The systemic inflammatory response syndrome (SIRS) is triggered by lipopolysaccharide (LPS) from Gram-negative bacteria. Insulin was shown to have a protective role in SIRS related to sepsis. Lungs are particularly affected in this condition and provide a second wave of mediators/cytokines which amplifies SIRS. The aim of the present study was to investigate the effect of insulin on the signaling pathways elicited by LPS in alveolar macrophages (AMs) and its consequence in cellular response to LPS measured as production of tumor necrosis factor (TNF). To this purpose, resident AMs from male Wistar rats were obtained by lung lavage and stimulated by LPS (100 ng/mL). Insulin (1 mU/mL) was added 10 min before LPS. Activation (phosphorylation) of signaling molecules by LPS was analyzed by western blot, 30 min after LPS stimulation. TNF was measured in the AMs culture supernatants by bioassay using L-929 tumor cells. Relative to controls, LPS induced a significant increase in the activation of ERK (3.6-fold), p38 (4.4-fold), Tyr-326 Akt (4.7-fold), Ser-473 Akt (6.9-fold), PKCα (4.7-fold) and PKCδ (2.3-fold). Treatment of AMs with insulin before LPS stimulation, significantly reduced the activation of ERK (54%), p38 (48%), Tyr-326 Akt (64%), Ser-473 Akt (41%), PKCα (62%) and PKCδ (39%). LPS induced TNF production in AMs which was also inhibited by insulin (60%). These results show that insulin down-regulates MAPK, PI3K and PKCs and inhibits a downstream effect of LPS, TNF production, in rat AMs stimulated with LPS and suggest that the protective effect of insulin in sepsis could be through modulation of signal transduction pathways elicited by LPS in lung macrophages.
The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin down-regulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E2, on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-ĸB) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-ĸB p65 activation and inhibitor of kappa B (I-ĸB)α phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE2 by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE2, and, in parallel, NF-ĸB p65 activation and cytoplasmic I-ĸBα phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE2 production and Nuclear NF-ĸB p65 activation. Insulin also prevented cytoplasmic I-ĸBα phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-ĸB, possibly by blocking I-ĸBα degradation, and supresses the production of NO and PGE2, two molecules that contribute to septic shock.
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