Changes in gene expression in PBMCs profiles of PPAR alpha target genes in obese and non-obese individuals during fasting

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identification and characterization of regions of difference between the Salmonella Gallinarum biovar Gallinarum and the Salmonella Gallinarum biovar Pullorum genomes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Circulating thyroid hormone levels in young pregnant rats and their fetuses: Effect of malnutrition

In order to evaluate some factors likely to be involved in the maternal and fetal growth impairment due to alimentary protein deficiency, the circulating levels of triiodothyronine (T 3) and thyroxine (T 4) were studied in 4 young (45-day-old) female rat groups: control and malnourished, both nonpregnant and pregnant; similarly schedules groups were studied using adult (100-day-old) rats. Circulating levels of T 4 were higher in nonpregnant, malnourished young rats in their corresponding controls. T 3 levels were higher in young malnourished animals and lower in adult malnourished animals, nonpregnant or pregnant, as compared to controls. Pups from young malnourished mothers showed significantly lower birth weights than those from controls. The present results suggest that there are age differences in thyroid function, as affected by protein-calorie malnutrition in pregnant and non-pregnant rats. On the other hand, the circulating thyroid hormone levels were not importantly affected by the mother dietary protein restriction under our experimental conditions.

Triiodothyronine and breast cancer

The thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), are essential for survival; they are involved in the processes of development, growth, and metabolism. In addition to hyperthyroidism or hypothyroidism, THs are involved in other diseases. The role of THs in the development and differentiation of mammary epithelium is well established; however, their specific role in the pathogenesis of breast cancer (BC) is controversial. Steroid hormones affect many human cancers and the abnormal responsiveness of the mammary epithelial cells to estradiol (E2) in particular is known to be an important cause for the development and progression of BC. The proliferative effect of T3 has been demonstrated in various types of cancer. In BC cell lines, T3 may foster the conditions for tumor proliferation and increase the effect of cell proliferation by E2; thus, T3 may play a role in the development and progression of BC. Studies show that T3 has effects similar to E2 in BC cell lines. Despite controversy regarding the relationship between thyroid disturbances and the incidence of BC, studies show that thyroid status may influence the development of tumor, proliferation and metastasis.

Hypothalamic energy metabolism is impaired by doxorubicin independently of inflammation in non-tumour-bearing rats

We sought to explore the effects of doxorubicin on inflammatory profiles and energy metabolism in the hypothalamus of rats. To investigate these effects, we formed two groups: a control (C) group and a Doxorubicin (DOXO) group. Sixteen rats were randomly assigned to either the control (C) or DOXO groups. The hypothalamus was collected. The levels of interleukin (IL)-1β, IL-6, IL-10, TNF-α and energy metabolism (malate dehydrogenase, complex I and III activities) were analysed in the hypothalamus. The DOXO group exhibited a decreased body weight (p < 0.01). Hypothalamic malate dehydrogenase activity was reduced when compared with control (p < 0.05). In addition, pro-inflammatory cytokine levels were unchanged. Therefore, our results demonstrate that doxorubicin leads to an impairment of \hypothalamic energy metabolism, but do not affect the inflammatory pathway. Copyright © 2015 John Wiley & Sons, Ltd. Conflict of Interest Significance paragraph The hypothalamus is a central organ that regulates a great number of functions, such as food intake, temperature and energy expenditure, among others. Doxorubicin can lead to deep anorexia and metabolic chaos; thus, we observed the effect of this chemotherapeutic drug on the inflammation and metabolism in rats after the administration of doxorubicin in order to understand the central effect in the hypothalamus. Drug treatment by doxorubicin is used as a cancer therapy; however the use of this drug may cause harmful alterations to the metabolism. Thus, further investigations are needed on the impact of drug therapy over the long term.

Sodium bicarbonate supplementation improved MAOD but is not correlated with 200- and 400-m running performances: a double-blind, crossover, and placebo-controlled study

The aim of the study was to investigate the effects of acute supplementation of sodium bicarbonate (NaHCO3) on maximal accumulated oxygen deficit (MAOD) determined by a single supramaximal effort (MAODALT) in running and the correlation with 200- and 400-m running performances. Fifteen healthy men (age, 23 ± 4 years; maximal oxygen uptake, 50.6 ± 6.1 mL·kg(-1)·min(-1)) underwent a maximal incremental exercise test and 2 supramaximal efforts at 110% of the intensity associated with maximal oxygen uptake, which was carried out after ingesting either 0.3 g·kg(-1) body weight NaHCO3 or a placebo (dextrose) and completing 200- and 400-m performance tests. The study design was double-blind, crossover, and placebo-controlled. Significant differences were found between the NaHCO3 and placebo conditions for MAODALT (p = 0.01) and the qualitative inference for substantial changes showed a very likely positive effect (98%). The lactic anaerobic contribution in the NaHCO3 ingestion condition was significantly higher (p < 0.01) and showed a very likely positive effect (99% chance), similar to that verified for peak blood lactate concentration (p < 0.01). No difference was found for time until exhaustion (p = 0.19) or alactic anaerobic contribution (p = 0.81). No significant correlations were observed between MAODALT and 200- and 400-m running performance tests. Therefore, we can conclude that both MAODALT and the anaerobic lactic metabolism are modified after acute NaHCO3 ingestion, but it is not correlated with running performance.

Nutritional and metabolic risk factors for insulin resistance in adults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The effects of high-intensity resistance exercise on the blood lipid profile and liver function in hypercholesterolemic hamsters

It is well established that atherogenic dyslipidemia, characterized by high levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL) cholesterol and low levels of high-density lipoprotein (HDL) cholesterol, constitutes important risk factors for cardiovascular disease. Regular exercise has been associated with a reduced risk for metabolic diseases. However, studies supporting the concept that resistance exercise is a modifier of blood lipid parameters are often contradictory. The aim of this study was to investigate the effects of high-intensity resistance exercise on the serum levels of TG, TC, HDL and non-HDL cholesterol, glucose, and the liver function enzymes alanine aminotransferase (ALT, EC 2.6.1.2) and aspartate aminotransferase (AST, EC 2.6.1.1) in golden Syrian hamsters (Mesocricetus auratus (Waterhouse, 1839)) fed a hypercholesterolemic diet. Sedentary groups (S) and exercise groups (E) were fed a standard diet (SS and ES) or a cholesterol-enriched diet (standard plus 1% cholesterol, SC and EC). Resistance exercise was performed by jumps in the water, carrying a load strapped to the chest, representing 10 maximum repetitions (10 RM, 30 s rest, five days per week for five weeks). Mean blood sample comparisons were made by ANOVA + Tukey or ANOVA + Kruskal-Wallis tests (p < 0.05) to compare parametric and nonparametric samples, respectively. There were no differences in blood lipids between the standard diet groups (SS and ES) (p > 0.05). However, the EC group increased the glucose, non-HDL, and TC levels in comparison with the ES group. Moreover, the EC group increased the TG levels versus the SC group (p < 0.05). In addition, the ALT levels were increased only by diet treatment. These findings indicated that high-intensity resistance exercise contributed to dyslipidemia in hamsters fed a hypercholesterolemic diet, whereas liver function enzymes did not differ in regards to the exercise protocol.

Omega-3 improves glucose tolerance but increases lipid peroxidation and DNA damage in hepatocytes of fructose-fed rats

The high consumption of fructose is linked to the increase in various characteristics of the metabolic syndrome. Fish oil is beneficial for the treatment of these comorbidities, such as insulin resistance, dyslipidemia, and hepatic steatosis. The objective of this study was to evaluate the consequences of the administration of fish oil concomitant to fructose ingestion during the experiment (45 days) and during the final 15 days in high-fructose-fed rats. Male Wistar rats were divided into 5 groups: control; those receiving 10% fish oil (FO); those receiving 60% fructose (Fr); those receiving 60% fructose and 10% fish oil for 45 days (FrFO); and those receiving fructose plus soybean oil for 30 days and fish oil for the final 15 days of the study (FrFO15). There was an increase in triacylglycerol, serum total cholesterol, and hepatic volume in the Fr group. The FO and FrFO groups experienced an increase in lipid peroxidation and a decrease in serum reduced glutathione. The FrFO group suffered greater hepatic injury, with increased alanine aminotransferase levels and DNA damage. Marked n-3 incorporation occurred in the groups receiving fish oil, favoring a better response to the oral glucose tolerance test. Fructose induced comorbidities of the metabolic syndrome, and the use of fish oil promoted a better glucose tolerance, although it was accompanied by more hepatocyte damage.

Low fatness, reduced fat intake and adequate plasmatic concentrations of LDL-cholesterol are associated with high bone mineral density in women: a cross-sectional study with control group

Background: Several parameters are associated with high bone mineral density (BMD), such as overweight, black background, intense physical activity (PA), greater calcium intake and some medications. The objectives are to evaluate the prevalence and the main aspects associated with high BMD in healthy women. Methods: After reviewing the database of approximately 21,500 BMD scans performed in the metropolitan area of Sao Paulo, Brazil, from June 2005 to October 2010, high BMD (over 1400 g/cm(2) at lumbar spine and/or above 1200 g/cm2 at femoral neck) was found in 421 exams. Exclusion criteria were age below 30 or above 60 years, black ethnicity, pregnant or obese women, disease and/or medications known to interfere with bone metabolism. A total of 40 women with high BMD were included and matched with 40 healthy women with normal BMD, paired to weight, age, skin color and menopausal status. Medical history, food intake and PA were assessed through validated questionnaires. Body composition was evaluated through a GE-Lunar DPX MD + bone densitometer. Radiography of the thoracic and lumbar spine was carried out to exclude degenerative alterations or fractures. Biochemical parameters included both lipid and hormonal profiles, along with mineral and bone metabolism. Statistical analysis included parametric and nonparametric tests and linear regression models. P < 0.05 was considered significant. Results: The mean age was 50.9 (8.3) years. There was no significant difference between groups in relation to PA, smoking, intake of calcium and vitamin D, as well as laboratory tests, except serum C-telopeptide of type I collagen (s-CTX), which was lower in the high BMD group (p = 0.04). In the final model of multivariate regression, a lower fat intake and body fatness as well a better profile of LDL-cholesterol predicted almost 35% of high BMD in women. (adjusted R2 = 0.347; p < 0.001). In addition, greater amounts of lean mass and higher IGF-1 serum concentrations played a protective role, regardless age and weight. Conclusion: Our results demonstrate the potential deleterious effect of lipid metabolism-related components, including fat intake and body fatness and worse lipid profile, on bone mass and metabolism in healthy women.

Trabecular bone loss after administration of the second-generation antipsychotic risperidone is independent of weight gain

Second generation antipsychotics (SGAs) have been linked to metabolic and bone disorders in clinical studies, but the mechanisms of these side effects remain unclear. Additionally, no studies have examined whether SGAs cause bone loss in mice. Using in vivo and in vitro modeling we examined the effects of risperidone, the most commonly prescribed SGA, on bone in C57BL6/J (B6) mice. Mice were treated with risperidone orally by food supplementation at a dose of 1.25 mg/kg daily for 5 and 8 weeks, starting at 3.5 weeks of age. Risperidone reduced trabecular BV/TV, trabecular number and percent cortical area. Trabecular histomorphometry demonstrated increased resorption parameters, with no change in osteoblast number or function. Risperidone also altered adipose tissue distribution such that white adipose tissue mass was reduced and liver had significantly higher lipid infiltration. Next, in order to tightly control risperidone exposure, we administered risperidone by chronic subcutaneous infusion with osmotic minipumps (0.5 mg/kg daily for 4 weeks) in 7 week old female B6 mice. Similar trabecular and cortical bone differences were observed compared to the orally treated groups (reduced trabecular BV/TV, and connectivity density, and reduced percent cortical area) with no change in body mass, percent body fat, glucose tolerance or insulin sensitivity. Unlike in orally treated mice, risperidone infusion reduced bone formation parameters (serum P1NP, MAR and BFR/BV). Resorption parameters were elevated, but this increase did not reach statistical significance. To determine if risperidone could directly affect bone cells, primary bone marrow cells were cultured with osteoclast or osteoblast differentiation media. Risperidone was added to culture medium in clinically relevant doses of 0, 2.5 or 25 ng/ml. The number of osteoclasts was significantly increased by addition in vitro of risperidone while osteoblast differentiation was not altered. These studies indicate that risperidone treatment can have negative skeletal consequences by direct activation of osteoclast activity and by indirect non-cell autonomous mechanisms. Our findings further support the tenet that the negative side effects of SGAs on bone mass should be considered when weighing potential risks and benefits, especially in children and adolescents who have not yet reached peak bone mass. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism. (C) 2011 Elsevier Inc. All rights reserved.

Pet food: quality and quality improvement

Today’s pet food industry is growing rapidly, with pet owners demanding high-quality diets for their pets. The primary role of diet is to provide enough nutrients to meet metabolic requirements, while giving the consumer a feeling of well-being. Diet nutrient composition and digestibility are of crucial importance for health and well being of animals. A recent strategy to improve the quality of food is the use of “nutraceuticals” or “Functional foods”. At the moment, probiotics and prebiotics are among the most studied and frequently used functional food compounds in pet foods. The present thesis reported results from three different studies. The first study aimed to develop a simple laboratory method to predict pet foods digestibility. The developed method was based on the two-step multi-enzymatic incubation assay described by Vervaeke et al. (1989), with some modification in order to better represent the digestive physiology of dogs. A trial was then conducted to compare in vivo digestibility of pet-foods and in vitro digestibility using the newly developed method. Correlation coefficients showed a close correlation between digestibility data of total dry matter and crude protein obtained with in vivo and in vitro methods (0.9976 and 0.9957, respectively). Ether extract presented a lower correlation coefficient, although close to 1 (0.9098). Based on the present results, the new method could be considered as an alternative system of evaluation of dog foods digestibility, reducing the need for using experimental animals in digestibility trials. The second parte of the study aimed to isolate from dog faeces a Lactobacillus strain capable of exert a probiotic effect on dog intestinal microflora. A L. animalis strain was isolated from the faeces of 17 adult healthy dogs..The isolated strain was first studied in vitro when it was added to a canine faecal inoculum (at a final concentration of 6 Log CFU/mL) that was incubated in anaerobic serum bottles and syringes which simulated the large intestine of dogs. Samples of fermentation fluid were collected at 0, 4, 8, and 24 hours for analysis (ammonia, SCFA, pH, lactobacilli, enterococci, coliforms, clostridia). Consequently, the L. animalis strain was fed to nine dogs having lactobacilli counts lower than 4.5 Log CFU per g of faeces. The study indicated that the L animalis strain was able to survive gastrointestinal passage and transitorily colonize the dog intestine. Both in vitro and in vivo results showed that the L. animalis strain positively influenced composition and metabolism of the intestinal microflora of dogs. The third trail investigated in vitro the effects of several non-digestible oligosaccharides (NDO) on dog intestinal microflora composition and metabolism. Substrates were fermented using a canine faecal inoculum that was incubated in anaerobic serum bottles and syringes. Substrates were added at the final concentration of 1g/L (inulin, FOS, pectin, lactitol, gluconic acid) or 4g/L (chicory). Samples of fermentation fluid were collected at 0, 6, and 24 hours for analysis (ammonia, SCFA, pH, lactobacilli, enterococci, coliforms). Gas production was measured throughout the 24 h of the study. Among the tested NDO lactitol showed the best prebiotic properties. In fact, it reduced coliforms and increased lactobacilli counts, enhanced microbial fermentation and promoted the production of SCFA while decreasing BCFA. All the substrates that were investigated showed one or more positive effects on dog faecal microflora metabolism or composition. Further studies (in particular in vivo studies with dogs) will be needed to confirm the prebiotic properties of lactitol and evaluate its optimal level of inclusion in the diet.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.

New analytical LC-mass spectrometry methodologies for the quali-quantitative determination of natural substances and drugs in complex matrices

This thesis reports an integrated analytical and physicochemical approach for the study of natural substances and new drugs based on mass spectrometry techniques combined with liquid chromatography. In particular, Chapter 1 concerns the study of Berberine a natural substance with pharmacological activity for the treatment of hepatobiliary and intestinal diseases. The first part focused on the relationships between physicochemical properties, pharmacokinetics and metabolism of Berberine and its metabolites. For this purpose a sensitive HPLC-ES-MS/MS method have been developed, validated and used to determine these compounds during their physicochemical properties studies and plasma levels of berberine and its metabolites including berberrubine(M1), demethylenberberine(M3), and jatrorrhizine(M4) in humans. Data show that M1, could have an efficient intestinal absorption by passive diffusion due to a keto-enol tautomerism confirmed by NMR studies and its higher plasma concentration. In the second part of Chapter 1, a comparison between M1 and BBR in vivo biodistribution in rat has been studied. In Chapter 2 a new HPLC-ES-MS/MS method for the simultaneous determination and quantification of glucosinolates, as glucoraphanin, glucoerucin and sinigrin, and isothiocyanates, as sulforaphane and erucin, has developed and validated. This method has been used for the analysis of functional foods enriched with vegetable extracts. Chapter 3 focused on a physicochemical study of the interaction between the bile acid sequestrants used in the treatment of hypercholesterolemia including colesevelam and cholestyramine with obeticolic acid (OCA), potent agonist of nuclear receptor farnesoid X (FXR). In particular, a new experimental model for the determination of equilibrium binding isotherm was developed. Chapter 4 focused on methodological aspects of new hard ionization coupled with liquid chromatography (Direct-EI-UHPLC-MS) not yet commercially available and potentially useful for qualitative analysis and for “transparent” molecules to soft ionization techniques. This method was applied to the analysis of several steroid derivatives.

Eye movement and diffusion tensor imaging analysis of treatment effects in a Niemann-Pick Type C patient

New treatment options for Niemann-Pick Type C (NPC) have recently become available. To assess the efficiency and efficacy of these new treatment markers for disease status and progression are needed. Both the diagnosis and the monitoring of disease progression are challenging and mostly rely on clinical impression and functional testing of horizontal eye movements. Diffusion tensor imaging (DTI) provides information about the microintegrity especially of white matter. We show here in a case report how DTI and measures derived from this imaging method can serve as adjunct quantitative markers for disease management in Niemann-Pick Type C. Two approaches are taken--first, we compare the fractional anisotropy (FA) in the white matter globally between a 29-year-old NPC patient and 18 healthy age-matched controls and show the remarkable difference in FA relatively early in the course of the disease. Second, a voxelwise comparison of FA values reveals where white matter integrity is compromised locally and demonstrate an individualized analysis of FA changes before and after 1year of treatment with Miglustat. This method might be useful in future treatment trials for NPC to assess treatment effects.