Intrauterine food restriction impairs the lipogenesis process in the mesenteric adipocytes from low-birth-weight rats into adulthood.

Background: Intrauterine food restriction (IFR) during pregnancy is associated with low birth weight (LBW) and obesity in adulthood. It is known that white adipose tissue (WAT) plays critical metabolic and endocrine functions; however, this tissue's behavior before weight gain and obesity into adulthood is poorly studied. Thus, we evaluated the repercussions of IFR on the lipogenesis and lipolysis processes in the offspring and described the effects on WAT inflammatory cytokine production and secretion. Methods: We induced IFR by providing gestating rats with 50% of the necessary chow daily amount during all gestational periods. After birth, we monitored the offspring for 12 weeks. The capacity of isolated fat cells from mesenteric white adipose tissue (meWAT) to perform lipogenesis (14C-labeled glucose incorporation into lipids) and lipolysis (with or without isoproterenol) was assessed. The expression levels of genes linked to these processes were measured by real-time PCR. In parallel, Multiplex assays were conducted to analyze pro-inflammatory markers, such as IL-1, IL-6, and TNF-α, in the meWAT. Results: Twelve-week-old LBW rats presented elevated serum triacylglycerol (TAG) content and attenuated lipogenesis and lipolysis compared to control animals. Inflammatory cytokine levels were increased in the meWAT of LBW rats, evidenced by augmented secretion by adipocytes and upregulated gene and protein expression by the tissue. However, there were no significant alterations in the serum cytokines content from the LBW group. Additionally, liver weight, TAG content in the hepatocytes and serum glucocorticoid levels were increased in the LBW group. Conclusion: The results demonstrate that IFR throughout pregnancy yields LBW offspring characterized by inhibited lipogenesis and lipolysis and reduced meWAT lipid storage at 12 weeks. The increased serum TAG content may contribute to the augmented synthesis and secretion of pro-inflammatory markers detected in the LBW group.

Bypass gastroplasty impacts oral health, salivary inflammatory biomarkers, and microbiota: a controlled study.

OBJECTIVES: Knowledge about the impact of gastroplasty on oral health and salivary biomarkers is limited. The aim was to prospectively evaluate oral health status, salivary inflammatory markers, and microbiota in patients undergoing gastroplasty compared with a control group undergoing a dietary program. MATERIALS AND METHODS: Forty participants with obesity class II/III were included (20 individuals in each sex-matched group; 23-44 years). Dental status, salivary flow, buffering capacity, inflammatory cytokines, and uric acid were assessed. Salivary microbiological analysis (16S-rRNA sequencing) assessed the abundance of genus, species, and alpha diversity. Cluster analysis and mixed-model ANOVA were applied. RESULTS: Oral health status, waist-to-hip ratio, and salivary alpha diversity were associated at baseline. A subtle improvement in food consumption markers was observed, although caries activity increased in both groups, and the gastroplasty group showed worse periodontal status after three months. IFNγ and IL10 levels decreased in the gastroplasty group at 3 months, while a decrease was observed in the control group at 6 months; IL6 decreased in both groups (p < 0.001). Salivary flow and buffering capacity did not change. Significant changes in Prevotella nigrescens and Porphyromonas endodontalis abundance were observed in both groups, while alpha diversity (Sobs, Chao1, Ace, Shannon, and Simpson) increased in the gastroplasty group. CONCLUSIONS: Both interventions changed in different degrees the salivary inflammatory biomarkers and microbiota, but did not improve the periodontal status after 6 months. CLINICAL RELEVANCE: Although the observed discrete improvement in dietary habits, caries activity increased with no clinical improvement in the periodontal status, emphasizing the need of oral health monitoring during obesity treatment.

Dexamethasone-Induced Adipose Tissue Redistribution and Metabolic Changes: Is Gene Expression the Main Factor? An Animal Model of Chronic Hypercortisolism.

Chronic hypercortisolism has been associated with the development of several metabolic alterations, mostly caused by the effects of chronic glucocorticoid (GC) exposure over gene expression. The metabolic changes can be partially explained by the GC actions on different adipose tissues (ATs), leading to central obesity. In this regard, we aimed to characterize an experimental model of iatrogenic hypercortisolism in rats with significant AT redistribution. Male Wistar rats were distributed into control (CT) and GC-treated, which received dexamethasone sodium phosphate (0.5 mg/kg/day) by an osmotic minipump, for 4 weeks. GC-treated rats reproduced several characteristics observed in human hypercortisolism/Cushing's syndrome, such as HPA axis inhibition, glucose intolerance, insulin resistance, dyslipidemia, hepatic lipid accumulation, and AT redistribution. There was an increase in the mesenteric (meWAT), perirenal (prWAT), and interscapular brown (BAT) ATs mass, but a reduction of the retroperitoneal (rpWAT) mass compared to CT rats. Overexpressed lipolytic and lipogenic gene profiles were observed in white adipose tissue (WAT) of GC rats as BAT dysfunction and whitening. The AT remodeling in response to GC excess showed more importance than the increase of AT mass per se, and it cannot be explained just by GC regulation of gene transcription.

Immune spleen cells attenuate the inflammatory profile of the mesenteric perivascular adipose tissue in obese mice.

The perivascular adipose tissue (PVAT) differs from other fat depots and exerts a paracrine action on the vasculature. The spleen has an important role in the immune response, and it was observed to have either a protective role or a contribution to obesity-related diseases. However, the relation between spleen and PVAT is elusive in obesity. We investigated the role of spleen in the inflammatory profile of the mesenteric PVAT (mPVAT) from mice fed a high-fat diet (HFD) for 16 weeks. Male C57Bl/6 mice were sham-operated or splenectomized (SPX) and fed a HFD for 16 weeks. mPVAT morphology was evaluated by hematoxylin and eosin staining, infiltrated immune cells were evaluated by flow cytometry, inflammatory cytokines were evaluated by ELISA and the splenic cell chemotaxis mediated by mPVAT was evaluated using a transwell assay. In SPX mice, HFD induced adipocyte hypertrophy and increased immune cell infiltration and proinflammatory cytokine levels in mPVAT. However, none of these effects were observed in mPVAT from sham-operated mice. Spleen from HFD fed mice presented reduced total leukocytes and increased inflammatory markers when compared to the spleen from control mice. Chemotaxis of spleen cells mediated by mPVAT of HFD fed mice was reduced in relation to standard diet fed mice. The spleen protects mPVAT against the effects of 16-week HFD. This information was missing, and it is important because PVAT is different from other fat depots and data cannot be extrapolated from any type of adipose tissue to PVAT.

Mitochondrial DNA: A new driver for sex differences in spontaneous hypertension.

The prevalence of arterial hypertension (AH) is higher in men than in premenopausal women of the same age. AH has been characterized as a chronic inflammatory disease and activation of Toll-like receptors (TLR) by damage-associated molecular patterns (DAMPs) is involved. Mitochondrial DNA (mtDNA) may be released by end-organ damage, which is recognized and activates TLR9. The serum level of mtDNA is increased in AH. The aim of this study was to compare the serum mtDNA levels between male and female spontaneously hypertensive rats (SHR) and to evaluate the sex differences in the effect of mtDNA on the function, inflammation and signaling pathway related to TLR9 in the vasculature. Male and female 15-week-old SHR and Wistar rats were used to evaluate the arterial blood pressure, serum mtDNA, contractile response, inflammatory markers and signaling pathway related to TLR9. Male SHR had higher arterial blood pressure values and serum mtDNA compared to female SHR and to male and female normotensive Wistar rats. In male SHR aorta, mtDNA incubation increased the contractile response to phenylephrine, which was blunted by inhibition of TLR9, and also increased pro-inflammatory molecules IL-6 and TNF-α. However, in female SHR aorta, mtDNA incubation did not change the contractile response, reduced pro-inflammatory molecules and prevented oxidative stress. mtDNA incubation did not change the expression of TLR9, MyD88 and eNOS neither in male nor in female SHR aorta, but it increased the phosphorylation of ERK1/2 in male and reduced in female SHR aorta. The mtDNA differential modulation of vascular response in male and female SHR might contribute to sex differences in AH. This study contributes to the understanding of a need for more personalized therapeutic strategies for men and women with hypertension. Keywords: Sex differences, Arterial hypertension, Mitochondrial DNA, Toll-Like receptor 9.

Increased chemical acetylation of peptides and proteins in rats after daily ingestion of diacetyl analyzed by Nano-LC-MS/MS.

BACKGROUND: Acetylation alters several protein properties including molecular weight, stability, enzymatic activity, protein-protein interactions, and other biological functions. Our previous findings demonstrating that diacetyl/peroxynitrite can acetylate L-lysine, L-histidine, and albumin in vitro led us to investigate whether diacetyl-treated rats suffer protein acetylation as well. METHODS: Wistar rats were administered diacetyl daily for four weeks, after which they were sacrificed, and their lung proteins were extracted to be analysed by Nano-LC-MS/MS (Q-TOF). A C18 reversed-phase column and gradient elution with formic acid/acetonitrile solutions from 2 to 50% over 150 min were used to separate the proteins. Protein detection was performed using a microTOF-Q II (QTOF) equipped with captive source and an electrospray-ionization source. The data from mass spectrometry were processed using a Compass 1.7 and analyzed using Protein Scape, software that uses Mascot algorithms to perform protein searches. RESULTS: A set of 3,162 acetylated peptides derived from 351 acetylated proteins in the diacetyl-treated group was identified. Among them, 23 targeted proteins were significantly more acetylated in the diacetyl-treated group than in the PBS control. Protein acetylation of the group treated with 540 mg/kg/day of diacetyl was corroborated by Western blotting analysis. CONCLUSIONS: These data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems.

1-[(2,3-Dihydro-1-benzofuran-2-yl) methyl]piperazines as novel anti-inflammatory compounds: Synthesis and evaluation on H3 R/H4 R.

The histamine receptors (HRs) are members of G-protein-coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H3 R and H4 R have been explored as targets for drug discovery, including in the search for dual-acting H3 R/H4 R ligands. The H4 R, the most recent histamine receptor, is a promising target for novel anti-inflammatory agents in several conditions such as asthma and other chronic inflammatory diseases. Due to similarity with previously reported ligands of HRs, a set of 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazines were synthesized and evaluated in competitive binding assays as H3 R/H4 R ligands herein. The results showed the compounds presented affinity (Ki ) for H3 R/H4 R in micromolar range, and they are more selective to H3 R. All the compounds showed no important cytotoxicity to mammalian cells. The phenyl-substituted compound LINS01005 has shown the higher affinity of the set for H4 R, but no considerable selectivity toward this receptor over H3 R. LINS01005 showed interesting anti-inflammatory activity in murine asthma model, reducing the eosinophil counts in bronchoalveolar lavage fluid, as well as the COX-2 expression. The presented compounds are valuable prototypes for further improvements to achieve better anti-inflammatory agents.

1-[(2,3-Dihydro-1-benzofuran-2-yl) methyl]piperazines as novel anti-inflammatory compounds: synthesis and evaluation on H3R/H4R

The histamine receptors (HRs) are members of G-protein-coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H3R and H4R have been explored as targets for drug discovery, including in the search for dual-acting H3R/H4R ligands. The H4R, the most recent histamine receptor, is a promising target for novel anti-inflammatory agents in several conditions such as asthma and other chronic inflammatory diseases. Due to similarity with previously reported ligands of HRs, a set of 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazines were synthesized and evaluated in competitive binding assays as H3R/H4R ligands herein. The results showed the compounds presented affinity (K-i) for H3R/H4R in micromolar range, and they are more selective to H3R. All the compounds showed no important cytotoxicity to mammalian cells. The phenyl-substituted compound LINS01005 has shown the higher affinity of the set for H4R, but no considerable selectivity toward this receptor over H3R. LINS01005 showed interesting anti-inflammatory activity in murine asthma model, reducing the eosinophil counts in bronchoalveolar lavage fluid, as well as the COX-2 expression. The presented compounds are valuable prototypes for further improvements to achieve better anti-inflammatory agents.

L-Arginine Transport and Nitric Oxide Production in Kinin Receptor B1-/- Endothelial Cells.

Kinins are important vasoactive peptides, but the role of the B1 receptor subtype in the vascular control is poorly understood. This study analyzed the nitric oxide (NO) release, L-arginine (L-Arg) uptake and the expression of the cationic amino acid transporter (CAT) -1 in endothelial cells obtained from B1 receptor knockout (B1-/-) and wild type (WT) mice. NO production was assessed through a fluorescent dye in living cells stimulated with acetylcholine. L-Arg uptake was determined indirectly in the culture medium by HPLC, in the presence or absence of the CAT-1 blocker N-ethylmaleimide (NEM). CAT-1 mRNA levels and protein expression were determined by qPCR and western blot, respectively. NO release was significantly reduced in B1-/- when compared to WT cells. This result was accompanied by a decreased rate in the L-Arg uptake by B1-/- cells. Incubation with NEM impaired the L-Arg uptake in WT, but had no effect in B1-/- cells. Protein expression and mRNA levels for CAT-1 were reduced in B1-/- in comparison to WT cells. These findings suggest an important role of the endothelial B1 receptor in the vascular control by interfering with CAT-1 expression, L-Arg uptake and NO release.

Lung remodeling in a mouse model of asthma involves a balance between TGF-ß1 and BMP-7.

A key event in chronic allergic asthma is the TGF-ß-induced activation of fibroblasts into α-SMA-positive myofibroblasts which synthesize type-I collagen. In the present study we investigated the effect of the anti-fibrotic molecule BMP-7 in asthma. Balb/c mice were immunized i.p. with ovalbumin in alum and challenged every 2 days with ovalbumin aerosol (two or six challenges for acute and chronic protocols, respectively). The lung was evaluated for: α-SMA and type-I collagen by immunohistochemistry; BMP-7 and TGF- ß1 gene expression by qRT-PCR; type-I collagen and Smads 2 and 3 by immunoblotting; mucus by PSA staining. Type-I collagen around bronchi, α-SMA, mucus secretion, TGF- ß1 and BMP-7 gene expression were all increased in asthma. The TGF- ß1/BMP-7 ratio was higher in the chronic group and correlated with higher levels of collagen. Fibroblasts isolated from asthmatic and healthy lungs produced type-I collagen upon stimulation with TGF- ß1 via phosphorylation of Smad-2, Smad-3. Pre-treatment of the fibroblasts with BMP-7 reduced collagen production and Smads phosphorylation. Intranasal treatment of asthmatic mice with recombinant BMP-7 during the immunization protocol reduced lung inflammation and type I collagen deposition. These results suggest a protective role for BMP-7 in lung allergic inflammation, opposing the pro-fibrotic effects of TGF- ß1.

Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice.

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

A selective cyclooxygenase-2 inhibitor suppresses the growth of endometriosis with an antiangiogenic effect in a rat model.

OBJECTIVE: To analyze the antiangiogenic effects of the selective cyclooxygenase-2 (COX-2) inhibitor parecoxib on the growth of endometrial implants in a rat model of peritoneal endometriosis. DESIGN: Pharmacologic interventions in an experimental model of peritoneal endometriosis. SETTING: Research laboratory in the Federal University of Rio de Janeiro. ANIMAL(S): Twenty female Sprague-Dawley rats with experimentally induced endometriosis. INTERVENTION(S): After implantation and establishment of autologous endometrium onto the peritoneum abdominal wall, rats were randomized into groups and treated with parecoxib or the vehicle by IM injection for 30 days. MAIN OUTCOME MEASURE(S): Vascular density, the expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1, the distribution of activated macrophages, the expression of COX-2, and the prostaglandin concentration in the endometriotic lesions treated with parecoxib were analyzed. RESULT(S): The treatment significantly decreased the implant size, and histologic examination indicated mostly atrophy and regression. A reduction in microvessel density and in the number of macrophages, associated with decreased expression of VEGF and Flk-1, also were observed. The treatment group showed a low concentration of prostaglandin E(2). CONCLUSION(S): These results suggest that the use of COX-2 selective inhibitors could be effective to suppress the establishment and growth of endometriosis, partially through their antiangiogenic activity.

Modulation of inflammatory response by selective inhibition of cyclooxygenase-1 and cyclooxygenase-2 in acute kidney injury.

OBJECTIVE AND DESIGN: This work explored the role of inhibition of cyclooxygenases (COXs) in modulating the inflammatory response triggered by acute kidney injury. MATERIAL: C57Bl/6 mice were used. TREATMENT: Animals were treated or not with indomethacin (IMT) prior to injury (days -1 and 0). METHODS: Animals were subjected to 45 min of renal pedicle occlusion and sacrificed at 24 h after reperfusion. Serum creatinine and blood urea nitrogen, reactive oxygen species (ROS), kidney myeloperoxidase (MPO) activity, and prostaglandin E2 (PGE(2)) levels were analyzed. Tumor necrosis factor (TNF)-alpha, t-bet, interleukin (IL)-10, IL-1beta, heme oxygenase (HO)-1, and prostaglandin E synthase (PGES) messenger RNA (mRNA) were studied. Cytokines were quantified in serum. RESULTS: IMT-treated animals presented better renal function with less acute tubular necrosis and reduced ROS and MPO production. Moreover, the treatment was associated with lower expression of TNF-alpha, PGE(2), PGES, and t-bet and upregulation of HO-1 and IL-10. This profile was mirrored in serum, where inhibition of COXs significantly decreased interferon (IFN)-gamma, TNF-alpha, and IL-12 p70 and upregulated IL-10. CONCLUSIONS: COXs seem to play an important role in renal ischemia and reperfusion injury, involving the secretion of pro-inflammatory cytokines, activation of neutrophils, and ROS production. Inhibition of COX pathway is intrinsically involved with cytoprotection.

Antineoplasic activity of Copaifera multijuga oil and fractions against ascitic and solid Ehrlich tumor.

The aim of this study was to investigate the effect of chronic treatment with C. multijuga oil on Ehrlich tumor evolution. C. multijuga was fractionated in a KOH impregnated silica gel column chromatography to give three distinct fractions, i.e., hexanic, chloroformic, and methanolic, mainly composed by hydrocarbon sesquiterpenes, oxygenated sesquiterpenes and acidic diterpenes, respectively. Results demonstrated that the C. multijuga oil, the hexanic, and chloroformic fractions did not develop toxic effects. The oil, hexanic and chloroformic fractions (doses varying between 100 and 200mg/kg) showed antineoplasic properties against Ehrlich ascitic tumor (EAT) and solid tumor during 10 consecutive days of treatment inhibiting ascitic tumor cell number, reverting medulla and blood cell counts to values similar to control group, and inhibiting the increase on several inflammatory mediators (total protein, PGE(2), nitric oxide, and TNF) on ascitic fluid. The treatment also inhibited the increase in paw volume on tumor-inoculated mice. In conclusion, C. multijuga as well as its fractions demonstrated antineoplasic effect even after oral administration confirming its use by traditional medicine.

Bronchoconstriction and endogenous nitric oxide in isolated lungs of spontaneously hypertensive rats.

Bronchoconstrictor responses were measured in lungs isolated from spontaneously hypertensive (SHR) and normotensive rats, perfused via the airways. Lungs from SHRs were more responsive than lungs from normotensive rats to methacholine, 5-hydroxytryptamine (5-HT), arachidonic acid or prostaglandin H(2). The responses of SHR airways to methacholine or 5-HT were unaffected by pretreatment in vivo with an inhibitor of nitric oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 30 mg kg(-1)), although responses in normotensive airways to methacholine, but not to 5-HT, were enhanced. Antigen challenge of isolated lungs from actively sensitized rats elicited bronchoconstriction, not different between strains. Pretreatment with L-NAME increased the response to antigen challenge only in normotensive lungs. Compound 48/80 induced bronchoconstriction in lungs from either strain, equally. These responses to compound 48/80 were unaffected by L-NAME pretreatment. Thus, SHR airways lack relaxing factors and degranulation of mast cells in SHR lungs was not affected by endogenous NO.