JNK3 is abundant in insulin-secreting cells and protects against cytokine-induced apoptosis.

AIMS/HYPOTHESIS: In insulin-secreting cells, activation of the c-Jun NH(2)-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines. METHODS: Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei. RESULTS: JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective. CONCLUSIONS/INTERPRETATION: In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.

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

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-1beta exposure. CONCLUSIONS/INTERPRETATION: JNK signalling participates in islet isolation- and IL-1beta-induced apoptosis in rat islets. Furthermore, the system we used might be more generally applicable for the persistent blockage (several days) of pro-apoptotic 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.

Human high-density lipoprotein particles prevent activation of the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreatic beta cells.

AIMS/HYPOTHESIS: We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. MATERIALS AND METHODS: Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. CONCLUSIONS/INTERPRETATION: These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.

Circadian regulation of islet genes involved in insulin production and secretion.

Both transcription factors albumin site d-binding protein (DBP) and thyrotroph embryonic factor (TEF) are elements of the "cell-clock". Their circadian accumulation in suprachiasmatic nucleus (SCN) and peripheral tissues such as liver, kidney and lung is thought to participate in controlling circadian regulation of downstream genes. TEF and DBP control elements have never been investigated in the insulin-secreting cells, but impairment of the circadian rhythm of the beta-cells might be involved in the development of diabetic state as type 2 diabetics have lost daily temporal variations of insulin secretion. We investigated the expression pattern of TEF and DBP in insulin-secreting cells. TEF and DBP transcripts are expressed at extremely high levels in human pancreatic islets compared to other tissues, suggesting a potentially important circadian regulation of these cells. Both TEF and DPB accumulate in a circadian way in insulin-secreting cells after a serum shock known to restore circadian rhythms in cultured cells. In addition, the expression of islet-specific genes involved in glucose sensing (glucose transporter 2 (Glut2), glucokinase), insulin production (insulin) and secretion (migration inhibitory factor (MIF), somatostatin and syntaxin 1A) were modulated in the same daily rhythm as well. The circadian deregulation of these genes could therefore participate in the diabetic state development.