Glucose, palmitate and pro-inflammatory cytokines modulate production and activity of a phagocyte-like NADPH oxidase in rat pancreatic islets and a clonal beta cell line.
OBJECTIVE
Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47(phox) component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS).


METHODS
Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47(phox) production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays.


RESULTS
Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47(phox) protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47(phox) was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1beta-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47(phox). Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation.


CONCLUSIONS
NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise.
Morgan
D
D
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1524, 05508.900, São Paulo, Brazil.
Oliveira-Emilio
H R
HR
Keane
D
D
Hirata
A E
AE
Santos da Rocha
M
M
Bordin
S
S
Curi
R
R
Newsholme
P
P
Carpinelli
A R
AR
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D016207:Cytokines / Q000494:pharmacology*; D017931:DNA Primers; D005260:Female; D005947:Glucose / Q000494:pharmacology*; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000201:enzymology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism*; D019308:Palmitic Acid / Q000494:pharmacology*; D010586:Phagocytes / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species; D014162:Transfection
D016207:Cytokines; D017931:DNA Primers; D017382:Reactive Oxygen Species; D019308:Palmitic Acid; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D005947:Glucose