Mitochondrial DNA as a Possible Ligand for TLR9 in Irinotecan-induced Small Intestinal Mucositis.

Cancer chemotherapy and radiotherapy may result in mucositis characterized by stem cell damage and inflammation in the gastrointestinal tract. The molecular mechanisms underlying this pathology remain unknown. Based on the assumption that mitochondrial CPG-DNA (mtDNA) released and sensed by TLR9 could underlie mucositis pathology, we analyzed the mtDNA levels in sera as well as inflammatory and disease parameters in the small intestine from wild-type (WT) and TLR9-deficient mice (TLR9-/-) in an experimental model of intestinal mucositis induced by irinotecan. Additionally, we verified the ability of WT and TLR9-/- macrophages to respond to CpG-DNA in vitro. WT mice injected with irinotecan presented a progressive increase in mtDNA in the serum along with increased hematocrit, shortening of small intestine length, reduction of intestinal villus:crypt ratio and increased influx of neutrophils, which were followed by higher expression of Nlrp3 and Casp1 mRNA and increased IL-1ß levels in the ileum when compared to vehicle-injected mice. TLR9-deficient mice were protected in all these parameters when compared to WT mice. Furthermore, TLR9 was required for the production of IL-1ß and NO after macrophage stimulation with CpG-DNA. Overall, our findings show that the amount of circulating free CpG-DNA is increased upon chemotherapy and that TLR9 activation is important for NLRP3 inflammasome transcription and further IL-1ß release, playing a central role in the development of irinotecan-induced intestinal mucositis. We suggest that TLR9 antagonism may be a new therapeutic strategy for limiting irinotecan-induced intestinal inflammation.

PAF signaling plays a role in obesity-induced adipose tissue remodeling.

BACKGROUND/OBJECTIVES: Platelet-activating factor receptor (PAFR) activation controls adipose tissue (AT) expansion in animal models. Our objective was twofold: (i) to check whether PAFR signaling is involved in human obesity and (ii) investigate the PAF pathway role in hematopoietic or non-hematopoietic cells to control adipocyte size. MATERIALS/SUBJECTS AND METHODS: Clinical parameters and adipose tissue gene expression were evaluated in subjects with obesity. Bone marrow (BM) transplantation from wild-type (WT) or PAFR-/- mice was performed to obtain chimeric PAFR-deficient mice predominantly in hematopoietic or non-hematopoietic-derived cells. A high carbohydrate diet (HC) was used to induce AT remodeling and evaluate in which cell compartment PAFR signaling modulates it. Also, 3T3-L1 cells were treated with PAF to evaluate fat accumulation and the expression of genes related to it. RESULTS: PAFR expression in omental AT from humans with obesity was negatively correlated to different corpulence parameters and more expressed in the stromal vascular fraction than adipocytes. Total PAFR-/- increased adiposity compared with WT independent of diet-induced obesity. Differently, WT mice receiving PAFR-/--BM exhibited similar adiposity gain as WT chimeras. PAFR-/- mice receiving WT-BM showed comparable augmentation in adiposity as total PAFR-/- mice, demonstrating that PAFR signaling modulates adipose tissue expansion through non-hematopoietic cells. Indeed, the PAF treatment in 3T3-L1 adipocytes reduced fat accumulation and expression of adipogenic genes. CONCLUSIONS: Therefore, decreased PAFR signaling may favor an AT accumulation in humans and animal models. Importantly, PAFR signaling, mainly in non-hematopoietic cells, especially in adipocytes, appears to play a significant role in regulating diet-induced AT expansion.

Eosinophil plays a crucial role in intestinal mucositis induced by antineoplastic chemotherapy.

Mucositis is a major clinical complication associated with cancer treatment and may limit the benefit of chemotherapy. Leukocytes and inflammatory mediators have been extensively associated with mucositis severity. However, the role of eosinophils in the pathophysiology of chemotherapy-induced mucositis remains to be elucidated. Here, using GATA-1-deficient mice, we investigated the role of eosinophils in intestinal mucositis. There was marked accumulation of eosinophils in mice given irinotecan and eosinophil ablation inhibited intestinal mucositis. Treatment with Evasin-4, a chemokine receptor antagonist, reduced the recruitment of eosinophils and decreased irinotecan-induced mucositis. Importantly, Evasin-4 did not interfere negatively with the antitumour effects of irinotecan. Evasin-4 was of benefit for mice given high doses of irinotecan once Evasin-4-treated mice presented delayed mortality. Altogether, our findings suggest that Evasin-4 may have significant mucosal-protective effects in the context of antineoplastic chemotherapy and may, therefore, be useful in combination with anticancer treatment in cancer patients.

Colonization by Enterobacteriaceae is crucial for acute inflammatory responses in murine small intestine via regulation of corticosterone production.

Although dysbiosis in the gut microbiota is known to be involved in several inflammatory diseases, whether any specific bacterial taxa control host response to inflammatory stimuli is still elusive. Here, we hypothesized that dysbiotic indigenous taxa could be involved in modulating host response to inflammatory triggers. To test this hypothesis, we conducted experiments in germ-free (GF) mice and in mice colonized with dysbiotic taxa identified in conventional (CV) mice subjected to chemotherapy-induced mucositis. First, we report that the absence of microbiota decreased inflammation and damage in the small intestine after administration of the chemotherapeutic agent 5-fluorouracil (5-FU). Also, 5-FU induced a shift in CV microbiota resulting in higher amounts of Enterobacteriaceae, including E. coli, in feces and small intestine and tissue damage. Prevention of Enterobacteriaceae outgrowth by treating mice with ciprofloxacin resulted in diminished 5-FU-induced tissue damage, indicating that this bacterial group is necessary for 5-FU-induced inflammatory response. In addition, monocolonization of germ-free (GF) mice with E. coli led to reversal of the protective phenotype during 5-FU chemotherapy. E. coli monocolonization decreased the basal plasma corticosterone levels and blockade of glucocorticoid receptor in GF mice restored inflammation upon 5-FU treatment. In contrast, treatment of CV mice with ciprofloxacin, that presented reduction of Enterobacteriaceae and E. coli content, induced an increase in corticosterone levels. Altogether, these findings demonstrate that Enterobacteriaceae outgrowth during dysbiosis impacts inflammation and tissue injury in the small intestine. Importantly, indigenous Enterobacteriaceae modulates host production of the anti-inflammatory steroid corticosterone and, consequently, controls inflammatory responsiveness in mice.

The Serotonin Neurotransmitter Modulates Virulence of Enteric Pathogens.

The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C. rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria.

l-Arginine sensing regulates virulence gene expression and disease progression in enteric pathogens.

Microbiota, host and dietary metabolites/signals compose the rich gut chemical environment, which profoundly impacts virulence of enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) engages a syringe-like machinery named type-III secretion system (T3SS) to inject effectors within host cells that lead to intestinal colonization and disease. We previously conducted a high-throughput screen to identify metabolic pathways that affect T3SS expression. Here we show that in the presence of arginine, the arginine sensor ArgR, identified through this screen, directly activates expression of the genes encoding the T3SS. Exogenously added arginine induces EHEC virulence gene expression in vitro. Congruently, a mutant deficient in arginine transport (ΔartP) had decreased virulence gene expression. ArgR also augments murine disease caused by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. The source of arginine sensed by C. rodentium is not dietary. At the peak of C. rodentium infection, increased arginine concentration in the colon correlated with down-regulation of the host SLC7A2 transporter. This increase in the concentration of colonic arginine promotes virulence gene expression in C. rodentium Arginine is an important modulator of the host immune response to pathogens. Here we add that arginine also directly impacts bacterial virulence. These findings suggest that a delicate balance between host and pathogen responses to arginine occur during disease progression.

SOCS2 modulates adipose tissue inflammation and expansion in mice.

INTRODUCTION: Obesity is usually triggered by a nutrient overload that favors adipocyte hypertrophy and increases the number of pro-inflammatory cells and mediators into adipose tissue. These mediators may be regulated by suppressors of cytokine signaling (SOCS), such as SOCS2, which is involved in the regulation of the inflammatory response of many diseases, but its role in obesity is not yet known. We aimed to investigate the role of SOCS2 in metabolic and inflammatory dysfunction induced by a high-refined carbohydrate-containing diet (HC). MATERIAL AND METHODS: Male C57BL/6 wild type (WT) and SOCS2 deficient (SOCS2-/-) mice were fed chow or an HC diet for 8 weeks. RESULTS: In general, SOCS2 deficient mice, independent of the diet, showed higher adipose tissue mass compared with their WT counterparts that were associated with decreased lipogenesis rate in adipose tissue, lipolysis in adipocyte culture and energy expenditure. An anti-inflammatory profile was observed in adipose tissue of SOCS2-/- by reduced secretion of cytokines, such as TNF and IL-6, and increased M2-like macrophages and regulatory T cells compared with WT mice. Also, SOCS2 deficiency reduced the differentiation/expansion of pro-inflammatory cells in the spleen but increased Th2 and Treg cells compared with their WT counterparts. CONCLUSION: The SOCS2 protein is an important modulator of obesity that regulates the metabolic pathways related to adipocyte size. Additionally, SOCS2 is an inflammatory regulator that appears to be essential for controlling the release of cytokines and the differentiation/recruitment of cells into adipose tissue during the development of obesity.

Paradoxical role of tumor necrosis factor on metabolic dysfunction and adipose tissue expansion in mice.

OBJECTIVES: Tumor necrosis factor (TNF) is a well-known cytokine that triggers insulin resistance during obesity development. On the other hand, it is also known that TNF induces a fat mass loss during acute diseases. However, whether TNF has a protective and physiological role to control adipose tissue expansion during obesity still needs to be verified. The aim of this study was to evaluate whether the ablation of TNF receptor 1 (TNFR1) alters fat mass and insulin resistance induced by a highly refined carbohydrate-containing (HC) diet. METHODS: Male C57 BL/6 wild-type (WT) mice and TNFR1 knockout (TNFR1-/-) mice were fed with chow or with the HC diet for 16 wk. RESULTS: TNFR1-/- mice gained more body weight than the WT groups independent of the diet composition. TNFR1-/- mice fed with the chow diet showed higher adiposity, accompanied by higher serum leptin levels. However, these mice showed lower non-esterified fatty acid levels. Furthermore, TNFR1-/- mice had suppressed TNF, interleukin (IL)-6, and IL-10 levels in adipose tissue compared with WT mice. TNFR1-/- mice fed with the HC diet were protected from increased adiposity and glucose intolerance induced by the HC diet and exhibited lower serum resistin levels. CONCLUSIONS: TNF signaling appears to have a paradoxical role on metabolism. Ablation of TNFR1 leads to a reduction of inflammatory cytokines in adipose tissue that is accompanied by higher adiposity in mice fed with chow diet. However, when these mice are given the HC diet, the loss of TNFR1 improves insulin sensitivity and protects mice against additional fat mass.

Sodium butyrate modulates adipocyte expansion, adipogenesis, and insulin receptor signaling by upregulation of PPAR-γ in obese Apo E knockout mice.

OBJECTIVES: Studies suggest that sodium butyrate reduces obesity-associated inflammation and insulin resistance in in vitro and in vivo models. Apo E-/- mice have high basal oxidative stress and naturally develop dyslipidemia and atherosclerosis. Because these disorders are present in obesity, the aim of this study was to determine whether Apo E-/- mice could be a more realistic model for studying obesity and insulin resistance. METHODS: We evaluated the action of orally administered sodium butyrate on adipose tissue expansion and insulin resistance using diet-induced obese Apo E-/- mice. RESULTS: Findings from the present study demonstrated that obese mice fed a sodium butyrate-supplemented diet presented a modest reduction of weight gain associated with reduction of adipocyte expansion, induction of adipogenesis and angiogenesis, and adiponectin production. Sodium butyrate also improved insulin sensitivity, by increasing insulin receptor expression associated with activation of Akt signaling pathway. These results were associated with increased peroxisome proliferator-activated receptor-γ expression and nuclear factor-κB downregulation. CONCLUSION: These results suggested that oral supplementation of butyrate could be useful as an adjuvant in the treatment of obesity, metabolic syndrome, and insulin resistance.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

TNF and IL-18 cytokines may regulate liver fat storage under homeostasis conditions.

The inflammation induced by obesogenic diets is associated with deposition of fat in the liver. On the other hand, anti-inflammatory and immunosuppressive therapies may impact in body fat storage and in liver lipid dynamics. It is important to study specific inflammatory mediators in this context, since their role on hepatic damage is not fully clarified. This study aimed to evaluate the role of interleukin (IL)-18 and tumor necrosis factor (TNF) receptor in liver dysfunction induced by diet. Male C57BL/6 wild-type (WT), IL-18, and TNF receptor 1 knockout mice (IL-18-/- and TNFR1-/-) were divided according to the experimental diets: chow diet or a high-refined carbohydrate-containing diet. Alanine aminotransferase was quantified by colorimetric analysis. Total fat content in the liver was determined by Folch methods. Levels of TNF, IL-6, IL-4, and IL-13 in liver samples were measured by ELISA assay. IL-18 and TNFR knockout mice fed with chow diet showed higher liver triglycerides deposition than WT mice fed with the same diet (WT: 131.9 ± 24.5; IL-18-/-: 239.4 ± 38.12*; TNF-/-: 179.6 ± 50.45*; *P < 0.01). Furthermore, these animals also showed a worse liver histopathological score and lower levels of TNF, IL-6, IL-4, and IL-13 in the liver. Interestingly, treatment with a high-carbohydrate diet did not exacerbate liver damage in IL-18-/- and TNFR1-/- mice. Our data suggest that IL-18 and TNF may be involved on hepatic homeostasis mainly in a context of a healthy diet.

Platelet-activating factor modulates fat storage in the liver induced by a high-refined carbohydrate-containing diet.

Hepatic diseases are comorbidities caused by obesity and are influenced by diet composition. The aim of this study was to evaluate the kinetics of metabolic and inflammatory liver dysfunction induced by a high-refined carbohydrate-containing (HC) diet and to determine how platelet-activating factor (PAF) modulates the liver lipid content of mice. BALB/c mice were fed a chow or HC diet for the following experimental periods: 1 and 3 days, 1, 2, 4, 6, 8, 10 and 12 weeks. Wild-type (WT) and PAF receptor-deficient (PAFR(-/-)) mice were fed the same diets for 8 weeks. Mice fed with HC diet showed higher triglycerides and cholesterol levels, fibrosis and inflammation in the liver. The number of neutrophils migrating into the liver was also increased in mice fed with HC diet. However, transaminase levels did not change. PAFR(-/-) mice fed with HC diet showed more steatosis, oxidative stress and higher transaminases levels associated with lower inflammation than WT mice. The consumption of HC diet altered the metabolic and inflammatory response in the liver and was worse in PAFR(-/-) mice. We suggest that PAF regulates liver lipid content and dyslipidemia, protecting the mice from lipotoxicity and liver damage.

Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.

UNLABELLED: Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-κB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9(-/-) mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. CONCLUSION: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue.

Distinct metabolic pathways trigger adipocyte fat accumulation induced by high-carbohydrate and high-fat diets.

OBJECTIVE: Several metabolic pathways may be associated with obesity development and may be differentially modulated by dietary constituents. The aim of this study was to access the adipocyte metabolic pathways that lead to lipid accumulation in fat cells of mice fed a high-carbohydrate or high-fat diet. METHODS: Male Swiss mice ages 7 to 8 wk were fed a standard laboratory rodent diet-chow diet, a high-carbohydrate (HC) diet, or a high-fat (HF) diet for 8 wk. RESULTS: Animals fed the HC diet exhibited a high lipogenesis rate and lipoprotein lipase activity even in a fasted state. Peroxisome proliferator-activated receptor gamma and sterol regulatory element-binding proteins-1 mRNAs were increased in adipose tissue. The HF diet did not promote the lipogenic pathways but attenuated lipolytic activity and glucose uptake in adipocytes. CONCLUSION: The HC diet induces constitutive expression of lipogenic transcription factors during fasting, whereas the HF diet decreases lipolysis in adipocytes. Both pathways may contribute to increase fat stores and maintain an obese state.

Inflammasome activation is reactive oxygen species dependent and mediates irinotecan-induced mucositis through IL-1ß and IL-18 in mice.

Irinotecan is a useful chemotherapeutic for the treatment of various cancers. Irinotecan treatment is associated with mucositis, which clearly limits the use of the drug. Mechanisms that account for mucositis are only partially known. This study assessed mechanisms and the role of inflammasome activation in irinotecan-induced mucositis. Mucositis in mice was induced by irinotecan injection in C57BL/6 wild-type, gp91phox(-/-), il-18(-/-), casp-1(-/-), and asc(-/-) mice once a day for 4 consecutive days. In some experiments, mice received apocynin to inhibit NADPH oxidase (NOX), IL-1 receptor antagonist, or IL-18 binding protein to prevent activation of IL-1 and IL-18 receptors, respectively. Mice were euthanized 7 days after the beginning of irinotecan treatment, and small intestines were collected for analysis. Irinotecan treatment resulted in increased IL-1ß and IL-18 production in ileum and NOX-2-dependent oxidative stress. gp91phox(-/-) and apocynin-treated mice had diminished oxidative stress and less severe mucositis. Furthermore, treatment with apocynin decreased caspase-1 activation and IL-1ß and IL-18 production in the ileum. asc(-/-) and casp-1(-/-) mice also had less intestinal injury and decreased IL-1ß and IL-18 production. Finally, both the absence of IL-18 and IL-1ß resulted in reduced inflammatory response and attenuated intestinal injury. NOX-2-derived oxidative stress mediates inflammasome activation and inflammasome-dependent production of IL-1ß and IL-18, which mediate tissue injury during irinotecan-induced mucositis in mice.

Acute intake of a high-fructose diet alters the balance of adipokine concentrations and induces neutrophil influx in the liver.

The postprandial state is a period of metabolic fluxes, biosynthesis and oxidative metabolism. A considerable amount is known about the inflammatory response to the chronic consumption of fructose, but little is known about its effects in the postprandial state. The aim of the present study was to investigate the inflammatory effects of a single meal containing fructose on healthy mice. Male BALB/c and LysM-eGFP mice at 12-14 weeks were divided into three groups: fasted, control (mice fed with a sucrose-containing diet) and fructose (mice fed with a fructose-containing diet). One, 2 or 4 h postprandial, the BALB/c mice were killed, and samples were collected. LysM-eGFP mice were submitted to intravital microscopy. The fed mice showed a low-grade inflammatory response apart from dietary composition, which was characterized by increased numbers of leukocytes and high serum concentrations of pentraxin 3, leptin and resistin. TNF-α and CCL2 concentrations rose in the liver after the meal. IL-6 concentration increased and IL-10 decreased in the adipose tissue of the fed mice. Mice fed with the fructose-containing diet showed an intensification of the inflammatory response. Furthermore, the adiponectin concentration dropped, and the liver influx of neutrophils increased after fructose intake. Overall, this study showed a rapid increase in the systemic and tissue-specific immune response after a balanced meal. The study also showed an increased neutrophil influx in liver associated with an imbalance of adipokine concentrations and an increase of cytokine in the liver and adipose tissue following a fructose-containing meal.

Evaluation of calcium supplementation with algae (Lithothamnion muelleri) on metabolic and inflammatory parameters in mice fed a high refined carbohydrate-containing diet.

The aim of the present study was to evaluate the potential of calcium supplementation from Lithothamnium muelleri algae on metabolic and inflammatory parameters in mice with increased adiposity. Male mice were fed and divided during 8 weeks in: control (C), a high refined carbohydrate-containing diet (HC), HC diet supplemented with 1% of Lithothamnion muelleri algae (HC + A) and HC diet supplemented with 0.9% calcium carbonate (HC + C). Animals fed HC diet had increased body weight gain and adiposity, serum glucose and cholesterol, glucose intolerance and decreased insulin sensitivity, compared to control diet. However, the HC + A and HC + C groups did not prevent these aspects and were not able to change the CD14 + cells population in adipose tissue of animals fed HC diet. Calcium supplementation with Lithothamnium muelleri algae and calcium carbonate had no protective effect against the development of adiposity, metabolic and inflammatory alterations induced by HC diet.

Increased expression of oxidative enzymes in adipose tissue following PPARα-activation.

OBJECTIVE: Evaluate the effect of fenofibrate treatment on the expression of PPARα and oxidative enzymes in adipose tissue. MATERIALS/METHODS: Wistar male rats were fed a balanced diet supplemented with 100mg.Kg-1 bw.day-1 fenofibrate (Sigma) during nine days. Plasma glucose, free fatty acids (FFA) leptin and insulin were determined. PPARα, ACO and CPT-1 mRNA expression and amount of PPARα and PPARγ protein were assessed in epididymal adipose tissue. Oral glucose tolerance test was evaluated into overnight fasted rats. Glucose uptake was measured in adipocytes isolated from epididymal fat pads in the presence or absence of insulin (25ng/mL). RESULTS: Fenofibrate treatment increased PPARα and PPARγ protein abundance in adipose tissue. In addition to it well- known effect on oxidative enzymes in liver, fenofibrate treatment also induces a high expression of Acyl CoA Oxidase (ACO) and Carnitine palmitoyltransferase 1 (CPT-1) in adipose tissue. Furthermore, we have shown that the fenofibrate treatment improves the glucose tolerance and enhance the glucose uptake by adipocytes. CONCLUSION: Altogether, the data suggest that fenofibrate have a direct effect in adipose tissue contributing to the low adiposity and improvement of glucose homeostasis.

Lack of platelet-activating factor receptor protects mice against diet-induced adipose inflammation and insulin-resistance despite fat pad expansion.

OBJECTIVE: The role of platelet-activating factor (PAF) on diet-induced inflammatory and metabolic dysfunction is unknown. The effects of diet-induced metabolic and inflammatory dysfunction in mice with deletion of the PAF receptor (PAFR(-/-) ) were evaluated in this study. METHODS: Wild-type and PAFR(-/-) mice were fed chow (WT-C and PAFR(-/-) -C) or high-refined carbohydrate-containing diet (WT-HC and PAFR(-/-) -HC). PAFR(-/-) - RESULTS: HC mice gained more weight and adiposity than PAFR(-/-) -C and WT-HC mice. Lipogenesis increased and hormone-sensitive lipase expression decreased in PAFR(-/-) -HC compared to WT-HC mice. WT-HC mice had impaired glucose tolerance and insulin sensitivity compared to WT-C mice. In contrast, glucose tolerance and insulin sensitivity in PAFR(-/-) -HC mice were similar to that of lean littermates. PAFR(-/-) -HC mice expressed significantly more peroxisome proliferator-activator receptor gamma (PPARγ) than PAFR(-/-) -C and WT-C mice. Resistin increased in WT-HC mice compared to WT-C mice. However, the levels of resistin were 35% lower in PAFR(-/-) -HC mice than WT-HC mice. PAFR(-/-) presented with less HC diet-induced adipose tissue inflammation than WT mice. Adipocytes isolated from PAFR(-/-) mice incubated in media containing normal or high levels of glucose secreted less interleukin-6 and tumor necrosis factor alpha and presented lower rate of lipolysis than WT mice. CONCLUSION: PAFR deficiency resulted in less inflammation in adipose tissue and improvement in glucose homeostasis when fed the HC diet. The higher adiposity observed in PAFR(-/-) mice fed HC diet could be owing to the maintenance of insulin sensitivity, decreased adipocyte lipolysis rate, high lipogenesis and PPARγ expression, and lower inflammatory milieu in adipose tissue.

Acute and sustained inflammation and metabolic dysfunction induced by high refined carbohydrate-containing diet in mice.

OBJECTIVE: The effects of high-refined carbohydrate-containing diet (HC) on inflammatory parameters and metabolic disarrangement of adipose tissue are poorly understood. The aim of this study was to evaluate the timing and progression of metabolic and inflammatory dysfunction induced by HC diet in mice. DESIGN AND METHODS: BALB/c mice were fed chow or HC diet for 1 and 3 days, 1, 2, 4, 6, 8, 10, and 12 weeks. RESULTS: Animals given HC diet exhibited acute and sustained increase in visceral adiposity, glucose intolerance, low insulin sensitivity, hyperlipemia, acute increase in mRNA expression of ACC, LPL, PPARγ, SREBP-1, and ChREBP and altered circulating levels of adiponectin, resistin, and leptin. There was leucocyte rolling and adhesion on adipose tissue microvessels already at 3 days and until 8 weeks of HC diet. Adipose tissue of mice had increased number of macrophages (M1 and M2), lymphocytes (CD8+ and CD4+ Foxp3+), and neutrophils (GR1+) already at 3 days after initiation of HC diet. Overall, concentration of cytokines and chemokines, TNF-α, IL-6, IL-10, TGF-ß1, CCL2, and CXCL1, in adipose tissue was elevated throughout the experimental period. Levels of IL-10 and TGF-ß1 tended to reach baseline levels at 12 weeks of HC diet. CONCLUSIONS: We describe a novel murine model of fat pad expansion induced by HC diet that is characterized by early onset and sustained adipose tissue inflammation and metabolic disarrangement. The acute inflammatory response in adipose tissue occurs very early and is sustained, suggesting that adipose tissue inflammation is a homeostatic mechanism to regulate nutrient overload and adipose expansion.