Murine Pancreatic Islets Research Articles

Imagerie d’îlots de Langerhans murins avec la microscopie à cohérence optique: l’avenir ?

Aims/hypothesis Structural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition.

Erratum to: Neural crest stem cells increase beta cell proliferation and improve islet function in co-transplanted murine pancreatic islets

Erratum to: Neural crest stem cells increase beta cell proliferation and improve islet function in co-transplanted murine pancreatic islets

Imagerie d’îlots de Langerhans murins avec la microscopie à cohérence optique: l’avenir ?

Structural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition. Ex vivo measurements on excised pancreases were performed and validated by standard immunohistochemistry to investigate the structures that can be observed with xfOCM. The influence of streptozotocin on the signature of the islets was investigated in a second step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo. xfOCM circumvents the fundamental physical limit that trades lateral resolution for depth of field, and achieves fast volumetric imaging with high-resolution in all three dimensions. It allows label-free visualisation of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans ex vivo and in vivo, and detects streptozotocin-induced islet destruction. Our results demonstrate the potential value of xfOCM in high-resolution in vivo studies to assess islet structure and function in animal models of diabetes, aiming towards its use in longitudinal studies of diabetes progression and islet transplants.

Fatty acids induce amylin expression and secretion by pancreatic β-cells

Amylin is the major component of pancreatic amyloid, which is implicated in the development of type 2 diabetes. It is costored with insulin in the secretory granules of pancreatic beta-cells and cosecreted with insulin following stimulation with glucose. Here, we investigate the effect of fatty acids (FAs) on amylin expression and secretion by beta-cells and explore the underlying mechanisms. Palmitate and oleate dose-dependently induced amylin mRNA accumulation in murine pancreatic beta-cell line MIN6 and primary pancreatic islets. the inductive effect of FAs on amylin expression is independent of glucose concentration. FAs upregulated amylin expression at the transcriptional level, and FAs must be metabolized to induce amylin expression. FAs also significantly induced human amylin promoter activation. Pretreatment of MIN6 cells with Ca(2+) chelator (EGTA, BAPTA-AM) PKC inhibitor Gö-6976 or protein synthesis inhibitor cycloheximide significantly inhibited FA-induced amylin mRNA expression. Transcription factors cAMP-responsive element-binding protein, pancreatic and duodenal homeobox factor-1, and peroxisome proliferator-activated receptor were not involved in FA-induced amylin expression. Palmitate and oleate both increased amylin and insulin release from MIN6 cells and stimulated amylin expression but had no effect on insulin expression. Mice refed with Intralipid had significantly higher levels of plasma FFA, amylin, and insulin than those refed with saline. These data demonstrate that FAs differently regulate amylin and insulin expression and induce both amylin and insulin release. Ca(2+) and PKC signaling pathways and de novo-synthesized protein(s) were involved in FA-induced amylin expression. Induction of amylin production and release by FA may contribute to its biological functions under physiological conditions.

Neural crest stem cells increase beta cell proliferation and improve islet function in co-transplanted murine pancreatic islets

Long-term graft survival after islet transplantation to patients with type 1 diabetes is insufficient, necessitating the development of new strategies to enhance transplant viability. Here we investigated whether co-transplantation of neural crest stem cells (NCSCs) with islets improves islet survival and function in normoglycaemic and diabetic mice. Islets alone or together with NCSCs were transplanted under the kidney capsule to normoglycaemic or alloxan-induced diabetic mice. Grafts were analysed for size, proliferation, apoptosis and insulin release. In diabetic recipients blood glucose levels were examined before and after graft removal. In mixed transplants NCSCs actively migrated and extensively associated with co-transplanted pancreatic islets. Proliferation of beta cells was markedly increased and transplants displayed improved insulin release in normoglycaemic mice compared with those receiving islet-alone transplants. Mixed grafts survived successfully and partially restored normoglycaemia in alloxan-induced diabetic mice. Co-grafting of NCSCs with pancreatic islets improved insulin release in mixed transplants and enhanced beta cell proliferation, resulting in increased beta cell mass. This co-transplantation model offers an opportunity to restore neural-islet interactions and improve islet functions after transplantation.

Sphingomyelinase dependent apoptosis following treatment of pancreatic beta-cells with amyloid peptides Aß1-42 or IAPP

Amyloid peptides interfere with survival of pancreatic beta-cells. In some cells apoptosis is paralleled by ceramide-dependent alterations of ion channel activity. The purpose of the present study was to elucidate the dependence of amyloid peptides Abeta(1-42) and islet amyloid polypeptide (IAPP)-induced cell death on ceramide formation and ion channel activity in murine pancreatic islet cells. As disclosed by TUNEL (terminal dUTP nick-end labelling) and cleaved caspase 3 staining, apoptotic cell death was induced by Abeta(1-42), IAPP and exogenously added C2-ceramide in islet cells from wild type mice. In islet cells from acid sphingomyelinase-deficient mice (ASMKO) Abeta(1-42) and IAPP but not exogenously added N-acetyl-D-sphingosine (C2-ceramide, 20 microM) failed to stimulate apoptosis. Immunofluorescent staining revealed a stimulatory effect of Abeta(1-42) on ceramide formation. According to patch clamp experiments, administration of Abeta(1-42) and IAPP significantly decreased outwardly rectifying whole cell currents in wild type but not in ASMKO islet cells. C2-ceramide but not inactive di-ceramide (20 microM) mimicked the inhibitory effect on Kv channel current. In conclusion, amyloid peptides induce apoptosis of pancreatic islet cells at least in part through activation of acid sphingomyelinase resulting in production of ceramide and subsequent inhibition of ion channel activity.

In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy

Structural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition. Ex vivo measurements on excised pancreases were performed and validated by standard immunohistochemistry to investigate the structures that can be observed with xfOCM. The influence of streptozotocin on the signature of the islets was investigated in a second step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo. xfOCM circumvents the fundamental physical limit that trades lateral resolution for depth of field, and achieves fast volumetric imaging with high resolution in all three dimensions. It allows label-free visualisation of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans ex vivo and in vivo, and detects streptozotocin-induced islet destruction. Our results demonstrate the potential value of xfOCM in high-resolution in vivo studies to assess islet structure and function in animal models of diabetes, aiming towards its use in longitudinal studies of diabetes progression and islet transplants.

Rapid and inexpensive fabrication of polymeric microfluidic devices via toner transfer masking

An alternative fabrication method is presented for production of masters for single- or multi-layer polymeric microfluidic devices in a standard laboratory environment, precluding the need for a cleanroom. This toner transfer masking (TTM) method utilizes an office laser printer to generate a toner pattern which is thermally transferred to a metal master to serve as a mask for etching. With master fabrication times as little as one hour (depending on channel depth) using commercially-available equipment and supplies, this approach should make microfluidic technology more widely accessible to the non-expert-even the non-scientist. The cost of fabrication consumables was estimated to be < $1 per master, over an order of magnitude decrease in consumable costs compared to standard photolithography. In addition, the use of chemical etching allows accurate control over the height of raised features (i.e., channel depths), allowing the flexibility to fabricate multiple depths on a single master with little added time. Resultant devices are shown capable of pneumatic valving, three-dimensional channel formation (using layer-connecting vias), droplet fluidics, and cell imaging and staining. The multiple-depth capabilities of the method are proven useful for cellular analysis by fabrication of handheld, disposable devices used for trapping and imaging of live murine pancreatic islets. The precise fluidic control provided by the microfluidic platform allows subsequent fixing and staining of these cells without significant movement, thus spatial correlation of imaging and staining is attainable-even with rare alpha cells that constitute only approximately 10% of the islet cells.

Pancreatic Islets Induce CD4+CD25−Foxp3+ T-Cell Regulated Tolerance to HY-Mismatched Skin Grafts

The ability to induce and maintain antigen-specific immunologic tolerance is the ultimate goal in allo-transplantation. Here, we report that the transplantation of unmanipulated murine pancreatic islets across the HY disparity induced transplantation tolerance that prevented HY-mismatched skin grafts being rejected. Three hundred islet equivalent of freshly isolated islets from male C57BL/6 donor mice was transplanted underneath the kidney capsule of syngeneic female recipients rendered diabetic by streptozotocin. Nephrectomy was carried out to remove the islet graft and retransplantation was performed using the contralateral kidney. For skin transplantation, donor tail skin was transplanted onto the lateral thorax. Islets from male C57BL/6 donors transplanted to syngeneic female recipients cured diabetes and the mice survived indefinitely. The acceptance of second grafts and rejection of third party islet grafts indicated antigen-specific transplantation tolerance. However, flow cytometry and ELISPOT analysis demonstrated that the HY-specific T cells were not deleted or anergized. A 2-fold increase of CD4+Foxp3+ regulatory T cells (Tregs) was observed in spleen and lymph nodes. Notably, CD25- Tregs increased threefold over levels in naïve mice. Adoptive transfer of CD4+ T cells to neonatal mice could transfer tolerance. At the graft site in long-term tolerant mice, CD4+ T cells, 40% of which were CD4+Foxp3+ Tregs (43% CD25-, 57% CD25+) infiltrated the peri-islet spaces. Unmanipulated pancreatic islets can induce immunologic tolerance associated with peripherally induced CD4+Foxp3+ Tregs, a significant proportion of which notably are CD25-.

Multifunctional Magnetic Nanocarriers for Image-Tagged SiRNA Delivery to Intact Pancreatic Islets

With the ultimate hope of finding a cure for diabetes, researches are looking into altering the genetic profile of the beta cell as a way to manage metabolic dysregulation. One of the most powerful new approaches for the directed regulation of gene expression uses the phenomenon of RNA interference. Here, we establish the feasibility of a novel technology centered around multifunctional magnetic nanocarriers, which concurrently deliver siRNA to intact pancreatic islets and can be detected by magnetic resonance and optical imaging. In the proof-of-principle studies described here, we demonstrate that, after in vitro incubation, magnetic nanoparticles carrying siRNA designed to target the model gene for enhanced green fluorescent protein are efficiently taken up by murine pancreatic islets, derived from egfp transgenic animals. This uptake can be visualized by magnetic resonance imaging and near-infrared fluorescence optical imaging and results in suppression of the target gene. These results illustrate the value of our approach in overcoming the challenges associated with genetic modification of intact pancreatic islets in a clinically acceptable manner. Furthermore, an added advantage of our technology derives from the combined capability of our magnetic nanoparticles for siRNA delivery and magnetic labeling of pancreatic islets.

Abscisic Acid Is an Endogenous Stimulator of Insulin Release from Human Pancreatic Islets with Cyclic ADP Ribose as Second Messenger

Abscisic acid (ABA) is a plant stress hormone recently identified as an endogenous pro-inflammatory cytokine in human granulocytes. Because paracrine signaling between pancreatic beta cells and inflammatory cells is increasingly recognized as a pathogenetic mechanism in the metabolic syndrome and type II diabetes, we investigated the effect of ABA on insulin secretion. Nanomolar ABA increases glucose-stimulated insulin secretion from RIN-m and INS-1 cells and from murine and human pancreatic islets. The signaling cascade triggered by ABA in insulin-releasing cells sequentially involves a pertussis toxin-sensitive G protein, cAMP overproduction, protein kinase A-mediated activation of the ADP-ribosyl cyclase CD38, and cyclic ADP-ribose overproduction. ABA is rapidly produced and released from human islets, RIN-m, and INS-1 cells stimulated with high glucose concentrations. In conclusion, ABA is an endogenous stimulator of insulin secretion in human and murine pancreatic beta cells. Autocrine release of ABA by glucose-stimulated pancreatic beta cells, and the paracrine production of the hormone by activated granulocytes and monocytes suggest that ABA may be involved in the physiology of insulin release as well as in its dysregulation under conditions of inflammation.

C-Jun N-terminal kinase 1 is deleterious to the function and survival of murine pancreatic islets

Inhibition of c-jun N-terminal kinase (JNK) favours pancreatic islet function and survival. Since two JNK isoforms are present in the pancreas (JNK1 and JNK2), we addressed their specific roles in experimental islet transplantation. C57BL/6J (wild-type [WT]), Jnk1 (also known as Mapk8)(-/-) and Jnk2 (also known as Mapk9)(-/-) mice were used as donor/recipients in a syngeneic islet transplantation model. Islet cell composition, function, viability, production of cytokines and of vascular endothelial growth factor (VEGF) were also studied in vitro. Jnk1 ( -/- ) islets secreted more insulin in response to glucose and were more resistant to cytokine-induced cell death compared with WT and Jnk2 (-/-) islets (p < 0.01). Cytokines reduced VEGF production in WT and Jnk2 (-/-) but not Jnk1 ( -/- ) islets; VEGF blockade restored Jnk1 ( -/- ) islet susceptibility to cytokine-induced cell death. Transplantation of Jnk1 ( -/- ) or WT islets into WT recipients made diabetic had similar outcomes. However, Jnk1 ( -/- ) recipients of WT islets had shorter time to diabetes reversal (17 vs 55 days in WT, p = 0.033), while none of the Jnk2 (-/-) recipients had diabetes reversal (0% vs 71% in WT, p = 0.0003). Co-culture of WT islets with macrophages from each strain revealed a discordant cytokine production. We have shown a deleterious effect of JNK2 deficiency on islet graft outcome, most likely related to JNK1 activation, suggesting that specific JNK1 blockade may be superior to general JNK inhibition, particularly when administered to transplant recipients.

Murine Pancreatic Islet Isolation

Murine Pancreatic Islet Isolation

Murine Pancreatic Islet Isolation

Murine Pancreatic Islet Isolation

Efficient gene transfer into murine pancreatic islets using adenovirus vectors

We investigated the efficiency of gene transduction into murine pancreatic islets using the adenovirus (Ad) vector. Western blotting analysis showed that mouse pancreatic islets express coxsackievirus and adenovirus receptor, a receptor for Ad. Nevertheless, gene expression after transduction of the Ad vector in vitro was observed only in the periphery of the islets, probably due to physical obstruction against Ad infection of the cells in the inside of islets. Ca 2+-free treatment before the Ad vector transduction enhanced transduction efficiency in the islets, but not the cells in the inside of islets. The Ad vector transduction through the celiac artery in vivo and then cultivation of islets in vitro resulted in efficient transduction even in the inside of islets. Thus we propose a new strategy for efficient gene transfer to pancreatic β-cells.

RNA Editing by ADAR2 Is Metabolically Regulated in Pancreatic Islets and β-Cells

RNA editing via the conversion of adenosine (A) to inosine (I) is catalyzed by two major families of adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. This genetic recoding process is known to play essential roles in the brain, due in part to changes in functional activities of edited neurotransmitter receptors and ion channels. Little is known, however, about the physiological regulation and function of A to I RNA editing in peripheral tissues and other biological processes. Here, we report that both ADAR1 and ADAR2 are expressed in the murine pancreatic islets, and ADAR2 is primarily localized in the islet endocrine cells. In contrast to ADAR1, ADAR2 transcripts in the pancreatic islets exhibit a nearly 2-fold increase in insulin-resistant mice chronically fed a high fat diet. Concurrent with this diet-induced metabolic stress, RNA editing in the islets is dramatically enhanced for the RNA transcripts encoding the ionotropic glutamate receptor subunit B. Moreover, ADAR2 protein expression is repressed in the islets under fuel deficiency condition during fasting, and this repression can be completely reversed by refeeding. We also show that, specifically in pancreatic beta-cell lines, not only the expression of ADAR2 but also the glutamate receptor subunit B editing and ADAR2 self-editing are markedly augmented in response to glucose at the physiological concentration for insulin secretion stimulation. Thus, RNA editing by ADAR2 in pancreatic islets and beta-cells is metabolically regulated by nutritional and energy status, suggesting that A to I RNA editing is most likely involved in the modulation of pancreatic islet and beta-cell function.

The Neuronal Ca2+ Sensor Protein Visinin-like Protein-1 Is Expressed in Pancreatic Islets and Regulates Insulin Secretion

Visinin-like protein-1 (VILIP-1) is a member of the neuronal Ca2+ sensor protein family that modulates Ca2+-dependent cell signaling events. VILIP-1, which is expressed primarily in the brain, increases cAMP formation in neural cells by modulating adenylyl cyclase, but its functional role in other tissues remains largely unknown. In this study, we demonstrate that VILIP-1 is expressed in murine pancreatic islets and beta-cells. To gain insight into the functions of VILIP-1 in beta-cells, we used both overexpression and small interfering RNA knockdown strategies. Overexpression of VILIP-1 in the MIN6 beta-cell line or isolated mouse islets had no effect on basal insulin secretion but significantly increased glucose-stimulated insulin secretion. cAMP accumulation was elevated in VILIP-1-overexpressing cells, and the protein kinase A inhibitor H-89 attenuated increased glucose-stimulated insulin secretion. Overexpression of VILIP-1 in isolated mouse beta-cells increased cAMP content accompanied by increased cAMP-responsive element-binding protein gene expression and enhanced exocytosis as detected by cell capacitance measurements. Conversely, VILIP-1 knockdown by small interfering RNA caused a reduction in cAMP accumulation and produced a dramatic increase in preproinsulin mRNA, basal insulin secretion, and total cellular insulin content. The increase in preproinsulin mRNA in these cells was attributed to enhanced insulin gene transcription. Taken together, we have shown that VILIP-1 is expressed in pancreatic beta-cells and modulates insulin secretion. Increased VILIP-1 enhanced insulin secretion in a cAMP-associated manner. Down-regulation of VILIP-1 was accompanied by decreased cAMP accumulation but increased insulin gene transcription.

Endogenous beta-galactosidase expression in murine pancreatic islets

To the Editor: The tissue-/cell-specific recombinase system, the Cre/loxP system, has become a very useful tool to conditionally inactivate or activate gene function [1–6]. This system is based on the ability of Cre to catalyse the excision of DNA flanked by loxP site recognition sequences. A number of studies on cell lineage in pancreatic cells have been performed by intercrossing cell-specific Cre-expressing transgenic mice and ROSAβgeo26 (ROSA26) mice, which carry the gene for bacterial beta-galactosidase (lacZ) [7]. In the double transgenic offspring, lacZ is expressed where Cre was expressed at a high enough level to excise the floxed stop codon. Commonly, X-gal staining is used to detect beta-galactosidase activity: beta-galactosidase catalyses the hydrolysis of X-gal to an indolyl alcohol, which then oxidises to form an intense blue indigo stain. While specific induction of lacZ by precise control of Cre, as well as leakage of beta-galactosidase by spontaneous Cre excision in double transgenic mouse, have been well studied, little work has dealt with the contribution of endogenous beta-galactosidase activity to false-positive results. Heart, liver, spleen, kidney, brain and skeletal muscle from mouse, rat and rabbit [8], and cultured human foetal pancreas [9], have been studied for endogenous betagalactosidase activity [8]. In cultured human foetal pancreas, the mRNA expression and enzyme activity of beta-galactosidase are developmentally regulated, peaking at 24 weeks of gestation and declining to low levels in the adult [9]. Endogenous beta-galactosidase activity in these organs was rarely detected following incubation at a pH greater than 7.5. In contrast, bacterial beta-galactosidase activity in the ROSAβgeo26 was strongly detected at pH 8.0–9.0 [8]. In the mouse pancreas, we evaluated endogenous beta-galactosidase expression and studied factors that may contribute to false-positive results. Firstly, to determine expression of the endogenous betagalactosidase mRNA in various tissues, we performed RTPCR on cDNAs from mouse islets, liver, kidney and spleen as well as from pancreatic beta cells from mouse insulin promoter I (MIP)–green fluorescent protein (GFP) mice [10], purified with FACS by their GFP expression (Fig. 1). The sizes of the PCR products for beta-galactosidase and the internal control TATA-binding protein were 330 and 190 bp, respectively. Pancreatic beta cells and islets as well as other organs expressed endogenous beta-galactosidase (Glb1) mRNA (Fig. 1). Since it is important to determine endogenous betagalactosidase enzyme activity, we performed X-gal staining on sections of pancreas and kidney from normal adult C57BL/6 mice; endogenous beta-galactosidase is known to be expressed in the kidney. From mice cardiac-perfused

Cathepsin S Controls Angiogenesis and Tumor Growth via Matrix-derived Angiogenic Factors

The cysteine protease cathepsin S is highly expressed in malignant tissues. By using a mouse model of multistage murine pancreatic islet cell carcinogenesis in which cysteine cathepsin activity has been functionally implicated, we demonstrated that selective cathepsin S deficiency impaired angiogenesis and tumor cell proliferation, thereby impairing angiogenic islet formation and the growth of solid tumors, whereas the absence of its endogenous inhibitor cystatin C resulted in opposite phenotypes. Although mitogenic vascular endothelial growth factor, transforming growth factor-beta1, and the anti-angiogenic endostatin levels in either serum or carcinoma tissue extracts did not change in cathepsin S- or cystatin C-null mice, tumor tissue basic fibroblast growth factor and serum type 1 insulin growth factor levels were higher in cystatin C-null mice, and serum type 1 insulin growth factor levels were also increased in cathepsin S-null mice. Furthermore, cathepsin S affected the production of type IV collagen-derived anti-angiogenic peptides and the generation of bioactive pro-angiogenic gamma2 fragments from laminin-5, revealing a functional role for cathepsin S in angiogenesis and neoplastic progression.

HUMAN UMBILICAL CORD BLOOD SERUM PROMOTES GROWTH, PROLIFERATION, AS WELL AS DIFFERENTIATION OF HUMAN BONE MARROW–DERIVED PROGENITOR CELLS

Fetal calf serum (FCS) is conventionally used for animal cell cultures due to its inherent growth-promoting activities. However animal welfare issues and stringent requirements for human transplantation studies demand a suitable alternative for FCS. With this view, we studied the effect of FCS, human AB serum (ABS), and human umbilical cord blood serum (UCBS) on murine islets of Langerhans and human bone marrow-derived mesenchymal-like cells (hBMCs). We found that there was no difference in morphology and functionality of mouse islets cultured in any of these three different serum supplements as indicated by insulin immunostaining. A comparative analysis of hBMCs maintained in each of these three different serum supplements demonstrated that UCBS supplemented media better supported proliferation of hBMCs. Moreover, a modification of adipogenic differentiation protocol using UCBS indicates that it can be used as a supplement to support differentiation of hBMCs into adipocytes. Our results demonstrate that UCBS not only is suitable for maintenance of murine pancreatic islets, but also supports attachment, propagation, and differentiation of hBMCs in vitro. We conclude that UCBS can serve as a better serum supplement for growth, maintenance, and differentiation of hBMCs, making it a more suitable supplement in cell systems that have therapeutic potential in human transplantation programs.