Insulin Secretion In Pancreatic Beta-cells Research Articles

Stem cell-derived tissue-associated regulatory T cells suppress autoimmune diabetes

Abstract CD4+ regulatory T cells (Tregs) are essential for normal immune surveillance, and their dysfunction can lead the development of autoimmune diseases, such as type-1 diabetes. Pluripotent stem cells (PSCs) can be utilized to obtain a renewable source of healthy Tregs to treat autoimmune diabetes as they have the ability to produce almost all cell types in the body, including Tregs. However, the right conditions for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) have not been fully defined. In this study, we developed a new approach to generate functional Ag-specific Tregs from induced PSC (iPSCs), i.e., iPSC-Tregs, which have the ability to suppress autoimmunity in a murine model of autoimmune diabetes in Ag-associated fashion. We retrovirally transduced murine iPSCs with a DsRed reporter construct containing genes of major histocompatibility complex (MHC) II (I-Ab)-restricted ovalbumin (OVA)-specific T cell receptor (TCR) and the transcriptional factor FoxP3. We in vitro differentiated the DsRed+ iPSC into OVA-specific iPSC-Tregs with an OP9 stromal cell line expressing Notch ligands DL1, DL4 and I-Ab in the presence of recombinant cytokines of rIL-7 and rFlt3L. We visualized the expression of CD3, TCR, CD4, CD25, and CTLA-4 on OVA-specific iPSC-Tregs. Moreover, adoptive transfer of such Tregs dramatically suppressed autoimmunity in a well-established OVA-induced model of autoimmune diabetes (RIP-OVA × OT-I double transgenic mice), including the inflammation and prevents the insulin secreting pancreatic beta cells from destruction. Our results indicate that PSCs can be used to develop Ag-specific Tregs, which have a therapeutic potential for autoimmune diabetes.

Mathematical Modeling of Human Pancreatic Alpha-Cells: Insight into the Role of SGLT2 in Glucagon Secretion

Pancreatic alpha-cells, which secrete the glucose promoting hormone glucagon, have regained renewed interest as it has become evident that dysfunctional glucagon secretion contributes to the development of diabetes. In contrast to the well-studied insulin-secreting pancreatic beta-cells, the control of glucagon release is still poorly understood. Recently it was shown that inhibitors of the sodium glucose co-transporters SGLT2, which are used as anti-diabetic drugs, increase glucagon secretion and hepatic glucose production. Following studies showed that pancreatic alpha-cells express SGLT2 and their inhibition augments glucagon secretion in isolated human pancreatic islets, suggesting a direct effect of the SGLT2 inhibitors on the alpha-cells.The mechanisms by which SGLT2 contribute to control of alpha-cell function are far from clear. Besides transporting glucose into the cell, SGLT2 are electrogenic. Here we investigate the electrophysiological effects of SGLT2 by building the first mathematical model of electrical activity in human pancreatic alpha-cells. The model is of Hodgkin-Huxley type, and includes all major currents of human alpha-cells. Individual currents were carefully fitted to published data from human alpha-cells. A six-state model of SGLT2 sodium-glucose co-transport and the related currents was also inserted in the model.We show that the SGLT2 current lowers the height of simulated action potentials (APs), and consequently, that SGLT2 inhibition allows full APs to develop, which would augment glucagon secretion. The underlying mechanisms of SGLT2-mediated reduction of AP peaks are complex, and involve inactivation of Na+ and Ca2+ currents contributing to the upstroke of the AP. Calculations of SGLT2-dependent glucose influx suggests that the major part of glucose uptake in alpha-cells is due to GLUT1 glucose transporters, and hence that SGLT2 inhibition primarily stimulates glucagon secretion via electrophysiological mechanisms, and not simply because of a reduction of glucose uptake.

An insulin signaling feedback loop regulates pancreas progenitor cell differentiation during islet development and regeneration

As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (β) cells. However, little is known about how insulin signaling feedback might influence neogenesis of β cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential β cell genes insulin and pdx1, and promoted β cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted β cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for β cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into β cells. Furthermore, the extent of this differentiation was dependent on the function of the β cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of β cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in β cell progenitors will reveal therapeutic targets for both in vivo and in vitro β cell generation.

Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

Aims/HypothesisSirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear.MethodsThis study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas.ResultsWe found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas.Conclusions/InterpretationThis study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells.

BackgroundDiabetes mellitus is a common metabolic disorder characterized by dysfunction of insulin-secreting pancreatic beta-cells. MicroRNAs are important regulators of beta-cell activities. These non-coding RNAs have recently been discovered to exert their effects not only inside the cell producing them but, upon exosome-mediated transfer, also in other recipient cells. This novel communication mode remains unexplored in pancreatic beta-cells. In the present study, the microRNA content of exosomes released by beta-cells in physiological and physiopathological conditions was analyzed and the biological impact of their transfer to recipient cells investigated.ResultsExosomes were isolated from the culture media of MIN6B1 and INS-1 derived 832/13 beta-cell lines and from mice, rat or human islets. Global profiling revealed that the microRNAs released in MIN6B1 exosomes do not simply reflect the content of the cells of origin. Indeed, while a subset of microRNAs was preferentially released in exosomes others were selectively retained in the cells. Moreover, exposure of MIN6B1 cells to inflammatory cytokines changed the release of several microRNAs. The dynamics of microRNA secretion and their potential transfer to recipient cells were next investigated. As a proof-of-concept, we demonstrate that if cel-miR-238, a C. Elegans microRNA not present in mammalian cells, is expressed in MIN6B1 cells a fraction of it is released in exosomes and is transferred to recipient beta-cells. Furthermore, incubation of untreated MIN6B1 or mice islet cells in the presence of microRNA-containing exosomes isolated from the culture media of cytokine-treated MIN6B1 cells triggers apoptosis of recipient cells. In contrast, exosomes originating from cells not exposed to cytokines have no impact on cell survival. Apoptosis induced by exosomes produced by cytokine-treated cells was prevented by down-regulation of the microRNA-mediating silencing protein Ago2 in recipient cells, suggesting that the effect is mediated by the non-coding RNAs.ConclusionsTaken together, our results suggest that beta-cells secrete microRNAs that can be transferred to neighboring beta-cells. Exposure of donor cells to pathophysiological conditions commonly associated with diabetes modifies the release of microRNAs and affects survival of recipient beta-cells. Our results support the concept that exosomal microRNAs transfer constitutes a novel cell-to-cell communication mechanism regulating the activity of pancreatic beta-cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0097-7) contains supplementary material, which is available to authorized users.

Epigenetic-mediated reprogramming of pancreatic endocrine cells.

Type 1 diabetes (T1D) results from cell-mediated autoimmune destruction of insulin-secreting pancreatic beta cells (β-cells). In the context of T1D, the scarcity of organ donors has driven research to alternate sources of functionally competent, insulin-secreting β-cells as substitute for donor islets to meet the clinical need for transplantation therapy. Experimental evidence of an inherent plasticity of pancreatic cells has fuelled interest in in vivo regeneration of β-cells. Transcriptional modulation and direct reprogramming of noninsulin secreting pancreatic α-cells to functionally mimic insulin-secreting β-cells is one of the promising avenues to the treatment of diabetes. Recent studies now show that adult progenitor and glucagon(+) α-cells can be converted into β-like cells in vivo, as a result of specific activation of the Pax4 gene in α-cells and curing diabetes in preclinical models. The challenge now is to understand the precise developmental transitions mediated by endocrine transcription factors and co-regulatory determinants responsible for pancreatic function and repair. Epigenetic-mediated regulation of transcription factor binding in pancreatic α-cells by specific drugs to direct reprogramming into functional insulin producing cells could be of potential innovative therapy for the treatment of T1D.

IL-21 production by CD4+ effector T cells and frequency of circulating follicular helper T cells are increased in type 1 diabetes patients.

Aims/hypothesisType 1 diabetes results from the autoimmune destruction of insulin-secreting pancreatic beta cells by T cells. Despite the established role of T cells in the pathogenesis of the disease, to date, with the exception of the identification of islet-specific T effector (Teff) cells, studies have mostly failed to identify reproducible alterations in the frequency or function of T cell subsets in peripheral blood from patients with type 1 diabetes.MethodsWe assessed the production of the proinflammatory cytokines IL-21, IFN-γ and IL-17 in peripheral blood mononuclear cells from 69 patients with type 1 diabetes and 61 healthy donors. In an additional cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. IL-21 and IL-17 production was also measured in peripheral blood mononuclear cells (PBMCs) from a subset of 46 of the 62 donors immunophenotyped for Tfh.ResultsWe found a 21.9% (95% CI 5.8, 40.2; p = 3.9 × 10−3) higher frequency of IL-21+ CD45RA− memory CD4+ Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). Consistent with this finding, we found a 14.9% increase in circulating Tfh cells in the patients (95% CI 2.9, 26.9; p = 0.016).Conclusions/interpretationThese results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-015-3509-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

The Role of NO-cGMP Signaling Pathway in Pancreatic Beta-cell Function

Insulin-secretion in pancreatic beta-cells is modulated by several second messengers and complex signaling pathways. Nitric oxide has been usually associated with beta-cell apoptosis, as part of the physiopathology of Type-2 diabetes. However there is increasing evidence that NO/cGMP signaling pathway is also involved in the secretory function through the activation of some target proteins such as the modulation of glucokinase activity, ionic channels, protein kinases, phosphodiesterases; and gene expression through the activation of transcription factors. These observations could be useful to establish new therapeutic strategies to maintain beta-cell optimal response before the development of islet dysfunction that leads to diabetes. Keywords: Ca2+ channels, cGMP, insulin secretion, insulin transcription, Nitric oxide, pancreatic beta-cell, PKG, soluble Guanylate Cyclase.

Cu(II) inhibits hIAPP fibrillation and promotes hIAPP-induced beta cell apoptosis through induction of ROS-mediated mitochondrial dysfunction

Human islet amyloid polypeptide (hIAPP), the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM), plays a central role in the loss of insulin-secreting pancreatic beta cells. Misfolded hIAPP fibrillating in islet beta cells may be one of the causations for T2DM. Studies have showed that fibrosis of hIAPP was inhibited by copper compounds while hIAPP-induced cytotoxicity was greatly stimulated. In this study, the suppression effects of three different forms of copper compounds CuCl2, CuSO4 and Cu(Gly)2 on amyloid fibril formation were examined in vitro. The results demonstrated that Cu(II) could interact with hIAPP to suppress the fibrosis without involvement of the anions. The fibrosis of hIAPP was inhibited by CuCl2, CuSO4 and Cu(Gly)2 with a similar degree. The particle size of hIAPP aggregates was decreased, which was further confirmed in atomic force microscopy (AFM) and transmission electron microscopy (TEM) images. Moreover, approximative cytotoxicity-enhancing levels between CuCl2, CuSO4 and Cu(Gly)2 on hIAPP were also observed in INS-1 cells. Studies on the action mechanisms displayed that copper compounds increased hIAPP-induced cytotoxicity by facilitating apoptosis-promoting effect of hIAPP, which was dominated mainly by cation. Furthermore, Cu(II)-promoted ROS overproduction and mitochondrial disruption might be the main reason for the enhanced apoptosis. Taken together, our studies demonstrate clear interaction mechanisms of Cu(II) and hIAPP in pancreatic beta cells, and provide useful information for our understanding and treatment of T2DM.

CFTR and Anoctamin 1 (ANO1) contribute to cAMP amplified exocytosis and insulin secretion in human and murine pancreatic beta-cells.

BackgroundMutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to the disease cystic fibrosis (CF). Although patients with CF often have disturbances in glucose metabolism including impaired insulin release, no previous studies have tested the hypothesis that CFTR has a biological function in pancreatic beta-cells.MethodsExperiments were performed on islets and single beta-cells from human donors and NMRI-mice. Detection of CFTR was investigated using PCR and confocal microscopy. Effects on insulin secretion were measured with radioimmunoassay (RIA). The patch-clamp technique was used to measure ion channel currents and calcium-dependent exocytosis (as changes in membrane capacitance) on single cells with high temporal resolution. Analysis of ultrastructure was done on transmission electron microscopy (TEM) images.ResultsWe detected the presence of CFTR and measured a small CFTR conductance in both human and mouse beta-cells. The augmentation of insulin secretion at 16.7 mM glucose by activation of CFTR by cAMP (forskolin (FSK) or GLP-1) was significantly inhibited when CFTR antagonists (GlyH-101 and/or CFTRinh-172) were added. Likewise, capacitance measurements demonstrated reduced cAMP-dependent exocytosis upon CFTR-inhibition, concomitant with a decreased number of docked insulin granules. Finally, our studies demonstrate that CFTR act upstream of the chloride channel Anoctamin 1 (ANO1; TMEM16A) in the regulation of cAMP- and glucose-stimulated insulin secretion.ConclusionOur work demonstrates a novel function for CFTR as a regulator of pancreatic beta-cell insulin secretion and exocytosis, and put forward a role for CFTR as regulator of ANO1 and downstream priming of insulin granules prior to fusion and release of insulin. The pronounced regulatory effect of CFTR on insulin secretion is consistent with impaired insulin secretion in patients with CF.

Unraveling the contribution of pancreatic beta-cell suicide in autoimmune type 1 diabetes.

In type 1 diabetes, an autoimmune disease mediated by autoreactive T-cells that attack insulin-secreting pancreatic beta-cells, it has been suggested that disease progression may additionally require protective mechanisms in the target tissue to impede such auto-destructive mechanisms. We hypothesize that the autoimmune attack against beta-cells causes endoplasmic reticulum stress by forcing the remaining beta-cells to synthesize and secrete defective insulin. To rescue beta-cell from the endoplasmic reticulum stress, beta-cells activate the unfolded protein response to restore protein homeostasis and normal insulin synthesis. Here we investigate the compensatory role of unfolded protein response by developing a multi-state model of type 1 diabetes that takes into account beta-cell destruction caused by pathogenic autoreactive T-cells and apoptosis triggered by endoplasmic reticulum stress. We discuss the mechanism of unfolded protein response activation and how it counters beta-cell extinction caused by an autoimmune attack and/or irreversible damage by endoplasmic reticulum stress. Our results reveal important insights about the balance between beta-cell destruction by autoimmune attack (beta-cell homicide) and beta-cell apoptosis by endoplasmic reticulum stress (beta-cell suicide). It also provides an explanation as to why the unfolded protein response may not be a successful therapeutic target to treat type 1 diabetes.

Quantitative-proteomic comparison of alpha and Beta cells to uncover novel targets for lineage reprogramming.

Type-1 diabetes (T1D) is an autoimmune disease in which insulin-secreting pancreatic beta cells are destroyed by the immune system. An emerging strategy to regenerate beta-cell mass is through transdifferentiation of pancreatic alpha cells to beta cells. We previously reported two small molecules, BRD7389 and GW8510, that induce insulin expression in a mouse alpha cell line and provide a glimpse into potential intermediate cell states in beta-cell reprogramming from alpha cells. These small-molecule studies suggested that inhibition of kinases in particular may induce the expression of several beta-cell markers in alpha cells. To identify potential lineage reprogramming protein targets, we compared the transcriptome, proteome, and phosphoproteome of alpha cells, beta cells, and compound-treated alpha cells. Our phosphoproteomic analysis indicated that two kinases, BRSK1 and CAMKK2, exhibit decreased phosphorylation in beta cells compared to alpha cells, and in compound-treated alpha cells compared to DMSO-treated alpha cells. Knock-down of these kinases in alpha cells resulted in expression of key beta-cell markers. These results provide evidence that perturbation of the kinome may be important for lineage reprogramming of alpha cells to beta cells.

Episodic hormone secretion: a comparison of the basis of pulsatile secretion of insulin and GnRH

Rhythms govern many endocrine functions. Examples of such rhythmic systems include the insulin-secreting pancreatic beta-cell, which regulates blood glucose, and the gonadotropin-releasing hormone (GnRH) neuron, which governs reproductive function. Although serving very different functions within the body, these cell types share many important features. Both GnRH neurons and beta-cells, for instance, are hypothesized to generate at least two rhythms endogenously: (1) a burst firing electrical rhythm and (2) a slower rhythm involving metabolic or other intracellular processes. This review discusses the importance of hormone rhythms to both physiology and disease and compares and contrasts the rhythms generated by each system.

Insulin Oversecretion in MSG-Obese Rats is Related to Alterations in Cholinergic Muscarinic Receptor Subtypes in Pancreatic Islets

Background/ Aims: Impaired pancreatic beta cell function and insulin secretion/action are a link between obesity and type 2 diabetes, which are worldwide public health burdens. We aimed to characterize the muscarinic acetylcholine receptor (mAChR) M₁-M₄ subtypes in isolated pancreatic islets from pre-diabetic obese rats that had been treated neonatally with monosodium L-glutamate (MSG). Methods: At 90 days of age, both the MSG and the control groups underwent biometric and biochemical evaluation. Anti-muscarinic drugs were used to study mAChR function either in vivo or in vitro. Results: The results demonstrated that atropine treatment reduced insulin secretion in the MSG-treated and control groups, whereas treatment with an M₂mAChR-selective antagonist increased secretion. Moreover, the insulinostatic effect of an M₃mAChR-selective antagonist was significantly higher in the MSG-treated group. M₁mAChR and M₃mAChR expression was increased in the MSG-obese group by 55% and 73%, respectively. In contrast, M₂mAChR expression decreased by 25% in the MSG group, whereas M₄mAChR expression was unchanged. Conclusions: Functional changes in and altered content of the mAChR (M₁-M₄) subtypes are pivotal to the demand for high pancreatic beta cell insulin secretion in MSG-obese rats, which is directly associated with vagal hyperactivity and peripheral insulin resistance.

Initial Cell Seeding Density Influences Pancreatic Endocrine Development During in vitro Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 104 cells/cm2 to 5.3 x 104 cells/cm2 followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1, PTF1a, NGN3, ARX, and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression, followed by pancreatic endocrine specification marker expression (BRN4, PAX4, ARX, NEUROD1, NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols.

Reporter islets in the eye reveal the plasticity of the endocrine pancreas

The islets of Langerhans constitute the endocrine part of the pancreas and are responsible for maintenance of blood glucose homeostasis. They are deeply embedded in the exocrine pancreas, limiting their accessibility for functional studies. Understanding regulation of function and survival and assessing the clinical outcomes of individual treatment strategies for diabetes requires a monitoring system that continuously reports on the endocrine pancreas. We describe the application of a natural body window that successfully reports on the properties of in situ pancreatic islets. As proof of principle, we transplanted "reporter islets" into the anterior chamber of the eye of leptin-deficient mice. These islets displayed obesity-induced growth and vascularization patterns that were reversed by leptin treatment. Hence, reporter islets serve as optically accessible indicators of islet function in the pancreas, and also reflect the efficacy of specific treatment regimens aimed at regulating islet plasticity in vivo.

Mechanisms of amino acid-stimulated insulin secretion in congenital hyperinsulinism

The role of amino acids in the regulation of insulin secretion in pancreatic beta-cells is highlighted in three forms of congenital hyperinsulinism (HI), namely gain-of-function mutations of glutamate dehydrogenase (GDH), loss-of-function mutations of ATP-dependent potassium channels, and a deficiency of short-chain 3-hydroxyacyl-CoA dehydrogenase. Studies on disease mouse models of HI suggest that amino acid oxidation and signaling effects are the major mechanisms of amino acid-stimulated insulin secretion. Amino acid oxidation via GDH produces ATP and triggers insulin secretion. The signaling effect of amino acids amplifies insulin release after beta-cell depolarization and elevation of cytosolic calcium.

Rapid and Simultaneous Analysis of Oral Antidiabetic Drug by Ultra-high-speed HPLC

Diabetes is a major cause of chronic kidney disease, and oral antidiabetic drugs are the mainstay of therapy for most patients with Type 2 diabetes. Type 2 diabetes is typically associated with insulin resistance and the dysfunction of insulin-secreting pancreatic beta-cells. Addressing these defects often requires therapy with a combination of differently acting antidiabetic agents. A potential novel combination in development brings together a DPP-4 inhibitor with the pioglitazone into a fixed-dose single-tablet combination. The former component acts mainly to increase prandial insulin secretion; the latter improves insulin sensitivity. Ultra-high-speed HPLC has become increasingly popular in analytical research fields. This novel analytical system provides fast and efficient chromatographic separation over a wide range of flow rate and pressure. In this study, we investigated the analysis of active ingredients in combination drugs using an ultra-high-speed HPLC system equipped with a photo-diode array detector. The HPLC system utilized in this study was a LaChrom Ultra system (Hitachi, Tokyo, Japan), equipped with a L-2455U diode array detector. A LaChrom Ultra C18 (particle size; 2 μm) was used as the analytical column. Rapid and simultaneous analysis of active ingredients in combination drugs was achieved.

Secreted Molecules from the Helminth Parasite Fasciola Hepatica Prevent Pro-Inflammatory Immune Responses to Prevent Autoimmune Diabetes

Introduction:Parasitic worms (helminths) have evolved mechanisms to regulate host protective immune responses, to ensure their own survival, and minimise immune-mediated tissue pathology for the host. Helminths achieve this by inducing potent anti-inflammatory (Th2) and regulatory immune responses, while simultaneously inhibiting the development of pro-inflammatory Th1 and Th17 immune responses. Understanding how helminths modulate the immune response will provide novel mechanisms to induce a regulatory immune environment, with significant clinical applications for the treatment of autoimmune/inflammatory diseases, and in transplantation. Methods:To investigate the therapeutic potential of helminths, we have isolated the excretory/secretory (ES) products of the liver fluke Fasciola hepatica, and have delivered them intraperitoneally to female nonobese diabetic (NOD) mice at 4 weeks of age (co-incident with the initiation of autoimmunity directed against the insulin-secreting pancreatic beta cells). Development of Type 1 diabetes (T1D) and the effects of ES-treatment on the developing (auto)immune response were both assessed. Blood glucose was monitored and mice were deemed diabetic when levels exceeded 14mmol/L on two consecutive occasions. Flow cytometry was used to phenotype immune cell populations within the pancreas, pancreatic lymph nodes and peritoneum after treatment with ES. The activation status of macrophages was determined using genetic markers for M1 and M2 phenotypes by RT-PCR. Cytokines secreted by T cells and the isotype of serum autoantibodies were determined by ELISA. Results: Treatment of NOD mice with parasite ES prevented the development of autoimmune diabetes in 80-86% of animals. By comparison, only 10-15% of NOD mice treated with PBS (vehicle control) remained normoglycaemic. Protection from autoimmune disease in ES-treated NOD mice was associated with the suppression of the normal secretion of IFN-γ from autoreactive T cells in response to stimulation with autoantigen, and a corresponding switch towards the production of a regulatory isotype (IgG1) of autoantibody. In addition, treatment with ES resulted in the expansion of a population of regulatory (IL-10 secreting) B cells within the pancreatic lymph nodes and the activation of regulatory (M2 phenotype) macrophages. Importantly, NOD mice treated with ES were capable of mounting an effective immune response to a third party antigen, suggesting that ES treatment did not cause immune suppression per se. Conclusion: Soluble products secreted by Fasciola hepatica prevent the development of antigen-specific inflammatory responses, thereby preventing the development of autoimmunity in a murine model of Type 1 diabetes. Our data indicates that protection is achieved via the induction of a regulatory immune environment in the pancreas (the site of beta cell destruction) and pancreatic lymph nodes (the site of T cell priming) at a time point co-incident with T cell priming events. Fasciola hepatica ES modulates the function of B cells and macrophages, which, via IL-10 secretion, suppress the polarisation of autoreactive T cells. These results suggest that ES contains novel immune-therapeutic agents which hold great potential for the treatment of autoimmune disease and transplant rejection, where a regulatory immune environment is required.

The pancreatic islet as a signaling hub

Over the last two decades we have focused on beta cell signal transduction, bringing many new insights, especially in the context of insulin signal transduction, the role of inositol polyphosphates and the regulation of cytoplasmic free Ca(2+) concentration. However, there has been a growing awareness that the beta cell, which is mandatory for insulin secretion, has a unique context within the micro-organ of the pancreatic Islet of Langerhans. In this environment the beta cell both mediates and receives paracrine regulation, critical for the control of blood glucose homeostasis. Failure of an appropriate beta cell function leads to the development of diabetes mellitus. In our quest to understand the molecular events maintaining beta cell function we have faced two key challenges. Firstly, whilst there are many similarities between signal transduction in pancreatic islets between the much used rodent models and humans there are some notable differences. Critical distinctions between rodent and primate can be made in the structure of the islet, including the arrangement of the islet cells, the innervation pattern and the microcirculation. This means that important signaling interactions between islets cells, mediated through for example insulin, glucagon, GABA, glutamate and ATP, will have a unique human framework. The second challenge was to be able to take the discoveries we have made using in vitro systems and examine them in an in vivo context. Advances in in vivo imaging achieved by utilizing the anterior chamber of the eye as a transplantation site for pancreatic islets make it possible for non-invasive, longitudinal studies at single cell resolution in real time of islet cell physiology and pathology. Thus it is becoming possible to study the insulin secreting pancreatic beta cell within the framework of the unique micro-organ, the Islet of Langerhans, for the first time in a physiological context, i.e. when being innervated and connected to the blood supply.