Extracellular matrix remodeling and matrix metalloproteinase inhibition in visceral adipose during weight cycling in mice.

Extracellular matrix (ECM) remodeling is necessary for a health adipose tissue (AT) expansion and also has a role during weight loss. We investigate the ECM alteration during weight cycling (WC) in mice and the role of matrix metalloproteinases (MMPs) was assessed using GM6001, an MMP inhibitor, during weight loss (WL). Obesity was induced in mice by a high-fat diet. Obese mice were subject to caloric restriction for WL followed by reintroduction to high-fat diet for weight regain (WR), resulting in a WC protocol. In addition, mice were treated with GM6001 during WL period and the effects were observed after WR. Activity and expression of MMPs was intense during WL. MMP inhibition during WL results in inflammation and collagen content reduction. MMP inhibition during WL period interferes with the period of subsequent expansion of AT resulting in improvements in local inflammation and systemic metabolic alterations induced by obesity. Our results suggest that MMPs inhibition could be an interesting target to improve adipose tissue inflammation during WL and to support weight cyclers.

Deferoxamine Interference in Fibro-inflammation: Additional Action in Control of Obese Adipose Tissue Dysfunction.

INTRODUCTION: Several studies demonstrated that deferoxamine, an iron chelator, can improve inflammatory alterations in adipose tissue induced by obesity. Obesity alterations in adipose tissue are also associated with tissue remodeling, and deferoxamine has anti-fibrosis action previously described in sites like the skin and liver. METHODS: In this work, we analyzed deferoxamine effects on adipose tissue fibro-inflammation during obesity induced by diet in mice. in vitro approaches with fibroblasts and macrophages were also carried out to elucidate deferoxamine activity. RESULTS: Our results demonstrated that in addition to exerting anti-inflammatory effects, reducing the cytokine production in adipose tissue of obese mice and by human monocyte differentiated in macrophage in vitro, deferoxamine can alter metalloproteinases expression and extracellular matrix production in vivo and in vitro. CONCLUSION: Deferoxamine could be an alternative to control fibro-inflammation in obese adipose tissue, contributing to the metabolic improvements previously described.

Infliximab modifies mesenteric adipose tissue alterations and intestinal inflammation in rats with TNBS-induced colitis.

OBJECTIVE: Infliximab is a monoclonal anti-TNF-α antibody that is used therapeutically to treat Crohn's disease (CD). High levels of pro-inflammatory cytokines, especially TNF-α, have been observed in the gastrointestinal tract of CD patients and were associated with alterations in the mesenteric adipose tissue, which also contributed to the high levels of adipokine release. The authors used a rat model of colitis that produces mesenteric adipose tissue alterations that are associated with intestinal inflammation to study the effects that infliximab treatment has on adipokine production, morphological alterations in adipose tissue and intestinal inflammation. MATERIAL AND METHODS: The ability of infliximab to neutralize rat TNF-α was evaluated in vitro using U937 cells. Colitis was induced by repeated intracolonic trinitrobenzene sulfonic acid instillations and was evaluated by macroscopic score, histopathological analysis, myeloperoxidase activity, TNF-α and IL-10 expression as well as iNOS (inducible NO synthase) expression and JNK phosphorylation in colon samples. The alterations in adipose tissue were assessed by TNF-α, IL-10, leptin, adiponectin and resistin levels as well as adipocyte size and peroxisome proliferator-activated receptor (PPAR)-γ expression. RESULTS: Infliximab treatment controlled intestinal inflammation, which reduced lesions and neutrophil infiltration. Inflammatory markers, such as iNOS expression and JNK phosphorylation, were also reduced. In mesenteric adipose tissue, infliximab increased the production of IL-10 and resistin, which was associated with the restoration of adipocyte morphology and PPAR-γ expression. CONCLUSIONS: Our results suggest that infliximab could contribute to the control of intestinal inflammation by modifying adipokine production by mesenteric adipose tissue.

Perinodal adipose tissue and mesenteric lymph node activation during reactivated TNBS-colitis in rats.

BACKGROUND: Colitis induced by trinitrobenzene sulfonic acid (TNBS) with reactivation is a good experimental model for studying inflammatory bowel disease pathogenesis and appropriate therapeutics. This experimental model allows the induction of colitis relapse and remission periods and the establishment of chronic disease features, such as the mesenteric adipose tissue alterations observed in Crohn's disease. Lymph node activation and the role of perinodal adipose tissue (PAT) have been poorly studied in this model. Thus, a study of the interactions of lymph nodes and PAT could help to elucidate the mechanisms behind IBD pathogenesis. AIMS: The purpose of this study was to examine lymph nodes and PAT alterations during reactivated TNBS-colitis in Wistar rats. METHODS: In this study, the alterations of PAT and lymph node cells during experimental colitis, induced by repeated intracolonic TNBS instillations, were evaluated, focusing on fatty acid and adipocytokine profile analysis and cytokines production, respectively. RESULTS AND CONCLUSION: Fatty acid analysis of PAT reveals an increase of ω-6 polyunsaturated fatty acids during colits, such as linoleic acid, gamma-linolenic acid and arachidonic acid. ω-6 arachidonic acid was not increased in lymph node cells or serum. PAT also produces elevated levels of pro- and anti-inflammatory adipokines during colitis. Lymph node cells release high levels of IFN-γ and TNF-α but not IL-10, characterizing the predominant Th-1 response associated with this disease. Nevertheless, T cells from animals with colitis demonstrated increased IFN-γ production via a COX-2-dependent mechanism after supplementation with ω-6 arachidonic acid, suggesting that PAT modification could contribute to the lymph node cell activation observed during colitis.

Effects of iron supplementation in mice with hypoferremia induced by obesity.

Iron is an important micronutrient, but it can also act as a dangerous element by interfering with glucose homeostasis and inflammation, two features that are already disturbed in obese subjects. In this work, we study the effects of systemic iron supplementation on metabolic and inflammatory responses in mice with hypoferremia induced by obesity to better characterize whether iron worsens the parameters that are already altered after 24 weeks of a high-fat diet (HFD). Mice were maintained on a control diet or a HFD for 24 weeks and received iron-III polymaltose (50 mg/kg/every 2 days) during the last two weeks. Glucose homeostasis (basal glucose and insulin test tolerance) and systemic and visceral adipose tissue (VAT) inflammation were assessed. Iron levels were measured in serum. The Prussian blue reaction was used in isolated macrophages to detect iron deposition. Iron supplementation resulted in an increased number of VAT macrophages that were positive for Prussian blue staining as well as increased serum iron levels. Systemic hepcidin, leptin, resistin, and monocyte chemoattractant protein-1 (MCP-1) levels were not altered by iron supplementation. Local adipose tissue inflammation was also not made worse by iron supplementation because the levels of hepcidin, MCP-1, leptin, and interleukin (IL)-6 were not altered. In contrast, iron supplementation resulted in an increased production of IL-10 by adipose tissue and VAT macrophages. Leukocytosis and VAT plasminogen activator inhibitor-1 (PAI-1) level were reduced, but insulin resistance was not altered after iron supplementation. In conclusion, systemic iron supplementation in mice with hypoferremia induced by obesity did not worsen inflammatory marker or adipose tissue inflammation or the metabolic status established by obesity. Iron deposition was observed in adipose tissue, mainly in macrophages, suggesting that these cells have mechanisms that promote iron incorporation without increasing the production of inflammatory mediators.

Role of pentoxifylline in non-alcoholic fatty liver disease in high-fat diet-induced obesity in mice.

AIM: To study pentoxifylline effects in liver and adipose tissue inflammation in obese mice induced by high-fat diet (HFD). METHODS: Male swiss mice (6-wk old) were fed a high-fat diet (HFD; 60% kcal from fat) or AIN-93 (control diet; 15% kcal from fat) for 12 wk and received pentoxifylline intraperitoneally (100 mg/kg per day) for the last 14 d. Glucose homeostasis was evaluated by measurements of basal glucose blood levels and insulin tolerance test two days before the end of the protocol. Final body weight was assessed. Epididymal adipose tissue was collected and weighted for adiposity evaluation. Liver and adipose tissue biopsies were homogenized in solubilization buffer and cytokines were measured in supernatant by enzyme immunoassay or multiplex kit, respectively. Hepatic histopathologic analyses were performed in sections of paraformaldehyde-fixed, paraffin-embedded liver specimens stained with hematoxylin-eosin by an independent pathologist. Steatosis (macrovesicular and microvesicular), ballooning degeneration and inflammation were histopathologically determined. Triglycerides measurements were performed after lipid extraction in liver tissue. RESULTS: Pentoxifylline treatment reduced microsteatosis and tumor necrosis factor (TNF)-α in liver (156.3 ± 17.2 and 62.6 ± 7.6 pg/mL of TNF-α for non-treated and treated obese mice, respectively; P < 0.05). Serum aspartate aminotransferase levels were also reduced (23.2 ± 6.9 and 12.1 ± 1.6 U/L for non-treated and treated obese mice, respectively; P < 0.05) but had no effect on glucose homeostasis. In obese adipose tissue, pentoxifylline reduced TNF-α (106.1 ± 17.6 and 51.1 ± 9.6 pg/mL for non-treated and treated obese mice, respectively; P < 0.05) and interleukin-6 (340.8 ± 51.3 and 166.6 ± 22.5 pg/mL for non-treated and treated obese mice, respectively; P < 0.05) levels; however, leptin (8.1 ± 0.7 and 23.1 ± 2.9 ng/mL for non-treated and treated lean mice, respectively; P < 0.05) and plasminogen activator inhibitor-1 (600.2 ± 32.3 and 1508.6 ± 210.4 pg/mL for non-treated and treated lean mice, respectively; P < 0.05) levels increased in lean adipose tissue. TNF-α level in the liver of lean mice also increased (29.6 ± 6.6 and 75.4 ± 12.6 pg/mL for non-treated and treated lean mice, respectively; P < 0.05) while triglycerides presented a tendency to reduction. CONCLUSION: Pentoxifylline was beneficial in obese mice improving liver and adipose tissue inflammation. Unexpectedly, pentoxifylline increased pro-inflammatory markers in the liver and adipose tissue of lean mice.

Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats.

AIM: To investigate hepcidin expression, interleukin-6 (IL-6) production and iron levels in the rat colon in the presence of trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: In rats, we evaluated the severity of colitis induced by repeated TNBS administration using macroscopic and microscopic scoring systems and myeloperoxidase activity measurements. The colonic levels of hepcidin, tumor necrosis factor alpha (TNF-α), IL-10 and IL-6 were measured by Enzyme-Linked Immunosorbent Assay, and hepcidin-25 expression and iron deposition were analyzed by immunohistochemistry and the Prussian blue reaction, respectively. Stat-3 phosphorylation was assessed by Western blot analysis. Hematological parameters, iron and transferrin levels, and transferrin saturation were also measured. Additionally, the ability of iron, pathogen-derived molecules and IL-6 to induce hepcidin expression in HT-29 cells was evaluated. RESULTS: Repeated TNBS administration to rats resulted in macroscopically and microscopically detectable colon lesions and elevated colonic myeloperoxidase activity. Hepcidin-25 protein levels were increased in colonic surface epithelia in colitic rats (10.2 ± 4.0 pg/mg protein vs 71.0 ± 8.4 pg/mg protein, P < 0.01). Elevated IL-6 levels (8.2 ± 1.7 pg/mg protein vs 14.7 ± 0.7 pg/mg protein, P < 0.05), TNF-α levels (1.8 ± 1.2 pg/mg protein vs 7.4 ± 2.1 pg/mg protein, P < 0.05) and Stat-3 phosphorylation were also observed. Systemic alterations in iron homeostasis, hepcidin levels and anemia were not detected in colitic rats. Iron deposition in the colon was only observed during colitis. Hepcidin gene expression was increased in HT-29 cells after IL-6 and lipopolysaccharide [a toll-like receptor 4 (TLR-4) ligand] treatment. Deferoxamine, ferric citrate and peptidoglycan (a TLR-2 ligand) were unable to alter the in vitro expression of hepcidin in HT-29 cells. CONCLUSION: Colitis increased local hepcidin-25 expression, which was associated with the IL-6/Stat-3 signaling pathway. An increase in local iron sequestration was also observed, but additional studies are needed to determine whether this sequestration is a defensive or pathological response to intestinal inflammation.

Mice that are fed a high-fat diet display increased hepcidin expression in adipose tissue.

Since the discovery that hepcidin is expressed in the adipose tissue of obese subjects, attention has been increasingly focused on alterations in iron homeostasis that are associated with adiposity. We examined the production of hepcidin, the expression of hepcidin-related genes and the iron content of the adipose tissue in obesity using Swiss mice fed a high-fat diet (HFD). The mice were maintained on a control diet or HFD for 12 or 24 wk, and body weight, adiposity and glucose homeostasis were evaluated. The expression of several genes (hepcidin, TfR1, TfR2, DMT1, FT-heavy, ferroportin, IRP-1, IRP-2 and HIF-1) and the protein expression of hepcidin and IL-6 were quantified. The iron level was assessed using a Prussian blue reaction in paraffin-embedded tissue. After 24 wk on the HFD, we observed increases in the levels of hepcidin in the serum and the visceral adipose tissue. The IL-6 levels also increased in the visceral adipose tissue. Adipocytes isolated from the visceral adipose tissues of lean and obese mice expressed hepcidin at comparable levels; however, isolated macrophages from the stromal vascular fraction expressed higher hepcidin levels. Adipose tissues from obese mice displayed increased tfR2 expression and the presence of iron. Our results indicate that IL-6 and iron may affect the signaling pathways governing hepcidin expression. Thus, the mice fed HFD for 24 wk represent a suitable model for the study of obesity-linked hepcidin alterations. In addition, hepcidin may play local roles in controlling iron availability and interfering with inflammation in adipose tissue.

Effects of methotrexate on inflammatory alterations induced by obesity: an in vivo and in vitro study.

Immunosuppressant drugs, such as methotrexate, are able to inhibit cytokine production and leukocyte migration to inflammatory foci; therefore, they could modify the establishment of inflammation in adipose tissue during obesity. Thus, we studied the effects of methotrexate in vivo on high-fat diet induced-obesity in mice and in vitro in isolated and co-cultured adipocytes and macrophages. Obese mice treated with methotrexate presented reduced serum levels of TNF-α, insulin and glucose, and an improvement of insulin sensitivity. Adipose tissue from these mice produced less proinflammatory (TNF-α, IL-6, leptin) and more anti-inflammatory adipokines (adiponectin and IL-10) associated with reduced macrophage infiltration and inflammation. Cytokine inhibition was also confirmed in isolated and co-cultured adipocytes and macrophages. Methotrexate presented anti-lipolytic effect in vivo and, in vitro through adenosine release. Drugs that combine anti-lipolytic effect and the ability to control inflammation in adipose tissue could play a role in the control of insulin resistance and other pathologies associated with obesity.

Estudo comparativo da atividade antiinflamatória, ulcerogênica e citotóxica do S-ibuprofeno e ibuprofeno racêmico / Comparative study of anti-inflammatory, ulcerogenic and cytotoxic activities of racemate and S-ibuprofen

Ibuprofen is widely commercialized in racemic form. Although metabolic chiral inversion occurs through the conversion of R(-)-ibuprofen to S(+)-ibuprofen and the latter enantiomer is considered the active form, clinical trials involving the administration of a racemate to S-enantiomer dosage ratio of 1:0.5 have demonstrated that S(+)-ibuprofen is as efficacious as the racemic formulation. Moreover, the R(-)-enantiomer has been implicated in adverse gastrointestinal effects found with the racemic form, but the mechanisms involved in this process are not yet fully understood. The aim of the present study was to evaluate the anti-inflammatory activity of a racemate to S(+)-ibuprofen dosage ratio of 1:0.5 using the carrageenan air pouch model of inflammation and determine both ulcerogenic activity and the chiral conversion rate in rats. An in vitro study of the cytotoxicity of racemate and S(+)-ibuprofen in gastric cells was also performed. Although the plasma level of S(+)-ibuprofen was raised after racemate administration, no significant difference was found in anti-inflammatory activity, as assessed by exudate formation, PGE2 production and leukocyte migration to the air pouches. Fewer gastric lesions were found after S(+)-ibuprofen administration, despite the low gastric PGE2 content. In the in vitro study, the racemic compound proved more cytotoxic than S(+)-ibuprofen. The present findings suggest that the S-enantiomer of ibuprofen could be considered a therapeutic alternative to minimize gastrointestinal side effects, since the chiral inversion of R(-)-ibuprofen to S(+)-ibuprofen did not result in an improved anti-inflammatory response.

O Ibuprofeno é normalmente comercializado na forma racêmica. Embora ocorra inversão quiral convertendo a forma R(-)- em S(+)-ibuprofeno e, a última seja considerada a forma ativa, a administração da proporção 1:0,5 (racemato: S-enantiômero) demonstrou que o S(+)-ibuprofeno é mais eficaz que a formulação racêmica. Adicionalmente, o R(-)-enantiômero está envolvido nos efeitos adversos gastrintestinais descritos para a formulação racêmica, embora os mecanismos não sejam complemente compreendidos. O objetivo deste estudo foi avaliar a atividade antiinflamatória da proporção 1:0,5 (racemato:S-ibuprofeno) utilizando o modelo experimental de bolsa de ar, a atividade ulcerogênica e a taxa de conversão quiral em ratos. Também estudamos in vitro, a citotoxicidade provocada pelo racemato e S(+)-ibuprofeno em células gástricas. Embora os níveis plasmáticos de S(+)-ibuprofeno tenham aumentado após a administração do racemato, a atividade antiinflamatória avaliada pela formação de exsudato, produção de PGE2 e migração de leucócitos para a bolsa de ar não foram diferentes. As lesões gástricas foram reduzidas após a administração de S(+)-ibuprofeno, apesar da inibição de PGE2 gástrica. In vitro, o composto racêmico foi mais citotóxico que o S(+)-ibuprofeno. Nossos resultados sugerem que o S-enantiômero do ibuprofeno pode ser considerado uma alternativa terapêutica visando a redução dos efeitos colaterais gastrintestinais, visto que a inversão quiral do R(-)- para o S(+)-ibuprofeno não resultou em melhora do efeito antiinflamatório observado.