miRNAs, target genes expression and morphological analysis on the heart in gestational protein-restricted offspring

Gestational protein restriction was associated with low birth weight, hypertension and higher prevalence of cardiac disorders in adults. Several mechanisms, including epigenetics, could be related with the cardiovascular phenotype on protein-restricted offspring. Thus, we investigated the morphological cardiac effects of gestational protein restriction and left ventricle miRNAs and target genes expression pattern in both 12-day and 16-week old gestational protein-restricted male offspring. Pregnant Wistar rats were allocated into two groups, according to protein supply during pregnancy: NP (normal protein diet-17%) or LP (low protein diet-6%). Dams on the gestational protein-restricted diet had lower body weight gain and higher food intake. Gestational protein-restricted offspring had low birth weight, followed by rapidly body weight recovery, hypertension, and increased myocytes cross-sectional area and collagen fraction at 16-week old age. At 12-days old, miR-184, miR-192, miR-376c, miR-380-3p, miR-380-5p, miR-451, and miR-582-3p had increased expression, and miR-547 and miR-743a had decreased expression in the gestational protein-restricted left ventricle. At 16-week old, let-7b, miR-125a-3p, miR-142-3p, miR-182 and miR-188-5p had increased expression and let-7g, miR-107, miR-127, miR-181a, miR-181c, miR-184, miR-324-5p, miR-383, miR-423-5p and miR-484 had decreased expression in gestational protein-restricted left ventricle. Target predicted gene expression analysis showed higher expression of Dnmt3a, Oxct1, Rictor and Trps1 and lower expression of Bbs1 and Calml3 in 12-day old protein-restricted offspring. 16-week old protein-restricted offspring had higher expression of Adrbk1, Bbs1, Dnmt3a, Gpr22, Inppl1, and Oxct1 genes. In conclusion, gestational protein restriction was related to offspring low birth weight, increased systolic blood pressure and morphological heart alterations that could be related to early heart miRNA expression changes that perpetuate into adulthood and which are associated with the regulation of essential genes involved in cardiovascular development, heart morphology, function, and metabolism144CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP9128/12-32013/12486-

Estresse oxidativo, alterações metabólicas e renais decorrentes da elevada ingestão de sacarose: efeito protetor da N-acetilcisteína (NAC)

Oxidative stress is related with physiopathology of diabetes mellitus type II and with its secondary complications, such as diabetic nephropathy. Thus, the purpose of this study was to examine the effects of n-acetylcysteine NAC, an antioxidant from Allium cepa, on oxidative stress, morphometrical and nutritional parameters and basal metabolism and energetic substrate utilization, serum glucose and oral glucose tolerance test (OGTT), and renal function of high-sucrose intake rats. Animals were initially divided into three groups. Rats in the control group (C; n=10) were given free access to a standard chow and water; (S; n=10) group received standard chow and 30% sucrose in its drinking water; (SN; n=5) group received standard chow and 2g/L NAC and 30% sucrose in its drinking water. After 25 days of treatments, rats were submitted to indirect calorimetry in fasted and feed states. After 30 days of treatments, rats from C and S groups were divided into four groups of five rats each. The (CC) and (SS) groups were given an intra-gastric dose 0,6mL saline (0,9%NaCl) and the (CNAC) and (SNAC) groups were treated with a intra-gastric dose of 0,6mL NAC (160g/day), and then everybody received a intra-gastric dose of glucose solution (20%) for the OGTT. Animals of S and SN groups have higher liquid consumption and lower food consumption than C group. Calorimetric analyses confirm that despite of the final body weight had not statistical difference among groups, S group have lower resting metabolic rate when compared to C and SN animals. Besides, S group has higher respiratory quotient, higher carbohydrate oxidation and lower lipid oxidation, both in fasted and feed states, than C and SN groups, evidencing the beneficial effect of NAC. Fasting plasma glucose is increased in SS and SNAC animals when compared to CC and CNAC, however SN group has glicemic level at 30 min before OGTT decreased when ...(Complete abstract click electronic access below)O estresse oxidativo está envolvido tanto na fisiopatologia do diabetes mellitus tipo II bem como de suas complicações secundárias, como a nefropatia. Sendo assim, o objetivo deste trabalho é verificar os efeitos da N-acetilcisteína (NAC), antioxidante da Allium cepa, sobre o estresse oxidativo, aspectos nutricionais e morfométricos, metabolismo basal e utilização de substrato energético, glicemia de jejum e teste oral de tolerância à glicose (TOTG), bem como na função renal de ratos com elevada ingestão de sacarose. Os animais foram inicialmente divididos em três grupos: C (n=10), recebendo dieta basal e água; S (n=10), recebendo dieta basal e solução de sacarose (30%) e grupo SN (n=5), recebendo dieta basal, solução de sacarose e de NAC (2g/L). Após 25 dias de experimento foram realizadas as calorimetrias no estado alimentado e de jejum, e após 30 dias de experimento, animais do grupo C foram divididos nos grupos CC (n=5) e CNAC (n=5) e os do grupo S, em SS (n=5) e SNAC (n=5), e então todos os animais receberam solução intra-gástrica de sacarose (20%) para a realização do TOTG. Além disso, animais dos grupos CNAC e SNAC receberam, além da solução de sacarose, 0,6 mL de NAC (160g/dia) por gavagem. Animais dos grupos S e SN apresentam maior ingestão de líquido a despeito de menor consumo de ração quando comparados a animais do grupo C. A análise da calorimetria mostra que apesar de o peso final não ter diferido significativamente entre os grupos, animais do grupo S apresentam menor taxa metabólica basal em relação aos grupos C e SN. Além disso, animais do grupo S têm maior QR, maior oxidação de carboidratos e menor oxidação de lipídios, tanto no estado alimentado como no jejum, ao serem comparados a animais dos grupos C e SN, mostrando efeito benéfico da NAC. A glicemia de jejum é elevada nos animais dos grupos SS e SNAC em relação aos ...(Resumo completo, clicar acesso eletrônico abaixo

Epigenetics in fetal programming by gestational protein restriction : global profile of microRNAs expression in the cardiac left ventricle

Orientadores: Patricia Aline Boer, Jose Antonio Rocha GontijoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Fatores nutricionais maternos são determinantes na plasticidade fetal e estão associados à fisiopatologia de doenças cardiovasculares na prole adulta. O modelo experimental de restrição proteica gestacional está relacionado ao baixo peso ao nascimento e ao desenvolvimento de alterações cardíacas e hipertensão no adulto. Diversos mecanismos, como a menor disponibilidade de substrato e a superexposição fetal aos glicocorticoides maternos, estão envolvidos nas alterações decorrentes da restrição proteica gestacional. Além disso, processos epigenéticos, dentre os quais a regulação da expressão gênica exercida pelos miRNAs, estão envolvidos na programação. Poucos estudos investigaram a importância da regulação dos miRNAs em modelo de programação por restrição proteica gestacional. Assim, o objetivo desta tese foi avaliar o padrão de expressão de miRNAs e de seus alvos de predileção no ventrículo esquerdo de ratos Wistar machos submetidos à restrição proteica gestacional. Ratas Wistar prenhes foram alocadas em dois grupos, de acordo com o conteúdo proteico da dieta oferecida durante a prenhes: NP (normal protein - 17%) ou LP (low-protein - 6%). A massa corporal e consumo alimentar foram avaliados semanalmente nas ratas prenhes e na prole. A pressão sistólica da prole foi aferida semanalmente a partir da 6ª a 16ª semana de vida; a massa do ventrículo esquerdo cardíaco, a área de miócitos, a fração de colágeno e a expressão de miRNAs e mRNAs foram avaliados na prole de machos após 12 dias (12d) e 16 semanas (16s) do nascimento. Nossos resultados corroboram dados da literatura que mostram que a restrição proteica gestacional está associada ao menor ganho de peso das ratas durante a prenhez, ao baixo peso ao nascer, com recuperação do peso corporal a partir da 3a semana de vida e ao desenvolvimento de hipertensão arterial a partir da 9a semana. Não encontramos diferença no peso do VE, entretanto, o grupo LP-16s apresentou maior área de miócitos e maior fração de colágeno em relação ao grupo NP-16s. O ventrículo esquerdo de animais do grupo LP-12d apresentou aumento significativo na expressão de mir-184, mir-192, mir-376c, mir-380-3p, mir-380-5p, mir-451, mir-582-3p e redução significativa na expressão de mir-547 e mir-743a em relação ao grupo NP-12d. O grupo LP-16s apresentou aumento na expressão de let-7b, mir-125a-3p, mir-142-3p, mir-182, mir-188-5p e redução significativa na expressão de let-7g, mir-107, mir-127, mir-181a, mir-181c, mir-184, mir-324-5p, mir-383, mir-423-5p, mir-484 em relação ao grupo NP-16s. Após a predição de mRNAs alvos para os miRNAs diferencialmente expressos temos que o grupo LP-12d apresentou aumento na expressão dos genes Dnmt3a, Oxct1, Rictor e Trps1 e redução na expressão dos genes Bbs1 e Calml3 em relação ao NP-12d. O grupo LP-16s apresentou aumento na expressão dos genes Adrkb1, Bbs1, Dnmt3a, Gpr22, Inppl1 e Oxct1 em relação ao NP-16s. Assim, concluímos que a restrição proteica gestacional determina redução no peso ao nascer da prole, elevação na pressão sistólica e alterações na morfologia do ventrículo esquerdo, sugestiva de injúria cardíaca, no adulto. Além disso, está relacionada a alterações tanto precoces quanto em adultos, na expressão de miRNAs cardíacos que estão associados à regulação de genes importantes durante o desenvolvimento cardiovascular, na regulação da morfologia, da função e do metabolismo cardíacoAbstract: Maternal nutritional factors determines fetal plasticity and are associated with the physiopathology of cardiovascular diseases in adult offspring. Gestational protein restriction experimental models are related to low birth weight, development of cardiac disorders and hypertension in adults. Several mechanisms, such as the lower substrate availability and the fetal overexposure to maternal glucocorticoids, are involved with the changes related to gestational protein restriction. Furthermore, epigenetic processes, among which the gene expression regulation by microRNA action, are involved in fetal programming. Few studies have investigated the importance of miRNA regulation on gestational protein restriction model. Thus, the aim of this thesis was to evaluate the miRNAs expression pattern and their predilection targets on cardiac left ventricle of gestational protein restricted rats. Pregnant Wistar rats were allocate in two groups, according to protein supply during pregnancy: NP (normal protein ¿ 17%) or LP (low-protein ¿ 6%). Body weight and food consumption were evaluated weekly in pregnant rats and offspring. Systolic blood pressure was measured weekly in offspring from the sixth to sixteenth week; cardiac left ventricle weight, myocytes cross sectional area, collagen fraction and the miRNA and mRNA expression were evaluated in male offspring after 12-days (12d) and 16-weeks (16w) after birth. Our results corroborate other studies showing that gestational protein restriction is associated with less weight gain of pregnant rats, offspring low birth weight, followed by body weight recovery on third week, and hypertension development from the ninth week. We found no difference on cardiac left ventricle weight; however, the LP-16w has increased myocytes cross sectional area and collagen fraction versus NP-16w. Left ventricle in LP-12d group has significantly increased expression of mir-184, mir-192, mir-376c, mir-380-3p, mir-380-5p, mir-451, mir-582-3p and decreased expression of mir-547, mir-743a versus NP-12d. LP-16w has increased expression of let-7b, mir-125a-3p, mir-142-3p, mir-182, mir-188-5p and decreased expression of let-7g, mir-107, mir-127, mir-181a, mir-181c, mir-184, mir-324-5p, mir-383, mir-423-5p, mir-484 versus NP-16w. After the mRNAs target prediction for the miRNAs differentially expressed, the LP-12d animals has increased expression of Dnmt3a, Oxct1, Rictor and Trps1 genes and decreased expression of Bbs1 and Calml3 genes compared to NP-12d. LP-16w animals has increased expression of Adrbk1, Bbs1, Dnmt3a, Gpr22, Inppl1 and Oxct1 genes compared to NP-16w. In conclusion, the gestational protein restriction leads to offspring low birth weight, increased systolic blood pressure and changes in cardiac left ventricle morphology suggestive of cardiac injuries in adults. Furthermore, it is related to early heart miRNA expression changes that are perpetuate into adulthood and which are associated with the regulation of important genes involved in cardiovascular development, heart morphology, function, and metabolismDoutoradoFisiopatologia MédicaDoutora em Ciências, 9128-12-3CAPE

Effect of gestational protein restriction on left ventricle hypertrophy and heart angiotensin II signaling pathway in adult offspring rats

Maternal protein restriction may be a risk factor for cardiovascular disorders in adulthood. The RAS (renin-angiotensin-system) plays a pivotal role in cardiac remodeling. Components of the RAS, including angiotensin II (AngII) and its receptors type 1 (AT1R) and 2 (AT2R) are expressed in the heart. This study investigates whether gestational protein restriction alters the expression and localization of AT1R and AT2R and RAS signaling pathway proteins in parallel with left ventricle hypertrophy and systemic hypertension in male offspring. Dams were kept on normal (NP, 17% protein) or low (LP, 6% protein) protein diet during pregnancy. Systolic blood pressure (SBP) of male offspring was measured from the 8th to 16th week and left ventricles of 16-wk-old rats were processed for histology, morphometric, immunoblotting and immunohistochemistry. LP offspring showed a significant reduction in birth body weight and SBP increased significantly from the 8th week. Left ventricle mass and cardiomyocytes area were also significantly higher in LP animals. Widespread perivascular fibrosis was not detected in the heart tissue. Analysis by immunoblotting and immunohistochemistry demonstrated a significant enhance in cardiomyocyte expression of AT1R and ERK1 in LP offspring. Expression of PI3K in LP was significantly reduced in cardiomyocytes and in the intramural coronary wall, while AT2R expression was unchanged in the NP group. We also found reduced LP expression of JAK2 and STAT3. In conclusion, our data also suggest that changes in the RAS may play a role in the ventricular growth through upregulation of the AT1-mediated ERK1/2 response, despite unchanged AT2R expression.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Microbiota determines insulin sensitivity in TLR2-KO mice

Environmental factors have a key role in the control of gut microbiota and obesity. TLR2 knockout (TLR2(-/-)) mice in some housing conditions are protected from diet-induced insulin resistance. However, in our housing conditions these animals are not protected from diet-induced insulin-resistance. Aim: The aim of the present study was to investigate the influence of our animal housing conditions on the gut microbiota, glucose tolerance and insulin sensitivity in TLR2(-/-) mice. Material and methods: The microbiota was investigated by metagenomics, associated with hyperinsulinemic euglycemic clamp and GTT associated with insulin signaling through immunoblotting. Results: The results showed that TLR2(-/-) mice in our housing conditions presented a phenotype of metabolic syndrome characterized by insulin resistance, glucose intolerance and increase in body weight. This phenotype was associated with differences in microbiota in TLR2(-/-) mice that showed a decrease in the Proteobacteria and Bacteroidetes phyla and an increase in the Firmicutesphylum, associated with and in increase in the Oscillospira and Ruminococcus genera. Furthermore there is also an increase in circulating LPS and subclinical inflammation in TLR2(-/-). The molecular mechanism that account for insulin resistance was an activation of TLR4, associated with ER stress and JNK activation. The phenotype and metabolic behavior was reversed by antibiotic treatment and reproduced in WT mice by microbiota transplantation. Conclusions: Our data show, for the first time, that the intestinal microbiota can induce insulin resistance and obesity in an animal model that is genetically protected from these processes234CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação465693/2014-8We also acknowledge the financial support INCT de Obesidade e Diabetes 465693/2014-8 (FAPESP) and CAPES/CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico)

N-acetylcysteine in high-sucrose diet-induced obesity: Energy expenditure and metabolic shifting for cardiac health

To study the effects of N-acetylcysteine (NAC, C(5)H(9)-NO(3)S) on high-sucrose diet-induced obesity and its effects on energy metabolism and cardiac oxidative stress, male Wistar 24 rats were divided into four groups (n = 6): (C) given standard chow and water; (N) receiving standard chow and 2 g/l N-acetylcysteine in its drinking water; (HS) given standard chow and 30% sucrose in its drinking water, and (HS-N) receiving standard chow, 30% sucrose and N-acetylcysteine in its drinking water.After 30 days of the treatment, obesity was evidenced in HS rats from enhanced body weight, respiratory quotient, hypertriglyceridemia. As well depressed resting metabolic rate, and oxygen consumption per surface area. HS rats had triacylglycerol accumulation, oxidative stress and metabolic shifting in cardiac tissue. NAC enhanced fat oxidation and energy expenditure, normalizing these adverse effects, comparing HS-N and HS rats. The beta-hydroxyacyl coenzymne-A dehydrogenase activity was higher in HS-N animals, indicating higher heart fatty acid degradation than in HS. NAC normalized myocardial glycogen and lactate dehydrogenase activity, comparing HS-N and HS rats, but had no effects on calorimetric and biochemical parameters in standard-fed rats, comparing N and C groups.In conclusion, N-acetylcysteine offers promising therapeutic value in prevention of high-sucrose induced-obesity and its effect on cardiac tissue. N-acetylcysteine reduced the oxidative stress and prevented the metabolic shifting in cardiac tissue, enhancing fatty acid oxidation and reducing anaerobic metabolism in high-sucrose-fed conditions. The application of this agent in food system via exogenous addition may be feasible and beneficial for antioxidant protection and energy metabolism in cardiac tissue. (C) 2008 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Oral Glutamine Supplementation Reduces Obesity, Pro-Inflammatory Markers, and Improves Insulin Sensitivity in DIO Wistar Rats and Reduces Waist Circumference in Overweight and Obese Humans

In the present study, we aimed to investigate whether chronic oral glutamine (Gln) supplementation may alter metabolic parameters and the inflammatory profile in overweight and obese humans as well as whether Gln may modulate molecular pathways in key tissues linked to the insulin action in rats. Thirty-nine overweight/obese volunteers received 30 g of Gln or alanine (Ala-control) for 14 days. Body weight (BW), waist circumference (WC), hormones, and pro-inflammatory markers were evaluated. To investigate molecular mechanisms, Gln or Ala was given to Wistar rats on a high-fat diet (HFD), and metabolic parameters, euglycemic hyperinsulinemic clamp with tracers, and Western blot were done. Gln reduced WC and serum lipopolysaccharide (LPS) in overweight volunteers. In the obese group, Gln diminished WC and serum insulin. There was a positive correlation between the reduction on WC and LPS. In rats on HFD, Gln reduced adiposity, improved insulin action and signaling, and reversed both defects in glucose metabolism in the liver and muscle. Gln supplementation increased muscle glucose uptake and reversed the increased hepatic glucose production, in parallel with a reduced glucose uptake in adipose tissue. This insulin resistance in AT was accompanied by enhanced IRS1 O-linked-glycosamine association in this tissue, but not in the liver and muscle. These data suggest that Gln supplementation leads to insulin resistance specifically in adipose tissue via the hexosamine pathway and reduces adipose mass, which is associated with improvement in the systemic insulin action. Thus, further investigation with Gln supplementation should be performed for longer periods in humans before prescribing as a beneficial therapeutic approach for individuals who are overweight and obese

Impact of the length of vitamin D deficiency on cardiac remodeling.

This study was aimed to evaluate the influence of vitamin D (VD) deficiency on cardiac metabolism, morphology, and function. Thus, we investigated the relationship of these changes with the length of the nutrient restriction. Male weanling Wistar rats were allocated into 4 groups: C2 (n=24), animals were fed an AIN-93G diet with 1000 IU VD/kg of chow and were kept under fluorescent light for 2 months; D2 (n=22), animals were fed a VD-deficient AIN-93G diet and were kept under incandescent light for 2 months; C4 (n=21) animals were kept in the same conditions of C2 for 4 months; and D4 (n=23) animals were kept in the same conditions of D2 for 4 months. Biochemical analyses showed lower β-hydroxyacyl coenzyme-A dehydrogenase activity and higher lactate dehydrogenase activity in VD-deficient animals. Furthermore, VD deficiency was related to increased cytokines release, oxidative stress, apoptosis, and fibrosis. Echocardiographic data showed left ventricular hypertrophy and lower fractional shortening and ejection fraction in VD-deficient animals. Difference became evident in the lactate dehydrogenase activity, left ventricular weight, right ventricle weight, and left ventricular mass after 4 months of VD deficiency. Our data indicate that VD deficiency is associated with energetic metabolic changes, cardiac inflammation, oxidative stress, fibrosis and apoptosis, cardiac hypertrophy, left chambers alterations, and systolic dysfunction. Furthermore, length of the restriction influenced these cardiac changes