Diabetes confers an increased propensity to atherosclerosis. Inflammation is pivotal in atherogenesis, and diabetes is a proinflammatory state. Interleukin (IL)-6, in addition to inducing the acute-phase response, contributes to insulin resistance. Monocytes from type 2 diabetic patients secrete increased IL-6. The aim of this study was to examine molecular mechanisms for increased IL-6 release from monocytes under hyperglycemia. Monocytic cells (THP-1) were cultured in the presence of 5.5

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... l (normal) or 15 mmol/l (high) glucose and mannitol. Secreted IL-6, intracellular IL-6, and IL-6 mRNA were significantly increased with hyperglycemia (P < 0.001). Incubation of cells with inhibitors of reactive oxygen species failed to affect high-glucose-induced IL-6 release. Pan-protein kinase C (PKC) inhibitors significantly decreased high-glucose-induced IL-6 release. A specific inhibitor of p38 mitogen-activated protein kinase (MAPK; SB 202190), but not the extracellular signal-regulated kinase inhibitor PD98059, significantly decreased highglucose-induced IL-6 release. Furthermore, the PKC-␣/␤2 inhibitor decreased p38MAPK and the resulting highglucose-induced IL-6 release. Both antisense oligos to PKC-␤ and -␣ as well as small interfering RNA (siRNA) to PKC-␣ and -␤ resulted in significantly decreased highglucose-induced IL-6 release. Nuclear factor-B (NF-B) inhibitors significantly decreased IL-6 mRNA and protein. siRNA to PKC-␤ and -␣ also significantly decreased NF-B activity and IL-6 release. The combination was not additive to either siRNA alone, suggesting that they work through a common pathway. Thus, IL-6 release from monocytes under hyperglycemia appears to be mediated via upregulation of PKC, through p38MAPK and NF-B, resulting in increased mRNA and protein for IL-6. Thus, inhibition of PKC-␣ and -␤ can ameliorate the proinflammatory state of diabetes. Diabetes 54:85-91, 2005 RESEARCH DESIGN AND METHODS Cells from the human monocytic cell line THP-1 were obtained from the American Type Culture Collection. Endotoxin-and glucose-free RPMI-1640 media and fetal bovine serum were purchased from Gibco (Carlsbad, CA). Antibiotics, glutamine, phenylmethylsulfonyl fluoride, glucose, HEPES, protease inhibitor cocktail, Triton X-100, dithiothreitol, polyethylene glycolsuperoxide dismutase (PEG-SOD), and Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP), were from Sigma. Antibodies to PKC, PKC-␣, and PKC-␤II, were obtained from Santa Cruz Biotechnology. 2,2Ј,3,3Ј,4,4Ј-hexahydroxy-1,1Ј-biphenyl-6,6Ј-dimethanol dimethyl ether (HBDDE), apocynin, and diphenylene iodonium chloride (DPI) were obtained from Calbiochem, and Eli Lilly kindly provided LY379196. Polyvinylidene difluoride membranes and Tris-glycine gels were from Invitrogen. A bicinchoninic acid kit was obtained from Pierce. Enhanced chemiluminescence and PKC activity kits were purchased from Amersham Pharmacia. Oligonucleotides were purchased from Integrated DNA Technologies. Cell culture. THP-1 cells were maintained in endotoxin-free RPMI-1640 (containing 5.5 mmol/l glucose, 50 mol/l mercaptoethanol, 10% fetal bovine serum, 2 mmol/l glutamine, 1 mmol/l sodium pyruvate, and 10 mmol/l HEPES) and used for experiments between the third and fifth passages, as described From the