Effects of different branched-chain amino acids supplementation protocols on the inflammatory response of LPS-stimulated RAW 264.7 macrophages.

Although branched-chain amino acids (BCAA) are commonly used as a strategy to recover nutritional status of critically ill patients, recent findings on their role as immunonutrients have been associated with unfavorable outcomes, especially in obese patients. The present study aimed to explore the effects of different BCAA supplementation protocols in the inflammatory response of LPS-stimulated RAW 264.7 macrophages. Cell cultures were divided into five groups, with and without BCAA supplementation, (2 mmol/L of each amino acid). Then, cell cultures followed three different treatment protocols, consisting of a pretreatment (PT), an acute treatment (AT), and a chronic treatment (CT) with BCAA and LPS stimulation (1 µg/mL). Cell viability was analyzed by MTT assay, NO production was assessed by the Griess reaction and IL-6, IL-10, TNF-α and PGE2 synthesis, was evaluated by ELISA. BCAA significantly increased cell viability in AT and CT protocols, and NO and IL-10 synthesis in all treatment protocols. IL-6 synthesis was only increased in PT and CT protocols. TNF-α and PGE2 synthesis were not altered in any of the protocols and groups. BCAA supplementation was able to increase both pro and anti-inflammatory mediators synthesis by RAW 264.7 macrophages, which was influenced by the protocol applied. Moreover, these parameters were significantly increased by isoleucine supplementation, highlighting a potential research field for future studies.

Macrophages from a type 1 diabetes mouse model present dysregulated Pl3K/AKT, ERK 1/2 and SAPK/JNK levels.

Diabetes causes dysregulation in signal transduction in immune cells leading to an impaired response to pathogens. Herein, we investigated the impact of type 1 diabetes (T1D) in bone marrow-derived macrophages (BMDM), using male non-diabetic and diabetic C57BL/6 mice (alloxan 60 mg/kg, i.v., CEUA/FCF/USP - 467). Diabetic BMDM expressed impaired phosphoinositide 3-kinase (PI3K), being lower p-PI3K p55 levels and higher levels of PI3K p110 alpha, whereas protein kinase B (PKB/Akt) (Ser-473 and Thr-308), extracellular signal-regulated kinases (ERK 1/2), and stress-activated protein kinase (SAPK/JNK) were enhanced compared to non-diabetic BMDM. Further evaluation of the responsiveness to lipopolysaccharide (LPS; 0.1 and 1 ug/mL), diabetic BMDM and peritoneal macrophage secreted dysregulated levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 levels. In 24 h, diabetic BMDM stimulated by LPS presented lower metabolic activity, with no differences in cell surveillance. Therefore, LPS re-stimulation (0.1 ug/mL) in diabetic BMDM resulted in higher secretion of TNF-α compared to non-diabetic BMDM. However, diabetic peritoneal macrophages secreted similar IL-6 levels in the first and additional 24 h of LPS stimulation. In general, our results demonstrated that diabetes exerts an impact in both BMDM and peritoneal macrophages ability to secrete cytokine under LPS stimulation.

High Glucose Environments Interfere with Bone Marrow-Derived Macrophage Inflammatory Mediator Release, the TLR4 Pathway and Glucose Metabolism.

Macrophages may be a crucial aspect of diabetic complications associated with the inflammatory response. In this study, we examined how hyperglycaemia, a common aspect of diabetes, modulates bone marrow-derived macrophages (BMDMs) under an inflammatory stimulus. To perform this study, BMDMs from non-diabetic and diabetic (60 mg/kg alloxan, i.v.) male C57BL/6 mice (CEUA/FCF/USP-488) were cultured under normal (5.5 mM) and high glucose (HG, 25 or 40 mM) conditions and stimulated or not stimulated with lipopolysaccharide (LPS, 100 ng/mL). Compared to the BMDMs from the normoglycaemic mice, the LPS-stimulated BMDMs from the diabetic mice presented reduced TLR4 expression on the cell surface, lower phagocytic capacity, and reduced secretion of NO and lactate but greater oxygen consumption and greater phosphorylation of p46 SAPK/JNK, p42 ERK MAPK, pAKT and pPKC-δ. When the BMDMs from the non-diabetic mice were cultured under high-glucose conditions and stimulated with LPS, TLR4 expression was reduced on the cell surface and NO and H2O2 levels were reduced. In contrast, the diabetic BMDMs cultured under high glucose conditions presented increased levels of lactate and reduced phosphorylation of AKT, PKC-δ and p46 SAPK/JNK but enhanced phosphorylation of the p46 subunit of SAPK/JNK after LPS stimulation. High glucose levels appear to modify macrophage behaviour, affecting different aspects of diabetic and healthy BMDMs under the same LPS stimulus. Thus, hyperglycaemia leaves a glucose legacy, altering the basal steady state of macrophages.

Colecalciferol regula os parâmetros hematológicos e a produção de citocinas pró-inflamatórias renais em camundongos diabéticos e nas células RAW 264. 7 / Colecalciferol regulates haematological parameters and the production of renal proinflammatory cytokines in diabetic mice and RAW 264. 7 cells

Os efeitos causados pelo tratamento em conjunto da insulina e do colecalciferol em indivíduos diabéticos não estão completamente elucidados. O presente trabalho avaliou o efeito de ambos os hormônios nos rins, no fígado, no coração e nos parâmetros hematológicos de camundongos machos (C57BL/6) sadios e diabéticos, bem como a ação do colecalciferol (in vitro) na resposta imunológica desenvolvida pelas células RAW 264.7 e pelos macrófagos peritoneais (MP) após estímulo com lipopolissacarídeo (LPS). Após dez dias da administração da aloxana (60 mg/kg), animais diabéticos exibiram redução do ganho de peso corporal e hiperglicemia quando comparados aos animais que receberam salina. No sétimo dia do período experimental, foi verificado que animais diabéticos que não receberam nenhum hormônio, em relação aos não diabéticos, exibiram redução do peso corporal, dos níveis de hemoglobina (Hb), hematócrito, hematimetria, insulina, TNF-α e IL-6 (coração) e aumento da glicemia, da relação peso corpóreo/peso rim esquerdo, das concentrações séricas de ureia, creatinina, Fosfatase Alcalina (FAL), Lactato desidrogenase (LDH) e lactato, fator de necrose tumoral (TNF)-α, interleucina (IL)-6 e IL-10 (no rim); o tratamento com insulina (1 UI/300 mg/dL glicemia), em relação aos animais diabéticos não tratados, promoveu aumento do peso corporal, das concentrações séricas de insulina e redução da glicemia, das concentrações séricas de ureia e da razão TNF-α/IL-10 (coração); o tratamento com colecalciferol (800 UI/dia), em relação aos animais diabéticos não tratados, promoveu aumento das concentrações séricas de 25-hidroxicolecalciferol [25(OH)D], Hb, hematócrito, hematimetria, IL-10 (coração) e reduziu IL-6, IL-10, TNF-α e EPO (rim); os animais diabéticos tratados com insulina, em relação aos animais diabéticos suplementados com colecalciferol apresentaram aumento do peso corpóreo, de ureia sérica, IL-6 e TNF-α (coração) e redução da glicemia, das concentrações séricas de lactato, de IL-6, TNF-α, IL-10 e EPO (rim); os animais -que receberam ambos os hormônios, em relação aos animais tratados com insulina, apresentaram aumento sérico de insulina e lactato; os animais diabéticos que receberam ambos os hormônios, em relação aos animais diabéticos tratados com colecalciferol, exibiram aumento sérico de 25(OH)D, de insulina, além da redução das concentrações de IL-10, da razão de TNF-α/IL-10 e TNF-α/IL-6 (coração); animais diabéticos que receberam ambos os hormônios, em relação aos diabéticos não suplementados com colecalciferol, exibiram: aumento de insulina sérica e redução das concentrações séricas de ureia e das razões renal e hepática de TNF-α/IL-6; células RAW 264.7 estimuladas pelo LPS e tratadas com 100 nM colecalciferol exibiram maior expressão da CYP27B1 e redução na liberação de mediadores inflamatórios quando comparadas ao grupo estimulado pelo LPS. Entretanto, não foi observado o mesmo efeito nos MP. Em conjunto, os resultados sugerem que: 1) em animais diabéticos, o colecalciferol pode modular parâmetros hematológicos e que a insulina pode melhorar a função renal, bem como a recuperação do peso corporal; 2) o colecalciferol pode ser metabolizado pelas células RAW 264.7 e modular a resposta imunológica desencadeada pelo LPS

The effects caused by the treatment of insulin and cholecalciferol in diabetic subjects are not completely elucidated. The present study evaluated the effect of both hormones on the kidneys, liver, heart and hematological parameters of healthy and diabetic male mice (C57BL/6), as well as the action of cholecalciferol (in vitro) on the immune response developed by the cells RAW 264.7 and peritoneal macrophages (MP) after stimulation with lipopolysaccharide (LPS). After ten days of alloxan administration (60 mg/kg), diabetic animals exhibited a reduction in body weight gain and hyperglycemia when compared to animals that received saline. On the seventh day of the experimental period, it was verified that diabetic animals that did not receive any hormones, in relation to non-diabetics, showed reduction of body weight, hemoglobin (Hb), hematocrit, hematimetry, insulin, TNF-α and IL- 6 (heart) and increased glycemia, body weight / left kidney weight, serum urea, creatinine, Phosphatase Alkaline, lactate dehydrogenase (LDH) and lactate levels, tumor necrosis factor (TNF) interleukin (IL) -6 and IL-10 (in the kidney); diabetic mice treated with insulin (1 IU / 300 mg/dL glycemia) in relation to untreated diabetic animals promoted increased body weight, serum insulin levels and blood glucose lowering, serum urea levels and TNF-α ratio / IL-10 (heart); diabetic animals treated with cholecalciferol (800 IU/day), in relation to untreated diabetic animals, exhibited increased serum levels of 25-hydroxycholecalciferol [25 (OH) D], Hb, hematocrit, hematimetry, IL-10 (heart) and reduced IL-6, IL-10, TNF-α and EPO (kidney);insulin-treated diabetic animals compared to diabetic animals supplemented with cholecalciferol exhibited an increase of body weight, serum urea, IL-6 and TNF-α (heart) and a reduction of glycaemia, serum lactate levels, IL-6, TNF- α, IL-10 and EPO (kidney); animals that received both hormones, compared to animals treated with insulin exhibited an increase of insulin and lactate serum levels; diabetic animals that received both hormones, compared to diabetic animals treated with cholecalciferol, exhibited an increase of 25(OH)D and insulin serum levels, and a reduction of IL-10, TNF-α/IL-10 and TNF-α/IL-6 ratios (heart); diabetic animals that received both hormones, compared to diabetic animals not supplemented with cholecalciferol, exhibited an increase of insulin and reduced urea serum levels and reduced renal and hepatic TNF-α/IL-6 ratios; LPS-stimulated RAW 264.7 cells and treated with 100 nM cholecalciferol exhibited greater CYP27B1 expression and reduced release of inflammatory mediators when compared to the LPS-stimulated group. However, the same effect was not observed in PM. Taken together, the results suggest that: 1) in diabetic animals, cholecalciferol may modulate hematological parameters and that insulin may improve renal function as well as recovery of body weight; 2) cholecalciferol can be metabolized by RAW 264.7 cells and modulate the immune response triggered by LPS

Hipovitaminose D em idosos institucionalizados tratados com anticonvulsivantes, uma associação frequente / Hypovitaminosis D in institutionalized elders treated with anticonvulsants, a common association

CONTEXTO: A população idosa apresenta alta prevalência de hipovitaminose D, sendo provável que, exposta ao uso de anticonvulsivantes, ocorra agravamento dessa condição.OBJETIVO: Avaliar a interferência do uso crônico de fármacos anticonvulsivantes nos níveis séricos de vitamina D em idosos institucionalizados com idade acima de 65 anos.MÉTODOS: Foram estudados 18 idosos institucionalizados tratados com anticonvulsivantes, por no mínimo 12 meses, comparados a 16 idosos não tratados.RESULTADOS: O estudo demonstrou que os dois grupos cursaram com deficiência de vitamina D, sendo mais pronunciada no grupo tratado com anticonvulsivantes. Embora não houvesse diferença estatisticamente significativa nos valores de paratormônio, nos idosos tratados foi observada uma tendência de níveis mais elevados, 53,44 ± 28,92 pg/ml em comparação aos idosos não tratados, 38,5 ± 10,08 pg/ml (P = 0,42). Foi observada diferença estatisticamente significativa entre os níveis séricos de 25-hidroxivitamina D nas pacientes do sexo feminino tratadas de 9,22 ± 3,80 ng/ml versus não tratadas, 18,78 ± 7,62 ng/ml (P = 0,03).CONCLUSÃO: Nossos achados sugerem que idosos institucionalizados apresentam menores níveis séricos de 25-hidroxivitamina D, configurando um estado de deficiência, e diferença significativa foi detectada nas mulheres tratadas com fármacos anticonvulsivantes.

BACKGROUND: Elderly people have a high prevalence of hypovitaminosis D, especially when they are exposed to anticonvulsants.OBJECTIVE: The present study evaluated the influence of chronic use of anticonvulsants on serum levels of vitamin D in institutionalized elders aged above 65 years.METHODS: Eighteen elderly subjects treated with anticonvulsants were studied for at least 12 months and compared to 16 untreated elders.RESULTS: Vitamin D deficiency was observed in both groups, but the group treated with anticonvulsants showed a more remarkable deficiency. Although there was no statistically significant difference in serum parathyroid hormone levels, elderly patients in treatment had a higher value (53.44 ± 28.92 pg/ml) compared to untreated elders (38.5 ± 10.8 pg/ml: p = 0.42). Statistically significant difference was observed between serum 25-hydroxyvitamin D in treated female patients (9.22 ± 3.80 ng/ml) compared to untreated female patients (18.78 ± 7.62 ng/ml: p = 0.03).DISCUSSION: The observed deficiency in both groups suggests that elderly subjects have lower serum 25-hydroxyvitamin D. Compared to untreated elderly women, elderly women treated with anticonvulsants showed a significantly lower serum level of vitamin D.