate which functional characteristics of these cells are critical by a computational model of islet electrophysiology. Based on the dynamics of [Ca2+] responses, first responder cells are distinct from previously identified functional subpopulations. The first-phase response time of {beta}-cells is spatially organized, dependent on the cells distance to the first responder cells, and consistent over time up to ~24 h. First responder cells showed characteristics of high membrane excitability and slightly lower than average coupling to their neighbors. When first responder cells were ablated, the first-phase [Ca2+] diminished, compared to ablating a random cell. We also observed a hierarchy of the first-phase response time, where cells that were next earliest to respond often take over the role of the first responder cell upon ablation. In summary, we discover and characterize a distinct first responder {beta}-cell subpopulation, based on [Ca2+] response timing, which is critical for the islet first-phase response to glucose.

Type2 diabetes results from failure of the β-cell to compensate for insulin resistance, such as in obesity. Insulin secretion is governed by a series of metabolic and electrical events which can fail during the progression of diabetes. β-cells are electrically coupled via Cx36 gap junction channels, thereby coordinating the pulsatile dynamics of electrical activity, Ca2+ and insulin release across the islet, enhancing insulin action. Pulsatile insulin release is disrupted in human type2 diabetes, although whether this disruption results from diminished gap junction coupling is unclear. Factors such as pro-inflammatory cytokines and free fatty acids disrupt gap junction coupling under invitro conditions. Here we test whether gap junction coupling and coordinated Ca2+ dynamics are disrupted in type2 diabetes, and whether recovery of gap junction coupling can recover islet function. We examine islets from healthy donors and those with type2 diabetes, as well as islets from db/db mice and islets treated with a cocktail of proinflammatory cytokines (TNF-α, IL-1β, IFN-γ) or free fatty acids (palmitate). We modulate gap junction coupling using Cx36 over-expression or pharmacological activation via modafinil. We also develop a peptide mimetic (S293) of the c-terminal regulatory tail of Cx36 designed to compete against its phosphorylation and downregulation. Cx36 gap junction permeability and coordinated Ca2+ dynamics were disrupted in islets from human donors with type2 diabetes, as well as in islets from db/db mice or treated with proinflammatory cytokines or palmitate. Cx36 over-expression, modafinil treatment and S293 peptide all enhanced Cx36 gap junction coupling and protected against declines in coordinated Ca2+ dynamics. Cx36 over-expression and S293 peptide also reduced apoptosis induced by proinflammatory cytokines. Critically S293 peptide rescued gap junction coupling and Ca2+ dynamics in islets from both db/db mice and a sub-set of T2D donors. Thus, recovering or enhancing Cx36 gap junction coupling can improve islet function in diabetes.

The islets of Langerhans exist as a multicellular network that is important for the regulation of blood glucose levels. The majority of cells in the islet are insulin-producing β-cells, which are excitable cells that are electrically coupled via gap junction channels. β-cells have long been known to display heterogeneous functionality. However, due to gap junction electrical coupling, β-cells show coordinated [Ca2+] oscillations when stimulated with glucose, and global quiescence when unstimulated. Small subpopulations of highly functional β-cells have been suggested to control the dynamics of [Ca2+] and insulin release across the islet. In this study, we investigated the theoretical basis of whether small subpopulations of β-cells can disproportionality control islet [Ca2+] dynamics. Using a multicellular model of the islet, we generated continuous or bimodal distributions of β-cell heterogeneity and examined how islet [Ca2+] dynamics depended on the presence of cells with increased excitability or increased oscillation frequency. We found that the islet was susceptible to marked suppression of [Ca2+] when a ∼10% population of cells with high metabolic activity was hyperpolarized; where hyperpolarizing cells with normal metabolic activity had little effect. However, when these highly metabolic cells were removed from the islet model, near normal [Ca2+] remained. Similarly, when ∼10% of cells with either the highest frequency or earliest elevations in [Ca2+] were removed from the islet, the [Ca2+] oscillation frequency remained largely unchanged. Overall these results indicate that small populations of β-cells with either increased excitability or increased frequency, or signatures of [Ca2+] dynamics that suggest such properties, are unable to disproportionately control islet-wide [Ca2+] via gap junction coupling. As such, we need to reconsider the physiological basis for such small β-cell populations or the mechanism by which they may be acting to control normal islet function.Author summaryMany biological systems can be studied using network theory. How heterogeneous cell subpopulations come together to create complex multicellular behavior is of great value in understanding function and dysfunction in tissues. The pancreatic islet of Langerhans is a highly coupled structure that is important for maintaining blood glucose homeostasis. β-cell electrical activity is coordinated via gap junction communication. The function of the insulin-producing β-cell within the islet is disrupted in diabetes. As such, to understand the causes of islet dysfunction we need to understand how different cells within the islet contribute to its overall function via gap junction coupling. Using a computational model of β-cell electrophysiology, we investigated how small highly functional β-cell populations within the islet contribute to its function. We found that when small populations with greater functionality were introduced into the islet, they displayed signatures of this enhanced functionality. However, when these cells were removed, the islet, retained near-normal function. Thus, in a highly coupled system, such as an islet, the heterogeneity of cells allows small subpopulations to be dispensable, and thus their absence is unable to disrupt the larger cellular network. These findings can be applied to other electrical systems that have heterogeneous cell populations.

Diabetes results from insufficient insulin secretion as a result of dysfunction to β-cells within the islet of Langerhans. Elevated glucose causes β-cell membrane depolarization and action potential generation, voltage gated Ca2+channel activation and oscillations in free-Ca2+activity ([Ca2+]), triggering insulin release. Nuclear Factor of Activated T-cell (NFAT) is a transcription factor that is regulated by increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems, and within the β-cell regulates proliferation and insulin granule biogenesis. However the link between the regulation of β-cell electrical activity and oscillatory [Ca2+], with NFAT activation and downstream transcription is poorly understood. In this study we tested whether dynamic changes to β-cell electrical activity and [Ca2+] regulates NFAT activation and downstream transcription. In cell lines, mouse islets and human islets, including those from donors with type2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate β-cell electrical activity. Both glucose-induced membrane depolarization and optogenetic-stimulation triggered NFAT activation, and increased transcription of NFAT targets and intermediate early genes (IEGs). Importantly only conditions in which slow sustained [Ca2+] oscillations were generated led to NFAT activation and downstream transcription. In contrast in human islets from donors with type2 diabetes NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. Thus, we gain insight into the specific patterns of electrical activity that regulate NFAT activation and gene transcription and how this is disrupted in diabetes.

Electrical activity of a pancreatic islet is globally activated under high glucose conditions and globally suppressed under low glucose conditions. This homogeneous behavior occurs due to the highly-coupled nature of the islet, despite heterogeneity of individual β-cells. Using hyperpolarization to silence β-cell activity, studies have shown that small subpopulations of highly functional β-cells can disproportionately control whole islet dynamics. Using a computational model of islet electrophysiology, we studied the theoretical basis of how small highly functional subpopulations of β-cells may regulate the global activity of islets. We modeled metabolic activity of β-cells as a continuous distribution and as a bimodal distribution. Then, in each case, we silenced the highest metabolically active cells and quantified the response of the islet. These highly metabolic cells (GKHigh) were silenced by hyperpolarization, similarly to experimental data. Only under a bimodal distribution, did a small (10%) population of GKHigh cells dramatically reduce islet activity, as experimentally demonstrated. Next, a simulation was run without GKHigh cells present in the islet. When GKHigh cells were absent, the islet remained globally active and had near normal duty cycle, suggesting that these cells are unnecessary for maintaining islet dynamics. Similarly, we investigated another functional subpopulation, β-cells with higher Ca2+ oscillation frequencies which could potentially act as pacemaker cells. When simulating the absence of 10% of these higher frequency cells, the global frequency of the islet was not significantly altered. These results indicate that small highly functional subpopulations are unable to drive islet function under normal conditions, but when targeted under hyperpolarizing conditions may cause dysfunction. Disclosure J.M. Dwulet: None. R.K. Benninger: None. Funding National Institutes of Health (R01DK106412, R01DK102950)

Glucose-stimulated insulin secretion from the pancreatic β-cells is driven by calcium (Ca2+) uptake. In mice Ca2+ is disproportionally controlled by functional subpopulations of β-cells, identified in affecting 2nd phase oscillatory Ca2+ response. Yet, the “1st-responder” cells that lead the 1st Ca2+phase disrupted in early diabetes were overlooked. It is unknown whether 1st responders are a distinct subpopulation, and if so, which factors underlie this distinction, and what is their role. We hypothesized that 1st responder metabolic and electrical parameters are distinct from the known β-cell subpopulations, and they play an important role in the 1st Ca2+phase of the islet response. We simultaneously measured Ca2+ dynamics and electrical coupling in intact islets using β-cells specific expression of the Ca2+ sensor GCaMP6s. We performed repeated glucose and Kapt channel blocker stimulation of the islet. We used femtosecond laser ablation of specific cells. This was complimented by multicellular islet electrophysiology model where we could remove specific cells from the islet and evaluate glucokinase rate, Katp and coupling conductance of the cells. We found no overlap of 1st responder with either “wave-initiator”, leading Ca2+ wave, or highly connected “hub” cell subpopulations, based on the oscillatory 2nd phase Ca2+ wave coordination. Interestingly 1st responders were less electrically coupled (p=0.0157), supported by simulations. 1st responders were consistent under repeatable glucose stimulation and remained 1st responders under the Katp channel blocker, supported by computational results of lower than average Katp channel conductance (p In conclusion, 1st responders are distinct β-cell sub-population, which influences the islet 1st phase Ca2+response. Disclosure V. Kravets: None. J.M. Dwulet: None. W. Schleicher: None. R.K. Benninger: None. Funding JDRF (63012150); National Institutes of Health (R0163012150)

In type1 diabetes (T1D) autoreactive T-cells infiltrate the islets of Langerhans, depleting insulin-secreting β-cells (insulitis). Insulitis arises during an asymptomatic phase, prior to clinical diagnosis of T1D. Methods to diagnose insulitis and β-cell mass changes during this asymptomatic phase are limited, precluding early therapeutic intervention. During T1D the islet microvasculature increases permeability, allowing nanoparticles to access the microenvironment. Contrast enhanced ultrasound (CEUS) uses shell-stabilized gas bubbles to provide acoustic backscatter in vasculature. Here, we report that sub-micron sized ‘nanobubble’ ultrasound contrast agents can be used to measure increased islet microvasculature permeability and indicate asymptomatic T1D. Through CEUS and histological analysis, pre-clinical models of T1D show accumulation of nanobubbles specifically within pancreatic islets, correlating with insulitis. Importantly, accumulation is detected early in disease progression and decreases with successful therapeutic intervention. Thus, sub-micron sized nanobubble ultrasound contrast agents provide a predicative marker for disease progression and therapeutic reversal early in asymptomatic T1D., Infiltration of immune cells to the pancreatic islets precedes clinical symptoms of type 1 diabetes, and lack of methods to detect this insulitis impedes early interventions. Here the authors report a contrast enhanced ultrasound method that can detect early insulitis in mouse models of type 1 diabetes, based on increased microvascular permeability.

In type 1 diabetes (T1D) immune-cell infiltration into the islets of Langerhans (insulitis) and β-cell decline occurs many years before diabetes presents. Non-invasively detecting insulitis and β-cell decline would allow diagnosis of eventual diabetes and provide a means to monitor the efficacy of therapeutic intervention. However, there is a lack of validated clinical approaches for non-invasively imaging disease progression leading to T1D. Islets have a dense microvasculature that reorganizes during diabetes. We previously demonstrated contrast-enhanced ultrasound measurements of pancreatic blood-flow dynamics could predict disease progression in T1D pre-clinical models. Here we test whether these measurements can predict successful therapeutic prevention of T1D. We performed destruction-reperfusion measurements using a small-animal ultrasound machine and size-isolated microbubbles, in NOD-scid mice receiving an adoptive transfer of diabetogenic splenocytes (AT mice). Mice received vehicle control or either of the following treatments: 1) antiCD4 to deplete CD4+ T cells; 2) antiCD3 to block T cell activation, 3) Verapamil to reduce β-cell apoptosis and 4) TUDCA to reduce ER stress. We compared measurements of pancreas blood-flow dynamics with subsequent progression to diabetes. In AT mice blood-flow dynamics were altered >2 weeks after splenocyte transfer. AntiCD4, antiCD3 and verapamil provided a significant delay in diabetes development. Treated AT mice with delayed or absent diabetes development showed significantly altered blood flow dynamics compared to untreated AT mice. Conversely, treated AT mice that developed diabetes, despite therapy, showed similar blood-flow dynamics to untreated AT mice. Thus, contrast-enhanced ultrasound measurement of pancreas blood-flow dynamics can predict the successful or unsuccessful delay or prevention of diabetes upon therapeutic treatments that target both immune activity or β-cell protection. This strategy may provide a clinically deployable predictive marker for disease progression and therapeutic reversal in asymptomatic T1D.

A major limitation of islet transplantation as a therapy for treating Type 1 Diabetes is eventual graft failure, which can be partially attributed to islet cell death. When cultured in vitro, cells in the center of large islets show increased necrosis and exhibit decreased viability and insulin secretion compared to smaller islets. Given the necessity of β-cell-to-β-cell coupling for the physiological response to glucose, a technique to re-aggregate primary islet cells or cells derived from progenitor cells into small clusters of defined sizes may prove advantageous for promoting function upon transplantation. Here, hydrogel microwell arrays were utilized to generate 3-dimensional pseudo-islets from primary murine islets. Pseudo-islets ranged from 50 to 100 μm in diameter as controlled through the microwell dimensions, and contained β-, α-, and δ-cells with ratios similar to those in whole murine islets. Over two weeks in culture, pseudo-islets remained highly viable and responsive to glucose. Intracellular calcium flux showed more robust and coordinated dynamics at high glucose and decreased activity at low glucose compared to age-matched wild-type islets. Therefore, microwell devices can control the aggregation of cells isolated from primary islets to produce islet-like clusters that are functionally similar to freshly isolated islets, and may provide a technique to create improved cellular therapies for Type 1 Diabetes.

In recent work, oxygen microbubbles (OMB) have been shown to oxygenate hypoxic tumors, increase radio-sensitivity and improve tumor control by radiation therapy. Compared to intra-tumoral injection, intravenous delivery of adjuvant agents such as OMBs for radiotherapy offers an attractive means of achieving true theranostic function in a minimally invasive manner via contrast-enhanced ultrasound (CEUS), while reducing the risk of injury, infection or displacing tumor cells. However, short intravascular circulation times with conventional DSPC-lipid OMBs may lead to premature off-target dissolution of OMBs with an associated reduction in tumoral oxygen delivery. Prior work on microbubble stability and gas exchange suggests that increasing phospholipid acyl-chain length of the encapsulating shell and OMB size may increase circulation persistence, delivery and dissolved oxygen content. In the following studies, we investigate the effect of two phospholipid shell compositions, DSPC (C18:0) and DBPC (C22:0), as well as three size distributions (0.5-2 µm, 2-10 µm and polydisperse) on OMB circulation persistence utilizing CEUS in the kidneys of live C57B1/6 male and female mice, six weeks of age. DBPC OMB formulations demonstrated increased circulation half-lives versus DSPC formulations (2.4 ± 1.0 vs. 0.6 ± 0.5 s, p

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets lose synchronized intra-islet Ca2+ oscillations between β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results will have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

KEY POINTS: The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca(2+) signalling and electrical coupling of the insulin producing β‐cells in the islet. We have previously shown that cytokines decrease β‐cell coupling and that compounds which increase cAMP can increase coupling. In both mouse and human islets exendin‐4, which increases cAMP, protected against cytokine‐induced decreases in coupling and in mouse islets preserved glucose‐stimulated calcium signalling by increasing connexin36 gap junction levels on the plasma membrane. Our data indicate that protein kinase A regulates β‐cell coupling through a fast mechanism, such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover. Increases in β‐cell coupling with exendin‐4 may protect against cytokine‐mediated β‐cell death as well as preserve insulin secretion dynamics during the development of diabetes. ABSTRACT: The pancreatic islets of Langerhans maintain glucose homeostasis. Insulin secretion from islet β‐cells is driven by glucose metabolism, depolarization of the cell membrane and an influx of calcium, which initiates the release of insulin. Gap junctions composed of connexin36 (Cx36) electrically couple β‐cells, regulating calcium signalling and insulin secretion dynamics. Cx36 coupling is decreased in pre‐diabetic mice, suggesting a role for altered coupling in diabetes. Our previous work has shown that pro‐inflammatory cytokines decrease Cx36 coupling and that compounds which increase cAMP can increase Cx36 coupling. The goal of this study was to determine if exendin‐4, which increases cAMP, can protect against cytokine‐induced decreases in Cx36 coupling and altered islet function. In both mouse and human islets, exendin‐4 protected against cytokine‐induced decreases in coupling and preserved glucose‐stimulated calcium signalling. Exendin‐4 also protected against protein kinase Cδ‐mediated decreases in Cx36 coupling. Exendin‐4 preserved coupling in mouse islets by preserving Cx36 levels on the plasma membrane. Exendin‐4 regulated Cx36 coupling via both protein kinase A (PKA)‐ and Epac2‐mediated mechanisms in cytokine‐treated islets. In mouse islets, modulating Epac2 had a greater impact in mediating Cx36 coupling, while in human islets modulating PKA had a greater impact on Cx36 coupling. Our data indicate that PKA regulates Cx36 coupling through a fast mechanism, such as channel gating, while Epac2 regulates slower mechanisms of regulation, such as Cx36 turnover in the membrane. Increases in Cx36 coupling with exendin‐4 may protect against cytokine‐mediated β‐cell dysfunction to insulin secretion dynamics during the development of diabetes.

Type 1 diabetes (T1D) is characterized by the infiltration of autoreactive T-cells into the islet of Langerhans, and depletion of insulin-secreting β-cells. This immune cell infiltration (insulitis) first occurs during an asymptomatic phase of T1D that can take place many years prior to clinical diagnosis. Methods to diagnose insulitis and changes in β-cell mass during this asymptomatic phase are limited, thus precluding early therapeutic intervention. While therapeutic treatments can delay T1D progression, treatment efficacy is limited and widely varying, and a method to track this efficacy is also lacking. During T1D progression, the islet microvasculature increases permeability as a result of insulitis, in both mouse models of T1D and humans with T1D. This increased permeability can allow nanoparticles, such as contrast agents for diagnostic imaging, to access the islet microenvironment. Contrast enhanced ultrasound (CEUS) uses shell-stabilized gas bubbles to provide high acoustic backscatter in vasculature and tissue and is clinically approved. A novel, sub-micron sized ‘nanobubble’ (NB) ultrasound contrast agent has been developed and shown to extravasate and accumulate in tumors, where microvascular permeability is high. To test whether CEUS can be used to measure increased islet microvasculature permeability and indicate the asymptomatic phase of T1D, we applied CEUS measurements with NBs in pre-clinical T1D models. NOD mice and mice receiving an adoptive-transfer of diabetogenic splenocytes showed accumulation of NBs specifically within the pancreatic islets, and only in the presence of insulitis. This accumulation was measured by both ultrasound contrast and histological analysis, and accumulation only occurred for sub-micron sized bubbles. Importantly, accumulation was detected as early as 4w in NOD mice. Thus, CEUS with sub-micron sized NB contrast agent may provide a predicative marker for disease progression early in asymptomatic T1D, as well as monitoring of disease prevention or reversal.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion; concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools enable high specificity in neural stimulation using cell-type specific targeting of opsins and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting vagal parasympathetic axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to both ultrasound image-guided optical stimulation of axon terminals in the pancreas and optical stimulation of axons of the cervical vagus nerve, together with ultrasound-based measures of pancreas blood flow. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and vagal stimulation, accompanying rapid reductions in glycemic levels. Additionally, a significant increase in pancreatic blood flow was measured following optical stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest therapeutic potential for the control of insulin secretion and glucose homeostasis.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing thisin vivoin the intact pancreas is challenging.Robo βKOmice, in which the genesRobo1andRobo2are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. CombiningRobo βKOmice with intravital microscopy, we show here thatRobo βKOislets lose synchronized intra-islet Ca2+oscillations between β cellsin vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

In type 1 diabetes (T1D), immune-cell infiltration into the islets of Langerhans (insulitis) and β-cell decline occurs many years before diabetes clinically presents. Non-invasively detecting insulitis and β-cell decline would allow the diagnosis of eventual diabetes, and provide a means to monitor therapeutic intervention. However, there is a lack of validated clinical approaches for specifically and non-invasively imaging disease progression leading to T1D. Islets have a denser microvasculature that reorganizes during diabetes. Here we apply contrast-enhanced ultrasound measurements of pancreatic blood-flow dynamics to non-invasively and predictively assess disease progression in T1D pre-clinical models. STZ-treated mice, NOD mice, and adoptive-transfer mice demonstrate altered islet blood-flow dynamics prior to diabetes onset, consistent with islet microvasculature reorganization. These assessments predict both time to diabetes onset and future responders to antiCD4-mediated disease prevention. Thus contrast-enhanced ultrasound measurements of pancreas blood-flow dynamics may provide a clinically deployable predictive marker for disease progression in pre-symptomatic T1D and therapeutic reversal., Non-invasive techniques to assess the progression of type 1 diabetes prior to clinical onset are needed. Here the authors apply a contrast-enhanced ultrasound measurement of mouse pancreatic blood flow to detect changes in the islet microvasculature that undergoes rearrangements during diabetes and predict disease progression.

Understanding how heterogeneous cells within a multicellular system interact and affect overall function is difficult without a means of perturbing individual cells or subpopulations. Here we apply optogenetics to understand how subpopulations of β-cells control the overall [Ca2+]i response and insulin secretion dynamics of the islets of Langerhans. We spatiotemporally perturbed electrical activity in β-cells of channelrhodopsin2-expressing islets, mapped the [Ca2+]i response, and correlated this with the cellular metabolic activity and an in silico electrophysiology model. We discovered organized regions of metabolic activity across the islet, and these affect the way in which β-cells electrically interact. Specific regions acted as pacemakers by initiating calcium wave propagation. Our findings reveal the functional architecture of the islet, and show how distinct subpopulations of cells can disproportionality affect function. These results also suggest ways in which other neuroendocrine systems can be regulated, and demonstrate how optogenetic tools can discern their functional architecture.

Aging is associated with increased risk for type 2 diabetes, resulting from reduced insulin sensitivity and secretion. Reduced insulin secretion can result from reduced proliferative capacity and reduced islet function. Mechanisms underlying altered β-cell function in aging are poorly understood in mouse and human islets, and the impact of aging on intraislet communication has not been characterized. Here, we examine how β-cell [Ca2+] and electrical communication are impacted during aging in mouse and human islets. Islets from human donors and from mice were studied using [Ca2+] imaging, static and perifusion insulin secretion assays, and gap junction permeability measurements. In human islets, [Ca2+] dynamics were coordinated within distinct subregions of the islet, invariant with islet size. There was a marked decline in the coordination of [Ca2+] dynamics, gap junction coupling, and insulin secretion dynamics with age. These age-dependent declines were reversed by pharmacological gap junction activation. These results show that human islet function declines with aging, which can reduce insulin action and may contribute to increased risk of type 2 diabetes.

Cells within the islet of Langerhans are heterogeneous. Camunas-Soler et al. (2020) implement a patch-seq technique to collect both transcriptomic and electrophysiological data from the same cell. By doing so, they discover new genes that correlate with functional heterogeneity and find that shifts in these correlations indicate β cell compensation in type 2 diabetes.

Here we report on an oxygen microbubble that has increased circulation and contrast persistence to treat tumor hypoxia during X-ray therapy. In recent work, oxygen microbubbles injected directly into tumors were shown to oxygenate hypoxic tumors and improve radiation therapy [1]. Here, we investigate oxygen microbubbles designed for intravenous, rather than intra-tumoral, injection. Oxygen microbubbles were designed with two phospholipid shell compositions, DSPC (C18:0) and DBPC (C22:0). DBPC microbubbles showed a significant increase in ultrasound contrast persistence in vivo in the mouse kidney, and this composition may be ideal for an intravascular microbubble injection to treat tumor hypoxia in radiation oncology.

T1D is characterized by a loss of insulin-producing β-cells in pancreatic islets. The immune destruction of β-cells is partly mediated by high levels of Th1 cytokines. Previously we have shown that cytokines alter islet function via protein kinase C δ (PKCδ) dependent mechanisms. Previous studies have suggested a role for PKCδ in mediating β-cell death, however little is known about the pro-apoptotic role of PKCδ in T1D. I hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis via translocation to the nucleus and cleavage into a constitutively active fragment. To test this hypothesis, we utilized mice with an inducible β-cell specific knockout of PKCδ (PKCδ-βKO). Isolated WT and PKCδ-βKO islets were treated with 10ng/ml TNF-α, 5ng/ml IL-1β, and 100ng/ml IFN-γ for 24h. Human islets were treated with 10μM of the PKCδ inhibitor δV1-1 for 24h with or without cytokines. PKCδ-βKO islets were virally transduced with GFP tagged PKCδ and incubated with or without cytokines for 3 or 24h. MIN6 cells were transfected with a FRET-based sensor for PKCδ and were incubated with or without cytokines. Cytokine treatment increased β-cell apoptosis in mouse (p=0.003) and human islets (p=0.007). Isolated PKCδ-βKO islets showed significantly reduced cytokine-induced apoptosis compared to WT controls (p=0.003). Human islets treated with δV1-1 had reduced cytokine-induced apoptosis (p=0.020) compared to cytokine treatment alone. After 24h cytokine treatment PKCδ translocated to the nucleus (p=0.003) where it showed increased activity as measured by FRET, and an increase in a ~40kDa cleavage product of PKCδ as measured by western blot. In conclusion, our results implicate a role for PKCδ in mediating cytokine-induced β-cell apoptosis via translocation to the nucleus and cleavage into a constitutively active fragment in both mouse and human islets. The role of PKCδ in mediating cytokine-induced death presents a novel β-cell specific mechanism which may be targeted for therapies to preserve β-cell mass in T1D. Disclosure N.L. Farnsworth: None. R.A. Piscopio: None. R.K. Benninger: None. Funding JDRF (5-CDA-2014-198-A-NJDRF, 3-APF-2019-749-A-N)

Glucose-stimulated insulin secretion is driven by the coordinated Ca2+ response in pancreatic islets following glucose elevation. In mouse islets, spatial and temporal Ca2+ dynamics are disproportionally controlled by subpopulations of β-cells, as identified by optogenetics. It is unknown which factors underlie this functional heterogeneity of β-cells in intact islets. Here, we tested if distinct β-cell subpopulations may be identified based on Ca2+ response to glucose elevation, and if their Ca2+ uptake efficiency, metabolic activity and electrical coupling are significantly different. We used MIP-CreER GCaMP6s mouse model which expresses Ca2+-sensitive GFP specifically in β-cells. We performed simultaneous Ca2+ dynamics and metabolic activity (NADH) response to [2-11] mM glucose elevation, along with gap junction permeability measurements in individual islets. We observed three functional subpopulations of β-cells distinct from an islet average: cells with 1) the fastest response to glucose elevation (1st responders); 2) the fastest 2nd phase oscillatory Ca2+ response (wave-initiators); and 3) the non-coordinated Ca2+ oscillations (un-coordinated). While 1st responders and wave-initiators were in different sub-regions of the islet, both subpopulations accumulated more Ca2+ during initial Ca2+ elevation (p=0.0403, p=0.0101) compared to the islet-average. Wave-initiators were more metabolically active (p=0.0223). The un-coordinated cells accumulated less Ca2+ (p In conclusion, the 1st phase Ca2+ uptake and 2nd phase Ca2+ oscillations emerge in spatially- and metabolically-distinct subregions of the islet. This is likely due to increased metabolic activity of the β-cells in these regions which leads to greater membrane depolarization and thus, disproportionately enhances the islet Ca2+response. (NIH R01 DK102950 R01 DK106412) Disclosure V. Kravets: None. R.A. Piscopio: None. W. Schleicher: None. R.K. Benninger: None. Funding National Institutes of Health

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.

Purpose of Review To discuss advances in our understanding of beta-cell heterogeneity and the ramifications of this for type 1 diabetes (T1D) and its therapy. Recent Findings A number of studies have challenged the long-standing dogma that the majority of beta cells are eliminated in T1D. As many as 80% are present in some T1D subjects. Why don’t these cells function properly to release insulin in response to high glucose? Other findings deploying single-cell “omics” to study both healthy and diseased cells—from patients with both T1D and type 2 diabetes (T2D)—have revealed cell subpopulations and heterogeneity at the transcriptomic/protein level between individual cells. Finally, our own and others’ findings have demonstrated the importance of functional beta-cell subpopulations for insulin secretion. Summary Heterogeneity may endow beta cells with molecular features that predispose them to failure/death during T1D.

Insulin-secreting β-cells are heterogeneous in their regulation of hormone release. While long known, recent technological advances and new markers have allowed the identification of novel subpopulations, improving our understanding of the molecular basis for heterogeneity. This includes specific subpopulations with distinct functional characteristics, developmental programs, abilities to proliferate in response to metabolic or developmental cues, and resistance to immune-mediated damage. Importantly, these subpopulations change in disease or aging, including in human disease. Although discovering new β-cell subpopulations has substantially advanced our understanding of islet biology, a point of caution is that these characteristics have often necessarily been identified in single β-cells dissociated from the islet. β-Cells in the islet show extensive communication with each other via gap junctions and with other cell types via diffusible chemical messengers. As such, how these different subpopulations contribute to in situ islet function, including during plasticity, is not well understood. We will discuss recent findings revealing functional β-cell subpopulations in the intact islet, the underlying basis for these identified subpopulations, and how these subpopulations may influence in situ islet function. Furthermore, we will discuss the outlook for emerging technologies to gain further insight into the role of subpopulations in in situ islet function.

The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose‐stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre‐diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose‐ and cAMP‐dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

The large Maf transcription factors, MafA and MafB, are expressed with distinct spatial–temporal patterns in rodent islet cells. Analysis of Mafa−/− and pancreas-specific Mafa∆panc deletion mutant mice demonstrated a primary role for MafA in adult β-cell activity, different from the embryonic importance of MafB. Our interests here were to precisely define when MafA became functionally significant to β-cells, to determine how this was affected by the brief period of postnatal MafB production, and to identify genes regulated by MafA during this period. We found that islet cell organization, β-cell mass, and β-cell function were influenced by 3 weeks of age in MafaΔpanc mice and compromised earlier in MafaΔpanc;Mafb+/− mice. A combination of genome-wide microarray profiling, electron microscopy, and metabolic assays were used to reveal mechanisms of MafA control. For example, β-cell replication was produced by actions on cyclin D2 regulation, while effects on granule docking affected first-phase insulin secretion. Moreover, notable differences in the genes regulated by embryonic MafB and postnatal MafA gene expression were found. These results not only clearly define why MafA is an essential transcriptional regulator of islet β-cells, but also why cell maturation involves coordinated actions with MafB.

Mutations to the ATP-sensitive K+ channel (KATP channel) that reduce the sensitivity of ATP inhibition cause neonatal diabetes mellitus via suppression of β-cell glucose-stimulated free calcium activity ([Ca2+]i) and insulin secretion. Connexin-36 (Cx36) gap junctions also regulate islet electrical activity; upon knockout of Cx36, β-cells show [Ca2+]i elevations at basal glucose. We hypothesized that in the presence of overactive ATP-insensitive KATP channels, a reduction in Cx36 would allow elevations in glucose-stimulated [Ca2+]i and insulin secretion to improve glucose homeostasis. To test this, we introduced a genetic knockout of Cx36 into mice that express ATP-insensitive KATP channels and measured glucose homeostasis and islet metabolic, electrical, and insulin secretion responses. In the normal presence of Cx36, after expression of ATP-insensitive KATP channels, blood glucose levels rapidly rose to >500 mg/dL. Islets from these mice showed reduced glucose-stimulated [Ca2+]i and no insulin secretion. In mice lacking Cx36 after expression of ATP-insensitive KATP channels, normal glucose levels were maintained. Islets from these mice had near-normal glucose-stimulated [Ca2+]i and insulin secretion. We therefore demonstrate a novel mechanism by which islet function can be recovered in a monogenic model of diabetes. A reduction of gap junction coupling allows sufficient glucose-stimulated [Ca2+]i and insulin secretion to prevent the emergence of diabetes.

Pancreatic islets are micro-organs which secrete hormones including insulin and glucagon to maintain glucose homeostasis. Regulated secretion of insulin is managed by β-cells when glucose is taken into the cell and enters the metabolic pathway to generate NADH and ATP. The increased ATP/ADP ratio triggers the closure of ATP sensitive potassium channels which activates voltage gated calcium channels, depolarizing the membrane and increasing cytosolic calcium levels [Ca2+] to induce insulin release. Oscillatory depolarizations of β-cells are synchronized by connexin 36 gap junctions that allow the passage of cations between cells. Travelling waves of depolarization and [Ca2+] influx emerge from one spatial origin and terminate at another, strengthening the collective effect of insulin secretion. Individual β-cells show large amounts of electrical heterogeneity; in both the level of glucose required to induce electrical oscillations and in oscillatory dynamics. To test if cells of similar heterogeneity are grouped together forming regions of spatial homogeneities a mouse line expressing the optogenetic tool Chanelrhodopsin-2 (ChR2) was created to induce membrane depolarizations in subregions of the islet. Two-photon NADH imaging was used with ChR2 activation to determine if subregions showed preferential excitability. β-cell electrical activity measured through ChR2 activation was found to be spatially variable, and this intra-islet variation corresponded with NADH levels in that subregion. A computational electrophysiological model of the islet was used to determine that variations in ChR2 stimulated spatial electrical activity and NADH levels could come from subregions of similar cells. Furthermore, ChR2 stimulated activity was less in the depolarizing wave origin – consistent with the computational model. These data suggest islets are grouped in neighborhood regions of like heterogeneity which provide functional control over electrical activity and dynamics.

Insulin is secreted from the islets of Langerhans in coordinated pulses. These pulses are thought to lead to plasma insulin oscillations, which are putatively more effective in lowering blood glucose than continuous levels of insulin. Gap-junction coupling of β-cells by connexin-36 coordinates intracellular free calcium oscillations and pulsatile insulin release in isolated islets, however a role in vivo has not been shown. We test whether loss of gap-junction coupling disrupts plasma insulin oscillations and whether this impacts glucose tolerance. We characterized the connexin-36 knockout (Cx36−/−) mouse phenotype and performed hyperglycemic clamps with rapid sampling of insulin in Cx36−/− and control mice. Our results show that Cx36−/− mice are glucose intolerant, despite normal plasma insulin levels and insulin sensitivity. However, Cx36−/− mice exhibit reduced insulin pulse amplitudes and a reduction in first-phase insulin secretion. These changes are similarly found in isolated Cx36−/− islets. We conclude that Cx36 gap junctions regulate the in vivo dynamics of insulin secretion, which in turn is important for glucose homeostasis. Coordinated pulsatility of individual islets enhances the first-phase elevation and second-phase pulses of insulin. Because these dynamics are disrupted in the early stages of type 2 diabetes, dysregulation of gap-junction coupling could be an important factor in the development of this disease.

Cell-cell communication in the islet of Langerhans is important for the regulation of insulin secretion. Gap-junctions coordinate oscillations in intracellular free-calcium ([Ca(2+)](i)) and insulin secretion in the islet following elevated glucose. Gap-junctions can also ensure that oscillatory [Ca(2+)](i) ceases when glucose is at a basal levels. We determine the roles of gap-junctions and other cell-cell communication pathways in the suppression of insulin secretion under basal conditions. Metabolic, electrical and insulin secretion levels were measured from islets lacking gap-junction coupling following deletion of connexion36 (Cx36(-/-)), and these results were compared to those obtained using fully isolated β-cells. K(ATP) loss-of-function islets provide a further experimental model to specifically study gap-junction mediated suppression of electrical activity. In isolated β-cells or Cx36(-/-) islets, elevations in [Ca(2+)](i) persisted in a subset of cells even at basal glucose. Isolated β-cells showed elevated insulin secretion at basal glucose; however, insulin secretion from Cx36(-/-) islets was minimally altered. [Ca(2+)](i) was further elevated under basal conditions, but insulin release still suppressed in K(ATP) loss-of-function islets. Forced elevation of cAMP led to PKA-mediated increases in insulin secretion from islets lacking gap-junctions, but not from islets expressing Cx36 gap junctions. We conclude there is a redundancy in how cell-cell communication in the islet suppresses insulin release. Gap junctions suppress cellular heterogeneity and spontaneous [Ca(2+)](i) signals, while other juxtacrine mechanisms, regulated by PKA and glucose, suppress more distal steps in exocytosis. Each mechanism is sufficiently robust to compensate for a loss of the other and still suppress basal insulin secretion.