A insulina regula as vias de sinalização induzidas por LPS em macrófagos derivados de medula óssea / Insulin regulates LPS-induced signaling pathways in macrophages derived from bone marrow

Pacientes diabéticos apresentam alterações no sistema imunológico que promovem, em parte, maior suscetibilidade de infecções bacterianas. O tratamento com insulina melhora a sobrevida e reduz o número de infecções recidivas no paciente com diabetes mellitus do tipo 1 (DM1). Pouco se sabe sobre os efeitos do diabetes e a ação da insulina nos macrófagos. Neste trabalho, investigamos a proteína fosfatidilinositol-3-quinase (PI3K), proteína quinase B (Akt) e as quinases ativadas por mitógenos (MAPK) em macrófagos derivados de medula óssea (BMDM) e sua participação no estímulo por lipopolissacarídeo (LPS) na presença ou não do tratamento com insulina através da secreção dos mediadores inflamatórios fator de necrose tumoral (TNF)-α, interleucina (IL)-6 e IL-10. Observamos que os BMDM de animais com DM1 apresentam aumento da expressão da subunidade catalítica PI3K p110alpha com redução na subunidade reguladora PI3K p55 e maior expressão da fosforilação das proteínas Akt (Serina-473 e Treonina-308), quinase regulada por sinal extracelular (ERK) 1/2 e quinase ativada por estresse/quinase Jun-amino-terminal (SAPK/JNK) MAPK. Observou-se alteração na concentração das citocinas TNF-α, IL-6 e IL-10 no sobrenadante da cultura de BMDM dos animais diabéticos após estímulo com LPS, menor taxa de metabolismo mitocondrial, no entanto, sem resultar em morte celular, tampouco na expressão do receptor do tipo Toll 4 na membrana celular. Já o reestímulo destas células com LPS promoveu aumento na concentração de TNF-α sem alteração das demais citocinas. Além disto, o tratamento com insulina, simultaneamente ao estímulo com LPS, dos BMDM oriundos de animais diabéticos aumentou a concentração de TNF-α, IL-6, da fosforilação de p38, ERK 1/2 e SAPK/JNK MAPK, PI3K p55 e da Akt (Serina-473), o que não ocorreu nos BMDM dos animais não diabéticos sob a mesma condição. Este efeito foi abolido pela inibição farmacológica da PI3K e da ERK 1/2, resultando em novo aumento da concentração de TNF-α e IL-6. A análise conjunta destes resultados indica que a insulina, através da modulação das vias PI3K, Akt, ERK 1/2 e SAPK/JNK, amplifica o aumento da concentração de TNF-α e IL-6 sob estímulo com LPS

Diabetic patients present alterations in the immune system that promote in part a greater susceptibility of bacterial infections. Insulin treatment improves survival and reduces the number of recurrent infections in patients with type 1 diabetes mellitus (DM1). Little is known about the effects of diabetes and the action of insulin on macrophages. In this work we investigated the phosphatidylinositol-3-kinase (PI3K) / protein kinase B (Akt) and mitogen-activated kinase (MAPK) proteins in bone marrow-derived macrophages (BMDM) and their participation in lipopolysaccharide (LPS) or treatment with insulin through the secretion of inflammatory mediators tumor necrosis factor (TNF) -α, interleukin (IL) -6 and IL-10. We observed that BMDM of animals with DM1 increased PI3K p110alpha catalytic subunit expression with a reduction in the PI3K p55 regulatory subunit and increased expression of the phosphorylation of the Akt (Serine-473 and Threonine-308), extracellular signal regulated kinase (ERK) 1/2 and Jun-amino-terminal stress-kinase / kinase (SAPK / JNK) MAPK. Changes in the concentration of TNF-α, IL-6 and IL-10 cytokines in the supernatant of the BMDM culture of diabetic animals after stimulation with LPS were observed, possibly due to a lower rate of mitochondrial metabolism, however, without resulting in cell death , so little in the expression of the Toll 4 receptor on the cell membrane. The re-stimulation of these cells with LPS promoted an increase in TNF-α concentration without alteration of the other cytokines. In addition, insulin and simultaneously LPS stimulation of BMDM from diabetic animals increased the concentration of TNF-α, IL-6, phosphorylation of p38, ERK 1/2 and SAPK / JNK MAPK, PI3K p55 and Akt (Serine-473), which did not occur in the BMDM of non-diabetic animals under the same condition. This effect was abolished by pharmacological inhibition of PI3K and ERK 1/2, resulting in a further increase in the concentration of TNF-α and IL-6. The analysis of these results indicate that insulin by modulating the PI3K, Akt, ERK 1/2 and SAPK / JNK pathways amplifies the concentration levels of TNF-α and IL-6 under stimulation with LPS

Transcription, expression and tissue binding in vivo of INGAP and INGAP-related peptide in normal hamsters.

We studied islet neogenesis-associated protein (INGAP) transcription and its immunocytochemical presence in and binding in vivo of (125)I-tyrosylated INGAP pentadecapeptide ((125)I-T-INGAP-PP) to different normal male hamster tissues. (125)I-T-INGAP-PP was injected intraperitoneally with or without unlabeled T-INGAP-PP (0-1 mg/100 g bw), drawing blood samples at different times after injection; radioactivity was measured in serum, brain, skeletal muscle, dorsal root ganglia, liver, kidney, small intestine and pancreas samples, expressing results as organ:serum ratio. INGAP transcription (RT-PCR) and immunopositive cells were investigated in liver, kidney, brain, small intestine and pancreas. Total serum radioactivity increased progressively as a function of time; whereas 71% of this activity was displaced by unlabeled T-INGAP-PP at 5, 10 and 20 min, only 9% was at 60 min. Only liver, pancreas and small intestine specifically bound (125)I-T-INGAP-PP. The pancreas tissue dose-response curve showed a 50% displacement at 3.9x10(4) ng/100 g bw, suggesting a low binding affinity of its receptor. INGAP-mRNA was only identified in pancreatic islets and exocrine tissue. Our results suggest that INGAP transcription/expression is probably restricted to pancreas cells exerting its effect in a paracrine fashion. INGAP would be released and circulate bound to a serum protein from where it is bound and inactivated by the liver. Tissue binding could also explain INGAP's immunocytochemical presence in small intestine, where it could affect epithelial cell turnover.