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Perinatal maternal hypercaloric diets increase the susceptibility to metabolic disorders in the offspring. We hypothesized that maternal intake of an isocaloric moderate-fat diet (mMFD) would disturb the glucose homeostasis and favor the β-cell failure in response to fructose overload in adult male offspring.Female Wistar rats received an isocaloric diet (3.9 kcal/g) containing 29 % (mMFD) or 9 % as fat (mSTD) prior mating and throughout gestation and lactation. After weaning, male offspring received standard chow and fructose-drinking water (15 %) between 120 and 150 days old.mMFD offspring had higher body weight, visceral adiposity and, fasting glycemia, with normal insulinemia. Fructose increased glycemia at 15 min from oral glucose administration, but only mMFD had returned to basal glucose levels at 120 min. Fructose increased HOMA-IR index regardless diet, but only mMFD exhibited hyperinsulinemia and a higher HOMA-β index. mMFD pancreatic islets showed increased area and insulin immunostaining density, suggesting β-cell hypertrophy. Fructose induced the expected compensatory hypertrophy in mSTD islets, while the opposite occurred in mMFD islets, associated with reduced insulin immunostaining, suggesting lower insulin storage. Pancreatic islets isolated from mMFD offspring exhibited higher glucose-stimulated insulin release at physiological concentrations. However, at higher glucose concentrations, the islets from fructose-treated mMFD reduced dramatically their insulin release, suggesting exhaustion.Isocaloric mMFD induced adaptive mechanism in the offspring allowing insulin hypersecretion, but under metabolic challenge with fructose, β-cell compensation shifts to exhaustion, favoring dysfunction. Therefore, a maternal MFD may contribute to developing diabetes under fructose overload in the adult offspring.

In collaboration with the American College of Veterinary Pathologists

The endocannabinoid system (ECS) increases food intake, appetite for fat and lipogenesis, while decreases energy expenditure (thermogenesis), contributing to metabolic dysfunctions. We demonstrated that maternal high-fat diet (HFD) alters cannabinoid signaling in brown adipose tissue (BAT) of neonate and weanling male rat offspring, which have increased adiposity but also higher energy expenditure in adulthood. In this study, the main objective was to investigate the ECS expression in thermogenic tissues as BAT and skeletal muscle of adult rats programmed by maternal HFD. We hypothesized that maternal HFD would modulate ECS and energy metabolism markers in BAT and skeletal muscle of adult male offspring.Female rats received standard diet (9.4 % of calories as fat) or isocaloric HFD (28.9 % of calories as fat) for 8 weeks premating and throughout gestation and lactation. Male offspring were weaned on standard diet and euthanatized in adulthood.Maternal HFD increased body weight, adiposity, glycemia, leptinemia while decreased testosterone levels in adult offspring. Maternal HFD did not change cannabinoid receptors in BAT or skeletal muscle as hypothesized but increased the content of uncoupling protein and tyrosine hydroxylase (thermogenic markers) in parallel to changes in mitochondrial morphology in skeletal muscle of adult offspring.In metabolic programming models, the ECS modulation in the BAT and skeletal muscle may be more important early in life to adapt energy metabolism during maternal dietary insult, and other mechanisms are possibly involved in muscle metabolism long-term regulation.

Diverse food safety programmes around the world are designed to help ensure production of safe food. To meet this need, the development and implementation of more efficient and effective analytical methods to monitor residues (pesticides and veterinary drugs) and contaminants in food is important. In this study, we report the validation results for a simple high-throughput mega-method for residual analysis of 213 pesticides and veterinary drugs, including 15 metabolites, plus 12 environmental contaminants (polychlorinated biphenyls) in tilapia muscle for implementation in routine laboratory analyses. The generic sample preparation method and analytical approach are known as QuEChERSER (more than QuEChERS). A small portion of the initial extract (204 µL) is taken for analysis by ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) covering 145 analytes, and the remaining extract undergoes a salting out step followed by an automated robotic instrument top sample preparation (ITSP) cleanup, also known as micro-solid-phase extraction (µSPE), plus fast low-pressure gas chromatography LPGC-MS/MS for 134 analytes (66 pesticides are targeted in both UHPLC-MS/MS and LPGC-MS/MS). The mega-method was validated in spiked tilapia samples at 5, 10, 15, and 20 ng/g with 10 replicates per level over two days (n = 80 overall), and 70-140% recoveries with RSDs ≤20% were achieved for 92% of the analytes in LC and 82% in GC. No significant matrix effects were observed for the analytes in LPGC-MS/MS, and only 5% of the analytes exceeded ±20% matrix effect in UHPLC-MS/MS. Analysis of standard reference materials (NIST SRMs 1946 and 1947) for contaminants in freeze-dried fish showed acceptable results, further demonstrating that the QuEChERSER mega-method can be implemented to expand analytical scope and increase laboratory efficiency compared to the QuEChERS method.

Perinatal maternal obesity and excessive fructose consumption have been associated with liver metabolic diseases. The study investigates whether moderate maternal high-fat diet affects the liver mitochondria responses to fructose intake in adult offspring.Wistar female rats have received a standard diet (mSTD) or high-fat diet (mHFD) (9% and 28.6% fat, respectively), before mating until the end of lactation. Male offspring were fed standard diet from weaning to adulthood and received water or fructose-drinking water (15%) from 120 to 150 days old. Fructose induces liver mitochondrial ultrastructural alterations with higher intensity in mHFD offspring, accompanied by reduced autophagy markers. Isolated mitochondria respirometry shows unaltered ATP-coupled oxygen consumption with increased Atp5f1b mRNA only in mHFD offspring. Fructose increases basal respiration and encoding complex I-III mRNA, only in mSTD offspring. Uncoupled respiration is lower in mHFD mitochondria that are unable to exhibit fructose-induced increase Ucp2 mRNA. Fructose decreases antioxidative defense markers, increases unfolded protein response and insulin resistance only in mHFD offspring without fructose-induced hepatic lipid accumulation.Mitochondrial dysfunction and homeostatic disturbances in response to fructose are early events evidencing the higher risk of fructose damage in the liver of adult offspring from dams fed an isocaloric moderate high-fat diet.

Neuromedin B, a bombesin-like peptide, and its receptor, are expressed in white adipose tissue with undefined roles. Female mice with disruption of neuromedin B receptor (NB-R) exhibited partial resistance to diet-induced obesity leading to our hypothesis that NB-R is involved in adipogenesis. Here, we showed that adipose stem/stromal cells (ASC) from perigonadal fat of female NB-R-knockout mice, exposed to a differentiation protocol in vitro, accumulated less lipid (45%) than wild type, suggesting reduced capacity to differentiate under adipogenic input. To further explore mechanisms, preadipocytes 3T3-L1 cells were incubated in the presence of NB-R antagonist (PD168368) during the first 3 days in culture. Cells were analyzed in the end of the treatment (Day 3) and later when fully differentiated (Day 21). NB-R antagonist induced lower number of cells at day 3 and 21 (33–39%), reduced cell proliferation at day 3 (−53%) and reduced lipid accumulation at day 21 (−86%). The mRNA expressions of several adipocyte differentiation markers were importantly reduced at both days: Cebpb and Pparg and Fabp4, Plin-1 and Adipoq, and additionally Lep mRNA at day 21. The antagonist had no effect when incubated with mature 3T3-L1 adipocytes. Therefore, genetically disruption of NB-R in mice ASC or pharmacological antagonism of NB-R in 3T3-L1 cells impairs adipogenesis. The mechanisms suggested by results in 3T3-L1 cells involve reduction of cell proliferation and of early gene expressions, leading to decreased number of mature adipocytes. We speculate that NB-R antagonism may be useful to limit the increase in adiposity due to pre-adipocyte differentiation.

AIM The aim of this study is to assess three protocols for removing a eugenol-based filling endodontic sealer from root dentin and the bond strength of fiberglass posts luted with resin cement. MATERIALS AND METHODS Sixty single-rooted bovine teeth were prepared and filled with the Endofill endodontic sealer and gutta-percha cones. After 7 days, 9 mm of the filling was removed and divided into five groups (n = 12) according to the cleaning protocol: G1 (negative control), unfilled; G2 (positive control), saline solution; G3, 95% alcohol; G4, amyl acetate; and G5, Largo bur. The canals were hybridized and fiberglass posts were luted with resin cement. They were cross-sectioned in slices of 1 mm and subjected to the push-out test. The other samples were sectioned longitudinally and visualized in a scanning electron microscope (SEM), and the existing chemical elements were quantified by energy dispersive spectroscopy (EDS). The statistical analysis used variance analysis (ANOVA)-one way and Fisher's test, at a 5% significance level. RESULTS The 95% alcohol was more effective for cleaning the canal, resulting in a clean dentinal surface and bond strength statistically similar to the negative control. The Largo bur was also statistically similar to the negative control, with only a few debris impregnated on the wall. Amyl acetate showed more sealer residues on the canal walls, with a consequent lower bond strength value than the other groups, only higher than the positive control. CONCLUSION The 95% alcohol and Largo burs may be used after removing the canal filling, so that the bond strength is improved when using the eugenol-based sealer.

Large amounts of by-products are generated during fish processing. The study aimed to assess whether tilapia scales are a potential source for obtaining glycosaminoglycans, as well as to determine their anticoagulant and cytotoxic/antiproliferative activities, against different tumor lines. The glycosaminoglycans were extracted, purified, and fractionated. The fractions that indicated the presence of uronic acid and sulfated GAGs were characterized by electrophoresis, NMR, and degree of sulfation (DS). The extraction process using the papain enzyme had a yield of 0.86%. Fraction V (FV) revealed the presence of chondroitin sulfate chains CS-A and CS-C, with DS of 0.146. FV demonstrated anticoagulant potential, as it was able to increase aPTT time. FV showed a cytotoxic effect for HTC metabolizing cells at 24, 48, and 72 h. However, it did not show activity for neuroblastoma cells in any of the evaluated times. The results indicate that the tilapia scales are a possible source for obtaining chondroitin sulfate, with potential use as anticoagulant and cytotoxic/antitumor.

Obesity and high-fat (HF) diet are associated with over activation of the endocannabinoid system (ECS). We have demonstrated that maternal HF diet induces early obesity and modulates cannabinoid signaling in visceral (VIS) and subcutaneous (SUB) white adipose tissue (WAT) in weanling rat offspring. We hypothesized that perinatal maternal HF diet would program the expression of ECS in adipose tissue in a long-term way in parallel to alterations in epigenetic markers and sex hormone signaling.Progenitor female rats received control diet (C, 9% fat) or isocaloric high-fat diet (HF, 28% fat) for 8 weeks before mating, gestation, and lactation. All pups were weaned to C diet and they were euthanized at 180 days old.Maternal HF diet induced overweight and increased SUB WAT mass of male and female adult offspring. Maternal HF diet induced hypertrophy of VIS and SUB adipocytes only in female offspring associated with increased type 1 cannabinoid receptor protein (CB1) and mRNA (Cnr1) levels. These changes were associated with increased estrogen receptor α binding to Cnr1 promoter in SUB WAT of adult female offspring, which may contribute to higher expression of Cnr1.Increased CB1 signaling in adipose tissue might contribute to higher adiposity programmed by maternal HF diet because endocannabinoids stimulate the accumulation of fat in the adipose tissue. Our findings provide molecular insights into sex-specific targets for anti-obesity therapies based on the endocannabinoid system.

Scope Non-alcoholic fatty liver disease (NAFLD) among adolescents has been related to fructose intake. Additionally, maternal high-fat diet (mHFD) increases the offspring susceptibility to NAFLD at adulthood. Here, it is hypothesized that mHFD may exacerbate the fructose impact in adolescent male rat offspring, by changing the response of contributing mechanisms to liver injury. Methods and results Female Wistar rats receive standard (mSTD: 9% fat) or high-fat diet (mHFD: 29% fat) prior mating throughout pregnancy and lactation. After weaning, offspring receive standard chow and, from the 25th to 45th day, receive water or fructose-drinking water (15%). At 46 days old, fructose groups show increased adiposity, increased serum and hepatic triglycerides, regardless of maternal diet. Fructose aggravates the hepatic imbalance of redox state already exhibited by mHFD offspring. The hepatic activation of cellular repair pathways by fructose, such as unfolded protein response and macroautophagy, is disrupted only in mHFD offspring. Fructose does not change the liver morphology of mSTD offspring. However, it intensifies the liver injury already present in mHFD offspring. Conclusion Fructose intake during adolescence accelerates the emergence of NAFLD observed previously at the adult life of mHFD offspring, and reveals a differentiated hepatic response to metabolic insult, depending on the maternal diet.

Perinatal maternal high-fat diet (HFD) increases susceptibility to obesity and fatty liver diseases in adult offspring, which can be attenuated by the potent hypolipidaemic action of fish oil (FO), ann-3 PUFA source, during adult life. Previously, we described that adolescent HFD offspring showed resistance to FO hypolipidaemic effects, although FO promoted hepatic molecular changes suggestive of reduced lipid accumulation. Here, we investigated whether this FO intervention only during the adolescence period could affect offspring metabolism in adulthood. Then, female Wistar rats received isoenergetic, standard (STD: 9 % fat) or high-fat (HFD: 28·6 % fat) diet before mating, and throughout pregnancy and lactation. After weaning, male offspring received the standard diet; and from 25 to 45 d old they received oral administration of soyabean oil or FO. At 150 d old, serum and hepatic metabolic parameters were evaluated. Maternal HFD adult offspring showed increased body weight, visceral adiposity, hyperleptinaemia and decreased hepatic pSTAT3/STAT3 ratio, suggestive of hepatic leptin resistance. FO intake only during the adolescence period reduced visceral adiposity and serum leptin, regardless of maternal diet. Maternal HFD promoted dyslipidaemia and hepatic TAG accumulation, which was correlated with reduced hepatic carnitine palmitoyl transferase-1a content, suggesting lipid oxidation impairment. FO intake did not change serum lipids; however, it restored hepatic TAG content and hepatic markers of lipid oxidation to STD offspring levels. Therefore, we concluded that FO intake exclusively during adolescence programmed STD offspring and reprogrammed HFD offspring male rats to a healthier metabolic phenotype in adult life, reducing visceral adiposity, serum leptin and hepatic TAG content in offspring adulthood.

Nutrition at early stages of life contributes to the alarming incidence of childhood obesity, insulin resistance and hepatoesteatosis. Cinnamaldehyde, major component of cinnamon, increases insulin sensitivity and modulates adiposity and lipid metabolism. The aim of this study was to analyze the impact of cinnamaldehyde treatment during adolescence in a rat model of early obesity. Litter size reduction was used to induce overfeeding and early obesity. At postnatal day 30 (adolescence), the male Wistar rats received cinnamaldehyde by gavage (40 mg/kg of body weight/day) for 29 days and were studied at the end of treatment at 60 days old or 4 months thereafter (180 days old). At 60 days of age, the treatment with cinnamaldehyde promoted reduced visceral adiposity, serum triacylglycerol, and attenuation of energy efficiency and insulin resistance. In the liver, it reduced lipid synthesis, stimulated autophagy and reduced ER stress. At 180 days of age, animals treated with cinnamaldehyde during the adolescence exhibited normalization of visceral adiposity and energy efficiency, and attenuation of hyperphagia, serum hypertriglyceridemia and hepatic triacylglycerol content, with molecular markers indicative of reduced hepatic synthesis. However, the beneficial effect observed at 60 days of age on glucose homeostasis, autophagy and ER stress was lost. Therefore, the cinnamaldehyde supplementation during the adolescence has short- and long-term metabolic beneficial effects, highlighting its potential as an adjuvant in the treatment of early obesity.

Taxifolin possesses gastroprotective property but is characterized by low water solubility, is instabile in alkaline medium, and is degraded by the intestinal bacteria flora. The purpose of the work was therefore to produce a gastroadhesive formulation to prolong taxifolin residence time and release in the stomach. We first demonstrated that taxifolin is stable in simulated gastric fluid with or without pepsin and mucus, and is able to cross pig gastric mucus layer and stomach mucosa. Next, gastromucoadhesive microparticles composed of Syloid® AL-1 mesoporous silica, chitosan and HPMC were produced using spray-drying. Microparticles were characterized by a spherical shape and a mean volume-equivalent diameter around 12 μm. The optimized microparticles were able to release taxifolin and to adhere to pig stomach mucosa for 5 h.

Neuromedin B (NB) and gastrin-releasing peptide (GRP) are bombesin-like peptides, found in the gastrointestinal tube and pancreas, among other tissues. Consistent data proposed that GRP stimulates insulin secretion, acting directly in pancreatic cells or in the release of gastrointestinal hormones that are incretins. However, the role of NB remains unclear. We examined the glucose homeostasis in mice with deletion of NB receptor (NBR-KO). Female NBR-KO exhibited similar fasting basal glucose with lower insulinemia (48.4%) and lower homeostasis model assessment of insulin resistance index (50.5%) than wild type (WT). Additionally, they were more tolerant to oral glucose, demonstrated by a decrease in the area under the glucose curve (18%). In addition, 15 min after an oral glucose load, female and male NBR-KO showed lower insulin serum levels (45.6 and 26.8%, respectively) than WT, even though blood glucose rose to similar levels in both groups. Single injection of NB, one hour before the oral glucose administration, tended to induce higher serum insulin in WT (28.9%, p=0.3), however the same did not occur in NBR-KO. They showed no changes in fasting insulin content in pancreatic islets by immunohistochemistry, however, the fasting serum levels of glucagon-like peptide, a potent incretin, exhibited a strong trend to reduction (40%, p=0.07). Collectively, mice with deletion of NB receptor have lower insulinemia, especially in response to oral glucose, and females also exhibited a better glucose tolerance, suggesting the involvement of NB and its receptor in regulation of insulin secretion induced by incretins, and also, in insulin sensitivity.

Scope Maternal high-fat diet (HFD) promotes obesity and metabolic disturbances in offspring at weaning and adult life. We investigated metabolic consequences of maternal HFD in adolescent rat offspring and the potential benefic effects of fish oil (FO) (n-3 polyunsaturated fatty acid source). Methods and results Female rats received isocaloric, standard diet (STD: 9% fat) or HFD (28.6%) before mating, and throughout pregnancy and lactation. After weaning, male offspring received standard diet and, from 25th to 45th day, received oral administration of soybean oil (SO) or FO. HFD offspring showed higher body weight and adiposity, which was not attenuated by FO. In STD offspring, FO reduced serum triglyceride and cholesterol, as expected, but not in HFD offspring. Liver of HFD offspring groups showed increased free cholesterol and FO-treated HFD group showed lower expression of Abcg8, suggesting decreased cholesterol biliary excretion. HFD offspring presented higher hepatic expression of lipogenic markers, Srebf1 mRNA and acetyl CoA carboxylase (ACC). Serum n-3 PUFA were decreased in FO-treated HFD compared to FO-treated STD offspring, which may explain the reduced hypolipidemic FO effect. Conclusion Maternal HFD impaired the ability of FO to reduce adiposity and serum lipids in adolescent offspring, suggesting a potential predisposition to future development of metabolic disorders.

Maternal diet plays a critical role in health development. Perinatal overnutrition induces metabolic dysfunctions and obesity in the offspring. Obesity is associated with endocannabinoid system (ECS) over activation and oxidative stress. Liver ECS activation induces hepatic steatosis, inflammation and fibrosis while the antagonism of cannabinoid receptors ameliorates these alterations. Here, we investigated the effect of perinatal maternal high-fat diet (HF, 29% of calories as fat) on the ECS and antioxidant system in liver of male and female adult rat offspring (180 days old). Maternal HF diet increased hepatic cannabinoid receptors, ECS metabolizing enzymes and triglyceride content, with male offspring more affected. ECS changes are likely independent of estradiol serum levels but associated with increased hepatic content of estrogen receptor, which can stimulate the expression of ECS components. Differently, maternal HF diet decreased the activity of the antioxidant enzymes glutathione peroxidase, superoxide dismutase and catalase, and increased oxidative stress markers in both sexes. Alterations in the redox homeostasis were associated with mitochondria damage but not with liver fibrosis. Our data suggest that maternal HF diet induces ECS over activation in adulthood, and that male offspring are at higher risk to develop liver disease compared with female rats.

Maternal nutritional imbalances trigger developmental adaptations involving early epigenetic mechanisms associated with adult chronic disease. Maternal high-fat (HF) diet promotes obesity and hypothalamic leptin resistance in male rat offspring at weaning and adulthood. Leptin resistance is associated with over activation of the endocannabinoid system (ECS). The ECS mainly consists of endocannabinoids derived from n-6 fatty acids and cannabinoid receptors (CB1 coded by Cnr1 and CB2 coded by Cnr2). The CB1 activation in hypothalamus stimulates feeding and appetite for fat while CB2 activation seems to play an immunomodulatory role. We demonstrated that maternal HF diet increases hypothalamic CB1 in male offspring while increases CB2 in female offspring at birth, prior to obesity development. However, the molecular mechanisms behind these changes remain unexplored. We hypothesized that maternal HF diet would down-regulate leptin signaling and up-regulate Cnr1 mRNA levels in the hypothalamus of the offspring at birth, associated with sex-specific changes in epigenetic markers and sex steroid signaling. To test our hypothesis, we used progenitor female rats that received control diet (C, 9% fat) or isocaloric high-fat diet (HF, 28% fat) from 8 weeks before mating until delivery. Blood, hypothalamus and carcass from C and HF male and female offspring were collected for biochemical and molecular analyses at birth. Maternal HF diet down-regulated the transcriptional factor STAT3 in the hypothalamus of male and female offspring, but induced hypoleptinemia only in males and decreased phosphorylated STAT3 only in female offspring. Because leptin acts through STAT3 pathway to inhibit central ECS, our results suggest that leptin pathway impairment might contribute to increased levels of Crn1 mRNA in hypothalamus of both sex offspring. Besides, maternal HF diet increased the histone acetylation percentage of Cnr1 promoter in male offspring and increased the androgen receptor binding to the Cnr1 promoter, which can contribute to higher expression of Cnr1 in newborn HF offspring. Maternal HF diet increased plasma n6 to n3 fatty acid ratio in male offspring, which is an important risk factor to metabolic diseases and might indicate an over activation of endocannabinoid signaling. Thus, although maternal HF diet programs a similar phenotype in adult offspring of both sexes (obesity, hyperphagia and higher preference for fat), here we showed that molecular mechanisms involving leptin signaling, ECS, epigenetic markers and sex hormone signaling were modified prior to obesity development and can differ between newborn male and female offspring. These observations may provide molecular insights into sex-specific targets for anti-obesity therapies.

Studies with foods, known to promote health benefits in addition to the nutritive value, show that their consumption by pregnant and/or lactating females could induce negative outcomes to the offspring. It is well characterized that cinnamon intake promotes benefits to energy homeostasis. The present study aimed to analyze the effects of the consumption of an aqueous extract of cinnamon by lactating female rats on the endocrine-metabolic outcomes in the adult offspring. Lactating dams (Wistar rats) were supplemented with cinnamon aqueous extract (400 mg/kg body weight/day) for the entire lactating period. The male adult offspring were evaluated at 180 days old (CinLac). The offspring presented visceral obesity (P = 0.001), hyperleptinemia (P = 0.002), and hyperinsulinemia (P = 0.016). In the liver, CinLac exhibited reduced p-IRβ (P = 0.018) suggesting insulin resistance. However, phosphorylation of IRS1 (P = 0.041) and AKT (P = 0.050) were increased. JAK2 (P = 0.030) and p-STAT3 (P = 0.015) expressions were higher, suggesting that the activation of IRS1/AKT in the CinLac group could have resulted from the increased activation of leptin signaling. Although we observed no changes in the gluconeogenic pathway, the CinLac group exhibited lower hepatic glycogen content (P = 0.005) accompanied by increased p-GSK3β (P = 0.011). In addition, the CinLac group showed increased hepatic triacylglycerol content (P = 0.049) and a mild steatosis (P = 0.001), accompanied by reduced PPARα mRNA expression (P = 0.005). We conclude that maternal intake of aqueous extract of cinnamon induces long-term molecular, metabolic, and hormonal changes in the adult progeny, including visceral obesity, higher lipid accumulation, and lower glycogen content in the liver.

The aim of this study was to evaluate the bond strength between the fibre post and root dentin after different cleaning protocols to remove eugenol from dentin. The residues and elemental composition of dentin were analysed using SEM and EDS. Bovine incisor roots were obturated with eugenol-based sealer. The post space was prepared, and the roots were divided into the following groups according to the cleaning protocol: unfilled (negative control), saline solution (positive control), 70% ethanol, acetone and 70% isopropyl alcohol. The fibre posts were cemented and push-out test realised. Chemical analysis of the dentin was performed using SEM and EDS. The bond strength values were analysed by anova/Fisher's and were as follows: negative control (7.30 ± 2.77) = 70% ethanol (6.64 ± 2.91) = 70% isopropyl alcohol (5.01 ± 3.15) = acetone (4.42 ± 1.53) >positive control (1.29 ± 0.56). It was concluded that 70% ethanol may be a good cleaning protocol to remove eugenol from dentin.

Early life inadequate nutrition triggers developmental adaptations and adult chronic disease. Maternal high-fat (HF) diet promotes visceral obesity and hypothalamic leptin resistance in male rat offspring at weaning and adulthood. Obesity is related to over active endocannabinoid system (ECS). The ECS consists mainly of endogenous ligands, cannabinoid receptors (CB1 and CB2), and the enzymes fatty acid anandamide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). We hypothesized that perinatal maternal HF diet would regulate offspring ECS in hypothalamus and brown adipose tissue (BAT) at birth, prior to visceral obesity development, and program food preference and energy expenditure of adult offspring. Female rats received control diet (C, 9% fat) or isocaloric high-fat diet (HF, 28% fat) for 8 weeks before mating, and throughout gestation and lactation. We evaluated C and HF offspring at birth and adulthood. At birth, maternal HF diet decreased leptinemia and increased hypothalamic CB1, orexin-A, and proopiomelanocortin while it decreased thyrotropin-releasing hormone (Trh) in male pups. Differentially, maternal HF diet increased hypothalamic CB2 in female pups. In BAT, maternal HF diet decreased CB1 and increased CB2 in male and female pups, respectively. Besides presenting different molecular ECS profile at birth, HF adult offspring developed overweight, higher adiposity and high-fat diet preference, independently of the sex, but only males presented hyperleptinemia and higher energy expenditure. In conclusion, maternal HF diet alters ECS components and energy metabolism targets in hypothalamus and BAT of offspring at birth, in a sex-specific manner, which may contribute for hyperphagia, food preference and higher adiposity later in life.

Background This study aimed to investigate the pathophysiology of hepatic microcirculatory dysfunction in non-alcoholic fatty liver disease (NAFLD). Methods In Wistar rats, NAFLD model was induced by 20 weeks of high-fat diet (HFD) feeding. Rolling and adhesion of leukocytes and tissue perfusion in hepatic microcirculation were examined using in vivo microscopic and laser speckle contrast imaging (LSCI), respectively. Oxidative stress and inflamatory parameters were analysed by TBARs, catalase enzyme activity, RT-PCR and ELISA. The participation of advanced glycation end-products (AGE) and its receptor RAGE was evaluated by the measurement of gene and protein expression of RAGE by RT-PCR and Western-blot, respectively and by liver and serum quantification of fluorescent AGEs. Results Wistar rats fed high-fat diet (HFD) showed increase in epididymal and abdominal fat content, systolic arterial blood pressure, fasting blood glucose levels, hepatic triglycerides and cholesterol, and impairment of glucose and insulin metabolisms. Liver histology confirmed the presence of steatosis and ultrasound analysis revealed increased liver size and parenchymal echogenicity in HFD-fed rats. HFD causes significant increases in leukocyte rolling and adhesion on hepatic microcirculation and decrease in liver microvascular blood flow. Liver tissue presented increase in oxidative stress and inflammtion. At 20 weeks, there was a significantly increase in AGE content in the liver and serum of HFD-fed rats and an increase in RAGE gene expression in the liver. Conclusion The increase in liver AGE levels and microcirculatory disturbances could play a role in the pathogenesis of liver injury and are key components of NAFLD.

The inhibition of maternal prolactin production in late lactation leads to metabolic syndrome and hypothyroidism in adult offspring. Physical training is a therapeutic strategy that could prevent or reverse this condition. We evaluated the effects of a short-duration low-intensity running wheel training program on the metabolic and hormonal alterations in rats. Lactating Wistar rats were treated with bromocriptine (Bro, 1 mg twice a day) or saline on days 19, 20, and 21 of lactation, and the training of offspring began at 35 days of age. Offspring were divided into sedentary and trained controls (C-Sed and C-Ex) and sedentary and trained Bro-treated rats (Bro-Sed and Bro-Ex). Chronic exercise delayed the onset of weight gain in Bro-Ex offspring, and the food intake did not change during the experimental period. At 180 days, visceral fat mass was higher (+46%) in the Bro-Sed offspring than in C-Sed and Bro-Ex rats. As expected, running capacity was higher in trained animals. Most parameters observed in the Bro-Sed offspring were consistent with hypothyroidism and metabolic syndrome and were reversed in the Bro-Ex group. Chronic exercise did not influence the muscle glycogen in the C-Ex group; however, liver glycogen was higher (+30%) in C-Ex group and was unchanged in both Bro offspring groups. Bro-Ex animals had higher plasma lactate dehydrogenase levels, indicating skeletal muscle damage and intolerance of the training program. Low-intensity chronic training is able to normalize many clinical aspects in Bro animals; however, these animals might have had a lower threshold for exercise adaptation than the control rats.

Thyroid hormones are important modulators of lipid metabolism because the liver is a primary hormonal target. The hypolipidaemic effects of thyroid hormones result from the balance between direct and indirect actions resulting in stimulation of lipid synthesis and lipid oxidation, which favours degradation pathways. Originally, it was believed that thyroid hormone activity was only transduced by alteration of gene transcription mediated by the nuclear receptor thyroid hormone receptors, comprising the classic action of thyroid hormone. However, the discovery of other effects independent of this classic mechanism characterised a new model of thyroid hormone action, the non-classic mechanism that involves other signalling pathways. To date, this mechanism and its relevance have been intensively described. Considering the increasing evidence for non-classic signalling of thyroid hormones and the major influence of these hormones in the regulation of lipid metabolism, we reviewed the role of thyroid hormone in cytosolic signalling cascades, focusing on the regulation of second messengers, and the activity of effector proteins and the implication of these mechanisms on the control of hepatic lipid metabolism.

Cinnamon supplementation has been associated with an improvement in glucose disposal and a reduction in fat mass in type 2 diabetes. Maternal nutrition during lactation impacts the health of the offspring throughout life. We hypothesize that cinnamon intake by lactating rats affects maternal physiology, leading to hormonal and metabolic changes in their offspring. To investigate this hypothesis, dams received aqueous cinnamon extract (400 mg cinnamon kgMaternal cinnamon intake did not affect the body mass gain or food intake of dams or their offspring, although it decreased visceral white adipose tissue mass in dams and in their adult offspring of both sexes. Cinnamon-treated dams exhibited no differences in serum insulin, adiponectin, leptin or estradiol levels, although they presented higher serum progesterone. At weaning, cinnamon male pups exhibited lower insulinemia, whereas cinnamon female pups exhibited lower glycemia. Interestingly, in adulthood, only the female offspring exhibited an altered hormonal profile, with reduced serum leptin, adiponectin and insulin levels accompanied by lower glycemia.The present study demonstrates that maternal cinnamon intake during lactation promotes mild changes in dams and can trigger sex-specific metabolic programming in pups that lasts into adulthood. © 2017 Society of Chemical Industry.

Made available in DSpace on 2018-11-26T17:12:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The aim of this study was to investigate the relationship between antibiotic residues found in the muscle of cage-farm-raised Nile tilapia (Oreochromisniloticus), the occurrence of resistant bacteria, and the sanitary practices adopted by farmers in Ilha Solteira reservoir, Brazil. Nine fish (three small fish, 40-200 g; three medium-sized fish, 200-500 g; and three large fish, 500-800 g) were collected from four cage farms every three months from April 2013 to January 2014. Ten antibiotic residues were determined using liquid chromatography-mass spectrometry, and bacteria were isolated and tested for antibiotic resistance to determine the multiple antibiotic resistance (MAR) index. Only three antibiotics (oxytetracycline, tetracycline, and florfenicol) were detected in the muscle of Nile tilapia, and their residues were the highest in small fish; however, the MAR index was higher in large fish. In addition, a direct positive relationship between the MAR index and the concentration of antibiotic residues in Nile tilapia was found. Overall, the adoption of prophylactic management practices improved the sanitary status of cage farms, reducing bacterial infections and hampering the development of antibiotic-resistant bacteria. Inst Biol, Sao Paulo, SP, Brazil Univ Sao Paulo, Ctr Nucl Energy Agr, Piracicaba, SP, Brazil Inst Fisheries, Votuporanga, SP, Brazil Sao Paulo State Univ, Ctr Aquaculture, Jaboticabal, SP, Brazil Inst Agron, Votuporanga, SP, Brazil Sao Paulo State Univ, Ctr Aquaculture, Jaboticabal, SP, Brazil FAPESP: 2011/18179-1

Aims To understand how thyroid hormone (TH) regulates tissue-specific gene expression in patients with the syndrome of resistance to TH (RTHβ), we used a mouse model that replicates the human RTHβ, specifically the ∆ 337T mutation in the thyroid hormone receptor β (THRβ). Main methods We investigated the expression of key TH target genes in the pituitary and liver of TRβ ∆ 337T and wild type THRβ mice by qPCR before and after a T 3 suppression test consisting of the administration of increasing concentrations of T 3 to hypothyroid mice. Key findings Pituitary Tshb and Cga expression decreased and Gh expression increased in TRβ ∆ 337T mice after T 3 suppression. The stimulation of positively regulated TH genes was heterogeneous in the liver. Levels of liver Me1 and Thsrp were elevated in TRβ ∆ 337T mice after T 3 administration. Slc16a2 and Gpd2 did not respond to T 3 stimulation in the liver of TRβ ∆ 337T mice whereas Dio1 response was lower than that observed in WT mice. Moreover, although Chdh and Upd1 genes were negatively regulated in the liver, the expression of these genes was elevated after T 3 suppression. We did not observe significant changes in THRα expression in the liver and pituitary, while THRβ levels were diminished in the pituitary and increased in the liver. Significance Using a model expressing a THRβ unable to bind T3, we showed the expression pattern of liver negative and positive regulated genes by T3.

OBJECTIVES: To evaluate the effect of Carbopol gel formulations containing pilocarpine on the morphology and morphometry of the vaginal epithelium of castrated rats. METHODS: Thirty-one female Wistar-Hannover rats were randomly divided into four groups: the control Groups I (n=7, rats in persistent estrus; positive controls) and II (n=7, castrated rats, negative controls) and the experimental Groups, III (n=8) and IV (n=9). Persistent estrus (Group I) was achieved with a subcutaneous injection of testosterone propionate on the second postnatal day. At 90 days postnatal, rats in Groups II, III and IV were castrated and treated vaginally for 14 days with Carbopol gel (vehicle alone) or Carbopol gel containing 5% and 15% pilocarpine, respectively. Next, all of the animals were euthanized and their vaginas were removed for histological evaluation. A non-parametric test with a weighted linear regression model was used for data analysis (p

Early weaning is associated with changes in the developmental plasticity. Here, we studied the adipocytes morphology, adipokines expression or content in adipose tissue as well as adrenal and thyroid function of neonate and adult offspring primed by early weaning. After birth, lactating rats were divided into 2 groups: EW (early weaning) – dams were wrapped with a bandage to block access to milk during the last 3 days of lactation, and Control – dams whose pups had free access to milk throughout lactation (21 days). At postnatal day (PN) 21, EW pups had lower visceral and subcutaneous adipocyte area ( − 67.7% and − 62%, respectively), body fat mass ( − 26%), and leptin expression in visceral adipocyte ( − 64%) but higher leptin expression in subcutaneous adipocyte (2.9-fold increase). Adrenal evaluations were normal, but neonate EW pups presented lower serum T3 ( − 55%) and TSH ( − 44%). At PN 180, EW offspring showed higher food intake, higher body fat mass (+21.6%), visceral and subcutaneous adipocyte area (both 3-fold increase), higher leptin (+95%) and ADRβ3 (2-fold increase) content in visceral adipose tissue, and higher adiponectin expression in subcutaneous adipose tissue (+47%) but lower in visceral adipose tissue ( − 40%). Adult EW offspring presented higher adrenal catecholamine content (+31%), but no changes in serum corticosterone or thyroid status. Thus, early weaning primed for hypothyroidism at weaning, which can be associated with the adipocyte hypertrophy at adulthood. The marked changes in catecholamine adrenal content and visceral adipocyte ADRB3 are generally found in obesity, contributing to the development of other cardiovascular and metabolic disturbances.

Neuropeptide Y (NPY) inhibits TRH neurons in fed state, and hypothalamic NPY higher expression during fasting has been proposed to be involved in fasting-induced suppression of the hypothalamus-pituitary–thyroid (HPT) axis. We investigated the role of central Y5 receptors in the control of thyrotropin (TSH) and thyroid hormone (TH) secretion. Fed and fasting rats received twice daily central injections (3rd ventricle) of Y5 receptor antagonist (CGP71683; 15nmol/rat) for 72h. Fasted rats also received a single central injection of CGP71683 (15nmol/rat) at the end of 72h of fasting. In fed rats, Y5 receptor blockade reduced total food intake by 32% and body mass by almost 10% (p

n-3 polyunsaturated fatty acids (n-3 PUFA) from fish oil (FO) exert important lipid-lowering effects, an effect also ascribed to thyroid hormones (TH) and TH receptor β1 (TRβ1)-specific agonists. n-3 PUFA effects are mediated by nuclear receptors, such as peroxisome proliferator-activated receptors (PPAR) and others. In this study, we investigated a role for TH signaling in n-3 PUFA effects. Euthyroid and hypothyroid adult rats (methimazole-treated for 5 weeks) received FO or soybean oil (control) by oral administration for 3 weeks. In euthyroid rats, FO treatment reduced serum triglycerides and cholesterol, diminished body fat, and increased protein content of the animals. In addition, FO-treated rats exhibited higher liver expression of TRβ1 and mitochondrial α-glycerophosphate dehydrogenase (mGPD), at protein and mRNA levels, but no alteration of glutathione S-transferase or type 1 deiodinase. In hypothyroid condition, FO induced reduction in serum cholesterol and increase in body protein content, but lost the ability to reduce triglycerides and body fat, and to induce TRβ1 and mGDP expression. FO did not change PPARα liver abundance regardless of thyroid state; however, hypothyroidism led to a marked increase in PPARα liver content but did not alter TRβ1 or TRα expression. The data suggest that part of the effect of n-3 PUFA from FO on lipid metabolism is dependent on TH signaling in specific steps and together with the marked upregulation of PPARα in liver of hypothyroid rats suggest importantin vivoconsequences of the cross-talking between those fatty acids and TH pathways in liver metabolism.

Mice bearing the genomic mutation Δ337T on the thyroid hormone receptor β (TRβ) gene present the classical signs of resistance to thyroid hormone (TH), with high serum TH and TSH. This mutant TR is unable to bind TH, remains constitutively bound to co-repressors, and has a dominant negative effect on normal TRs. In this study, we show that homozygous (TRβΔ337T) mice for this mutation have reduced body weight, length, and body fat content, despite augmented relative food intake and relative increase in serum leptin. TRβΔ337T mice exhibited normal glycemia and were more tolerant to an i.p. glucose load accompanied by reduced insulin secretion. Higher insulin sensitivity was observed after single insulin injection, when the TRβΔ337T mice developed a profound hypoglycemia. Impaired hepatic glucose production was confirmed by the reduction in glucose generation after pyruvate administration. In addition, hepatic glycogen content was lower in homozygous TRβΔ337T mice than in wild type. Collectively, the data suggest that TRβΔ337T mice have deficient hepatic glucose production, by reduced gluconeogenesis and lower glycogen deposits. Analysis of liver gluconeogenic gene expression showed a reduction in the mRNA of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme, and of peroxisome proliferator-activated receptor-γ coactivator 1α, a key transcriptional factor essential to gluconeogenesis. Reduction in both gene expressions is consistent with resistance to TH action via TRβ, reproducing a hypothyroid phenotype. In conclusion, mice carrying the Δ337T-dominant negative mutation on the TRβ are leaner, exhibit impaired hepatic glucose production, and are more sensitive to hypoglycemic effects of insulin.

Hyperleptinemia during lactation programs for higher serum leptin in 30-day-old and adult rats, associated with metabolic changes. Here we evaluated the inhibition of serum leptin at 29 and 30 days on the metabolic phenotype of rats programmed with leptin during lactation. Pups from Wistar rats were saline-injected or leptin-injected from postnatal day 1 to day 10. At 29 and 30 days old, animals were injected with anti-leptin antibody (LA and CA) or saline (LS and CS). In adult animals, higher visceral (+53%) and total fat mass (+33%), hyperleptinemia (+67%), hypertriglyceridemia (+47%), and hypoadiponectinemia (-44%) observed in LS group compared to CS were prevented by immunoneutralization of leptin, since LA group had those parameters values similar to CS group. However, immunoblockade of leptin in normal animals led to the same metabolic changes seen in leptin-treated animals, in addition to lower serum adiponectin (-77% vs. CS) and higher insulin resistance index (+37%). Liver sirtuin1 (SIRT1) was higher (+41%) only in LA group, suggesting a role for SIRT1 in the prevention of leptin programming. Hypothalamic OBR was lower and SOCS3 higher in LS group and these changes were normalized in LA group. In conclusion, blocking leptin action one week after weaning seems to revert most of the alterations observed in rats programmed by neonatal hyperleptinemia. Higher liver SIRT1 expression may be one of the mechanisms involved, leading to a better glucose and lipid metabolism. Our data suggest that the lack or the excess of leptin programs an adverse metabolic phenotype in adulthood.

Previous studies have shown that alterations in thyroid status may lead to changes in serum leptin and adiponectin, both in humans and rodents. The mechanisms, especially for adiponectin, are unclear. In the present study, we investigated the effect of triiodothyronine (T3) on the expression of adiponectin mRNA and the release of leptin and adiponectin by white adipose tissue (WAT) explants obtained from epididymal (visceral) or inguinal (subcutaneous) depots from normal rats. We also analyzed the effects of other known regulators of adiponectin and leptin release, such as rosiglitazone and dexamethasone. T3 acted directly at rat WAT explants in a depot-specific manner and in a unique fashion to each hormone. T3 was able to inhibit leptin release only by epididymal explants, and to reduce adiponectin mRNA expression only in inguinal explants. However, T3 was incapable of modifying adiponectin release by both explants. Additionally, rosiglitazone exhibited an inhibitory effect on adiponectin release by both WAT explants, even though adiponectin mRNA was importantly upregulated only in inguinal explants. Rosiglitazone acted as an inhibitor of leptin release by both studied fat depots, while only epididymal explants responded to the stimulatory effect of dexamethasone on leptin release. Therefore, the present model of isolated rat white adipose tissue explants highlights the fact that the regulation of hormonal production by white adipose tissue depends on the type of depot and its anatomical location. In this context, our results show for the first time a potential inhibitory effect of T3 on adiponectin mRNA expression specifically on WAT from a subcutaneous depot.

Thyroid hormone (TH) regulates many cardiac genes via nuclear thyroid receptors, and hyperthyroidism is frequently associated with atrial fibrillation. Electrical activity propagation in myocardium depends on the transfer of current at gap junctions, and connexins (Cxs) 40 and 43 are the predominant junction proteins. In mice, Cx40, the main Cx involved in atrial conduction, is restricted to the atria and fibers of the conduction system, which also express Cx43. We studied cardiac expression of Cx40 and Cx43 in conjunction with electrocardiogram studies in mice overexpressing the dominant negative mutant thyroid hormone receptor-β Δ337T exclusively in cardiomyocytes [myosin heavy chain (MHC-mutant)]. These mice develop the cardiac hypothyroid phenotype in the presence of normal serum TH. Expression was also examined in wild-type mice rendered hypothyroid or hyperthyroid by pharmacological treatment. Atrial Cx40 mRNA and protein levels were decreased (85 and 55%, respectively; P < 0.001) in MHC-mt mice. Atrial and ventricular Cx43 mRNA levels were not significantly changed. Hypothyroid and hyperthyroid animals showed a 25% decrease and 40% increase, respectively, in Cx40 mRNA abundance. However, MHC-mt mice presented very low Cx40 mRNA expression regardless of whether they were made hypothyroid or hyperthyroid. Atrial depolarization velocity, as represented by P wave duration in electrocardiograms of unanesthetized mice, was extremely reduced in MHC-mt mice, and to a lesser extent also in hypothyroid mice (90 and 30% increase in P wave duration). In contrast, this measure was increased in hyperthyroid mice (19% decrease in P wave duration). Therefore, this study reveals for the first time that Cx40 mRNA is up-regulated by TH acting in cardiac atria via the TH receptor and that this may be one of the mechanisms contributing to atrial conduction alterations in thyroid dysfunctions.Cardiac-specific expression of a mutant thyroid hormone receptor unable to bind thyroid hormone profoundly reduces atrial connexin 40 expression in association with prolonged atrial conduction.

Neuromedin B (NB), a neuropeptide highly concentrated in pituitary, has been proposed to be an inhibitor of thyrotropin (TSH) secretion. Previous study showed that mice with disruption of neuromedin B receptor (NBR-KO) have higher TSH release in response to thyrotropin-releasing hormone (TRH), although TSH seems to have decreased bioactivity. Here we examined in NBR-KO mice the response of TSH to thyroid hormone (TH) deprivation, obtained by methimazole treatment, or excess, obtained by acute and chronic TH administration. In response to hypothyroidism NBR-KO mice exhibited a lower magnitude increase in serum TSH compared to wild-type (WT) mice (1.7 vs. 3.3-times increase compared to euthyroid values, respectively, P 0.001). One hour after a single T4 injection (0.4μg/100 g BW), WT and NBR-KO hypothyroid mice presented similar degree of serum TSH reduction (54%, P 0.05). However, 3 h after T4 administration, WT mice presented serum TSH similar to hypothyroid baseline, while NBR-KO mice still had decreased serum TSH (30% reduced in comparison to hypothyroid baseline P 0.05). T3 treatment of euthyroid mice for 21 days, with progressively increasing doses, significantly reduced serum TSH similarly in WT and NBR-KO mice. Also, serum T4 exhibited the same degree of suppression in WT and NBR-KO. In conclusion, disruption of neuromedin B receptor did not interfere with the sensitivity of thyroid hormone-mediated suppression of TSH release, but impaired the ability of thyrotroph to increase serum TSH in hypothyroidism, which highlights the importance of NB in modulating the set point of the hypothalamus–pituitary–thyroid axis at hypothyroidism.

Made available in DSpace on 2013-09-27T14:54:07Z (GMT). No. of bitstreams: 1 WOS000267218200025.pdf: 446263 bytes, checksum: 7232c4fbcfe19ade6252554576e5cb49 (MD5) Previous issue date: 2008-01-01 Made available in DSpace on 2013-09-30T18:15:24Z (GMT). No. of bitstreams: 1 WOS000267218200025.pdf: 446263 bytes, checksum: 7232c4fbcfe19ade6252554576e5cb49 (MD5) Previous issue date: 2008-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:31:44Z No. of bitstreams: 1 WOS000267218200025.pdf: 446263 bytes, checksum: 7232c4fbcfe19ade6252554576e5cb49 (MD5) Made available in DSpace on 2014-05-20T13:31:44Z (GMT). No. of bitstreams: 1 WOS000267218200025.pdf: 446263 bytes, checksum: 7232c4fbcfe19ade6252554576e5cb49 (MD5) Previous issue date: 2008-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) We examined the genotoxic and mutagenic effects of a crude extract of Clusia alata ( a potential medicinal plant) on peripheral leukocyte and bone marrow cells of mice, using the comet and chromosome aberration assays. Extracts at doses of 1000, 1500 and 2000 mg/kg were administered by gavage, and a positive control, N-nitroso-N-ethylurea (50 mg/kg) was injected intraperitoneally. Peripheral blood leukocytes were collected 4 and 24 h after the treatments for the comet assay, and bone marrow cells were collected 24 h after the treatments, for the chromosome aberration assay. The comet assay showed that C. alata extract causes an increase in damage to DNA in the peripheral blood leukocytes, but it was significant only with the 2000 mg/kg dose after 24 h; the extract also induced a small but significant increase in the mean number of chromosome aberrations in the bone marrow cells at doses of 1500 and 2000 mg/kg. No evidence of a significant decrease in the mitotic index was observed. Acute consumption of high concentrations of C. alata extract produced some mutagenic effects in bone marrow cells. Univ Estadual Paulista, Fac Filosofia & Ciencias, Dept Fonoaudiol, Marilia, SP, Brazil Univ Fed Amapa, Lab Pesquisa Farmacos, Macapa, AP, Brazil Univ Estadual Paulista, Fac Filosofia & Ciencias, Dept Fonoaudiol, Marilia, SP, Brazil CNPq: 306544/2006-7 FAPESP: 06/57514-2

Leptin has been shown to modulate deiodinase type 1 (D1) and type 2 (D2) enzymes responsible for thyroxine (T4) to triiodothyronine (T3) conversion. Previously, it was demonstrated that a single injection of leptin in euthyroid fed rats rapidly increased liver, pituitary, and thyroid D1 activity, and simultaneously decreased brown adipose tissue (BAT) and hypothalamic D2 activity. We have now examined D1 and D2 activities, two hours after a single subcutaneous injection of leptin (8 microg/100 g BW) into hypo- and hyperthyroid rats. In hypothyroid rats, leptin did not modify pituitary, liver and thyroid D1, and thyroid D2 activity, while pituitary D2 was decreased by 41% (p

Thyroid hormone and leptin are essential regulators of energy homeostasis. Both hormones stimulate energy expenditure but have opposite effects on appetite. The mechanisms behind food intake regulation in thyroid dysfunctions are poorly understood. It has been shown that hypothyroid rats exhibited impaired leptin anorexigenic effect and signaling in total hypothalamus, even though they were hypophagic. It was hypothesized that hypothyroidism modulates the expression of neuropeptides: orexigenic neuropeptide Y (NPY) and anorexigenic proopiomelanocortin (POMC), independently of inducing nuclei-specific changes in hypothalamic leptin signaling.Adult male rats were rendered hypothyroid by administration of 0.03% methimazole in the drinking water for 21 days. Protein content of NPY, POMC, and leptin signaling (the signal transducer and activator of transcription 3 [STAT3] pathway) were evaluated by Western blot, and mRNA levels by real time reverse transcription polymerase chain reaction in arcuate (ARC), ventromedial (VMN), and paraventricular (PVN) hypothalamic nuclei isolated from euthyroid (eu) and hypothyroid (hypo) rats. Leptin anorexigenic effect was tested by recording food intake for two hours after intracerebroventricular (i.c.v.) administration of leptin. Statistical differences were considered significant at p ≤ 0.05.Hypothyroidism was confirmed by decreased serum triiodothyronine, thyroxine, and increased thyrotropin, in addition to increased levels of pro-TRH mRNA in PVN and Dio2 mRNA in the ARC of hypo rats. Hypothyroidism decreased body weight and food intake associated with decreased protein content of NPY and increased content of POMC in the ARC. Conversely, hypothyroidism induced central resistance to the acute anorexigenic effect of leptin, since while euthyroid rats displayed reduced food intake after leptin i.c.v. injection, hypothyroid rats showed no response. Hypothyroid rats exhibited decreased leptin receptor (ObRb) protein content in ARC and VMN but not in PVN nucleus. ObRb protein changes were concomitant with decreased phosphorylated STAT3 in the ARC, and decreased total STAT3 in VMN and PVN. However, hypothyroidism did not affect mRNA levels of Lepr or Stat3 in the hypothalamic nuclei.Experimental hypothyroidism induced a negative energy balance accompanied by decreased NPY and increased POMC protein content in the ARC, resulting in predominance of anorexigenic pathways, despite central leptin resistance and impairment of the leptin signaling cascade in a nuclei-specific manner.

In models of metabolic disorders, cinnamon improves glucose and lipid metabolism. This study explores the effect of chronic supplementation with aqueous cinnamon extract (CE) on the lipid metabolism of rats. Male adult Wistar rats were separated into a control group (CTR) receiving water and a CE Group receiving aqueous cinnamon extract (400 mg of cinnamon per kg body mass per day) by gavage for 25 consecutive days. Cinnamon supplementation did not change the food intake or the serum lipid profile but promoted the following changes: lower body mass gain (P = 0.008), lower relative mass of white adipose tissue (WAT) compartments (P = 0.045) and higher protein content (percentage of the carcass) (P = 0.049). The CE group showed lower leptin mRNA expression in the WAT (P = 0.0017) and an important tendency for reduced serum leptin levels (P = 0.059). Cinnamon supplementation induced lower mRNA expression of SREBP1c (sterol regulatory element-binding protein 1c) in the WAT (P = 0.001) and liver (P = 0.013) and lower mRNA expression of SREBP2 (P = 0.002), HMGCoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) (P = 0.0003), ACAT1 (acetyl-CoA acetyltransferase 1) (P = 0.032) and DGAT2 (diacylglycerol O-acyltransferase 2) (P = 0.03) in the liver. These changes could be associated with the reduced esterified cholesterol and triacylglycerol content detected in this tissue. Our results suggest that chronic ingestion of aqueous cinnamon extract attenuates lipogenic processes, regulating the expression of key enzymes and transcriptional factors and their target genes, which are directly involved in lipogenesis. These molecular changes possibly promote adaptations that would prevent an increase in circulating cholesterol and triacylglycerol levels and prevent lipid accumulation in tissues, such as liver and WAT. Therefore, we speculate that cinnamon may also be useful for preventing or retarding the development of lipid disorders.

Cinnamon has several effects on energy metabolism. However, no data exist on the impact of cinnamon intake on thyroid hormone serum concentrations and action, since thyroid hormones (THs) play a major role in metabolism.Male rats were treated with cinnamon water extract (400 mg kg(-1) body weight, 25 days). Cinnamon supplementation resulted in a lower serum total T3 level accompanied by normal serum T4 and TSH levels. The cinnamon-treated rats did not exhibit significant differences in TSHβ subunit, TRβ or deiodinase type 2 mRNA expression in the pituitary. In the liver, cinnamon did not change the TRβ protein expression or the deiodinase type 1 mRNA expression, suggesting that there were no changes in T3 signaling or metabolism in this organ. However, mitochondrial GPDH, a target gene for T3 in the liver, exhibited no changes in mRNA expression, although its activity level was reduced by cinnamon. In the cardiac ventricle, T3 action was markedly reduced by cinnamon, as demonstrated by the lower TRα mRNA and protein levels, reduced SERCA2a and RyR2 and increased phospholamban mRNA expression.This study has revealed that TH action is a novel target of cinnamon, demonstrating impairment of T3 signaling in the cardiac ventricles. © 2015 Society of Chemical Industry.

Background/Aims: Thyroid hormone (TH) signalling is critical for heart function. The heart expresses thyroid hormone receptors (THRs); THRα1 and THRβ1. We aimed to investigate the regulation mechanisms of the THRβ isoform, its association with gene expression changes and implications for cardiac function. Methods: The experiments were performed using adult male mice expressing TRβΔ337T, which contains the Δ337T mutation of the human THRB gene and impairs ligand binding. Cardiac function and RNA expression were studied after hypo-or hyperthyroidism inductions. T3-induced cardiac hypertrophy was not observed in TRβΔ337T mice, showing the fundamental role of THRβ in cardiac hypertrophy. Results: We identified a group of independently regulated THRβ genes, which includes Adrb2, Myh7 and Hcn2 that were normally regulated by T3 in the TRβΔ337T group. However, Adrb1, Myh6 and Atp2a2 were regulated via THRβ. The TRβΔ337T mice exhibited a contractile deficit, decreased ejection fraction and stroke volume, as assessed by echocardiography. In our model, miR-208a and miR-199a may contribute to THRβ-mediated cardiac hypertrophy, as indicated by the absence of T3-regulated ventricular expression in TRβΔ337T mice. Conclusion: THRβ has important role in the regulation of specific mRNA and miRNA in T3-induced cardiac hypertrophic growth and in the alteration of heart functions.

We investigated the influence of hypo- and hyperthyroidism on the ability of leptin to modulate TSH secretion. Two hours after receiving leptin (8 μg leptin/100 g BW; s.c.), hyperthyroid rats (10 μg thyroxine (T4)/100 g body weight (BW) for 5 days) showed a 1.7-fold increase in serum TSH (P−9 and 10−7M leptin showed reductions in TSH release of 40 and 50% respectively (PPPin vivo and in vitro responsiveness of TSH to leptin is abolished in hypothyroidism and is preserved in short-term hyperthyroidism, in comparison to previous reports in euthyroidism. In addition, the inhibitory action of pituitary leptin is enhanced in hyperthyroid glands, which may suggest a role for locally produced leptin in the suppression of TSH release associated with hyperthyroidism.

Pathogenic mycobacteria survive inside macrophages and deactivate these cells, using a mechanism that is still poorly understood. Mycobacterial cell wall lipids constitute the first contact with the host cell. Although Mycobaterium leprae and M. bovis BCG share common antigens, they induce opposite inflammatory responses. Apolar M. leprae lipids have been shown to be anti-inflammatory by down-regulating macrophage activation and T-cell functions. We wonder if these lipids would influence cellular migration to BCG or to other inflammatory agent. We investigated the effect of M. leprae, its lipids or delipidated bacteria on acute and chronic BCG- or carrageenan-induced pleurisy. Previous injection of intact or delipidated M. leprae did not alter either the BCG- or carrageenan-induced pleural inflammatory reaction. However, M. leprae lipids enhanced carrageenan-induced acute cellular migration without impairing BCG inflow; moreover, they reduced BCG chronic response. Together these data suggest distinct mechanisms for intracellular deactivation and pleural cell recruitment exerted by mycobacterial structures.

Thyroid hormones, T4 and T3, regulate their own production by feedback inhibition of TSH and TRH synthesis in the pituitary and hypothalamus when T3 binds to thyroid hormone receptors (TRs) that interact with the promoters of the genes for the TSH subunit and TRH. All TR isoforms are believed to be involved in the regulation of this endocrine axis, as evidenced by the massive dysregulation of TSH production in mice lacking all TR isoforms. However, the relative contributions of TR isoforms in the pituitary vs. the hypothalamus remain to be completely elucidated. Thus, to determine the relative contribution of pituitary expression of TR-α in the regulation of the hypothalamic-pituitary-thyroid axis, we selectively impaired TR-α function in TR-β null mice (TR-β−/−) by pituitary restricted expression of a dominant negative TR-β transgene harboring a Δ337T mutation. These animals exhibited 10-fold and 32-fold increase in T4 and TSH concentrations, respectively. Moreover, the negative regulation of TSH by exogenous T3 was completely absent and a paradoxical increase in TSH concentrations and TSH-β mRNA was observed. In contrast, prepro-TRH expression levels in T3-treated TR-β−/− were similar to levels observed in the Δ337/TR-β−/− mice, and ligand-independent activation of TSH in hypothyroid mice was equivalently impaired. Thus, isolated TR-β deficiency in TRH paraventricular hypothalamic nucleus neurons and impaired function of all TRs in the pituitary recapitulate the baseline hormonal disturbances that characterize mice with complete absence of all TRs.

ClC-2 is a CLC family member of chloride channels sensitive to changes in cell volume, pH and voltage. The ClC-2 is widely distributed along the nephron although in the kidney its role still not well understood. Aldosterone studies suggest that ClC-2 expression in the kidney may be hormonally regulated. To explore the possibility that estrogen control ClC-2 expression, we investigated whether its expression changed in the kidney of female Wistar rats subjected to ovariectomy with or without near-physiological or high doses of 17beta-estradiol benzoate treatment for 10 days. Total RNA isolated from rat kidney and dissected nephron segments was analyzed by ribonuclease protection assay and/or a semi-quantitative RT-PCR. The renal ClC-2 protein expression was analyzed by Western blot. The decreased renal expression of ClC-2 mRNA and protein observed in ovariectomized rats was restored to control levels after treatment with low doses of estradiol. Higher dose estradiol lead to an even greater increase in ClC-2 mRNA and protein expression. This change in overall expression was shown to be caused by the modulation of ClC-2 mRNA expression in the proximal tubule. These results suggest that ClC-2 may be involved in estrogen-induced Cl(-) transport in rat kidney.

We investigated the effect of acute cold exposure, leptin, and the somatostatin analog octreotide (OCT) on thyroid type I (D1) and II (D2) deiodinase activities. Microsomal D1 and D2 activities were measured by the release of 125I from 125I-reverse triiodothyronine (rT3) under different assay conditions. Rats exposed to 4°C (15, 30, 60, and 120 min) showed progressive reduction in thyroidal D1 and D2, reaching ∼40% at 2 h ( P < 0.05) despite increased circulating TSH ( P < 0,05) associated with the higher thyroid D1 and D2 in hypothyroid rats. A single injection of leptin (8 μg/100 g body wt sc) induced increased thyroid and liver D1 ( P < 0.05), but not thyroid D2, activities at 30 and 120 min, independently of the serum TSH rise shown only at 2 h. OCT (1 μg/kg body wt sc) increased D1 and D2 activity significantly 24 h after a single injection, with no changes in serum TSH. Therefore, leptin and somatostatin are potential physiological upregulators of thyroid deiodinases, and their low secretion during acute cold exposure may be a potential mechanism contributing to cold-induced reduction in thyroid deiodinase activity.

We have shown that protein restriction during lactation is associated with higher levels of serum and milk tri-iodothyronine (T(3)) with lower serum thyroxine (T(4)), suggesting an increased T(4) to T(3) conversion. To investigate this hypothesis, the activity of type 1 (D1) and/or type 2 (D2) iodothyronine deiodinases was evaluated on days 4, 12 and 21 of lactation in several tIssues of dams fed an 8% protein-restricted (PR) diet and controls fed a 23% protein diet. Serum TSH, T(3) and T(4) were measured by radioimmunoassay. Deiodinase activity was determined by the release of (125)I from (125)I-reverse T(3), under specific conditions for D1 or D2. PR dams had a transitory reduction in liver D1 activity (P

Neuromedin B(NB), a bombesin-like peptide, has been shown to inhibit thyrotropin (TSH) release in pituitary explants of male rats and to stimulate Prolactin (PRL) release in male pituitary cell cultures. We investigated the effect of estrogen status of female rats on the response of thyrotrophs and lactotrophs to neuromedin B (NB) in vitro. Ovariectomized rats were treated with near-physiological or high doses of 17beta estradiol benzoate (0.7 or 14 EB microg/100 gBW/daily, 10 days) or with vehicle (OVX). EB treatment induced a dose-dependent increase in serum prolactin and an increase in pituitary NB content, measured by specific RIA, that was similar in both EB groups (P0.05). TSH release from isolated hemipituitaries of OVX rats was significantly reduced (P0.05) in the presence of 10(-7) M NB. OVX + EB0.7 glands responded to NB with a not statistically significant dose-dependent decrease in TSH release. However, glands from hyperestrogenized rats (OVX + EB14) required a higher dose (10(-5) M) of NB to inhibit TSH release (P0.05). PRL release was highly increased (p0.001) by the presence of 10(-5) M NB only in glands of hyperestrogenized rats, while no effect of NB was observed in the other groups. In conclusion, estrogen status of female rats modulates the inhibitory effect of NB on TSH release in vitro and hyperestrogenism is required for stimulatory effect of NB on PRL release in vitro. It is suggested that the induction of PRL release by neuromedin B is a pharmacological rather than a physiological effect, but neuromedin B may contribute to the increased release of PRL associated with hyperestrogenism.

Leptin has been shown to stimulate the hypothalamus-pituitary-thyroid axis in fasting rodents; however, its role in thyroid axis regulation under physiological conditions is still under investigation. Here it was investigated in freely fed rats whether leptin modulates thyrotroph function in vivo and whether leptin has direct pituitary effects on TSH release. Since leptin is produced in the pituitary, the possibility was also investigated that leptin may be a local regulator of TSH release. TSH was measured by specific RIA. Freely fed adult rats 2 h after being injected with a single s.c. injection of 8 microg leptin/100 g body weight showed a 2-fold increase in serum TSH (P

A rapid and sensitive method using liquid chromatography coupled with mass spectrometry triple quadrupole direct aqueous injection for analysis of atrazine and ametrine herbicides in surface waters was developed. According to the validation method, water samples from six different locations in the Piracicaba River were collected monthly from February 2011 to January 2012 and injected into a liquid chromatographer/dual mass spectrometer without the need for sample extraction. The method was validated and shown to be precise and accurate; limits of detection and quantification were 0.07 and 0.10 µg L(-1) for atrazine and 0.09 and 0.14 µg L(-1) for ametrine. During the sampling period, concentrations of atrazine ranged from 0.11 to 1.92 µg L(-1) and ametrine from 0.25 to 1.44 µg L(-1). After analysis of the herbicides, Danio rerio were exposed a range of concentrations found in the river water to check the induction of micronuclei and nuclear abnormalities (NAs) in erythrocytes. Concentrations of atrazine and ametrine1.0 and 1.5 µg L(-1), respectively, induced MN formation in D. rerio. Ametrine was shown to be more genotoxic to D. rerio because a greater incidence of NAs was observed compared with atrazine. Therefore, environmentally relevant concentrations of atrazine and ametrine found in the Piracicaba River are dangerous to the aquatic biota.