Tilo Moede

Interactions between endocrine α and β cells are critical to their secretory function in vivo. The interactions are highly regulated, although yet to be fully understood. In this study, we aim to assess the impact of α and β cell co-culture on hormone secretion. Mouse clonal cell lines α-TC6-1 (α cell line) and MIN-6 (β cell line) were cultured independently or in combination in a medium containing 5.5, 11.1, or 25 mM glucose, respectively. After 72 h, hormone release was measured using insulin and glucagon secretion assays, the cell distribution was visualized by inverted microscopy and an immunocytochemistry assay, and changes in gene expressions were assessed using the RT-PCR technique. The co-culture of the two cell lines caused a decrease in glucagon secretion from α-TC1-6 cells, while no effect on insulin secretion from MIN-6 cells was revealed. Both types of cells were randomly scattered throughout the culture flask, unlike in mice islets in vivo where β cells cluster in the core and α cells are localized at the periphery. During the α–β cell co-culture, the gene expression of glucagon (Gcg) decreased significantly. We conclude that islet β cells suppress glucagon secretion from α cells, apparently via direct cell-to-cell contact, of which the molecular mechanism needs further verification.

The pancreatic islets of Langerhans consist of endocrine cells that secrete peptide hormones into the blood circulation in response to metabolic stimuli. When transplanted into the anterior chamber of the eye (ACE), pancreatic islets engraft and maintain morphological features of native islets as well as islet-specific vascularization and innervation patterns. In sufficient amounts, intraocular islets are able to maintain glucose homeostasis in diabetic mice. Islet organoids (pseudo-islets), which are formed by self-reassembly of islet cells following disaggregation and genetic manipulation, behave similarly to native islets. Here, we tested the hypothesis that genetically engineered intraocular islet organoids can serve as production sites for leptin. To test this hypothesis, we chose the leptin-deficient ob/ob mouse as a model system, which becomes severely obese, hyperinsulinemic, hyperglycemic, and insulin resistant. We generated a Tet-OFF-based beta-cell-specific adenoviral expression construct for mouse leptin, which allowed efficient transduction of native beta-cells, optical monitoring of leptin expression by co-expressed fluorescent proteins, and the possibility to switch-off leptin expression by treatment with doxycycline. Intraocular transplantation of islet organoids formed from transduced islet cells, which lack functional leptin receptors, to ob/ob mice allowed optical monitoring of leptin expression and ameliorated their metabolic phenotype by improving bodyweight, glucose tolerance, serum insulin, and C-peptide levels.

Loss of pancreatic β-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of β-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional β-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of β-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents.

The secretion of glucagon by pancreatic alpha cells is regulated by a number of external and intrinsic factors. While the electrophysiological processes linking a lowering of glucose concentrations to an increased glucagon release are well characterized, the evidence for the identity and function of the glucose sensor is still incomplete. In the present study we aimed to address two unsolved problems: (1) do individual alpha cells have the intrinsic capability to regulate glucagon secretion by glucose, and (2) is glucokinase the alpha cell glucose sensor in this scenario. Single cell RT-PCR was used to confirm that glucokinase is the main glucose-phosphorylating enzyme expressed in rat pancreatic alpha cells. Modulation of glucokinase activity by pharmacological activators and inhibitors led to a lowering or an increase of the glucose threshold of glucagon release from single alpha cells, measured by TIRF microscopy, respectively. Knockdown of glucokinase expression resulted in a loss of glucose control of glucagon secretion. Taken together this study provides evidence for a crucial role of glucokinase in intrinsic glucose regulation of glucagon release in rat alpha cells.

The islet of Langerhans is a complex endocrine micro-organ consisting of a multitude of endocrine and non-endocrine cell types. The two most abundant and prominent endocrine cell types, the beta and the alpha cells, are essential for the maintenance of blood glucose homeostasis. While the beta cell produces insulin, the only blood glucose-lowering hormone of the body, the alpha cell releases glucagon, which elevates blood glucose. Under physiological conditions, these two cell types affect each other in a paracrine manner. While the release products of the beta cell inhibit alpha cell function, the alpha cell releases factors that are stimulatory for beta cell function and increase glucose-stimulated insulin secretion. The aim of this review is to provide a comprehensive overview of recent research into the regulation of beta cell function by alpha cells, focusing on the effect of alpha cell-secreted factors, such as glucagon and acetylcholine. The consequences of differences in islet architecture between species on the interplay between alpha and beta cells is also discussed. Finally, we give a perspective on the possibility of using an in vivo imaging approach to study the interactions between human alpha and beta cells under in vivo conditions.

The dynamics of cytoplasmic free Ca ²⁺ concentration ([Ca ²⁺ ] _i ) in pancreatic β cells is central to our understanding of β‐cell physiology and pathology. In this context, there are numerous in vitro studies available but existing in vivo data are scarce. We now critically evaluate the anterior chamber of the eye as an in vivo, non‐invasive, imaging site for measuring [Ca ²⁺ ] _i dynamics longitudinally in three dimensions and at single‐cell resolution. By applying a fluorescently labeled glucose analogue 2‐( N ‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)Amino)‐2‐Deoxyglucose in vivo, we followed how glucose almost simultaneously distributes to all cells within the islet volume, resulting in [Ca ²⁺ ] _i changes. We found that almost all β cells in healthy mice responded to a glucose challenge, while in hyperinsulinemic, hyperglycemic mice about 80% of the β cells could not be further stimulated from fasting basal conditions. This finding indicates that our imaging modality can resolve functional heterogeneity within the β‐cell population in terms of glucose responsiveness. Importantly, we demonstrate that glucose homeostasis is markedly affected using isoflurane compared to hypnorm/midazolam anesthetics, which has major implications for [Ca ²⁺ ] _i measurements. In summary, this setup offers a powerful tool to further investigate in vivo pancreatic β‐cell [Ca ²⁺ ] _i response patterns at single‐cell resolution in health and disease.

Although convincing in genetic models, the relevance of β‐cell insulin resistance in diet‐induced type 2 diabetes (T2DM) remains unclear. Exemplified by diabetes‐prone, male, C57B1/6J mice being fed different combinations of Western‐style diet, we show that β‐cell insulin resistance occurs early during T2DM progression and is due to a combination of lipotoxicity and increased β‐cell workload. Within 8 wk of being fed a high‐fat, high‐sucrose diet, mice became obese, developed impaired insulin and glucose tolerances, and displayed noncompensatory insulin release, due, at least in part, to reduced expression of syntaxin‐1A. Through reporter islets transplanted to the anterior chamber of the eye, we demonstrated a concomitant loss of functional β‐cell mass. When mice were changed from diabetogenic diet to normal chow diet, the diabetes phenotype was reversed, suggesting a remarkable plasticity of functional β‐cell mass in the early phase of T2DM development. Our data reinforce the relevance of diet composition as an environmental factor determining different routes of diabetes progression in a given genetic background. Employing the in vivo reporter islet–monitoring approach will allow researchers to define key times in the dynamics of reversible loss of functional β‐cell mass and, thus, to investigate the underlying, molecular mechanisms involved in the progression toward T2DM manifestation.—Paschen, M., Moede, T., Valladolid‐Acebes, I., Leibiger, B., Moruzzi, N., Jacob, S., García‐Prieto, C. F., Brismar, K., Leibiger, I. B., Berggren, P.‐O. Diet‐induced β‐cell insulin resistance results in reversible loss of functional β‐cell mass. FASEB J. 33, 204–218 (2019). www.fasebj.org

Insulin resistance contributes to the development of cardio-vascular disease and diabetes. An important but unresolved task is to study the dynamics of insulin resistance in selective cell types of insulin target tissues in vivo. Here we present a novel technique to monitor insulin resistance dynamics non-invasively and longitudinally in vivo in a cell type-specific manner, exemplified by the pancreatic β-cell situated within the micro-organ the islet of Langerhans. We utilize the anterior chamber of the eye (ACE) as a transplantation site and the cornea as a natural body-window to study the development and reversibility of insulin resistance. Engrafted islets in the ACE that express a FoxO1-GFP-based biosensor in their β-cells, report on insulin resistance measured by fluorescence microscopy at single-cell resolution in the living mouse. This technique allows monitoring of cell type specific insulin sensitivity/resistance in real-time in the context of whole body insulin resistance during progression and intervention of disease.

Significance: Insulin resistance and β-cell failure are the major defects in type 2 diabetes. We now demonstrate that local insulin resistance-induced increase in apolipoprotein CIII (apoCIII) within pancreatic islets causes promotion of an intraislet inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca ²⁺ concentration ([Ca ²⁺ ] _i ), and apoptosis. Decreasing apoCIII in vivo in animals with insulin resistance improves glucose tolerance, and apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of apoCIII, demonstrate a normal [Ca ²⁺ ] _i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes.