<p>(A) The percentage of X-gal-labeled cells that co-express insulin. Beta cell tracing in ... more <p>(A) The percentage of X-gal-labeled cells that co-express insulin. Beta cell tracing in RIP-CreER/R26-LacZ mice showed that almost all X-gal-positive cells are insulin positive in CTRL. A decrease of insulin positivity was observed in ALX and ALX+EGF/G on d3. Insulin positivity was recovered in ALX+EGF/G on d8. (B) X-gal-labeling of total beta cells (percentage beta cells labeled with X-gal). Total beta cell population includes INS<sup>-</sup> beta cell fraction. Percentage labeled beta cells in ALX and ALX+EGF/G was significantly reduced compared to CTRL on d8. (C) Pancreata of RIP-CreER/R26-LacZ mice were stained for insulin and X-gal. (D) The percentage insulin-positive clusters labeled with X-gal. The percentage X-gal-labeled insulin-positive clusters remained unchanged between d3 and d8. n = 4–6 per group per time point. Symbol * represents the statistical significance of each condition compared to CTRL. The horizontal bar denotes the significant difference between the experimental groups. +, P < 0.05; **, P < 0.01; ***,+++, P < 0.001.</p
Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) play... more Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) plays a pivotal role in supporting MM progression. In MM, MSC exhibit a different genomic profile and dysregulated cytokine secretion compared to normal MSC, however the mechanisms involved in these changes are not fully understood. Here, we examined the miRNA changes in human MSC after culture with conditioned medium of MM cells and found 19 dysregulated miRNAs, including upregulated miR-146a. Moreover, exosomes derived from MM cells contained miR-146a and could be transferred into MSC. After overexpressing miR-146a in MSC, secretion of several cytokines and chemokines including CXCL1, IL6, IL-8, IP-10, MCP-1, and CCL-5 was elevated, resulting in the enhancement of MM cell viability and migration. DAPT, an inhibitor of the endogenous Notch pathway, was able to abrogate the miR-146ainduced increase of cytokines in MSC, suggesting the involvement of the Notch pathway. Taken together, our results demonstrate a positive feedback loop between MM cells and MSC: MM cells promote the increase of miR146a in MSC which leads to more cytokine secretion, which in turn favors MM cell growth and migration.
Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates... more Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates the anti-apoptotic effects of IGF-I in human neutrophils independently of its downstream target Akt. In this study, we investigated whether IGF-I regulates Tec kinase, an alternative downstream target of PI 3 K, in neutrophils and whether this molecule is able to affect apoptosis. Design: We investigated the translocation of Tec kinases in neutrophils after stimulation with IGF-I. Furthermore, we transiently and stably transfected Hek293T cells with constructs expressing different forms of Tec kinase and measured the level of cell survival and apoptosis/necrosis through trypan blue exclusion test and Annexin-V/propidium iodide labelling, respectively. Results: We show that IGF-I stimulates the translocation of Tec kinase to the membrane in neutrophils in a PI 3 K dependent matter. Overexpression of Tec kinase augments cell survival by inhibition of necrosis. The pro-survival effect is attenuated by the deletion of the kinase domain but not by inactivation of this domain by a single amino acid substitution. Conclusion: Tec kinase can act as a prosurvival factor and is regulated by IGF-I in human neutrophils through PI 3 K activation.
Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalath... more Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalathus linearis) extract had anti-hyperglycemic activity and significant protective effects on the pancreatic beta cell mass in a chronic diet-induced diabetes model. The present study evaluated the cytoprotective effect of the phytochemical on beta cells exposed to acute cell stress. Synthetically prepared PPAG was administered orally in mice treated with a single dose of streptozotocin to acutely induce beta cell death and hyperglycemia. Its effect was assessed on beta cell mass, proliferation and apoptotic cell death. Its cytoprotective effect was also studied in vitro on INS-1E beta cells and on human pancreatic islet cells. Treatment with the phytochemical PPAG protected beta cells during the first days after the insult against apoptotic cell death, as evidenced by TUNEL staining, and prevented loss of expression of anti-apoptotic protein BCL2 in vivo. In vitro, PPAG protected INS-1E be...
One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to indu... more One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, how-ever it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treat-ment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogen-esis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experimen...
Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) play... more Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) plays a pivotal role in supporting MM progression. In MM, MSC exhibit a different genomic profile and dysregulated cytokine secretion compared to normal MSC, however the mechanisms involved in these changes are not fully understood. Here, we examined the miRNA changes in human MSC after culture with conditioned medium of MM cells and found 19 dysregulated miRNAs, including upregulated miR-146a. Moreover, exosomes derived from MM cells contained miR-146a and could be transferred into MSC. After overexpressing miR-146a in MSC, secretion of several cytokines and chemokines including CXCL1, IL6, IL-8, IP-10, MCP-1, and CCL-5 was elevated, resulting in the enhancement of MM cell viability and migration. DAPT, an inhibitor of the endogenous Notch pathway, was able to abrogate the miR-146a-induced increase of cytokines in MSC, suggesting the involvement of the Notch pathway. Taken together, our res...
To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we... more To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm 2 , corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/ or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing colocalization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition.
Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a cruci... more Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a crucial role in MM pathogenesis by secreting growth factors, cytokines, and other functional components. Exosomes are 30-100nm diameter membranous vesicles constitutively released by several cell types including reticulocytes, cytotoxic T lymphocytes, B lymphocytes, epithelial and endothelial cells. Exosomes mediate local cell-cell communication by transferring mRNAs, miRNAs and proteins. Due to their ability to transfer functional components, exosomes play multiple roles by stimulating target cells, transferring membrane receptors, delivering proteins, and inducing epigenetic changes in recipient cells. Although the promotion of MM growth and survival induced by BMSCs has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here, we investigated the effect and mechanisms of BMSC-derived exosomes on the proliferation and survival of MM cells using the murine 5T33MM ...
The insulin-like growth factor IGF-I is an important fetal and postnatal growth factor, which is ... more The insulin-like growth factor IGF-I is an important fetal and postnatal growth factor, which is also involved in tissue homeostasis via regulation of proliferation, differentiation, and cell survival. To understand the role of IGF-I in the pathophysiology of a variety of disorders, including growth disorders, cancer, and neurodegenerative diseases, a detailed knowledge of IGF-I signal transduction is required. This knowledge may also contribute to the development of new therapies directed at the IGF-I receptor or other signaling molecules. In this review, we will address IGF-I receptor signaling through the JAK/STAT pathway in IGF-I signaling and the role of cytokine-induced inhibitors of signaling (CIS) and suppressors of cytokine signaling (SOCS). It appears that, in addition to the canonical IGF-I signaling pathways through extracellular-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K)-Akt, IGF-I also signals through the JAK/STAT pathway. Activation of this pathway may lead to induction of SOCS molecules, well-known feedback inhibitors of the JAK/STAT pathway, which also suppress of IGF-I-induced JAK/STAT signaling. Furthermore, other IGF-I-induced signaling pathways may also be modulated by SOCS. It is conceivable that the effect of these classical inhibitors of cytokine signaling directly affect IGF-I receptor signaling, because they are able to associate to the intracellular part of the IGF-I receptor. These observations indicate that CIS and SOCS molecules are key to cross-talk between IGF-I receptor signaling and signaling through receptors belonging to the hematopoietic/cytokine receptor superfamily. Theoretically, dysregulation of CIS or SOCS may affect IGF-I-mediated effects on body growth, cell differentiation, proliferation, and cell survival.
Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We prev... more Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We previously showed that insulinlike growth factor-1 (IGF1) delays spontaneous neutrophil apoptosis without influencing the secretion of cytokines by these cells. In the present study, we further addressed the role of IGF1 in regulating neutrophil survival in the presence of other factors present during inflammation, and the mechanism involved in delaying apoptosis. We show that IGF1 delays neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11 and that the effect of IGF1 is comparable in magnitude to that of the acknowledged anti-apoptotic cytokines interferon-g (IFNG) and granulocyte-macrophage colony-stimulating factor (GM-CSF; now known as CSF2). Furthermore, IGF1 exerted additional effects on cell survival in the presence of these cytokines. IGF1 did not affect Fas expression or activation by anti-Fas of caspase-8, but inhibited the depolarization of the mitochondrial membrane. Inhibitor studies indicate that the phosphatidylinositol-3 kinase (PI3K) pathway, but not the MEK-ERK pathway, mediates the effects of IGF1. However, in contrast to CSF2, IGF1 did not induce phosphorylation and translocation to the membrane of AKT, the canonical downstream target of PI3K. We therefore speculate that other downstream targets of PI3K are involved in the delay of neutrophil apoptosis by IGF1, possibly through stabilization of the mitochondrial membrane.
The regenerative medicine field is expanding with great successes in laboratory and pre-clinical ... more The regenerative medicine field is expanding with great successes in laboratory and pre-clinical settings. Pancreatic acinar cells in diabetic mice were recently converted into beta cells by treatment with ciliary neurotrophic factor and epidermal growth factor. This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly loose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of beta cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifica...
One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to indu... more One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The glucose-lowering effect of the treatment might play an important role in the regeneration process.
A major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic be... more A major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic beta cell mass. Here, we examined the effect of a phenylpropenoic acid glucoside (PPAG) on the beta cell mass, and via which mechanism this effect is established. Methods and results: Mice were fed a high-fat and fructose-containing diet to induce obesity and hyperglycemia. PPAG treatment protected obese mice from diet-induced hyperglycemia and resulted in a tripling of beta cell mass. The effect of the phytochemical on beta cell mass was neither due to increased proliferation, as determined by Ki67 immunostaining, nor to neogenesis, which was assessed by genetic lineage tracing. TUNEL staining revealed suppressed apoptosis in PPAG-treated obese mice. In vitro, PPAG protected beta cells from palmitate-induced apoptosis. It protected beta cells against ER stress by increasing expression of antiapoptotic B-cell lymphoma 2 (BCL2) protein without affecting proapoptotic signals.
Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We prev... more Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We previously showed that insulinlike growth factor-1 (IGF1) delays spontaneous neutrophil apoptosis without influencing the secretion of cytokines by these cells. In the present study, we further addressed the role of IGF1 in regulating neutrophil survival in the presence of other factors present during inflammation, and the mechanism involved in delaying apoptosis. We show that IGF1 delays neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11 and that the effect of IGF1 is comparable in magnitude to that of the acknowledged anti-apoptotic cytokines interferon-g (IFNG) and granulocyte-macrophage colony-stimulating factor (GM-CSF; now known as CSF2). Furthermore, IGF1 exerted additional effects on cell survival in the presence of these cytokines. IGF1 did not affect Fas expression or activation by anti-Fas of caspase-8, but inhibited the depolarization of the mitochondrial membrane. Inhibitor studies indicate that the phosphatidylinositol-3 kinase (PI3K) pathway, but not the MEK-ERK pathway, mediates the effects of IGF1. However, in contrast to CSF2, IGF1 did not induce phosphorylation and translocation to the membrane of AKT, the canonical downstream target of PI3K. We therefore speculate that other downstream targets of PI3K are involved in the delay of neutrophil apoptosis by IGF1, possibly through stabilization of the mitochondrial membrane.
... Ron Kooijman Corresponding Author Contact Information , a , E-mail The Corresponding Author ,... more ... Ron Kooijman Corresponding Author Contact Information , a , E-mail The Corresponding Author , Eddy Himpe a , Saranyapin Potikanond a and Astrid Coppens ... the human IGF-IR cDNA driven by the SV40 promoter [27]) was a gift from Dr. Baserga (Thomas Jefferson University ...
Objective: Several studies showed that high serum levels of insulin-like growth factor-I (IGF-I) ... more Objective: Several studies showed that high serum levels of insulin-like growth factor-I (IGF-I) correlate with an increased risk for prostate cancer, although the causal role of IGF-I remains to be established. In this study, we addressed the role of IGF-I as a serum factor on the growth of two androgen-independent cell lines (Du145 and PC3) and one androgen-dependent cell line (LNCaP). Design: We investigated the effects of a blocking antibody against the IGF-I receptor (αIR3) on DNA synthesis in prostate cancer cells cultured in the presence of recombinant human IGF-I or normal human serum (NHS). Results: We show that in all three prostate cancer cell lines, NHS exerts a markedly stronger stimulating effect on DNA synthesis than IGF-I, and that the effect of NHS can be completely abrogated by an antibody against the IGF-I receptor (αIR3). Using pharmacological inhibitors of the two canonical IGF-I receptor signaling pathways, we show that the phosphatidylinositol-3′-kinase (PI3K) and the Mek-Erk pathways are not required for the stimulating effect of NHS. Conclusion: Our observations indicate that the stimulating effect of NHS is completely dependent on IGF-I receptor signaling transduction and that IGF-I stimulates DNA synthesis in prostate cancer cells in strong synergy with other serum factors. We speculate that the role of other serum factors could explain the discrepancy between the results observed in different animal models to study the function of IGF-I in prostate cancer.
Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates... more Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates the anti-apoptotic effects of IGF-I in human neutrophils independently of its down-stream target Akt. In this study, we investigated whether IGF-I regulates Tec kinase, an alternative down-stream target of PI 3 K, in neutrophils and whether this molecule is able to affect apoptosis. Design: We investigated the translocation of Tec kinases in neutrophils after stimulation with IGF-I. Furthermore, we transiently and stably transfected Hek293T cells with constructs expressing different forms of Tec kinase and measured the level of cell survival and apoptosis/necrosis through trypan blue exclusion test and Annexin-V/propidium iodide labelling, respectively. Results: We show that IGF-I stimulates the translocation of Tec kinase to the membrane in neutrophils in a PI 3 K dependent matter. Overexpression of Tec kinase augments cell survival by inhibition of necrosis. The pro-survival effect is attenuated by the deletion of the kinase domain but not by inactivation of this domain by a single amino acid substitution. Conclusion: Tec kinase can act as a prosurvival factor and is regulated by IGF-I in human neutrophils through PI 3 K activation.
<p>(A) The percentage of X-gal-labeled cells that co-express insulin. Beta cell tracing in ... more <p>(A) The percentage of X-gal-labeled cells that co-express insulin. Beta cell tracing in RIP-CreER/R26-LacZ mice showed that almost all X-gal-positive cells are insulin positive in CTRL. A decrease of insulin positivity was observed in ALX and ALX+EGF/G on d3. Insulin positivity was recovered in ALX+EGF/G on d8. (B) X-gal-labeling of total beta cells (percentage beta cells labeled with X-gal). Total beta cell population includes INS<sup>-</sup> beta cell fraction. Percentage labeled beta cells in ALX and ALX+EGF/G was significantly reduced compared to CTRL on d8. (C) Pancreata of RIP-CreER/R26-LacZ mice were stained for insulin and X-gal. (D) The percentage insulin-positive clusters labeled with X-gal. The percentage X-gal-labeled insulin-positive clusters remained unchanged between d3 and d8. n = 4–6 per group per time point. Symbol * represents the statistical significance of each condition compared to CTRL. The horizontal bar denotes the significant difference between the experimental groups. +, P < 0.05; **, P < 0.01; ***,+++, P < 0.001.</p
Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) play... more Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) plays a pivotal role in supporting MM progression. In MM, MSC exhibit a different genomic profile and dysregulated cytokine secretion compared to normal MSC, however the mechanisms involved in these changes are not fully understood. Here, we examined the miRNA changes in human MSC after culture with conditioned medium of MM cells and found 19 dysregulated miRNAs, including upregulated miR-146a. Moreover, exosomes derived from MM cells contained miR-146a and could be transferred into MSC. After overexpressing miR-146a in MSC, secretion of several cytokines and chemokines including CXCL1, IL6, IL-8, IP-10, MCP-1, and CCL-5 was elevated, resulting in the enhancement of MM cell viability and migration. DAPT, an inhibitor of the endogenous Notch pathway, was able to abrogate the miR-146ainduced increase of cytokines in MSC, suggesting the involvement of the Notch pathway. Taken together, our results demonstrate a positive feedback loop between MM cells and MSC: MM cells promote the increase of miR146a in MSC which leads to more cytokine secretion, which in turn favors MM cell growth and migration.
Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates... more Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates the anti-apoptotic effects of IGF-I in human neutrophils independently of its downstream target Akt. In this study, we investigated whether IGF-I regulates Tec kinase, an alternative downstream target of PI 3 K, in neutrophils and whether this molecule is able to affect apoptosis. Design: We investigated the translocation of Tec kinases in neutrophils after stimulation with IGF-I. Furthermore, we transiently and stably transfected Hek293T cells with constructs expressing different forms of Tec kinase and measured the level of cell survival and apoptosis/necrosis through trypan blue exclusion test and Annexin-V/propidium iodide labelling, respectively. Results: We show that IGF-I stimulates the translocation of Tec kinase to the membrane in neutrophils in a PI 3 K dependent matter. Overexpression of Tec kinase augments cell survival by inhibition of necrosis. The pro-survival effect is attenuated by the deletion of the kinase domain but not by inactivation of this domain by a single amino acid substitution. Conclusion: Tec kinase can act as a prosurvival factor and is regulated by IGF-I in human neutrophils through PI 3 K activation.
Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalath... more Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalathus linearis) extract had anti-hyperglycemic activity and significant protective effects on the pancreatic beta cell mass in a chronic diet-induced diabetes model. The present study evaluated the cytoprotective effect of the phytochemical on beta cells exposed to acute cell stress. Synthetically prepared PPAG was administered orally in mice treated with a single dose of streptozotocin to acutely induce beta cell death and hyperglycemia. Its effect was assessed on beta cell mass, proliferation and apoptotic cell death. Its cytoprotective effect was also studied in vitro on INS-1E beta cells and on human pancreatic islet cells. Treatment with the phytochemical PPAG protected beta cells during the first days after the insult against apoptotic cell death, as evidenced by TUNEL staining, and prevented loss of expression of anti-apoptotic protein BCL2 in vivo. In vitro, PPAG protected INS-1E be...
One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to indu... more One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, how-ever it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treat-ment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogen-esis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experimen...
Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) play... more Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) plays a pivotal role in supporting MM progression. In MM, MSC exhibit a different genomic profile and dysregulated cytokine secretion compared to normal MSC, however the mechanisms involved in these changes are not fully understood. Here, we examined the miRNA changes in human MSC after culture with conditioned medium of MM cells and found 19 dysregulated miRNAs, including upregulated miR-146a. Moreover, exosomes derived from MM cells contained miR-146a and could be transferred into MSC. After overexpressing miR-146a in MSC, secretion of several cytokines and chemokines including CXCL1, IL6, IL-8, IP-10, MCP-1, and CCL-5 was elevated, resulting in the enhancement of MM cell viability and migration. DAPT, an inhibitor of the endogenous Notch pathway, was able to abrogate the miR-146a-induced increase of cytokines in MSC, suggesting the involvement of the Notch pathway. Taken together, our res...
To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we... more To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm 2 , corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/ or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing colocalization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition.
Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a cruci... more Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a crucial role in MM pathogenesis by secreting growth factors, cytokines, and other functional components. Exosomes are 30-100nm diameter membranous vesicles constitutively released by several cell types including reticulocytes, cytotoxic T lymphocytes, B lymphocytes, epithelial and endothelial cells. Exosomes mediate local cell-cell communication by transferring mRNAs, miRNAs and proteins. Due to their ability to transfer functional components, exosomes play multiple roles by stimulating target cells, transferring membrane receptors, delivering proteins, and inducing epigenetic changes in recipient cells. Although the promotion of MM growth and survival induced by BMSCs has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here, we investigated the effect and mechanisms of BMSC-derived exosomes on the proliferation and survival of MM cells using the murine 5T33MM ...
The insulin-like growth factor IGF-I is an important fetal and postnatal growth factor, which is ... more The insulin-like growth factor IGF-I is an important fetal and postnatal growth factor, which is also involved in tissue homeostasis via regulation of proliferation, differentiation, and cell survival. To understand the role of IGF-I in the pathophysiology of a variety of disorders, including growth disorders, cancer, and neurodegenerative diseases, a detailed knowledge of IGF-I signal transduction is required. This knowledge may also contribute to the development of new therapies directed at the IGF-I receptor or other signaling molecules. In this review, we will address IGF-I receptor signaling through the JAK/STAT pathway in IGF-I signaling and the role of cytokine-induced inhibitors of signaling (CIS) and suppressors of cytokine signaling (SOCS). It appears that, in addition to the canonical IGF-I signaling pathways through extracellular-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K)-Akt, IGF-I also signals through the JAK/STAT pathway. Activation of this pathway may lead to induction of SOCS molecules, well-known feedback inhibitors of the JAK/STAT pathway, which also suppress of IGF-I-induced JAK/STAT signaling. Furthermore, other IGF-I-induced signaling pathways may also be modulated by SOCS. It is conceivable that the effect of these classical inhibitors of cytokine signaling directly affect IGF-I receptor signaling, because they are able to associate to the intracellular part of the IGF-I receptor. These observations indicate that CIS and SOCS molecules are key to cross-talk between IGF-I receptor signaling and signaling through receptors belonging to the hematopoietic/cytokine receptor superfamily. Theoretically, dysregulation of CIS or SOCS may affect IGF-I-mediated effects on body growth, cell differentiation, proliferation, and cell survival.
Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We prev... more Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We previously showed that insulinlike growth factor-1 (IGF1) delays spontaneous neutrophil apoptosis without influencing the secretion of cytokines by these cells. In the present study, we further addressed the role of IGF1 in regulating neutrophil survival in the presence of other factors present during inflammation, and the mechanism involved in delaying apoptosis. We show that IGF1 delays neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11 and that the effect of IGF1 is comparable in magnitude to that of the acknowledged anti-apoptotic cytokines interferon-g (IFNG) and granulocyte-macrophage colony-stimulating factor (GM-CSF; now known as CSF2). Furthermore, IGF1 exerted additional effects on cell survival in the presence of these cytokines. IGF1 did not affect Fas expression or activation by anti-Fas of caspase-8, but inhibited the depolarization of the mitochondrial membrane. Inhibitor studies indicate that the phosphatidylinositol-3 kinase (PI3K) pathway, but not the MEK-ERK pathway, mediates the effects of IGF1. However, in contrast to CSF2, IGF1 did not induce phosphorylation and translocation to the membrane of AKT, the canonical downstream target of PI3K. We therefore speculate that other downstream targets of PI3K are involved in the delay of neutrophil apoptosis by IGF1, possibly through stabilization of the mitochondrial membrane.
The regenerative medicine field is expanding with great successes in laboratory and pre-clinical ... more The regenerative medicine field is expanding with great successes in laboratory and pre-clinical settings. Pancreatic acinar cells in diabetic mice were recently converted into beta cells by treatment with ciliary neurotrophic factor and epidermal growth factor. This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly loose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of beta cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifica...
One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to indu... more One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The glucose-lowering effect of the treatment might play an important role in the regeneration process.
A major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic be... more A major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic beta cell mass. Here, we examined the effect of a phenylpropenoic acid glucoside (PPAG) on the beta cell mass, and via which mechanism this effect is established. Methods and results: Mice were fed a high-fat and fructose-containing diet to induce obesity and hyperglycemia. PPAG treatment protected obese mice from diet-induced hyperglycemia and resulted in a tripling of beta cell mass. The effect of the phytochemical on beta cell mass was neither due to increased proliferation, as determined by Ki67 immunostaining, nor to neogenesis, which was assessed by genetic lineage tracing. TUNEL staining revealed suppressed apoptosis in PPAG-treated obese mice. In vitro, PPAG protected beta cells from palmitate-induced apoptosis. It protected beta cells against ER stress by increasing expression of antiapoptotic B-cell lymphoma 2 (BCL2) protein without affecting proapoptotic signals.
Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We prev... more Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We previously showed that insulinlike growth factor-1 (IGF1) delays spontaneous neutrophil apoptosis without influencing the secretion of cytokines by these cells. In the present study, we further addressed the role of IGF1 in regulating neutrophil survival in the presence of other factors present during inflammation, and the mechanism involved in delaying apoptosis. We show that IGF1 delays neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11 and that the effect of IGF1 is comparable in magnitude to that of the acknowledged anti-apoptotic cytokines interferon-g (IFNG) and granulocyte-macrophage colony-stimulating factor (GM-CSF; now known as CSF2). Furthermore, IGF1 exerted additional effects on cell survival in the presence of these cytokines. IGF1 did not affect Fas expression or activation by anti-Fas of caspase-8, but inhibited the depolarization of the mitochondrial membrane. Inhibitor studies indicate that the phosphatidylinositol-3 kinase (PI3K) pathway, but not the MEK-ERK pathway, mediates the effects of IGF1. However, in contrast to CSF2, IGF1 did not induce phosphorylation and translocation to the membrane of AKT, the canonical downstream target of PI3K. We therefore speculate that other downstream targets of PI3K are involved in the delay of neutrophil apoptosis by IGF1, possibly through stabilization of the mitochondrial membrane.
... Ron Kooijman Corresponding Author Contact Information , a , E-mail The Corresponding Author ,... more ... Ron Kooijman Corresponding Author Contact Information , a , E-mail The Corresponding Author , Eddy Himpe a , Saranyapin Potikanond a and Astrid Coppens ... the human IGF-IR cDNA driven by the SV40 promoter [27]) was a gift from Dr. Baserga (Thomas Jefferson University ...
Objective: Several studies showed that high serum levels of insulin-like growth factor-I (IGF-I) ... more Objective: Several studies showed that high serum levels of insulin-like growth factor-I (IGF-I) correlate with an increased risk for prostate cancer, although the causal role of IGF-I remains to be established. In this study, we addressed the role of IGF-I as a serum factor on the growth of two androgen-independent cell lines (Du145 and PC3) and one androgen-dependent cell line (LNCaP). Design: We investigated the effects of a blocking antibody against the IGF-I receptor (αIR3) on DNA synthesis in prostate cancer cells cultured in the presence of recombinant human IGF-I or normal human serum (NHS). Results: We show that in all three prostate cancer cell lines, NHS exerts a markedly stronger stimulating effect on DNA synthesis than IGF-I, and that the effect of NHS can be completely abrogated by an antibody against the IGF-I receptor (αIR3). Using pharmacological inhibitors of the two canonical IGF-I receptor signaling pathways, we show that the phosphatidylinositol-3′-kinase (PI3K) and the Mek-Erk pathways are not required for the stimulating effect of NHS. Conclusion: Our observations indicate that the stimulating effect of NHS is completely dependent on IGF-I receptor signaling transduction and that IGF-I stimulates DNA synthesis in prostate cancer cells in strong synergy with other serum factors. We speculate that the role of other serum factors could explain the discrepancy between the results observed in different animal models to study the function of IGF-I in prostate cancer.
Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates... more Objective: Previously, we showed that the phosphatidylinositol-3 kinase (PI 3 K) pathway mediates the anti-apoptotic effects of IGF-I in human neutrophils independently of its down-stream target Akt. In this study, we investigated whether IGF-I regulates Tec kinase, an alternative down-stream target of PI 3 K, in neutrophils and whether this molecule is able to affect apoptosis. Design: We investigated the translocation of Tec kinases in neutrophils after stimulation with IGF-I. Furthermore, we transiently and stably transfected Hek293T cells with constructs expressing different forms of Tec kinase and measured the level of cell survival and apoptosis/necrosis through trypan blue exclusion test and Annexin-V/propidium iodide labelling, respectively. Results: We show that IGF-I stimulates the translocation of Tec kinase to the membrane in neutrophils in a PI 3 K dependent matter. Overexpression of Tec kinase augments cell survival by inhibition of necrosis. The pro-survival effect is attenuated by the deletion of the kinase domain but not by inactivation of this domain by a single amino acid substitution. Conclusion: Tec kinase can act as a prosurvival factor and is regulated by IGF-I in human neutrophils through PI 3 K activation.
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Papers by Eddy Himpe