Mouse Pancreatic Islets Research Articles

Regulator of G protein signaling complex, Gβ5‐R7, promotes glucose‐ and extracellular signal‐stimulated insulin secretion

Glucose‐stimulated insulin secretion (GSIS) is essential for maintaining energy homeostasis. G protein‐coupled receptors (GPCRs) are important modulators of this process by extracellular signals such as hormones, neurotransmitters and non‐glucose metabolites. We investigated the role of Gβ5‐R7, the protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling (RGS) of the R7 family. To analyze the effects of Gnb5 knockout on insulin secretion we used mouse insulinoma MIN6 cell line and pancreatic islets isolated from the Gnb5−/− mice. Consistent with previous work, Gnb5knockout diminished insulin secretion evoked by cholinergic agonist Oxo‐M. Here, we found that Gnb5 knockout also attenuated activity of other GPCR agonists ADP, AVP, GLP‐1, forskolin and, surprisingly, the response to high glucose. Our experiments also provided evidence that Gnb5 knockout eliminated the positive effect of cell adhesion on Oxo‐M‐stimulated GSIS, likely through adhesion GPCR GPR56. Total insulin content and the amount of insulin released in response to K+‐evoked membrane depolarization in Gnb5 −/− islets or MIN6 cells was normal. The pleiotropic effect on several GPCRs led us to the hypothesis that Gβ5‐R7 may play a non‐canonical role in insulin secretory pathway downstream of G protein signaling and glucose‐activated membrane depolarization. We found that Gnb5 knockout does not influence cortical actin depolymerization, but influences activity of protein kinase C, and thus far identified 14‐3‐3ɛ as one of the affected substrates. Together, these results allow us to propose that the Gβ5‐R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway, downstream of G protein signaling and actin depolymerization, but upstream of insulin granule release.Support or Funding InformationThis work was supported by National Institutes of Health Grants R01DK1054127(to V.S.), RO1‐DK073716, DK084236 (to E.B.M), EY11373 (to J.C.).

Darbepoetin-\u03b1 increases the blood volume flow in transplanted pancreatic islets in mice

AimsThe minimal-invasive transplantation of pancreatic islets is a promising approach to treat diabetes mellitus type 1. However, islet transplantation is still hampered by the insufficient process of graft revascularization, leading to a poor clinical outcome. Accordingly, the identification of novel compounds, which accelerate and improve the revascularization of transplanted islets, is of great clinical interest. Previous studies have shown that darbepoetin (DPO)-α, a long lasting analogue of erythropoietin, is capable of promoting angiogenesis. Hence, we investigated in this study whether DPO improves the revascularization of transplanted islets.MethodsIslets were isolated from green fluorescent protein-positive FVB/N donor mice and transplanted into dorsal skinfold chambers of FVB/N wild-type animals, which were treated with DPO low dose (2.5 µg/kg), DPO high dose (10 µg/kg) or vehicle (control). The revascularization was assessed by repetitive intravital fluorescence microscopy over an observation period of 14 days. Subsequently, the cellular composition of the grafts was analyzed by immunohistochemistry.ResultsThe present study shows that neither low- nor high-dose DPO treatment accelerates the revascularization of free pancreatic islet grafts. However, high-dose DPO treatment increased the blood volume flow of the transplanted islet.ConclusionsThese findings demonstrated that DPO treatment does not affect the revascularization of transplanted islets. However, the glycoprotein increases the blood volume flow of the grafts, which results in an improved microvascular function and may facilitate successful transplantation.

Alcohol ingestion induces pancreatic islet dysfunction and apoptosis via mediation of FGF21 resistance.

BackgroundDisruption of β-cell insulin secretion and viability caused by excessive ethanol consumption increases type 2 diabetes mellitus (T2DM) pathogenesis risk. Fibroblast growth factor 21 (FGF21) plays a significant role in regulating lipid and glucose homeostasis. Recently, FGF21, best known for its role in lipid and glucose homeostasis regulation, and its obligate co-receptor β-klotho have been shown to inhibit ethanol ingestion and metabolism. It remains unclear whether heavy ethanol intake modulates islet FGF21 expression and function. This study investigated the relationship between ethanol exposure, FGF21, and islet function in vivo/ex vivo islet and in vitro cell models.MethodsMice were gavaged with 3.5 g/kg ethanol or saline for 1–3 weeks (long-term exposure). Human MIN6 cells and isolated islets were cultured and treated with 80 mM ethanol for 24 h (short-term exposure) to mimic excessive ethanol consumption. We applied the oral glucose tolerance test (OGTT), blood glucometry, enzyme-linked immunosorbent assay (ELISAs) for insulin and FGF21, glucose stimulated insulin secretion (GSIS) testing, reverse-transcription (RT)-polymerase chain reaction (PCR), and western blot experiments.ResultsLong-term ethanol treatment induced FGF21 resistance in mouse pancreatic islets. Moreover, ethanol exposure damaged insulin secretory ability and glucose homeostasis. In vitro and ex vivo experiments showed that short-term ethanol treatment upregulated the expression of FGF21 signaling pathway-related genes and proteins, without affecting β-cell survival or function.ConclusionsLong-term ethanol consumption induces FGF21 resistance-mediated pancreatic β-cell dysfunction, and thus diabetes pathogenesis risk.

Glucagon-Like Peptide 1 Shows Glucose Dependence in Regulating Islet Hormone Secretion

Background: GLP-1, glucagon-like peptide 1, is an incretin secreted by intestinal L-cells, responsible for stimulating insulin and decreasing glucagon secretion. Analogs of this hormone such as Liraglutide are approved for treating Type II diabetes (T2D). Despite its clinical success, much about how GLP-1 inhibits the alpha-cell remains unknown. GLP-1 receptor is expressed in both beta and delta cells, but is absent from the alpha-cell. As somatostatin reduces alpha-cell Ca2+ influx, paracrine signaling between delta and alpha cells may contribute to GLP-1 mediated inhibition of glucagon secretion. Therefore, we hypothesize that altered delta-cell activity is involved in the inhibitory effects of GLP-1 on the alpha-cell. Methods: Genetically encoded cAMP indicator cADDIS or a Ca2+ dye together with mouse pancreatic islets that express red fluorescent protein in either alpha or delta cells were used. Islets were perfused in glucose with or without addition of Liraglutide while imaged in real-time on Zeiss LSm780. ImageJ was used for analysis. Results: GLP-1 reduced delta-cell Ca2+ influx in low glucose, possibly by enhancing electrical coupling between beta and delta cells. GLP-1 increased alpha-cell cAMP levels under low glucose, further suggesting that delta cell somatostatin secretion is also reduced in these conditions. In high glucose, we found increased synchronicity and frequency of Ca2+ oscillations between beta and delta cells but decreased alpha-cell cAMP. Conclusion: The data suggests that GLP-1 reduces delta-cell Ca2+ influx in low glucose and increases Ca2+ influx in high glucose, which may provide a mechanism for glucose-dependent modulation of alpha-cell glucagon secretion by GLP-1.

Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+ Progenitors

It has generally proven challenging to produce functional β cells invitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing ∼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. β cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.

Intra-islet GLP-1, but not CCK, is necessary for \u03b2-cell function in mouse and human islets

Glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK) are gut-derived peptide hormones known to play important roles in the regulation of gastrointestinal motility and secretion, appetite, and food intake. We have previously demonstrated that both GLP-1 and CCK are produced in the endocrine pancreas of obese mice. Interestingly, while GLP-1 is well known to stimulate insulin secretion by the pancreatic β-cells, direct evidence of CCK promoting insulin release in human islets remains to be determined. Here, we tested whether islet-derived GLP-1 or CCK is necessary for the full stimulation of insulin secretion. We confirm that mouse pancreatic islets secrete GLP-1 and CCK, but only GLP-1 acts locally within the islet to promote insulin release ex vivo. GLP-1 is exclusively produced in approximately 50% of α-cells in lean mouse islets and 70% of α-cells in human islets, suggesting a paracrine α to β-cell signaling through the β-cell GLP-1 receptor. Additionally, we provide evidence that islet CCK expression is regulated by glucose, but its receptor signaling is not required during glucose-stimulated insulin secretion (GSIS). We also see no increase in GSIS in response to CCK peptides. Importantly, all these findings were confirmed in islets from non-diabetic human donors. In summary, our data suggest no direct role for CCK in stimulating insulin secretion and highlight the critical role of intra-islet GLP-1 signaling in the regulation of human β-cell function.

De novo discovery of metabolic heterogeneity with immunophenotype-guided imaging mass spectrometry

BackgroundImaging mass spectrometry enables in situ label-free detection of thousands of metabolites from intact tissue samples. However, automated steps for multi-omics analyses and interpretation of histological images have not yet been implemented in mass spectrometry data analysis workflows. The characterization of molecular properties within cellular and histological features is done via time-consuming, non-objective, and irreproducible definitions of regions of interest, which are often accompanied by a loss of spatial resolution due to mass spectra averaging.Methods: We developed a new imaging pipeline called Spatial Correlation Image Analysis (SPACiAL), which is a computational multimodal workflow designed to combine molecular imaging data with multiplex immunohistochemistry (IHC). SPACiAL allows comprehensive and spatially resolved in situ correlation analyses on a cellular resolution. To demonstrate the method, matrix-assisted laser desorption-ionization (MALDI) Fourier-transform ion cyclotron resonance (FTICR) imaging mass spectrometry of metabolites and multiplex IHC staining were performed on the very same tissue section of mouse pancreatic islets and on human gastric cancer tissue specimens. The SPACiAL pipeline was used to perform an automatic, semantic-based, functional tissue annotation of histological and cellular features to identify metabolic profiles. Spatial correlation networks were generated to analyze metabolic heterogeneity associated with cellular features.Results: To demonstrate the new method, the SPACiAL pipeline was used to identify metabolic signatures of alpha and beta cells within islets of Langerhans, which are cell types that are not distinguishable via morphology alone. The semantic-based, functional tissue annotation allows an unprecedented analysis of metabolic heterogeneity via the generation of spatial correlation networks. Additionally, we demonstrated intra- and intertumoral metabolic heterogeneity within HER2/neu-positive and -negative gastric tumor cells.Conclusions: We developed the SPACiAL workflow to provide IHC-guided in situ metabolomics on intact tissue sections. Diminishing the workload by automated recognition of histological and functional features, the pipeline allows comprehensive analyses of metabolic heterogeneity. The multimodality of immunohistochemical staining and extensive molecular information from imaging mass spectrometry has the advantage of increasing both the efficiency and precision for spatially resolved analyses of specific cell types. The SPACiAL method is a stepping stone for the objective analysis of high-throughput, multi-omics data from clinical research and practice that is required for diagnostics, biomarker discovery, or therapy response prediction.

Immunoreactivity of cytotoxic T-lymphocyte antigen 2 alpha in mouse pancreatic islet cells.

Cells of the pancreatic islets produce several molecules including insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells), pancreatic polypeptide (PP cells), ghrelin (epsilon cells), serotonin (enterochromaffin cells), gastrin (G cells) and small granules of unknown content secreted by the P/D1 cells. Secretion mechanism of some of these molecules is still poorly understood. However, Cathepsin L is shown to regulate insulin exocytosis in beta cells and activate the trypsinogen produced by the pancreatic serous acini cells into trypsin. The structure of the propeptide region of Cathepsin L is homologous to Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2 alpha) which is also shown to exhibit selective inhibitory activities against Cathepsin L. It was thought that if CTLA-2 alpha was expressed in the pancreas; then, it would be an important regulator of protease activation and insulin secretion. The purpose of this study was, therefore, to examine by immunohistochemistry the cellular localization and distribution pattern of CTLA-2 alpha in the pancreas. Results showed that strong immunoreactivity was specifically detected in the pancreatic islets (endocrine pancreas) but not in the exocrine pancreas and pancreatic stroma. Immunostaining was further performed to investigate more on localization of Cathepsin L in the pancreas. Strong immunoreactivity for Cathepsin L was detected in the pancreatic islets, serous cells and the pancreas duct system. These findings suggest that CTLA-2 alpha may be involved in the proteolytic processing and secretion of insulin through regulation of Cathepsin L and that the regulated inhibition of Cathepsin L may have therapeutic potential for type 1 diabetes.

Anti-hyperglycaemic activity of H. rosa-sinensis leaves is partly mediated by inhibition of carbohydrate digestion and absorption, and enhancement of insulin secretion

Ethnopharmacological relevanceHibiscus rosa-sinensis (HRS) is a tropical flowery plant, widely distributed in Asian region and an important traditional medicine used in many diseases including cough, diarrhoea and diabetes. Aim of this studyHibiscus rosa-sinensis (HRS) leaves have been reported to possess anti-hyperglycaemic activity, but little is known concerning the underlying mechanism. This study investigated effects of ethanol extract of HRS on insulin release and glucose homeostasis in a type 2 diabetic rat model. Materials & methodsEffects of ethanol extract of grinded H. rosa-sinensis (HRS) leaves on insulin release, membrane potential and intracellular calcium were determined using rat clonal β-cells (BRIN-BD11 cells) and isolated mouse pancreatic islets. Effects on DPP-IV enzyme activity were investigated in vitro. Acute effects of HRS on glucose tolerance, gut perfusion in situ, sucrose content, intestinal disaccharidase activity and gut motility were measured. Streptozotocin induced type 2 diabetic rats treated for 28 days with ethanol extract of HRS leaf (250 and 500 mg/kg) were used to assess glucose homeostasis. ResultsHRS, significantly increased insulin release from clonal rat BRIN-BD11 cells and this action was confirmed using isolated mouse pancreas islets with stimulatory effects equivalent to GLP-1. HRS induced membrane depolarization and increased intracellular Ca2+ in BRIN BD11 cells and significantly inhibited DPP-IV enzyme activity in vitro. HRS administration in vivo improved glucose tolerance in type 2 diabetic rats, inhibited both glucose absorption during gut perfusion and postprandial hyperglycaemia and it reversibly increased unabsorbed sucrose passage through the gut following sucrose ingestion. HRS decreased intestinal disaccharidase activity and increased gastrointestinal motility in non-diabetic rats. In a chronic 28-day study with type 2 diabetic rats, HRS, at 250 or 500 mg/kg, significantly decreased serum glucose, cholesterol, triglycerides and increased circulating insulin, HDL cholesterol and hepatic glycogen without increasing body weight. ConclusionThese data suggest the antihyperglycaemic activity of HRS is mediated by inhibiting carbohydrate digestion and absorption, while significantly enhancing insulin secretion in a dose dependent manner. This suggests that HRS has potential as a novel antidiabetic therapy or a dietary supplement for the treatment of type 2 diabetes.

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells.

Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA—mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

AE804, AE806, AE999, AF017, AF041 and AF131 antibodies label mouse insulin-secreting beta cells by immunohistochemistry

The recombinant antibodies AE804, AE806, AE999, AF017, AF041 and AF131 detect by immunohistochemistry the insulin-secreting beta cells in mouse pancreatic islets. The most efficient staining was obtained with AE999.

Targeted Optical Imaging of the Glucagonlike Peptide 1 Receptor Using Exendin-4-IRDye 800CW.

The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intraoperative detection can be challenging. This challenge could be overcome with intraoperative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagonlike peptide 1 receptor (GLP-1R)–positive lesions, using the GLP-1 agonist exendin-4 labeled with IRDye 800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye 800CW binds GLP-1R with a half-maximal inhibitory concentration of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice, and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These data provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R–positive lesions. Intraoperative lesion delineation using exendin-4-IRDye 800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

The AE804 and AE806 antibodies label mouse insulin-secreting beta cells by immunofluorescence in histological frozen sections

The AE804 and AE806 antibodies detect the insulin-secreting beta cells by immunofluorescence in mice pancreatic islets.

The AK247 and AK248 antibodies label mouse glucagon-secreting alpha cells by immunofluorescence in histological frozen sections

The AK247 and AK248 antibodies detect the glucagon-secreting alpha cells by immunofluorescence in mice pancreatic islets.

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from β-cells), glucagon (α-cells) and somatostatin (δ-cells). β-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, α-cells and δ-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest. Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of β-cells is minimized. Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase. Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Piezo1 channel activation mimics high glucose as a stimulator of insulin release

Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic β-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat β-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca2+ measurements were performed using the fura-2 Ca2+ indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both β-cell lines and mouse islets. Yoda1 evoked Ca2+ entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from β-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca2+ and enhanced Yoda1 Ca2+ influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from β-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release.

Mouse Pancreas Stem/Progenitor Cells Get Augmented by Streptozotocin and Regenerate Diabetic Pancreas After Partial Pancreatectomy.

Existence of stem cells in adult pancreas remains contentious. Single cells suspensions obtained by collagenase and trypsin digestion separately from adult mouse pancreas and pancreatic islets were spun at 1000rpm (250g) to collect the cells. At this speed the stem/ progenitor cells remained buoyant and were further enriched by spinning the supernatant at 3000rpm (1000g). Two distinct populations of stem cells were detected including pluripotent, very small (2-6μm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct-4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2-6μm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings.

Compact fluidic system for functional assessment of pancreatic islets.

Transplantation of pancreatic islets is becoming a promising therapy for people with type I diabetes. In this study, we present a compact fluidic system that enables assessment of islet functionality ex vivo for efficient islet transplantation. The fluidic system includes a micromesh sheet-embedded chip. Islets can be loaded easily on the micromesh sheet and observed clearly by microscopy. Islets on the mesh sheet mainly remained in place during perfusion and did not get damaged by hydraulic pressure because of high porosity of the micromesh sheet. The fluidic system was assembled with a sample fraction chip of polydimethylsiloxane. The chip includes a channel and columns, both having surfaces that were super-hydrophilized so that solutions could flow smoothly within the chip by gravity. Using mouse pancreatic islets, a dynamic glucose-stimulated insulin secretion test was performed to examine the performance of the fluidic system. The system successfully analyzed levels and patterns of insulin secretion upon exposure of the islets to low- and high-glucose solutions in turns, thus demonstrating its capacity to assess islet functions more easily and cost-effectively.

Genetically Encoded, Photostable Indicators to Image Dynamic Zn2+ Secretion of Pancreatic Islets.

As an essential element for living organisms, zinc (Zn2+) exerts its biological functions both intracellularly and extracellularly. Previous studies have reported a number of genetically encoded Zn2+ indicators (GEZIs), which have been widely used to monitor Zn2+ in the cytosol and intracellular organelles. However, it is challenging to localize existing GEZIs to the extracellular space to detect secreted Zn2+. Herein, we report two photostable, green fluorescent protein (GFP) based indicators, ZIBG1 and ZIBG2, which respond to Zn2+ selectively and have affinities suited for detecting Zn2+ secretion from intracellular vesicles. In particular, ZIBG2 can be effectively targeted to the extracellular side of plasma membrane. We applied cell surface-localized ZIBG2 to monitor glucose-induced dynamic Zn2+ secretion from mouse insulinoma MIN6 cells and primary mouse and human pancreatic islets. Because Zn2+ is co-released with insulin from β-cells, the fluorescence of cell surface-localized ZIBG2 was shown to be a strong indicator for the functional potency of islets. Our work here has thus expanded the use of GEZIs to image dynamic Zn2+ secretion in live tissue. Because it is convenient to use genetically encoded indicators for expression over extended periods and for in vivo delivery, we envision future applications of ZIBG2 in development of induced β-cells or islets to advance cell replacement therapies for diabetes and in direct imaging of Zn2+ secretion dynamics in vivo.