Interleukin-1β-induced Rat Pancreatic Islet Nitric Oxide Synthesis Requires Both the p38 and Extracellular Signal-regulated Kinase 1/2 Mitogen-activated Protein Kinases

Abstract

Interleukin-1␤ (IL-1␤) is cytotoxic to rat pancreatic ␤-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1␤ induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1␤ was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1␤-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1␤-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1␤ but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1␤-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1␤-mediated ␤-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1␤ in ␤-cells.

Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

“…Islet isoloation and preculture Primary neonatal rat islets were isolated from 3-to 6-day-old Wistar Furth Rats (Charles River, Sulzfeldt, Germany) and precultured for 7 days as previously described [21,22] in complete medium (RPMI 1640 with 100 U/ml penicillin and 100 μg/ml streptomycin) supplemented with 10% newborn calf serum (Invitrogen/Gibco).…”

IL-1β-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

“…Rat islet isolation and pre-culture Rat islet isolation and pre-culture were performed as described previously [5]. Pancreata from 5-to 7-day-old Wistar Furth rats (Taconic, Rye, Denmark) were collagenase-digested and islets were isolated.…”

“…Islet, neuron and INS-1 cell lysis and measurement of protein concentration After exposure, cells were lysed for 30 min as described by Larsen et al [5]. The protein concentrations were measured by the Bradford method according to the manufacturer's instructions (BioRad, Hercules, CA, USA).…”

“…In vitro kinase assay was performed as described previously [5]. Whole-cell lysates were incubated with a reaction buffer containing glutathione-S-transferase (GST)-Elk1, GST-c-Jun, and GST-heat-shock protein (Hsp)25 (substrates for ERK, JNK, and the p38-activated kinase MAPKAPKII, respectively) and [γ-32 P]ATP for 30 min at 30°C.…”

“…The pro-apoptotic signalling pathways induced by IL-1β involve the family of mitogen-activated protein kinases (MAPK) as well as the nuclear factor-κB (NF-κB) and protein kinase C (PKC) pathways [2]. The MAPK family consists of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (p38), all of which are activated after phosphorylation by IL-1β in beta cells [5]. JNK especially is in focus because inhibition of this MAPK blocks IL-1β-mediated beta cell death in vitro [6].…”