High glucose increases nitric oxide generation in lipopolysaccharide-activated macrophages by enhancing activity of protein kinase C-α/δ and NF-κB.
OBJECTIVE
Although several mechanisms by which hyperglycemia modulate inflammation have been proposed, it remains unclear how hyperglycemia regulates inflammation induced by lipopolysaccharide (LPS).


METHODS
We hypothesized that hyperglycemia might interplay with LPS to modulate the generation of an inflammatory mediator. RAW 264.7 macrophages cultured in medium containing either normal glucose (5.5-mM) or high glucose (HG) (15- and 25-mM) were treated with LPS. The nitric oxide (NO) generation, inducible NO synthase (iNOS) expression and cytokine release were then quantified by Griess reaction, western blot, and enzyme-linked immunosorbent assay (ELISA) respectively. The effect of HG on the activation of kinase and Nuclear Factor-Kappa B (NF-κB) were measured by western blot and NF-κB reporter assay respectively.


RESULTS
Without LPS stimulation, HG alone did not induce NO generation and cytokine secretion; but LPS-induced NO generation, iNOS expression, and interleukin-1beta (IL-1β) secretion were higher in HG-cultured cells than in normal glucose-cultured cells. In contrast, LPS-induced interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) secretion were lower in HG-cultured cells than in normal glucose-cultured cells. Furthermore, HG increased iNOS expression and NO generation by enhancing phosphorylation levels of protein kinase C-alpha (PKC-α), protein kinase C-delta (PKC-δ), and p38 phosphorylation and NF-κB transcriptional activity.


CONCLUSIONS
This study revealed a possible role of PKC-α and PKC-δ potentially involved in diabetes-promoted inflammation.
Institute of Biotechnology, National Ilan University, Ilan, Taiwan.
Kuo-Feng
KF
Hua
Szu-Hsuan
SH
Wang
Wei-Chih
WC
Dong
Chai-Yi
CY
Lin
Chen-Lung
CL
Ho
Tzu-Hua
TH
Wu
D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D008070:Lipopolysaccharides; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000378:metabolism*
D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose