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, 7 (1), e2065

Inhibition of the MAP3 Kinase Tpl2 Protects Rodent and Human β-Cells From Apoptosis and Dysfunction Induced by Cytokines and Enhances Anti-Inflammatory Actions of exendin-4

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Inhibition of the MAP3 Kinase Tpl2 Protects Rodent and Human β-Cells From Apoptosis and Dysfunction Induced by Cytokines and Enhances Anti-Inflammatory Actions of exendin-4

E M Varin et al. Cell Death Dis.

Abstract

Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells.

Figures

Figure 1
Figure 1
Tpl2 is expressed in β-cells and rodent pancreatic islets and activated by proinflammatory cytokines. Tpl2 protein expression in (a) INS-1E cell line, mouse, rat and human islets and macrophage RAW264.7 cell line; or in (c) INS-1E treated for the indicated period of time with a cytokine mix (CK); or in (d) INS-1E treated for 20 min with glucose (Glc, 10 mM). (b) Tpl2 phosphorylation (Ser400) in INS-1E stimulated for 10 min with the CK. (e and f) Western blot analysis of phosphorylated and total proteins for ERK1/2 and p90RSK in INS-1E cells, pretreated without (black bars) or with (white bars) a Tpl2 inhibitor (Tpl2-I, 3 μM) for 2 h and then unstimulated (Basal) or stimulated with CK (e) or Glc 10 mM (f). (g and h) Western blot analysis of phosphorylated and total proteins for ERK1/2 in mouse islets (g) or human islets (h) treated with a Tpl2 inhibitor as in (e), and stimulated with CK. Representative immunoblots and quantification of three to six independent experiments are shown and expressed as a percentage of the ratio of p-Tpl2 to total Tpl2 (b), of Tpl2 to β-actin protein amount in untreated cells (c and d) or as ratio of phosphorylated to total protein amount and fold of phosphorylation over basal in cells without treatment (e–h). Data are presented as mean±S.E.M. *P<0.05, **P<0.01, and ***P<0.001 versus corresponded indicated controls (t-test (b, d) or one-way analysis of variance (c, eh))
Figure 2
Figure 2
The siRNA-mediated silencing of Tpl2 in INS-1E β-cells decreases ERK1/2 activation induced by cytokines without affecting glucose effect. (a) Western blot analysis of Tpl2 expression with β-actin as loading control in INS-1E cells treated with control (Ctr) or Tpl2 siRNA (Tpl2). (b and c) Western blot analysis of phosphorylated and total proteins for ERK1/2 in INS-1E cells treated with Ctr or Tpl2 siRNA and incubated without (Basal) or with cytokines (CK) (b) or with glucose (Glc: 10 mM) (c) for 20 min in KRB buffer. Representative immunoblots and quantification of four to five independent experiments are shown and expressed as a percentage of the ratio of Tpl2 to β-actin protein amount in untreated cells (a), or as ratio of phosphorylated to total protein amount and fold of phosphorylation over basal in cells without treatment (b and c). Data are presented as mean±S.E.M. ***P<0.001 versus corresponding indicated controls (t-test (a) or one-way analysis of variance (b and c))
Figure 3
Figure 3
Tpl2 expression is increased by prolonged exposure of β-cells and human islets to inflammatory cytokines. (a) Tpl2 protein expression in INS-1E treated for the indicated period of time with interleukin-1β alone (IL-1β) (n=5). (b) Same as (a) except than cells were treated with a cytokine mix (CK) (n=6). (c) Tpl2 protein expression in human islets treated for 72 h with CK (n=4). Representative immunoblots and quantification of four to six independent experiments are shown and expressed as mean±S.E.M. of Tpl2 relative to β-actin. *P<0.05, **P<0.01 and ***P<0.001 versus the corresponding indicated controls (t-test (c) or one-way analysis of variance (a, b))
Figure 4
Figure 4
Tpl2 inhibition protects INS-1E β-cells from cytokine-induced apoptosis. (a) Level of expression of the cleaved forms of caspase-3 and PARP in INS-1E cells treated without or with Tpl2 inhibitor (Tpl2-I, 3 μM) and in the absence (Control, Ctrl) or in the presence of IL-1β (20 ng/ml, 48 h, n=3). (b) Western blot analysis of phosphorylated and total proteins for JNK and p38 in INS-1E cells treated as in (a). (c) Level of the cleaved form of caspase-3 in INS-1E cells incubated without or with the indicated cytokines alone or in combination for 24 h. (d) Expression levels of the cleaved forms of caspase-3 and PARP in INS-1E cells treated without or with Tpl2 inhibitor as in (a) and incubated without (Ctrl) or with a cytokine mix (CK) (n=10). (e) Level of expression of the cleaved and total forms of caspase-3 in INS-1E cells pretreated without or with Tpl2 inhibitor (Tpl2-I, 5 μM) for 1 h and exposed for 24 h to a control medium (Ctrl M) or a conditioned medium (Cond. M) from RAW macrophages activated with LPS (0.5 ng/ml, 24 h, n=3). Representative immunoblots and mean±S.E.M. of 3 to 10 independent experiments are shown. Data are expressed as ratio of cleaved caspase-3 or cleaved PARP to loading control: β-actin (a, c and d) or HSP90 (e) and normalized to cells without treatment. *P<0.05, **P<0.01, and ***P<0.001 versus the corresponding indicated control by one-way ANOVA
Figure 5
Figure 5
Tpl2 inhibition protects mouse islets from cytokine-induced death and alteration of glucose-induced insulin secretion. (a) Measurement of caspase-3/7 activity in mouse islets pretreated for 2 h without or with Tpl2-I (3 μM), and then incubated for 24 h without (Control, Ctrl) or with a cytokine mix (CK). (b) Measurement of DNA fragmentation in mouse islets treated as in (a). (c) Glucose-stimulated insulin secretion in mouse islets treated as in (a and b) (inclined and horizontal hatched boxes: glucose 3 mM (G3), white and black solid boxes: glucose 15 mM (G15)). Each column represents mean±S.E.M. of 6 to 8 mice analyzed. *P<0.05, and **P <0.01 versus relative indicated controls by one-way ANOVA. (d and e) Intracellular calcium concentration ([Ca2+]c) changes monitored in perfused control islets incubated without or with 3 μM of Tpl2-I (d) or in perfused islets treated for 24 h with the cytokine mix, and incubated without or with Tpl2-I (3 μM) during 2 h before and during the CK treatment (e). Data are mean±S.E.M. of three experiments with three mice per group. (f) Correlation between insulin secretion and [Ca2+]c
Figure 6
Figure 6
Combined use of Tpl2 inhibitor and GLP-1 analogs produces a powerful anti-apoptotic effect on INS-1E β-cells and in human islets and protects human islets from cytokine-induced alteration of glucose-induced insulin secretion. (a) Level of expression of the cleaved and total forms of caspase-3 and PARP and phosphorylated and total form of p38 in INS-1E cells treated without or with Tpl2 inhibitor (Tpl2-I, 3 μM), and with or without Exendin-4 (Ex-4, 20 nM) and in the absence (Control, Ctrl) or in the presence of the cytokine mix (CK, 24 h). Representative immunoblots and mean±S.E.M. of 3 to 10 independent experiments are shown. Data are expressed as ratio of cleaved caspase-3 or cleaved PARP to loading control and normalized to cells without treatment. (b) Measurement of DNA fragmentation in supernatant of human islets pretreated without or with the Tpl2-I (3 μM) and Exendin-4 (Ex-4, 20 nM) for 2 h and then cultured in the absence (Control, Ctrl) or in presence of the cytokine mix (CK) for 72 h in the presence or absence of Tpl2-I (3 μM) and Ex-4 (20 nM). (c) Glucose-stimulated insulin secretion in the human islets treated as in (b). Black boxes represent glucose 2.8 mM (G2.8) and white boxes represent glucose 20 mM (G20). (d) Stimulatory index of insulin secretion from the same conditions as (c). Each column represents the mean±S.E.M. of 15 replicates (5 donors, each experiment in triplicate). *P<0.05, **P<0.01, and ***P<0.001 between low and high glucose (c) or versus related indicated controls (a, b and d) by one-way ANOVA

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