The c-Jun N-terminal kinase JNK participates in cytokine- and isolation stress-induced rat pancreatic islet apoptosis

Abstract

Aims/hypothesis The protocols used for the preparation of human pancreatic islets immediately induce a sustained and massive activation of the c-Jun-N-terminal kinase (JNK). JNK, which participates in apoptosis of insulin-secreting cells, is activated by mechanical stresses, as well as by exposure to pro-inflammatory cytokines. Here, we investigated whether the delivery of a protease-resistant JNK inhibitory peptide (D-JNKI) through a protein transduction system during pancreatic digestion might impair JNK signalling throughout the transplantation procedure. Methods Rat pancreases were treated with D-JNKI through the pancreatic duct and cells then isolated by enzymatic digestion. Protein extracts were prepared to determine JNK activity by kinase assays and total RNA was extracted to measure gene expressions by a Light-Cycler technique. Cell apoptosis rate was determined by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and by scoring cells displaying pycnotic nuclei.Results Our data establish that the peptide transduction system used here efficiently transfects islets, allowing for stable in vivo (up to 2 days) transfection of human islets transplanted under the kidney capsule. Further, D-JNKI decreases intracellular JNK signalling during isolation and following cytokine exposure in both human and rat islets, as measured by kinase assays and reduced c-fos expression; D-JNKI also confers protection against apoptosis induced during the rat islet preparation and subsequent to IL-1β exposure. Conclusions/interpretation JNK signalling participates in islet isolation-and IL-1β-induced apoptosis in rat islets. Furthermore, the system we used might be more generally applicable for the persistent blockage (several days) of proapoptotic pathways in the transplanted islets; this days-long protection might potentially be an absolute prerequisite to help transplanted islets better survive the first wave of the non-specific inflammatory attack.

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

“…This suggests that the protective effect of JNK in the first overnight culture might be very different from the one observed after cytokine exposure in culture and might involve factors responsible for the protection of islet disruption. It is conceivable that the observed effects of L-JNKI could be markedly improved by use of the protease-resistant D-JNKI, which has recently been shown to be safe and effective in human islets even when used at higher concentrations [6].…”

“…JNK is markedly activated in several tissues from diabetic mice [5]. Moreover, JNK inhibition markedly improved rat islet viability [6] and islet engraftment in a syngeneic animal model [6]. Thus, targeting JNK may improve functional beta cell mass via several mechanisms.…”

“…We found that anti-oxidative stress proteins such as haeme oxygenase-1 [10] and anti-apoptotic proteins such as B cell lymphoma associated X protein (Bcl-XL) [11] can be effectively delivered to human islets when coupled to a TAT-derived PTD. Similarly, it has recently been shown that a protease-resistant form of TAT-derived PTD inhibitor of JNK (D-JNKI) can effectively transduce rat and human islets [6]. However, PTDs have a length-and dose-dependent toxicity [12] particularly when used in the D-isoform [6,8].…”

“…However, PTDs have a length-and dose-dependent toxicity [12] particularly when used in the D-isoform [6,8]. Although D-JNKI appears safer in human than in rodent islets [6], further investigations are warranted prior to their introduction to clinical islet transplantation.…”

C/EBP homologous protein contributes to cytokine-induced pro-inflammatory responses and apoptosis in β-cells

“…The mechanisms regulating cytokine-mediated b-cell apoptosis and pro-inflammatory responses are intricate and include, but are not restricted to, the activation of the transcription factors nuclear factor-kB (NF-kB) and STAT-1, the c-Jun N-terminal kinases (JNK), endoplasmic reticulum (ER) stress pathways and the intrinsic mitochondrial apoptotic pathways. [3][4][5][6][7] NF-kB activation is due to cytokine-dependent activation of the inhibitor of k-light polypeptide gene enhancer in B-cells (IkB) kinase (IKK) complex (IKKa, IKKb and IKKg), which leads to IkB phosphorylation, ubiquitination and proteasomal degradation, releasing NF-kB for migration to the nucleus where it can drive gene expression. 8 NF-kB stimulates the expression of several chemokines and cytokines in b-cells, thereby contributing to islet inflammation in T1D.…”