Bmi-1 regulates the Ink4a/Arf locus to control pancreatic β-cell proliferation

The molecular mechanisms that regulate the age-induced increase of p16^INK4a expression associated with decreased β-cell proliferation and regeneration are not well understood. We report that in aged islets, derepression of the Ink4a/Arf locus is associated with decreased Bmi-1 binding, loss of H2A ubiquitylation, increased MLL1 recruitment, and a concomitant increase in H3K4 trimethylation. During β-cell regeneration these histone modifications are reversed resulting in reduced p16^INK4a expression and increased proliferation. We suggest that PcG and TrxG proteins impart a combinatorial code of histone modifications on the Ink4a/Arf locus to control β-cell proliferation during aging and regeneration.     

The pancreatic β-cell in ageing: Implications in age-related diabetes

The prevalence of type 2 diabetes (T2D) and impaired glucose tolerance (IGT) increases with ageing. T2D generally results from progressive impairment of the pancreatic islets to adapt β-cell mass and function in the setting of insulin resistance and increased insulin demand. Several studies have shown an age-related decline in peripheral insulin sensitivity. However, a precise understanding of the pancreatic β-cell response in ageing is still lacking. In this review, we summarize the age-related alterations, adaptations and/or failures of β-cells at the molecular, morphological and functional levels in mouse and human. Age-associated alterations include processes such as β-cell proliferation, apoptosis and cell identity that can influence β-cell mass. Age-related changes also affect β-cell function at distinct steps including electrical activity, Ca²⁺ signaling and insulin secretion, among others. We will consider the potential impact of these alterations and those mediated by senescence pathways on β-cells and their implications in age-related T2D. Finally, given the great diversity of results in the field of β-cell ageing, we will discuss the sources of this heterogeneity. A better understanding of β-cell biology during ageing, particularly at older ages, will improve our insight into the contribution of β-cells to age-associated T2D and may boost new therapeutic strategies.     

Dynamin-related protein 1 is implicated in endoplasmic reticulum stress-induced pancreatic β-cell apoptosis

Pancreatic β-cell dysfunction is a critical component in the pathogenesis of diabetes. Endoplasmic reticulum (ER) stress is one of the factors that induces pancreatic β-cell dysfunction, but the underlying mechanisms have not been well elucidated. In this study, we report that a mitochondrial fission modulator, dynamin-related protein 1 (DRP-1), plays an important role in ER stress-induced β-cell apoptosis. Induction of DRP-1 expression significantly promoted ER stress-induced apoptosis in the DRP-1 WT (DRP-1 wild- type) inducible β-cell line, but not in the DRP-1 K38A (a dominant negative mutant of DRP-1) inducible β-cell line. We further demonstrated that the mitochondrial membrane potential decreased, and that cytochrome c release, caspase-3 activation and generation of reactive oxygen species (ROS) were enhanced by induction of DRP-1 WT, but prevented by DRP-1 K38A in pancreatic β-cells under ER stress conditions. These results indicate that DRP-1 mediates ER stress-induced pancreatic β-cell apoptosis.     

Non-invasive in vivo imaging of pancreatic β-cell function and survival - a perspective

A major problem in medical research is to translate in vitro observations into the living organism. In this perspective, we discuss ongoing efforts to non-invasively image pancreatic islets/β-cells by techniques, such as magnetic resonance imaging and positron emission tomography, and present an experimental platform, which allows in vivo imaging of pancreatic β-cell mass and function longitudinally and at the single-cell level. Following transplantation of pancreatic islets into the anterior chamber of the eye of mice and rats, these islets are studied by functional microscopic imaging. This imaging platform can be utilized to address fundamental aspects of pancreatic islet cell biology in vivo in health and disease. These include the dynamics of pancreatic islet vascularization, islet cell innervation, signal-transduction, change in functional β-cell mass and immune responses. Moreover, we discuss the feasibility of studying human islet cell physiology and pathology in vivo as well as the potential of using the anterior chamber of the eye as a site for therapeutic transplantation in type 1 diabetes mellitus.     

Altered autonomic inputs as a cause of pancreatic β-cell amyloid

A partial loss of β-cell mass and β-cell dysfunction in Type 2 Diabetes Mellitus (T2DM) is associated with amyloid deposition but whether it is causal or consequential is debated. Although the in vitro polymerization of amylin has been studied in detail, the exact trigger for the mechanism in vivo has not been identified. One suggestion is that an increased load on β-cells results in inefficient handling of proteins leading to misfolding and aggregation, but this hypothesis is faced with certain paradoxes. We suggest an alternative mechanism based on the assumption that polymerization is a spontaneous process. The concentration of the polypeptide in β-cell granules is shown to be sufficient to allow polymerization. However if the rate of turnover in normal cells is greater than the rate of polymerization, amyloid deposition will not be observed. If this is true, it follows that amyloid deposition could be a result of increased retention time of amylin in the β-cell granules. In T2D, the sympathetic inputs are known to increase which could result in suppression of the secretion process. The increase in the retention time due to this suppression can allow polymerization. In addition to this in a prediabetic state parasympathetic stimulation increases β-cell proliferation. This reduces the insulin demand per cell thereby increasing the mean retention time. Thus a combination of contrasting actions of sympathetic and parasympathetic systems could lead to increase in the amyloid deposition. We suggest testable predictions of the alternative hypotheses and the lines of research needed to test them.     

Association of variants in genes involved in pancreatic β-cell development and function with type 2 diabetes in North Indians

Variants in genes involved in pancreatic β-cell development and function are known to cause monogenic forms of type 2 diabetes and are also associated with complex form. In this study, we studied the genetic association of polymorphisms in such important genes with type 2 diabetes in the high-risk Indians. We genotyped 91 polymorphisms in 19 genes (ABCC8, HNF1A, HNF1B, HNF4A, INS, INSM1, ISL1, KCNJ11, MAFA, MNX1, NEUROD1, NEUROG3, NKX2.2, NKX6.1, PAX4, PAX6, PDX1, USF1 and WFS1) in 2025 unrelated North Indians of Indo-European ethnicity comprising of 1019 diabetic and 1006 non-diabetic subjects. HNF4A promoter P2 polymorphisms rs1884613 and rs2144908, which are in high linkage disequilibrium, showed significant association with type 2 diabetes (odds ratio (OR)=1.37 (95% confidence interval (CI) 1.19-1.57), P=9.4 × 10(-6) for rs1884613 and OR=1.37 (95%CI 1.20-1.57), P=6.0 × 10(-6) for rs2144908), as previously shown in other populations. We observed body mass index-dependent association of these variants with type 2 diabetes in normal-weight/lean subjects. Variants in USF1, ABCC8, ISL1 and KCNJ11 showed nominal association, while haplotypes in these genes were significantly associated. rs3812704 upstream of NEUROG3 significantly increased risk for type 2 diabetes in normal-weight/lean subjects (OR=1.68 (95%CI 1.25-2.24), P=4.9 × 10(-4)). Thus, pancreatic β-cell development and function genes contribute to susceptibility to type 2 diabetes in North Indians.     

Dihydroartemisinin inhibits cell proliferation via AKT/GSK3β/cyclinD1 pathway and induces apoptosis in A549 lung cancer cells

Lung cancer is the most common cause of cancer-related death in the world. The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC); non small cell lung carcinoma (NSCLC) includes squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma, Non small cell lung carcinoma accounts for about 80% of the total lung cancer cases. Dihydroartemisinin (DHA) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effects of DHA on cell growth and proliferation in lung cancer cells remain to be elucidated. Here, we demonstrate that DHA inhibited cell proliferation in the A549 lung cancer cell line through suppression of the AKT/Gsk-3β/cyclin D1 signaling pathway. DHA significantly inhibited cell proliferation of A549 cells in a concentration and time dependent manner as determined by MTS assay. Flow cytometry analysis demonstrated that DHA treatment of A549 cells resulted in cell cycle arrest at the G1 phase, which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1. These results suggest that DHA is a potential natural product for the treatment of lung cancer.     

A novel pancreatic β-cell targeting bispecific-antibody (BsAb) can prevent the development of Type 1 diabetes in NOD mice

To prepare a novel Bispecific Antibody (BsAb) as a potential targeted therapy for T1D, we produced a “functionally inert” monoclonal antibody (mAb) against Glucose transporter-2 (GLUT-2) expressed on β-cells to serve as an anchoring antibody. The therapeutic arm is an agonistic mAb against Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), a negative regulator of T-cell activation expressed on activated CD4 + T-cells. A BsAb was prepared by chemically coupling an anti-GLUT2 mAb to an agonistic anti-CTLA-4 mAb. This BsAb was able to bind to GLUT2 and CTLA-4 in vitro, and to pancreatic islets, both in vitro and in vivo. We tested the safety and efficacy of this BsAb by treating Non-Obese Diabetes (NOD) mice and found that it could delay the onset of diabetes with no apparent undesirable side effects. Thus, engagement of CTLA-4 on activated T cells from target tissue can be an effective way to treat type-1 diabetes.     

Role of stromal cell-derived factor 1α pathway in bone metastatic prostate cancer

Metastatic prostate cancer is one of the leading causes of cancer-related death in men. The primary site of metastasis from prostate cancers is the bone. During the last decade, multiple studies have pointed to the role of the stromal cell-derived factor 1 alpha (SDF1α)/CXCR4 axis in the metastatic spread of the disease, but the mechanisms that underlie this effect are still incompletely understood. In this review, we summarize the current understanding of the role of the SDF1α/CXCR4 pathway in bone metastatic prostate cancer. We also discuss the therapeutic potential of disrupting the interaction between prostate tumor cells and bone environment with focus on the SDF1α pathway.     

Amino acid taste receptor regulates insulin secretion in pancreatic β-cell line MIN6 cells

Amino acids such as L-glutamate and L-arginine are potent stimuli for insulin secretion from pancreatic β-cells. However, the precise molecular mechanisms of amino acid-induced insulin secretion have only partly understood. In this study, we show here that mouse pancreatic β-cell line MIN6 cells expressed amino acid taste receptor (heterodimer of type 1 taste G protein-coupled receptor member 1 and member 3; Tas1R1 and Tas1R3, respectively). We found that administration of L-glutamate or L-arginine to MIN6 cells caused the increase in free intracellular concentrations of both inositol-1,4,5-triphosphate (IP(3)) and Ca(2+) , and umami substance inocinate enhanced the effects of l-glutamate. Effects of amino acids on intracellular IP(3) and Ca(2+) concentration were diminished by application of lactisole, a Tas1R3 receptor antagonist. Furthermore, we investigated the effect of amino acids on the insulin release from MIN6 cells by both ELISA and total internal reflection fluorescence microscopy. Application of L-glutamate or L-arginine significantly increased the release of insulin, whereas inhibited by the application of lactisole. Based on these findings, we propose that heterodimer of Tas1R1 and Tas1R3 is the fundamental receptor for the sensing amino acids and regulates the amino acid-induced insulin secretion in pancreatic β-cells.     

Polycomb protein Ezh2 regulates pancreatic β-cell Ink4a/Arf expression and regeneration in diabetes mellitus

Proliferation of pancreatic islet β cells is an important mechanism for self-renewal and for adaptive islet expansion. Increased expression of the Ink4a/Arf locus, which encodes the cyclin-dependent kinase inhibitor p16^INK4a and tumor suppressor p19^Arf, limits β-cell regeneration in aging mice, but the basis of β-cell Ink4a/Arf regulation is poorly understood. Here we show that Enhancer of zeste homolog 2 (Ezh2), a histone methyltransferase and component of a Polycomb group (PcG) protein complex, represses Ink4a/Arf in islet β cells. Ezh2 levels decline in aging islet β cells, and this attrition coincides with reduced histone H3 trimethylation at Ink4a/Arf, and increased levels of p16^INK4a and p19^Arf. Conditional deletion of β-cell Ezh2 in juvenile mice also reduced H3 trimethylation at the Ink4a/Arf locus, leading to precocious increases of p16^INK4a and p19^Arf. These mutant mice had reduced β-cell proliferation and mass, hypoinsulinemia, and mild diabetes, phenotypes rescued by germline deletion of Ink4a/Arf. β-Cell destruction with streptozotocin in controls led to increased Ezh2 expression that accompanied adaptive β-cell proliferation and re-establishment of β-cell mass; in contrast, mutant mice treated similarly failed to regenerate β cells, resulting in lethal diabetes. Our discovery of Ezh2-dependent β-cell proliferation revealed unique epigenetic mechanisms underlying normal β-cell expansion and β-cell regenerative failure in diabetes pathogenesis.     

Modification of K-ATP channels in pancreatic β-cells by trypsin

The inside-out configuration of the patchclamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic -cells. Trypsin (20 g/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg²⁺ was observed: however, the protease did reduce the inhibitory effect of Mg²⁺ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 M. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.     

Pyruvate kinase,muscle isoform 2 promotes proliferation and insulin secretion of pancreatic β-cells via activating Wnt/CTNNB1 signaling

Failure of pancreatic β-cells is closely associated with type 2 diabetes mellitus (T2DM), an intractable disease affecting numerous patients. Pyruvate kinase, muscle isoform 2 (PKM2) is a potential modulator of insulin secretion in β-cells. This study aims at revealing roles and possible mechanisms of PKM2 in pancreatic β-cells. Mouse pancreatic β-cell line NIT-1 was used for high glucose treatment and PKM2 overexpression by its specific expression vector. Cell proliferation by Thiazolyl blue assay, cell apoptosis by annexin V-fluorescein isothiocyanate/prodium iodide staining and insulin secretion assay by ELISA were performed in each group. The mRNA and protein levels of related factors were analyzed by real-time quantitative PCR and western blot. Results showed that Pkm2 was inhibited under high glucose conditions compared to the untreated cells (P < 0.01). Its overexpression significantly suppressed NIT-1 cell apoptosis (P < 0.01), and induced cell proliferation (P < 0.05) and insulin secretion (P < 0.05). Related factors showed consistent mRNA expression changes. Protein levels of β-catenin (CTNNB1), insulin receptor substrate 1 (IRS1) and IRS2 were all promoted by PKM2 overexpression (P < 0.01), indicating the activated Wnt/CTNNB1 signaling. These results indicated the inductive roles of PKM2 in pancreatic β-cell NIT-1, including promoting cell proliferation and insulin secretion, and inhibiting cell apoptosis, which might be achieved via activating the Wnt/CTNNB1 signaling and downstream factors. This study offers basic information on the role and mechanism of PKM2 in pancreatic β-cells, and lays the foundation for using PKM2 as a potential therapeutic target in T2DM.     

ClC-3 chloride channel modulates the proliferation and migration of osteosarcoma cells via AKT/GSK3β signaling pathway

In cultured human osteosarcoma (OS) cells, we recently demonstrated that insulin-like growth factors (IGF-1)-induced MG-63 and 143B human OS cells proliferation were consistent with increasing ClC-3 expression, and ClC-3 was up-regulated in cells with high metastatic potency. Blockade of ClC-3 greatly suppressed the phosphorylation activation of Akt/GSK3β. We also found that blockade of ClC-3 effectively down-regulated the expression of cyclin D1 and cyclin E, and caused activation of p27^KIP and p21^CIP. The synthesized effects on these proteins which play a major role in cell cycle regulation bring about G0/G1 cell cycle arrest in MG-63 cells, and finally abrogate the cell proliferation. Besides, ClC-3 deletion attenuates OS cell migration via down-regulation the expression of MMP-2 and MMP-9. Such information suggests that ClC-3 might be a potential target for anti-OS.     

Soluble β-glucan from Grifola frondosa induces proliferation and Dectin-1/Syk signaling in resident macrophages via the GM-CSF autocrine pathway

MD-Fraction, a highly purified, soluble β-(1,3) (1,6)-glucan obtained from Grifola frondosa (an oriental edible mushroom), has been reported to inhibit tumor growth by modulating host immunity. β-Glucan, a major component of the fungal cell wall, is generally recognized by PRRs expressed on macrophages and DCs, such as Dectin-1, and the ability of β-glucans to modulate host immunity is influenced by their structure and purity. Most cellular studies have used particulate β-glucans, such as yeast zymosan (crude β-glucan) and curdlan (purified β-glucan). However, little is known about the cellular mechanism of soluble β-glucans, including MD-Fraction, despite significant therapeutic implications. In this study, we investigated the cellular mechanism of MD-Fraction in murine resident macrophages and compared it with two well-known β-glucan particles. MD-Fraction induced GM-CSF production rapidly through Dectin-1-independent ERK and p38 MAPK activation. Subsequently, MD-Fraction-induced GM-CSF enhanced proliferation and Dectin-1 expression, which permitted Dectin-1-mediated TNF-α induction through the Syk pathway. Curdlan induced not only the proliferation and activation of Dectin-1/Syk signaling in a manner similar to MD-Fraction but also the uncontrolled, proinflammatory cytokine response. Contrastingly, zymosan reduced proliferation and Dectin-1 expression significantly, indicating that the mechanism of macrophage activation by MD-Fraction differs from that of zymosan. This is the first study to demonstrate that purified β-glucans, such as MD-Fraction and curdlan, induce GM-CSF production directly, resulting in Dectin-1/Syk activation in resident macrophages. In conclusion, we demonstrated that MD-Fraction induces cell proliferation and cytokine production without excessive inflammation in resident macrophages, supporting its immunotherapeutic potential.