Low Concentration of Interleukin-1β Induces FLICE-Inhibitory Protein–Mediated β-Cell Proliferation in Human Pancreatic Islets
Kathrin Maedler 1 ,
Desiree M. Schumann 2 ,
Nadine Sauter 2 ,
Helga ...Ellingsgaard 2 ,
Domenico Bosco 3 ,
Reto Baertschiger 3 ,
Yoichiro Iwakura 4 ,
José Oberholzer 5 ,
Claes B. Wollheim 6 ,
Benoit R. Gauthier 6 and
Marc Y. Donath 2
1 Larry L. Hillblom Islet Research Center, University of California, Los Angeles, California
2 Division of Endocrinology and Diabetes and Center for Integrative Human Physiology, University Hospital Zurich, Zurich, Switzerland
3 Department of Surgery, University Medical Center, Geneva, Switzerland
4 Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan
5 Division of Transplantation, University of Illinois at Chicago, Chicago, Illinois
6 Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland
Address correspondence and reprint requests to Marc Y. Donath, MD, Division of Endocrinology and Diabetes, Department of Medicine,
University Hospital, CH-8091 Zurich, Switzerland. E-mail: marc.donath{at}usz.ch
Abstract
High glucose concentrations have a dual effect on β-cell turnover, inducing proliferation in the short-term and apoptosis
in the long-term. Hyperglycemia leads to β-cell production of interleuking (IL)-1β in human pancreatic islets. Fas, a death
receptor regulated by IL-1β, is involved in glucose-induced β-cell apoptosis. Fas engagement can be switched from death signal
to induction of proliferation when the caspase 8 inhibitor, FLICE-inhibitory protein (FLIP), is active. Here, we show that
IL-1β at low concentrations may participate in the mitogenic actions of glucose through the Fas-FLIP pathway. Thus, exposure
of human islets to low IL-1β concentrations (0.01–0.02 ng/ml) stimulated proliferation and decreased apoptosis, whereas increasing
amounts of IL-1β (2–5 ng/ml) had the reverse effects. A similarly bimodal induction of FLIP, pancreatic duodenal homeobox
(PDX)-1, and Pax4 mRNA expression, as well as glucose-stimulated insulin secretion, was observed. In contrast, Fas induction
by IL-1β was monophasic. Low IL-1β also induced the IL-1 receptor antagonist (IL-1Ra), suppression of which by RNA interference
abrogated the beneficial effects of low IL-1β. The Fas antagonistic antibody ZB4 and small interfering RNA to FLIP prevented
low IL-1β–stimulated β-cell proliferation. Consistent with our in vitro results, IL-1β knockout mice displayed glucose intolerance
along with a decrease in islet Fas, FLIP, Pax4, and PDX-1 transcripts. These findings indicate that low IL-1β levels positively
influence β-cell function and turnover through the Fas-FLIP pathway and that IL-1Ra production prevents harmful effects of
high IL-1β concentrations.
FLIP, FLICE-inhibitory protein
GSIS, glucose-stimulated insulin secretion
IL, interleukin
IL-1Ra, IL-1 receptor antagonist
IRS, insulin receptor substrate
KRBB, Krebs-Ringer bicarbonate buffer
PDX, pancreatic duodenal homeobox
rh, recombinant human
siFLIP, siRNA directed to FLIP
siIL-1Ra, siRNA directed to IL-1Ra
siRNA, small interfering RNA
TUNEL, transferase-mediated dUTP nick-end labeling
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted July 7, 2006.
Received November 2, 2005.
DIABETES
Blockade of β1 Integrin–Laminin-5 Interaction Affects Spreading and Insulin Secretion of Rat β-Cells Attached on Extracellular
Matrix
Geraldine Parnaud 1 ,
Eva Hammar 1 ,
Dominique G. Rouiller 1 ,
...Mathieu Armanet 2 ,
Philippe A. Halban 1 and
Domenico Bosco 2
1 Department of Genetic Medicine and Development, University Medical Center, Geneva, Switzerland
2 Cell Isolation and Transplantation Center, Division of Surgical Research, Department of Surgery, University Hospital, Geneva,
Switzerland
Address correspondence and reprint requests to Geraldine Parnaud, Department of Genetic Medicine and Development, University
Medical Center, 1 rue Michel-Servet, 1211 Geneva-4, Switzerland. E-mail: geraldine.parnaud{at}medecine.unige.ch
Abstract
When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic β-cells spread in response
to glucose and secrete more insulin compared with cells attached on poly- l -lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor β1 integrin are implicated
in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G
matrix responsible for the effect of 804G matrix on β-cell function and spreading. When expression of two well-known laminin-5
ligands, β1 and β4 integrin, was assessed by Western blot and RT-PCR, only the β1 integrin was detected in β-cells. Anti–β1
integrin antibody reduced the spreading of β-cells on 804G matrix. Blockade of the interaction between β1 integrins and laminin-5
resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti–β1 integrin antibody also inhibited focal adhesion
kinase phosphorylation induced by 804G matrix. In conclusion, anti–β1 integrin and –laminin-5 antibodies interfere with spreading
of β-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling
via engagement of β1 integrins by laminin-5 is an important component of normal β-cell function.
DMEM, Dulbecco’s modified Eagle’s medium
FAK, focal adhesion kinase
HRP, horseradish peroxidase
KRBH, Krebs-Ringer bicarbonate HEPES buffer
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted February 6, 2006.
Received October 25, 2005.
DIABETES
As shown in a large clinical prospective trial, inhibition of the renin‐angiotensin system (RAS) can delay the onset of type 2 diabetes in high‐risk individuals. We evaluated the beneficial effects ...of RAS inhibition on β‐cell function under glucotoxic conditions. Human islets from 13 donors were cultured in 5.5 mM (controls) or 16.7 mM glucose high glucose (HG) for 4 d with or without losartan (5 μM), a selective AT1Rblocker, and/or U73122 (2 μM), a selective PLC inhibitor, during the last 2 d. HG induced RAS activation with overexpression of AT1R (P<0.05) and angiotensinogen (P< 0.001) mRNAs. HG increased endoplasmic reticulum (ER) stress markers (P< 0.001) such as GRP78, sXBP1, and ATF4 mRNAs and Grp78 protein levels (P<0.01). HG also decreased reticular calcium concentration (P<0.0001) and modified protein expressions of ER calcium pumps with reduction of SERCA2b (P<0.01) and increase of IP3R2 (P<0.05). Losartan prevented these deleterious effects and was associated with improved insulin secretion despite HG exposure. AT1R activation triggers the PLC‐IP3‐calcium pathway. Losartan prevented the increase of PLC β1 and γ1 protein levels induced by HG (P<0.05). U73122 reproduced all the protective effects of losartan. AT1R blockade protects human islets from the deleterious effects of glucose through inhibition of the PLC‐IP3‐calcium pathway.—Madec, A‐M., Cassel, R., Dubois, S., Ducreux, S., Vial, G., Chauvin, M.‐A., Mesnier, A., Chikh, K., Bosco, D., Rieusset, J., Van Coppenolle, F., Thivolet, C., Losartan, an angiotensin II type 1 receptor blocker, protects human islets from glucotoxicity through the phospholipase C pathway. FASEB J. 27, 5122–5130 (2013). www.fasebj.org
The aim of this study was to assess whether the expression of E-cadherin at the surface of rat β-cells is regulated by insulin secretagogues and correlates with insulin secretion. When cultured under ...standard conditions, virtually all β-cells expressed E-cadherin observed by immunofluorescence, but heterogeneous staining was observed. Using fluorescence-activated cell sorting (FACS), two β-cell sub-populations were sorted: one that was poorly labeled (‘ECad-low’) and another that was highly labeled (‘ECad-high’). After 1-h stimulation with 16.7 mM glucose, insulin secretion (reverse hemolytic plaque assay) from individual ECad-high β-cells was higher than that from ECad-low β-cells. Ca2+-dependent β-cell aggregation was increased at 16.7 mM glucose when compared with 2.8 mM glucose. E-cadherin at the surface of β-cells was increased after 18 h at 11.1 and 22.2 mM glucose when compared with 2.8 mM glucose, with the greatest increase at 22.2 mM glucose + 0.5 mM isobutylmethylxanthine (IBMX). While no labeling was detected on freshly trypsinized cells, the proportion of stained cells increased in a time-dependent manner during culture for 1, 3, and 24 h. This recovery was faster when cells were incubated at 16.7 vs 2.8 mM glucose. Cycloheximide inhibited expression of E-cadherin at 2.8 mM glucose, but not at 16.7 mM, while depolymerization of actin by either cytochasin B or latrunculin B increased surface E-cadherin at low glucose. In conclusion, these results show that expression of E-cadherin at the surface of islet β-cells is controlled by secretagogues including glucose, correlates with insulin secretion, and can serve as a surface marker of β-cell function.
Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection ...mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.
Flavescence dorée (FD) is a quarantine grapevine disease caused by a phytoplasma transmitted by the leafhopper Scaphoideus titanus Ball. FD management relies on compulsory insecticide treatments, ...roguing of infected plants, and substitution with certified material. Some grapevine cultivars show a spontaneous remission of symptoms (recovery). To determine if recovery is a suitable strategy to co-exist with disease in areas of strong infestation, the qualitative aspects of grapes, musts, and wines obtained from recovered Barbera and Chardonnay grapevines were investigated in two productive vineyards. Following field observations, about 1500 plants in each vineyard were divided into healthy (asymptomatic and negative in phytoplasma molecular diagnosis) and recovered (asymptomatic the year of observation but infected the year before). Maturation curves and microvinification tests followed by oenological and sensory analyses showed that maturation trends of recovered grapes were in line with those from healthy plants and the final qualities of wines were comparable. The spread of FD has strongly increased in Piedmont (Italy) in recent decades. Management strategies to cope with the disease are necessary to preserve traditional wine production. Despite the yield from recovered grapevines is quantitatively lower than that from healthy ones, we showed here that the wine quality is, however, preserved.
Following a request from the European Commission, the EFSA Panel on Plant Health (PLH) performed a quantitative analysis of the risk posed by the Flavescence dorée phytoplasma (FDp) in the EU ...territory. Three scenarios were analysed, one with current measures in place (scenario A0), one designed to improve grapevine propagation material phytosanitary status (scenario A1) and one with reinforced eradication and containment (scenario A2). The potential for entry is limited, FDp being almost non‐existent outside the EU. FDp and its major vector, Scaphoideus titanus, have already established over large parts of the EU and have the potential to establish in a large fraction of the currently unaffected EU territory. With the current measures in place (A0), spread of FDp is predicted to continue with a progression of between a few and ca 20 newly infested NUTS 2 regions during the next 10 years, illustrating the limitations of the current control measures against spread. FDp spread is predicted to be roughly similar between scenarios A1 and A2, but more restricted than under scenario A0. However, even with reinforced control scenarios, stabilisation or reduction in the number of infested NUTS 2 regions has only relatively low probability. Under scenario A0, FDp has a 0.5–1% impact on the overall EU grapes and wine production, reflecting the effectiveness of the current control measures against impact. Under both scenarios A1 and A2, FDp impact is predicted to be reduced, by approximately one‐third (A1) to two‐thirds (A2) as compared to A0, but the associated uncertainties are large. The generalised use of hot water treatment for planting material produced in infected zones has the most important contribution to FDp impact reduction in scenario A1 and has high feasibility. Both increased eradication and containment measures contribute to impact reduction under scenario A2 but the overall feasibility is lower.
Institut Georges Lopez-1 (IGL-1) is a preservation solution similar to University of Wisconsin (UW) with reversed Na/K contents. In this study, we assessed the impact of IGL-1, UW, and Celsior (CS) ...solutions on islet isolation and transplant outcome.
We retrospectively analyzed 376 islet isolations from pancreases flushed and transported with IGL-1 (n=95), UW (n=204), or CS (n=77). We determined isolation outcome and β-cell function in vitro. Transplanted patients were divided into three groups depending on preservation solution of pancreas, and islet graft function was assessed by decrease in daily insulin needs, C-peptide/glucose ratios, β-scores, and transplant estimated function at 1- and 6-month follow-up.
IGL-1, UW, and CS groups were similar according to donor age, body mass index, and pancreas weight. There was no difference in islet yields between the three groups. Success rates, transplant rates, β-cell secretory function, and viability were similar for all three groups. We observed no difference in decreased insulin needs, C-peptide glucose ratios, β-scores, and transplant estimated function at 1- and 6-month follow-up between IGL-1, UW, and CS groups.
Our study shows that IGL-1 is equivalent to UW or CS solutions for pancreas perfusion and cold storage before islet isolation and transplantation.
Genome-wide association studies have identified susceptibility genes for development of type 2 diabetes. We aimed to examine whether a subset of these (comprising FTO, IDE, KCNJ11, PPARG and TCF7L2) ...were transcriptionally restricted to or enriched in human beta cells by sorting islet cells into alpha and beta - specific fractions. We also aimed to correlate expression of these transcripts in both alpha and beta cell types with phenotypic traits of the islet donors and to compare diabetic and non-diabetic cells.
Islet cells were sorted using a previously published method and RNA was extracted, reverse transcribed and used as the template for quantitative PCR. Sorted cells were also analysed for insulin and glucagon immunostaining and insulin secretion from the beta cells as well as insulin, glucagon and GLP-1 content. All five genes were expressed in both alpha and beta cells, with significant enrichment of KCNJ11 in the beta cells and of TCF7L2 in the alpha cells. The ratio of KCNJ11 in beta to alpha cells was negatively correlated with BMI, while KCNJ11 expression in alpha cells was negatively correlated with age but not associated with BMI. Beta cell expression of glucagon, TCF7L2 and IDE was increased in cells from islets that had spent more time in culture prior to cell sorting. In beta cells, KCNJ11, FTO and insulin were positively correlated with each other. Diabetic alpha and beta cells had decreased expression of insulin, glucagon and FTO.
This study has identified novel patterns of expression of type 2 diabetes susceptibility genes within sorted islet cells and suggested interactions of gene expression with age or BMI of the islet donors. However, expression of these genes in islets is less associated with BMI than has been found for other tissues.
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