Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells.
OBJECTIVE
The immune-mediated elimination of pancreatic beta cells in type 1 diabetes involves release of cytotoxic cytokines such as IL-1beta and IFNgamma, which induce beta cell death in vitro by mechanisms that are both dependent and independent of nitric oxide (NO). Nuclear factor kappa B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone deacetylases (HDAC), and positive effects of HDAC inhibition have been obtained in several inflammatory diseases. Thus, the aim of this study was to investigate whether HDAC inhibition protects beta cells against cytokine-induced toxicity.


METHODS
The beta cell line, INS-1, or intact rat islets were precultured with HDAC inhibitors suberoylanilide hydroxamic acid or trichostatin A in the absence or presence of IL-1beta and IFNgamma. Effects on insulin secretion and NO formation were measured by ELISA and Griess reagent, respectively. iNOS levels and NFkappaB activity were measured by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone-DNA complex ELISA.


RESULTS
HDAC inhibition reduced cytokine-mediated decrease in insulin secretion and increase in iNOS levels, NO formation and apoptosis. IL-1beta induced a bi-phasic phosphorylation of inhibitor protein kappa Balpha (IkappaBalpha) with the 2nd peak being sensitive to HDAC inhibition. No effect was seen on IkappaBalpha degradation and NFkappaB DNA binding.


CONCLUSIONS
HDAC inhibition prevents cytokine-induced beta cell apoptosis and impaired beta cell function associated with a downregulation of NFkappaB transactivating activity.
Larsen
L
L
Steno Diabetes Center, 2 Niels Steensens Vej, 2820, Gentofte, Denmark.
Tonnesen
M
M
Ronn
S G
SG
Størling
J
J
Jørgensen
S
S
Mascagni
P
P
Dinarello
C A
CA
Billestrup
N
N
Mandrup-Poulsen
T
T
D000818:Animals; D017209:Apoptosis; D002470:Cell Survival; D016207:Cytokines / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000737:chemistry / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction
D016207:Cytokines; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D016328:NF-kappa B; D011994:Recombinant Proteins; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D006655:Histone Deacetylases