Cátia Lira do Amaral

Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice

The aim of this study was to investigate whether treatment with tributyrin (Tb; a butyrate prodrug) results in protection against diet-induced obesity and associated insulin resistance. C57BL/6 male mice fed a standard chow or high-fat diet were treated with Tb (2 g/kg body wt, 10 wk) and evaluated for glucose homeostasis, plasma lipid profile, and inflammatory status. Tb protected mice against obesity and obesity-associated insulin resistance and dyslipidemia without food consumption being affected. Tb attenuated the production of TNFα and IL-1β by peritoneal macrophages and their expression in adipose tissue. Furthermore, in the adipose tissue, Tb reduced the expression of MCP-1 and infiltration by leukocytes and restored the production of adiponectin. These effects were associated with a partial reversion of hepatic steatosis, reduction in liver and skeletal muscle content of phosphorylated JNK, and an improvement in muscle insulin-stimulated glucose uptake and Akt signaling. Although part of the beneficial effects of Tb are likely to be secondary to the reduction in body weight, we also found direct protective actions of butyrate reducing TNFα production after LPS injection and in vitro by LPS- or palmitic acid-stimulated macrophages and attenuating lipolysis in vitro and in vivo. The results, reported herein, suggest that Tb may be useful for the treatment and prevention of obesity-related metabolic disorders.

DNA Methylation Pattern in Overweight Women under an Energy-Restricted Diet Supplemented with Fish Oil

Dietary factors modulate gene expression and are able to alter epigenetic signatures in peripheral blood mononuclear cells (PBMC). However, there are limited studies about the effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on the epigenetic mechanisms that regulate gene expression. This research investigates the effects of n-3-rich fish oil supplementation on DNA methylation profile of several genes whose expression has been reported to be downregulated by n-3 PUFA in PBMC: CD36, FFAR3, CD14, PDK4, and FADS1. Young overweight women were supplemented with fish oil or control in a randomized 8-week intervention trial following a balanced diet with 30% energy restriction. Fatty acid receptor CD36 decreased DNA methylation at CpG +477 due to energy restriction. Hypocaloric diet-induced weight loss also reduced the methylation percentages of CpG sites located in CD14, PDK4, and FADS1. The methylation patterns of these genes were only slightly affected by the fish oil supplementation, being the most relevant to the attenuation of the weight loss-induced decrease in CD36 methylation after adjusting by baseline body weight. These results suggest that the n-3 PUFA-induced changes in the expression of these genes in PBMC are not mediated by DNA methylation, although other epigenetic mechanisms cannot be discarded.

Evaluation of the antihypertensive properties of yellow passion fruit pulp (Passiflora edulis Sims f. flavicarpa Deg.) in spontaneously hypertensive rats.

Various species of the genus Passiflora have been extensively used in traditional medicine as sedatives, anxiolytics, diuretics and analgesics. In the present study, after the identification and quantification of phytochemical compounds from yellow passion fruit pulp by liquid chromatography‐photodiode array‐mass spectrometry (HPLC‐PDA‐MS/MS), its antihypertensive effect was investigated on spontaneously hypertensive rats. Additionally, the renal function, evaluated by kidney/body weight, serum creatinine, proteinuria, urinary flow, reduced glutathione (GSH) levels and thiobarbituric acid‐reactive substances (TBARS) and mutagenicity in bone marrow cells were assessed to evaluate the safety of passion fruit consumption. Yellow passion fruit pulp (5, 6 or 8 g/kg b.w.) was administered by gavage once a day for 5 consecutive days. HLPC‐PDA‐MS/MS analysis revealed that yellow passion fruit pulp contains phenolic compounds, ascorbic acid, carotenoids and flavonoids. The highest dose of passion fruit pulp significantly reduced the systolic blood pressure, increased the GSH levels and decreased TBARS. There were no changes in renal function parameters or the frequency of micronuclei in bone marrow cells. In conclusion, the antihypertensive effect of yellow passion fruit pulp, at least in part, might be due to the enhancement of the antioxidant status. The exact mechanisms responsible by this effect need further investigation. Copyright

Sunflower Oil Supplementation Has Proinflammatory Effects and Does Not Reverse Insulin Resistance in Obesity Induced by High-Fat Diet in C57BL/6 Mice

High consumption of polyunsaturated fatty acids, such as sunflower oil has been associated to beneficial effects in plasma lipid profile, but its role on inflammation and insulin resistance is not fully elucidated yet. We evaluated the effect of sunflower oil supplementation on inflammatory state and insulin resistance condition in HFD-induced obese mice. C57BL/6 male mice (8 weeks) were divided in four groups: (a) control diet (CD), (b) HFD, (c) CD supplemented with n-6 (CD + n-6), and (d) HFD supplemented with n-6 (HFD + n-6). CD + n-6 and HFD + n-6 were supplemented with sunflower oil by oral gavage at 2 g/Kg of body weight, three times per week. CD and HFD were supplemented with water instead at the same dose. HFD induced whole and muscle-specific insulin resistance associated with increased inflammatory markers in insulin-sensitive tissues and macrophage cells. Sunflower oil supplementation was not efficient in preventing or reducing these parameters. In addition, the supplementation increased pro-inflammatory cytokine production by macrophages and tissues. Lipid profile, on the other hand, was improved with the sunflower oil supplementation in animals fed HFD. In conclusion, sunflower oil supplementation improves lipid profile, but it does not prevent or attenuate insulin resistance and inflammation induced by HFD in C57BL/6 mice.

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diets effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diets effects on genomic stability and DNA methylation.

Fish oil prevents changes induced by a high-fat diet on metabolism and adipokine secretion in mice subcutaneous and visceral adipocytes

Fish oil (FO), rich in omega‐3 polyunsaturated fatty acids, has beneficial effects on changes induced by obesity and partially prevents associated comorbidities. The effects of FO on adipocytes from different adipose tissue depots in high‐fat (HF) diet induced obese mice have not been uninvestigated. This is the first study to examine the effects of FO on changes in metabolism and adipokine production in adipocytes from s.c. (inguinal; ING) or visceral (retroperitoneal; RP) white adipose depots in a HF diet‐induced obese mice. Unlike most studies performed previously, FO supplementation was initiated 4 weeks before the induction of obesity. HF diet caused marked changes in ING (glucose uptake and secretion of adiponectin, tumour necrosis factor‐α and interleukin‐6 in ING) and RP (lipolysis, de novo lipogenesis and secretion of pro‐inflammatory cytokines) adipose depots. Previous and concomitant FO administration prevented the changes in ING and RP adipocytes induced by the HF diet.

DNA Methylation Changes Induced by a High-Fat Diet and Fish Oil Supplementation in the Skeletal Muscle of Mice

Background/Aims: To investigate the global changes in DNA methylation and methylation of the promoter region of the peroxisome proliferator-activated receptor gamma transcript variant 2 (Pparg2) gene resulting from a high-fat diet (HFD) and/or fish oil supplementation. Methods: Fish oil, rich in omega-3 polyunsaturated fatty acids, or water was orally administered to male mice for 12 weeks. After the first 4 weeks, the animals were fed a control diet or an HFD until the end of the experimental protocol, when the epididymal fat, gastrocnemius muscle and liver were excised. Results:Pparg2 mRNA expression was upregulated by obesity and downregulated by fish oil supplementation in the liver. In the gastrocnemius muscle, diet-induced obesity increased global DNA methylation. Fish oil prevented the decrease in Pparg2 promoter methylation induced by obesity in the gastrocnemius muscle. Regardless of the diet given, fish oil supplementation increased Pparg2 promoter methylation at CpG-263 in muscle and adipose tissue. Conclusion: HFD and fish oil modified global and Pparg2 promoter DNA methylation in a tissue-specific manner. Fish oil supplementation attenuated body weight gain, abolished the increase in Pparg2 expression in the liver and prevented the decrease in Pparg2 promoter methylation in the muscle induced by the HFD.

Combination of a high-fat diet with sweetened condensed milk exacerbates inflammation and insulin resistance induced by each separately in mice

Obesogenic diets increase body weight and cause insulin resistance (IR), however, the association of these changes with the main macronutrient in the diet remains to be elucidated. Male C57BL/6 mice were fed with: control (CD), CD and sweetened condensed milk (HS), high-fat (HF), and HF and condensed milk (HSHF). After 2 months, increased body weight, glucose intolerance, adipocyte size and cholesterol levels were observed. As compared with CD, HS ingested the same amount of calories whereas HF and HSHF ingested less. HS had increased plasma AST activity and liver type I collagen. HF caused mild liver steatosis and hepatocellular damage. HF and HSHF increased LDL-cholesterol, hepatocyte and adipocyte hypertrophy, TNF-α by macrophages and decreased lipogenesis and adiponectin in adipose tissue (AT). HSHF exacerbated these effects, increasing IR, lipolysis, mRNA expression of F4/80 and leptin in AT, Tlr-4 in soleus muscle and IL-6, IL-1β, VCAM-1, and ICAM-1 protein in AT. The three obesogenic diets induced obesity and metabolic dysfunction. HS was more proinflammatory than the HF and induced hepatic fibrosis. The HF was more detrimental in terms of insulin sensitivity, and it caused liver steatosis. The combination HSHF exacerbated the effects of each separately on insulin resistance and AT inflammatory state.

Attenuation of obesity and insulin resistance by fish oil supplementation is associated with improved skeletal muscle mitochondrial function in mice fed a high-fat diet.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to improve insulin sensitivity and glucose homeostasis in animal models of insulin resistance, but the involved mechanisms still remain unresolved. In this study, we evaluated the effects of fish oil (FO), a source of n-3 PUFAs, on obesity, insulin resistance and muscle mitochondrial function in mice fed a high-fat diet (HFD). C57Bl/6 male mice, 8 weeks old, were divided into four groups: control diet (C), high-fat diet (H), C+FO (CFO) and H+FO (HFO). FO was administered by oral gavage (2 g/kg b.w.), three times a week, starting 4 weeks before diet administration until the end of the experimental protocol. HFD-induced obesity and insulin resistance associated with impaired skeletal muscle mitochondrial function, as indicated by decreased oxygen consumption, tricarboxylic acid cycle intermediate (TCAi) contents (citrate, α-ketoglutarate, malate and oxaloacetate), oxidative phosphorylation protein content and mitochondrial biogenesis. These effects were associated with elevated reactive oxygen species production, decreased PGC1-a transcription and reduced Akt phosphorylation. The changes induced by the HFD were partially attenuated by FO, which decreased obesity and insulin resistance and increased mitochondrial function. In the H group, FO supplementation also improved oxygen consumption; increased TCAi content, and Akt and AMPK phosphorylation; and up-regulated mRNA expression of Gpat1, Pepck, catalase and mitochondrial proteins (Pgc1α, Pparα, Cpt1 and Ucp3). These results suggest that dietary FO attenuates the deleterious effects of the HFD (obesity and insulin resistance) by improving skeletal muscle mitochondrial function.

Methionine-supplemented diet affects the expression of cardiovascular disease-related genes and increases inflammatory cytokines in mice heart and liver

ABSTRACT Some important environmental factors that influence the development of cardiovascular diseases (CVD) include tobacco, excess alcohol, and unhealthy diet. Methionine obtained from the diet participates in the synthesis of DNA, proteins, lipids and affects homocysteine levels, which is associated with the elevated risk for CVD development. Therefore, the aim of this study was to investigate the manner in which dietary methionine might affect cellular mechanisms underlying CVD occurrence. Swiss albino mice were fed either control (0.3% DL-methionine), methionine-supplemented (2% DL-methionine), or a methionine-deprived diet (0% DL-methionine) over a 10-week period. The parameters measured included plasma homocysteine concentrations, oxidative stress by reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, levels of inflammatory cytokines IL-1ß, TNF-α, and IL-6, as well as expression of genes associated with CVD. The levels of apolipoprotein A5 (APOA5), a regulator of plasma triglycerides, were measured. The methionine-supplemented diet increased oxidative stress by lowering the GSH/GSSG ratio in heart tissues and decreased expression of the genes Apob, Ctgf, Serpinb2, Spp1, Il1b, and Sell, but elevated expression of Thbs4, Tgfb2, Ccr1, and Vegfa. Methionine-deprived diet reduced expression of Col3a1, Cdh5, Fabp3, Bax, and Hbegf and increased expression of Sell, Ccl5, Itga2, Birc3, Msr1, Bcl2a1a, Il1r2, and Selp. Methionine-deprived diet exerted pro-inflammatory consequences as evidenced by elevated levels of cytokines IL-1ß, TNF-α, and IL-6 noted in liver. Methionine-supplemented diet increased hepatic IL-6 and cardiac TNF-α. Both methionine supplementation and deprivation lowered hepatic levels of APOA5. In conclusion, data demonstrated that a methionine-supplemented diet modulated important biological processes associated with high risk of CVD development.