Β-cell Population Research Articles

Heterogeneity of autophagic status in pancreatic β cells under metabolic stress

Autophagy in β cells has been demonstrated to play a pivotal role in cellular homeostasis and the progression of glucose intolerance. Although autophagic activity is affected by metabolic stress both in vivo and in vitro, it remains unclear as to what extent the autophagic status in each β cell is different from its neighboring cells.To address this question, GFP-LC3 reporter mice, which can visualize the autophagic status of each β cell as green-fluorescent puncta, were crossed with obese diabetic db/db mice. Imaging of green-fluorescent puncta in the islets of GFP-LC3 mice revealed that β cells are a heterogeneous population, as the density of GFP-LC3 puncta in each cell was variable. Furthermore, the variability was greater in GFP-LC3; db/db mice than in non-diabetic GFP-LC3; db/+ mice. Furthermore, when GFP-LC3 mice were treated with a low dose of S961, which antagonizes insulin signaling without inducing overt hyperglycemia, the number of β cells with a high density of GFP puncta was increased, suggesting that insulin resistance affects autophagic status independently of glucose profiles. These results suggest that pancreatic β cells under metabolic stress are heterogeneous regarding their autophagic status, which provides insights into the cellular dynamics of each β cell rather than the whole β-cell population.

Corrigendum to “Protective Effects of the Mushroom Lactarius deterrimus Extract on Systemic Oxidative Stress and Pancreatic Islets in Streptozotocin-Induced Diabetic Rats”

[This corrects the article DOI: 10.1155/2015/576726.].

Substance P preserves pancreatic β-cells in streptozotocin-induced type 1 diabetic mice

Preservation of the pancreatic β-cell population is required for the development of therapies for diabetes, which is caused by a decrease in β-cells. Here, we demonstrate the antidiabetic effects of substance P (SP) in type 1 diabetes (T1D) mice induced with streptozotocin. SP enhanced the compensatory proliferation of β-cells in order to restore β-cells in response to acute injury induced by a single high-dose of streptozotocin. However, SP affected neither the basal proliferation of β-cells nor their apoptosis. In vitro studies by using the INS-1 pancreatic β-cell line showed that SP mediated the increase in the proliferation of β-cells via the activation of Akt. Chronic systemic treatment with SP restored the mass of β-cells and inhibited insulitis in T1D mice induced with multiple low-doses of streptozotocin. Therefore, systemic treatment with SP may be a promising therapeutic strategy for treating diabetes in patients with loss of functional β-cells.

High-Resolution Recording of the Circadian Oscillator in Primary Mouse α- and β-Cell Culture.

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

Innovative Microcapsules for Pancreatic β-Cells Harvested from Mature Double-Transgenic Mice: Cell Imaging, Viability, Induced Glucose-Stimulated Insulin Measurements and Proinflammatory Cytokines Analysis.

Recently we demonstrated that microencapsulation of a murine pancreatic β-cell line using an alginate-ursodeoxycholic acid (UDCA) matrix produced microcapsules with good stability and cell viability. In this study, we investigated if translation of this formulation to microencapsulation of primary β-cells harvested from mature double-transgenic healthy mice would also generate stable microcapsules with good cell viability. Islets of Langerhans were isolated from Ngn3-GFP/RIP-DsRED mice by intraductal collagenase P digestion and density gradient centrifugation, dissociated into single cells and the β-cell population purified by Fluorescence Activated Cell Sorting. β-cells were microencapsulated using either alginate-poly-l-ornithine (F1; control) or alginate-poly-l-ornithine-UDCA (F2; test) formulations. Microcapsules were microscopically examined and microencapsulated cells were analyzed for viability, insulin and cytokine release, 2days post-microencapsulation. Microcapsules showed good uniformity and morphological characteristics and even cell distribution within microcapsules with or without UDCA. Two days post microencapsulation cell viability, mitochondrial ATP and insulin production were shown to be optimized in the presence of UDCA whilst production of the proinflammatory cytokine IL-1β was reduced. Contradictory to our previous studies, UDCA did not reduce production of any other pro-inflammatory biomarkers. These results suggest that UDCA incorporation improves microcapsules' physical and morphological characteristics and improves the viability and function of encapsulated mature primary pancreatic β-cells.

Microbiome: Expanding through the microbiota.

This paper shows that the intestinal microbiota is required for normal expansion of the pancreatic β-cell population in zebrafish during early larval development.

Pancreatic Mesenchyme Regulates Islet Cellular Composition in a Patched/Hedgehog-Dependent Manner.

Pancreas development requires restrained Hedgehog (Hh) signaling activation. While deregulated Hh signaling in the pancreatic mesenchyme has been long suggested to be detrimental for proper organogenesis, this association was not directly shown. Here, we analyzed the contribution of mesenchymal Hh signaling to pancreas development. To increase Hh signaling in the pancreatic mesenchyme of mouse embryos, we deleted Patched1 (Ptch1) in these cells. Our findings indicate that deregulated Hh signaling in mesenchymal cells was sufficient to impair pancreas development, affecting both endocrine and exocrine cells. Notably, transgenic embryos displayed disrupted islet cellular composition and morphology, with a reduced β-cell portion. Our results indicate that the cell-specific growth rates of α- and β-cell populations, found during normal development, require regulated mesenchymal Hh signaling. In addition, we detected hyperplasia of mesenchymal cells upon elevated Hh signaling, accompanied by them acquiring smooth-muscle like phenotype. By specifically manipulating mesenchymal cells, our findings provide direct evidence for the non-autonomous roles of the Hh pathway in pancreatic epithelium development. To conclude, we directly show that regulated mesenchymal Hh signaling is required for pancreas organogenesis and establishment of its proper cellular composition.

Intra-islet lesions and lobular variations in β-cell mass expansion in ob/ob mice revealed by 3D imaging of intact pancreas.

The leptin deficient ob/ob mouse is a widely used model for studies on initial aspects of metabolic disturbances leading to type 2 diabetes, including insulin resistance and obesity. Although it is generally accepted that ob/ob mice display a dramatic increase in β-cell mass to compensate for increased insulin demand, the spatial and quantitative dynamics of β-cell mass distribution in this model has not been assessed by modern optical 3D imaging techniques. We applied optical projection tomography and ultramicroscopy imaging to extract information about individual islet β-cell volumes throughout the volume of ob/ob pancreas between 4 and 52 weeks of age. Our data show that cystic lesions constitute a significant volume of the hyperplastic ob/ob islets. We propose that these lesions are formed by a mechanism involving extravasation of red blood cells/plasma due to increased islet vessel blood flow and vessel instability. Further, our data indicate that the primary lobular compartments of the ob/ob pancreas have different potentials for expanding their β-cell population. Unawareness of the characteristics of β-cell expansion in ob/ob mice presented in this report may significantly influence ex vivo and in vivo assessments of this model in studies of β-cell adaptation and function.

β-Cell adaptation in pregnancy.

Pregnancy in placental mammals places unique demands on the insulin-producing β-cells in the pancreatic islets of Langerhans. The pancreas anticipates the increase in insulin resistance that occurs late in pregnancy by increasing β-cell numbers and function earlier in pregnancy. In rodents, this β-cell expansion depends on secreted placental lactogens that signal through the prolactin receptor. Then at the end of pregnancy, the β-cell population contracts back to its pre-pregnancy size. In the current review, we focus on how glucose metabolism changes during pregnancy, how β-cells anticipate these changes through their response to lactogens and what molecular mechanisms guide the adaptive compensation. In addition, we summarize current knowledge of β-cell adaptation during human pregnancy and what happens when adaptation fails and gestational diabetes ensues. A better understanding of human β-cell adaptation to pregnancy would benefit efforts to predict, prevent and treat gestational diabetes.

Physiology: Pancreatic β-cell heterogeneity revisited.

Two analyses of insulin-producing β-cells reveal differences in what has long been considered a homogeneous population. These differences might reflect changes during maturation or ageing, or distinct cell lineages. See Letter p.430 Pancreatic β-cells are functionally heterogeneous, and differences in their characteristics may be important in the context of insulin-dependent diabetes. Heiko Lickert and colleagues have found a marker, the Fltp reporter gene, that is expressed in mature β-cell populations but not in proliferative β-cells. Fltp expression is triggered by Wnt signalling and polarization of β-cells as they organize into three-dimensional islets and the authors show that this change in architecture is sufficient to induce mouse and human β-cell maturation.

Hypoglycaemic activity of ethanolic extract of Garcinia mangostana Linn. in normoglycaemic and streptozotocin-induced diabetic rats.

BackgroundVarious parts of Garcinia mangostana Linn., including its pericarp, have been traditionally used to treat a variety of ailments. In an attempt to establish its medicinal value, the present study was carried out to determine the hypoglycaemic potential of G. mangostana pericarp ethanolic extract (GME) using the streptozotocin-induced (STZ) diabetic rats.MethodsGME at 2,000 mg/kg was subjected to a single-dose acute toxicity test. Following this, the effect of GME (50, 100, and 200 mg/kg) on blood glucose level of normoglycaemic and STZ-induced diabetic rats was determined using single-dose (acute) and multiple-dose (subacute) approaches. Subsequent to the multiple-dose study, serum biochemical analysis and liver histopathological examination were also performed. Throughout the experiments, the effect of GME was compared against the standard hypoglycaemic drug, glibenclamide.ResultsGME was safe for oral consumption up to the dose of 2,000 mg/kg. In both single- and multiple-dose studies, GME significantly (p < 0.05) reduced the blood glucose level in normoglycaemic rats and STZ-induced diabetic rats when compared against the normal control group or diabetic control group, respectively. Moreover, GME also significantly (p < 0.05) increased the rats’ body weight in comparison to the diabetic control group in the multiple-dose study. GME also significantly (p < 0.05) reduced the levels of certain biochemical parameters [i.e., triglycerides (TG), total cholesterol (TC), low density lipoprotein (LDL), very low density lipoprotein (VLDL), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), urea, and creatinine] while increased the others [i.e., high density lipoprotein (HDL) and total protein (TP)] when compared to the diabetic control group. Histopathological assessment of the collected liver revealed a mild increase in the population of β-cells in the diabetic rats.ConclusionGME exerts the hypoglycaemic activity possibly by increasing the population of insulin-producing β-cells. This activity could be attributed to the presence of antioxidant-bearing tannins like epicathecin, and xanthones like α-mangostin. Thus, the findings demonstrated that GME could be a potential candidate in the management of diabetes owing to its hypoglycaemic effect.

Heterogeneity and nearest-neighbor coupling can explain small-worldness and wave properties in pancreatic islets.

Many multicellular systems consist of coupled cells that work as a syncytium. The pancreatic islet of Langerhans is a well-studied example of such a microorgan. The islets are responsible for secretion of glucose-regulating hormones, mainly glucagon and insulin, which are released in distinct pulses. In order to observe pulsatile insulin secretion from the β-cells within the islets, the cellular responses must be synchronized. It is now well established that gap junctions provide the electrical nearest-neighbor coupling that allows excitation waves to spread across islets to synchronize the β-cell population. Surprisingly, functional coupling analysis of calcium responses in β-cells shows small-world properties, i.e., a high degree of local coupling with a few long-range "short-cut" connections that reduce the average path-length greatly. Here, we investigate how such long-range functional coupling can appear as a result of heterogeneity, nearest-neighbor coupling, and wave propagation. Heterogeneity is also able to explain a set of experimentally observed synchronization and wave properties without introducing all-or-none cell coupling and percolation theory. Our theoretical results highlight how local biological coupling can give rise to functional small-world properties via heterogeneity and wave propagation.

A Role for the Host in the Roadmap to Diabetes Stem Cell Therapy.

Stem cells represent an unlimited source for cell therapy (1), and considerable efforts have been made to overcome barriers to introducing this revolutionary therapy into clinical practice. Briefly, the following actions must be taken: 1 ) design in vitro differentiation strategies to generate either mature postmitotic β-cells or β-cell progenitors that may be safely implanted into the host (e.g., without uncontrolled proliferation), 2 ) devise selection methods to produce a pure β-cell population, 3 ) validate standard characterization protocols to determine the real differentiation stage of the cells ready to be transplanted, 4 ) obtain encapsulation devices to implant the cells, 5 ) develop preclinical controls in representative animal models, and 6 ) define cell-host interactions (for a recent review see ref. 2). A breakthrough for in vitro differentiation and selection strategies was reported in Diabetes when it was shown for the first time that the process was doable (3). A combination of directed differentiation and gene-trapping strategies succeeded in manufacturing insulin-producing cells derived from rodent embryonic stem cells that normalized blood glucose when implanted into streptozotocin (STZ)-induced diabetic mice. Further reports improved the system by introducing new differentiation strategies, such as inhibiting sonic hedgehog or selecting cells expressing a gene also expressed in islet progenitor cells (Nkx6.1) (4). It was also shown that differentiated cells do not form teratomas (4,5) and they mature 30 days after transplantation (4). Subsequently, in vitro differentiation protocols for human …

Bioengineering Strategies for the Treatment of Type I Diabetes

Diabetes mellitus, the third most common disease in the world, is a chronic metabolic disorder caused by a failure of insulin production and/or an inability to respond to insulin. Specifically, type 1 diabetes is a disease resulted by the autoimmune destruction of a patient's β-cell population within the pancreatic islets of Langerhans. The current primary treatment for type 1 diabetes is daily multiple insulin injections. However, this treatment cannot provide sustained physiological release, and the insulin amount is not finely tuned to glycemia. Pancreatic transplants or islet transplants would be the preferred treatment method but the lack of donor tissue and immunoincompatibility has been shown to be a roadblock to their widespread use. Bioengineering strategies are poised to combat these challenges. In this review, bioengineering approaches for the treatment of type 1 diabetes, including insulin controlled release systems, strategies for immunoisolation of transplanted islets, and cell-based therapies, such as β-cells and stem cells, are discussed.

Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells

This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), β-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type-specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system.

A Unifying Organ Model of Pancreatic Insulin Secretion.

The secretion of insulin by the pancreas has been the object of much attention over the past several decades. Insulin is known to be secreted by pancreatic β-cells in response to hyperglycemia: its blood concentrations however exhibit both high-frequency (period approx. 10 minutes) and low-frequency oscillations (period approx. 1.5 hours). Furthermore, characteristic insulin secretory response to challenge maneuvers have been described, such as frequency entrainment upon sinusoidal glycemic stimulation; substantial insulin peaks following minimal glucose administration; progressively strengthened insulin secretion response after repeated administration of the same amount of glucose; insulin and glucose characteristic curves after Intra-Venous administration of glucose boli in healthy and pre-diabetic subjects as well as in Type 2 Diabetes Mellitus. Previous modeling of β-cell physiology has been mainly directed to the intracellular chain of events giving rise to single-cell or cell-cluster hormone release oscillations, but the large size, long period and complex morphology of the diverse responses to whole-body glucose stimuli has not yet been coherently explained. Starting with the seminal work of Grodsky it was hypothesized that the population of pancreatic β-cells, possibly functionally aggregated in islets of Langerhans, could be viewed as a set of independent, similar, but not identical controllers (firing units) with distributed functional parameters. The present work shows how a single model based on a population of independent islet controllers can reproduce very closely a diverse array of actually observed experimental results, with the same set of working parameters. The model’s success in reproducing a diverse array of experiments implies that, in order to understand the macroscopic behaviour of the endocrine pancreas in regulating glycemia, there is no need to hypothesize intrapancreatic pacemakers, influences between different islets of Langerhans, glycolitic-induced oscillations or β-cell sensitivity to the rate of change of glycemia.

Mathematical modeling of gap junction coupling and electrical activity in human β-cells

Coordinated insulin secretion is controlled by electrical coupling of pancreatic β-cells due to connexin-36 gap junctions. Gap junction coupling not only synchronizes the heterogeneous β-cell population, but can also modify the electrical behavior of the cells. These phenomena have been widely studied with mathematical models based on data from mouse β-cells. However, it is now known that human β-cell electrophysiology shows important differences to its rodent counterpart, and although human pancreatic islets express connexin-36 and show evidence of β-cell coupling, these aspects have been little investigated in human β-cells. Here we investigate theoretically, the gap junction coupling strength required for synchronizing electrical activity in a small cluster of cells simulated with a recent mathematical model of human β-cell electrophysiology. We find a lower limit for the coupling strength of approximately 20 pS (i.e., normalized to cell size, ∼2 pS pF−1) below which spiking electrical activity is asynchronous. To confront this theoretical lower bound with data, we use our model to estimate from an experimental patch clamp recording that the coupling strength is approximately 100–200 pS (10–20 pS pF−1), similar to previous estimates in mouse β-cells. We then investigate the role of gap junction coupling in synchronizing and modifying other forms of electrical activity in human β-cell clusters. We find that electrical coupling can prolong the period of rapid bursting electrical activity, and synchronize metabolically driven slow bursting, in particular when the metabolic oscillators are in phase. Our results show that realistic coupling conductances are sufficient to promote synchrony in small clusters of human β-cells as observed experimentally, and provide motivation for further detailed studies of electrical coupling in human pancreatic islets.

Calcium modulation of exocytosis-linked plasma membrane potential oscillations in INS-1 832/13 cells.

In the presence of high glucose or pyruvate, INS-1 832/13 insulinoma cells undergo stochastic oscillations in plasma membrane potential (Δψp) leading to associated fluctuations in cytosolic free Ca(2+) concentration ([Ca(2+)]c). Oscillations are not driven by upstream metabolic fluctuations, but rather by autonomous ionic mechanisms, the details of which are unclear. We have investigated the nature of the oscillator, with simultaneous fluorescence monitoring of Δψp, [Ca(2+)]c and exocytosis at single-cell resolution, combined with analysis of the occurrence, frequency and amplitude of Δψp oscillations. Oscillations were closely coupled to exocytosis, indicated by coincident synaptopHluorin fluorescence enhancement. L-type Ca(2+) channel inhibitors enhanced Δψp and [Ca(2+)]c oscillation frequency in the presence of pyruvate, but abolished the sustained [Ca(2+)]c response following KCl depolarization. The L-type Ca(2+) channel inhibitor isradipine did not inhibit oscillation-linked exocytosis. The T-type Ca(2+) channel inhibitor NNC 55-0396 inhibited Δψp and [Ca(2+)]c oscillations, implying that T-type Ca(2+) channels trigger oscillations and consequent exocytosis. Since distinct ion channels operate in oscillating and non-oscillating cells, quantitative analysis of Δψp and [Ca(2+)]c oscillations in a β-cell population may help to improve our understanding of the link between metabolism and insulin secretion.

The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans.

Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets.

Different Raman spectral patterns of primary rat pancreatic β cells and insulinoma cells.

As a noninvasive and label-free analytical technique, Raman spectroscopy has been widely used to study the difference between malignant cells and normal cells. Insulinomas are functional β-cell tumors of pancreatic islet cells. They exhibit many structural and immunohistochemical features in common with normal pancreatic β cells; thus, they are typically difficult to distinguish under the microscope, especially in vivo. We investigated insulinoma and primary rat pancreatic β-cell populations using Raman spectroscopy. The details of the optical heterogeneity between these two populations were determined based on different Raman regions primarily involving nucleic acid and protein contents, which are the most distinct cellular contents in these two types of cells. Using principal component analysis–linear discriminant analysis, these two cell types can be readily separated. The results of this work indicate that Raman spectroscopy is a promising tool for the noninvasive and label-free differentiation of insulinoma cells and normal pancreatic β cells.