A novel dual-color reporter for identifying insulin-producing beta-cells and classifying heterogeneity of insulinoma cell lines

Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+)) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+) phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+) and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.     

Activation of peroxisome proliferator-activated receptor-gamma protects pancreatic beta-cells from cytokine-induced cytotoxicity via NF kappaB pathway

Diabetes mellitus is characterized by cytokine-induced insulitis and a deficit in beta-cell mass. Ligands for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) have been shown to have anti-inflammatory effects in various experimental models. We questioned whether activation of endogenous PPAR-gamma by either PPAR-gamma ligands or adenoviral-directed overexpression of PPAR-gamma (Ad-PPAR-gamma) could inhibit cytokine-induced beta-cell death in RINm5F (RIN) cells, a rat insulinoma cell line. Treatment of RIN cells with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) induced beta-cell damage through NF kappaB-dependent signaling pathways. Activation of PPAR-gamma by PPAR-gamma ligands or Ad-PPAR-gamma inhibited IL-1 beta and IFN-gamma-stimulated nuclear translocation of the p65 subunit and DNA binding activity. NF kappaB target gene expression and their product formation, namely inducible nitric oxide synthase and cyclooxygenase-2 were decreased by PPAR-gamma activation, as established by real-time PCR, Western blots and measurements of NO and PGE(2). The mechanism by which PPAR-gamma activation inhibited NF kappaB-dependent cell death signals appeared to involve the inhibition of I kappa B alpha degradation, evidenced by inhibition of cytokine-induced NF kappaB-dependent signaling events by Ad-I kappaB alpha (S32A, S36A), non-degradable I kappaB alpha mutant. I kappaB beta mutant, Ad-I kappaB beta (S19A, S23A) was not effective in preventing cytokine toxicity. Furthermore, a protective effect of PPAR-gamma ligands was proved by assaying for normal insulin secreting capacity in response to glucose in isolated rat pancreatic islets. The beta-cell protective function of PPAR-gamma ligands might serve to counteract cytokine-induced beta-cell destruction.     

Indirubin modulates CD4+ T‐cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an immune mediated decrease in platelet number. Disturbance of CD4⁺ T‐cell homeostasis with simultaneous decrease of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4⁺ effector T cells underpin the pathophysiology of ITP. Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. which is clinically used for the treatment of ITP patients. Whether indirubin targets the Tregs/effector T cell‐axis to restore platelet number is unknown. In our in vitro studies, Indirubin could significantly enhance the number and function of Tregs and meanwhile dampen the activation of effector T cells in a dose‐dependent manner. Indirubin was observed to restore the expression of programmed cell‐death 1 (PD1) and phosphatase and tensin homolog (PTEN) on the CD4⁺ T cells of ITP patients, leading to the subsequent attenuation of the AKT/mTOR pathway. Furthermore, these observations were recapitulated in an active murine model of ITP with a prominent platelet response. Thus, our results identified a potentially novel mechanism of the therapeutic action of indirubin in the treatment of ITP through regulating the homeostasis of CD4⁺ T cells in a PD1/PTEN/AKT signalling pathway.     

Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.     

Deciphering role of FGFR signalling pathway in pancreatic cancer

Recently, fibroblast growth factors are identified to play a vital role in the development and progression of human pancreatic cancer. FGF pathway is critical involved in numerous cellular processes through regulation of its downstream targets, including proliferation, apoptosis, migration, invasion, angiogenesis and metastasis. In this review article, we describe recent advances of FGFR signalling pathway in pancreatic carcinogenesis and progression. Moreover, we highlight the available chemical inhibitors of FGFR pathway for potential treatment of pancreatic cancer. Furthermore, we discuss whether targeting FGFR pathway is a novel therapeutic strategy for pancreatic cancer clinical management.     

A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium

Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP-binding proteins (GbpA- GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP-phosphodiesterase activity and a 10-fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA(-)B(-) cells is associated with increased myosin II phosphorylation compared with wild-type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process.     

Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus

Diabetes is caused by impaired insulin secretion in pancreatic beta-cells and peripheral insulin resistance. Overload of pancreatic beta-cells leads to beta-cell exhaustion and finally to the development of diabetes. Reduced beta-cell mass is evident in type 2 diabetes, and apoptosis is implicated in this process. One characteristic feature of beta-cells is highly developed endoplasmic reticulum (ER) due to a heavy engagement in insulin secretion. The ER serves several important functions, including post-translational modification, folding, and assembly of newly synthesized secretory proteins, and its proper function is essential to cell survival. Various conditions can interfere with ER function and these conditions are called ER stress. Recently, we found that nitric oxide (NO)-induced apoptosis in beta-cells is mediated by the ER-stress pathway. NO causes ER stress and leads to apoptosis through induction of ER stress-associated apoptosis factor CHOP. The Akita mouse with a missense mutation (Cys96Tyr) in the insulin 2 gene has hyperglycemia and a reduced beta-cell mass. This mutation disrupts a disulfide bond between A and B chains of insulin and may induce its conformational change. In the development of diabetes in Akita mice, mRNAs for an ER chaperone Bip and CHOP were induced in the pancreas. Overexpression of the mutant insulin in mouse MIN6 beta-cells induced CHOP expression and led to apoptosis. Targeted disruption of the CHOP gene did not delay the onset of diabetes in the homozygous Akita mice, but it protected islet cells from apoptosis and delayed the onset of diabetes in the heterozygous Akita mice. We conclude that ER overload in beta-cells causes ER stress and leads to apoptosis via CHOP induction. These results highlight the importance of chronic ER stress in beta-cell apoptosis in type 2 diabetes, and suggest a new target to the management of the disease.     

Cytotoxicity and enzymatic activity inhibition in cell lines treated with novel iminosugar derivatives

Iminosugars are monosaccharide analogues that have been demonstrated to be specific inhibitors for glycosidases and are currently used therapeutically in several human disorders. N-alkylated derivatives of d-fagomine and (2R,3S,4R,5S)-2-(hydroxymethyl)-5-methylpyrrolidine-3,4-diol with aliphatic chains were tested in eight human cancer cell lines to analyze their cytotoxicity and the inhibitory effect in the activities of specific glycosidases. Results indicate that these compounds were more cytotoxic as the length of the alkyl chain increases. N-dodecyl-d-fagomine inhibited specifically the α-d-glucosidase activity in cell lysates, whereas no effect was detected in other glycosidases. The N-dodecyl derivative of (2R,3S,4R,5S)-2-(Hydroxymethyl)-5-methylpyrrolidine-3,4-diol induced specific inhibition against α-l-fucosidase in cell lysates. Our results indicated that the length of the alkyl chain linked to the iminosugars determine their cytotoxicity as well as the inhibitory effect on the enzymatic activities of specific glycosidases, in human cancer cell lines.     

Daunorubicin induces cell death via activation of apoptotic signalling pathway and inactivation of survival pathway in muscle-derived stem cells

Daunorubicin (as well as other anthracyclines) is known to be toxic to heart cells and other cells in organism thus limiting its applicability in human cancer therapy. To investigate possible mechanisms of daunorubicin cytotoxicity, we used stem cell lines derived from adult rabbit skeletal muscle. Recently, we have shown that daunorubicin induces apoptotic cell death in our cell model system and distinctly influences the activity of MAP kinases. Here, we demonstrate that two widely accepted antagonistic signalling pathways namely proapoptotic JNK and prosurvival PI3K/AKT participate in apoptosis. Using the Western blot method, we observed the activation of JNK and phosphorylation of its direct target c-Jun along with inactivation of AKT and its direct target GSK in the course of programmed cell death. By means of small-molecule kinase inhibitors and transfection of cells with the genes of the components of these pathways, c-Jun and AKT, we confirm that JNK signalling pathway is proapoptotic, whereas AKT is antiapoptotic in daunorubicin-induced muscle cells. These findings could contribute to new approaches which will result in less toxicity and fewer side effects that are currently associated with the use of daunorubicin in cancer therapies.     

Tumor suppressor gene PTEN affects anoikis via both PKB and FAK in human lung carcinoma cell lines

PTEN was identified in 1997 as a new tumor suppressor gene on human chromosome 10q23, deletions and mutations of this gene were associated with a variety of human cancers such as glioblastoma and prostate cancer. In this study Northern blot analysis was carried out and one major band at 2 kb region was observed in all 8 HLCC samples, consistent with previous reports. The result showed that the PTEN gene were expressed and its mRNA level similar in all cell lines tested. To determine whether the PTEN mRNA level reflects the parallel level of protein, the level of PTEN protein was examined by Western blot.PTEN protein level was high in H460 and detectable in A549, A4, A7, L1 cells, not detectable in 95C, 95D, A1     

A novel GIP receptor splice variant influences GIP sensitivity of pancreatic beta-cells in obese mice

Gastric inhibitory polypeptide (GIP) is an incretin that potentiates insulin secretion from pancreatic beta-cells by binding to GIP receptor (GIPR) and subsequently increasing the level of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). We have identified a novel GIPR splice variant in mouse beta-cells that retains intron 8, resulting in a COOH-terminal truncated form (truncated GIPR). This isoform was coexpressed with full-length GIPR (wild-type GIPR) in normal GIPR-expressing tissues. In an experiment using cells transfected with both GIPRs, truncated GIPR did not lead to cAMP production induced by GIP but inhibited GIP-induced cAMP production through wild-type GIPR (n = 3-4, P < 0.05). Wild-type GIPR was normally located on the cell surface, but its expression was decreased in the presence of truncated GIPR, suggesting a dominant negative effect of truncated GIPR against wild-type GIPR. The functional relevance of truncated GIPR in vivo was investigated. In high-fat diet-fed obese mice (HFD mice), blood glucose levels were maintained by compensatory increased insulin secretion (n = 8, P < 0.05), and cAMP production (n = 6, P < 0.01) and insulin secretion (n = 10, P < 0.05) induced by GIP were significantly increased in isolated islets, suggesting hypersensitivity of the GIPR. Total GIPR mRNA expression was not increased in the islets of HFD mice, but the expression ratio of truncated GIPR to total GIPR was reduced by 32% compared with that of control mice (n = 6, P < 0.05). These results indicate that a relative reduction of truncated GIPR expression may be involved in hypersensitivity of GIPR and hyperinsulinemia in diet-induced obese mice.     

Protein traffic from the secretory pathway to the endosomal system in pancreatic beta-cells

Constitutive-like secretion involves vesicular trafficking corresponding kinetically and biochemically with a post-trans-Golgi network (TGN) origin. In pancreatic beta-cells, the budding of AP-1/clathrin-coated vesicles, a portion of which is derived from immature secretory granules, has been hypothesized to initiate constitutive-like trafficking. However, approximately 30 min after release of a 20 degrees C intracellular transport block in pancreatic beta-cells (to synchronize protein egress from the TGN), addition of brefeldin A (BFA) (which inhibits AP-1 recruitment) was reported not to block subsequent constitutive-like secretion. To further explore post-TGN trafficking in pancreatic beta-cell lines, we have followed the fate of pulse-labeled procathepsin B (ProB, a lysosomal proenyzme) after postpulse wortmannin treatment or the BFA treatment described above. We find that continuous wortmannin treatment allows ProB to reach immature secretory granules but inhibits its egress from maturing granules. Remarkably, BFA treatment causes augmented unstimulated secretion of newly synthesized ProB that is not paralleled by insulin. This effect requires a delay of 25-35 min after release from the 20 degrees C block. Further, when ProB delivery to endosomes is inhibited, its BFA-augmented secretion is eliminated. We hypothesize that the constitutive-like pathway involves an endosomal intermediate.     

Porphyromonas gingivalis infection promotes inflammation via inhibition of the AhR signalling pathway in periodontitis

Porphyromonas gingivalis (P. gingivalis) is a key pathogen of chronic periodontitis. Aryl hydrocarbon receptor (AhR) is essential in immune homeostasis via modulation of pro‐inflammatory cytokines production and indoleamine 2,3‐dioxygenase (IDO). In this study, it is demonstrated that P. gingivalis may regulate AhR signalling in periodontitis, which provides a potential target for further immune regulation studies in periodontitis. Experimental periodontitis was induced in C57BL/6 mice by silk ligature and P. gingivalis oral inoculation. The alveolar bone resorption was examined using Micro‐CT. Histological structures were observed and related cytokines involved in AhR signalling pathway were analysed. RAW264.7 cells were pretreated with AhR agonist (FICZ) and antagonist ({"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191) and infected with P. gingivalis subsequently. The levels of IDO, AhR and other related cytokines were measured. To demonstrate IDO activity, the concentrations of tryptophan (Trp) and kynurenine (Kyn) were assessed by HPLC. Histological analysis of periodontitis mice showed distinct alveolar bone resorption and inflammatory cell infiltration. The level of AhR and its downstream target factors were significantly decreased in inflamed gingival tissue. Furthermore, RAW 264.7 cells incubated by P. gingivalis exhibited increased pro‐inflammatory cytokines production and decreased AhR, CYP1A1, CYP1B1, and IDO expression. Decreased IDO activity was observed as decreased Kyn/Trp ratio in the supernatant. Moreover, FICZ decreased the pro‐inflammatory cytokines levels in P. gingivalis infected cells. It is concluded that P. gingivalis may promote inflammatory responses via inhibiting the AhR signalling pathway in periodontitis.

The schematic figure illustrating P. gingivalis inhibits Aryl hydrocarbon receptor (AhR) signalling pathway in periodontitis. P. gingivalis infection suppressed AhR and its downstream indoleamine 2,3‐dioxygenase (IDO) expression in periodontitis, which is responsible for the degradation of tryptophan (Trp) to kynurenine (Kyn). The downregulation of AhR signalling may increase IL‐6 and IL‐1β production by activating NF‐κB and NLRP3 inflammasome.